JP2005532067A - Nucleic acid composition for stimulating immune response - Google Patents

Abstract

The present invention provides immunostimulatory nucleic acids comprising CpG motifs and their use in immunostimulation. The invention is based, in part, on the surprising discovery of a new family of nucleic acids that induce higher levels of immune stimulation than known nucleic acids. This finding is surprising, in part because one more than 100 nucleic acid sequences were screened prior to the discovery of the nucleic acid sequences disclosed herein. The immunostimulatory effects of bacterial DNA can be mimicked by synthetic oligodeoxynucleotides (ODNs) containing these CpG motifs.

Description

Field of the Invention
The present invention relates generally to immunostimulatory nucleic acids, compositions thereof, and methods of using such immunostimulatory nucleic acids.

BACKGROUND OF THE INVENTION
Bacterial DNA has an immunostimulatory effect to activate B cells and natural killer cells, but vertebrate DNA has no such effect (Tokunaga, T. et al., 1988. Jpn. J. Cancer Res) Tokunaga, T. et al., 1984, JNCI 72: 955-962; Messina, JP et al., 1991, J. Immunol. 147: 1759-1764; and Krieg, 1998, Applied Oligonucleotide Technology. , C. A. Stein and A. M. Krieg, (Eds.), John Wiley and Sons, Inc., New York, NY, pp. 431-448). Currently, these immunostimulatory effects of bacterial DNA are unmethylated CpG dinucleotides, in particular common bacterial base DNA (CpG motif) is present in bacterial DNA, but not in vertebrate DNA. It is understood that sufficient CpG dinucleotides are present (Krieg et al., 1995 Nature 374: 546-549; Krieg, 1999 Biochim. Biophys. Acta 93321: 1- 10).

  The immunostimulatory effects of bacterial DNA can be mimicked by synthetic oligodeoxynucleotides (ODNs) containing these CpG motifs. Such CpG-ODNs have a high stimulatory effect on human and mouse leukocytes, such as B cell proliferation, cytokine and immunoglobulin secretion, natural killer (NK) cell lytic activity and IFN. -Γ secretion, as well as activation of dendritic cells (DCs) and other antigen presenting cells, which express costimulatory molecules and which are important in promoting the expression of cytokines (especially Thl-like cell responses) Secrete cytokines).

  The immunostimulatory effect of the naturally occurring phosphodiester backbone CpG-ODN is the methylation of the CpG motif, the change to GpC, or otherwise the effect is essentially eliminated when the CpG motif is removed or modified. CpG specificity is high (Krieg et al., 1995 Nature 374: 546-549; Hartmann et al., 1999 Proc. Natl. Acad. Sci USA 96: 9305-10). The phosphodiester CpG-ODN can be formulated with lipids, alum, or other vehicles with retention properties or improved cellular uptake to enhance the immune effect (Yamamoto et al., 1994 Microbiol. Immunol. 38: 831-836 Gramzinski et al., 1998 Mol. Med. 4: 109-118).

  In earlier studies, it was thought that the immunostimulatory CpG motif was in accordance with the formula; purine-purine-CpG-pyrimidine- pyrimidine (Krieg et al., 1995 Nature 374: 546-549; Pisetsky, 1996 J. Immunol. 156: 421-. Hacker et al., 1998 EMBO J. 17: 6230-6240; Lipford et al., 1998 Trends in Microbiol. 6: 496-500). However, mouse lymphocyte react fairly well to phosphodiester CpG which does not follow this "formula" (Yi et al., 1998 J. Inununol. 160: 5898-5906), and the same thing is true. It is now revealed that also applies to B cells and dendritic cells (Hartmann et al., 1999 Proc. Natl. Acad. Sci USA 96: 9305-10; Liang, 1996 J. Clin. Invest. 98: 1119-1129). There is.

Several past investigators examined whether the nucleotide content of an ODN had an effect unrelated to the sequence of the ODN. Interestingly, when the antisense ODN is usually high in content of GG, CCC, CC, CAC, and CG sequences, while the use of bases is compared to what is expected to be random, It has been found that the frequency of TT or TCC nucleotide sequences is decreasing (Smetsers et al., 1996 Antisense Nucleic Acid Drug Develop. 6: 63-67). This means that the over-represented sequences may contain the preferred target elements for antisense oligonucleotides and vice versa. One reason to avoid the use of thymidine-rich ODN for antisense experiments is the release of thymidine, which competes with ³ H-thymidine, which is degraded by nucleases present in cells and is often used in experiments to assess cell proliferation. ((Matson et al., 1992 Antisense Research and Development 2: 325-330).).

Summary of the Invention
The invention is based, in part, on the surprising discovery of a new family of nucleic acids that induce higher levels of immune stimulation than known nucleic acids. This finding is surprising, in part because one more than 100 nucleic acid sequences were screened prior to the discovery of the nucleic acid sequences disclosed herein.

  One aspect of the present invention is the nucleotide sequence of SEQ ID NO: 1 (ODN 10102), SEQ ID NO: 19 (ODN 10103), SEQ ID NO: 45 (ODN 10104), SEQ ID NO: 118 (ODN 10105), or SEQ ID NO: 141 (ODN 10106) Provided a composition comprising an immunostimulatory nucleic acid molecule having

  Another aspect of the invention is a method of stimulating an immune response in a subject in need thereof, the method comprising stimulating the immune response in an effective amount of SEQ ID NO: 1 (ODN 10102), SEQ ID NO: Administering to the subject an immunostimulatory nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO: 19 (ODN 10103), SEQ ID NO: 45 (ODN 10104), SEQ ID NO: 118 (ODN 10105), or SEQ ID NO: 141 (ODN 10106) Provide a way that includes

  Various embodiments of the invention apply equally to the aspects described herein, some of which are described below.

  In one embodiment, the immunostimulatory nucleic acid molecule is SEQ ID NO: 1 (ODN 10102), SEQ ID NO: 19 (ODN 10103), SEQ ID NO: 45 (ODN 10104), SEQ ID NO: 118 (ODN 10105), or SEQ ID NO: 141 (ODN 10106). The nucleotide sequence of

  In another embodiment, the composition further comprises an antigen. Alternatively, the subject to be treated is additionally administered an antigen. The antigen can be selected from the group consisting of microbial antigens, autoantigens, cancer antigens, and allergens. However, it is not limited thereto. In one embodiment, the microbial antigen is selected from the group consisting of bacterial antigens, viral antigens, fungal antigens, and parasitic antigens. In another embodiment, the antigen is encoded by a nucleic acid vector. In related embodiments, the nucleic acid vector is separated from the immunostimulatory nucleic acid. The antigen may be a peptide antigen.

  In another embodiment, the composition further comprises an adjuvant, or the subject is further administered an adjuvant. The adjuvant may be, but is not limited to, a mucosal adjuvant.

  In another embodiment, the composition further comprises a cytokine, or the subject is further administered a cytokine.

  In yet another embodiment, the composition further comprises a therapeutic agent selected from the group consisting of an antibacterial agent, an anticancer agent, an allergy / asthmatic agent, or the subject is further selected from the same group It is administered. In related embodiments, the antimicrobial agent is selected from the group consisting of an antimicrobial agent, an antiviral agent, an antifungal agent, and an antiparasitic agent. In another embodiment, the anticancer agent is selected from the group consisting of a chemotherapeutic agent, a cancer vaccine, and an immunotherapeutic agent. In yet another related embodiment, the allergy / asthmatic agent is a PDE-4 inhibitor, a bronchodilator / β2 agonist, a K + channel opener, a VLA-4 antagonist, a neurokinin antagonist, TXA2 synthesis inhibitor, xanthanine, arachidonic acid antagonist, 5 lipoxygenase inhibitors, thromboxin A2 receptor antagonist, thromboxane A2 antagonist, 5-lipox activation protein inhibitor, And protease inhibitors? It is selected.

  In some embodiments, an immunostimulatory nucleic acid can have a nucleotide backbone comprising at least one backbone modification. In one embodiment, the backbone modification is a phosphorothioate modification. In another embodiment, the nucleotide backbone is chimeric. In one embodiment, the nucleotide backbone is fully modified.

  In one embodiment, the composition further comprises a pharmaceutically acceptable carrier.

  In one embodiment, the immunostimulatory nucleic acid does not comprise methylated CpG dinucleotides. In another embodiment, the immunostimulatory nucleic acid comprises at least four CpG motifs. In yet another embodiment, the immunostimulatory nucleic acid is T-rich. In a related embodiment, the immunostimulatory nucleic acid comprises a poly T sequence. In another embodiment, the immunostimulatory nucleic acid comprises a poly G sequence.

  In certain embodiments, the immunostimulatory nucleic acid is formulated in various ways. In one embodiment, the immunostimulatory nucleic acid is formulated for oral administration. The immunostimulatory nucleic acid may be formulated as a nutritional supplement. In a related embodiment, the nutritional supplement is formulated as a capsule, a pill, or a sublingual tablet. In another embodiment, the immunostimulatory agent is formulated for topical administration. The immunostimulatory nucleic acid may be formulated for parenteral administration, or may be formulated in a sustained release device. The sustained release device can be microparticles, but is not limited thereto. In another embodiment, the immunostimulatory nucleic acid is formulated for delivery to a mucosal surface. The mucosal surface can be selected from the group consisting of an oral surface, a nasal surface, a vaginal surface, and an ocular surface, but is not limited thereto.

  In one embodiment, the immunostimulatory nucleic acid stimulates a mucosal immune response. In another embodiment, the immunostimulatory nucleic acid stimulates a systemic immune response. In important embodiments, the immunostimulatory nucleic acid stimulates both mucosal and systemic immune responses. In some embodiments, the immune response is an antigen specific immune response. In related embodiments, the immunostimulatory nucleic acid is provided in an amount effective to stimulate a mucosal immune response. In other embodiments, the immunostimulatory nucleic acid is provided in an amount effective to stimulate a systemic immune response. In still other embodiments, the immunostimulatory nucleic acid is provided in an amount effective to stimulate an innate immune response.

    In many embodiments, the immunostimulatory nucleic acid is for the treatment or prevention of various diseases. Thus, in one embodiment, the immunostimulatory nucleic acid is provided in an amount effective to treat or prevent an infectious disease. In another embodiment, the immunostimulatory nucleic acid is provided in an amount effective to treat or prevent allergy. In yet another embodiment, the immunostimulatory nucleic acid is provided in an amount effective to treat or prevent asthma. In yet another embodiment, the immunostimulatory nucleic acid is provided in an amount effective to treat or prevent cancer.

  In a related embodiment, the infection is herpes simplex virus infection. In another embodiment, the immunostimulatory nucleic acid is for administration to a subject suffering from or at risk of developing an infection. The infection can be selected from the group consisting of bacterial infections, viral infections, fungal infections, and parasitic infections. In one embodiment, the viral infection is human immunodeficiency virus (HIV-1 and HIV-2), human type I T lymphoproliferative virus (HTLV-I), human type II T lymphoproliferative virus (HTLV-II) , Type I herpes simplex virus (HSV-1), type 2 herpes simplex virus (HSV-2), human papilloma virus (multiple type), hepatitis A virus, hepatitis B virus, hepatitis C virus and hepatitis D It is selected from the group consisting of hepatitis virus, Epstein-Barr virus (EBV), cytomegalovirus, and Molluscum infectious virus. In important embodiments, the viral infection is herpes simplex virus infection.

  In another embodiment, the infection is a microbial species selected from the group consisting of herpesvirus, retrovirus, orthomyxoviridae, toxoplasma, hemophilus, campylobacter, clostridia, E. coli, and staphylococci It is an infectious disease. In a related embodiment, the antigen contained in the antigen or composition administered to the subject is from one of the aforementioned species.

  In certain embodiments, the infection is SARS infection or sarpox infection.

  In another embodiment, the immunostimulatory nucleic acid is a subject that presents or is at risk of developing allergy, a subject that presents or is at risk of developing asthma, or a cancer. For administration to a subject who is or is at risk of developing cancer.

  In an embodiment related to treatment of a subject, the method further comprises isolating an immune cell from the subject and an effective amount of an immunostimulatory nucleic acid and the immune cell to activate the immune cell. It may further include contacting and re-administering the activated immune cells to the subject. In one embodiment, the immune cell is a white blood cell. In another embodiment, the immune cell is a dendritic cell. In another embodiment, the method further comprises contacting an immune cell with an antigen.

  In important embodiments, the subject is a human. In another embodiment, the subject is selected from the group consisting of dogs, cats, horses, cows, pigs, sheep, goats, birds, monkeys, and fish.

  Thus, the methods provided herein can be used on a subject suffering from or at risk of developing an infection, said method treating an infection in the subject. Or a way to prevent. The method can also be used on a subject suffering from or at risk of developing asthma, said method being a method for treating or preventing asthma. The method can be used on a subject suffering from or at risk of developing an allergy, said method being a method for treating or preventing an allergy. Furthermore, this method can be used on a subject suffering from or at risk of developing cancer, said method being a method for treating or preventing cancer. In one embodiment, the cancer is biliary cancer, bone cancer, brain and central nervous system (CNS) cancer, breast cancer, cervical cancer, choroidal cancer, colon cancer, connective tissue cancer, endometrial cancer, esophageal cancer, eyes Cancer, gastric cancer, Hodgkin's lymphoma, carcinoma in situ cancer, laryngeal cancer, lymphoma, liver cancer, lung cancer (eg small cell and non-small cell), melanoma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer , Rectal cancer, sarcoma, skin cancer, testicular cancer, thyroid cancer, and kidney cancer.

  In yet another embodiment of the methods for treatment or prevention provided herein, the method further comprises administering an antibody specific for a cell surface antigen, wherein the immune response is antigen dependent. Provides cytotoxic activity (ADCC).

  In another aspect of the invention, a method of preventing disease in a subject comprises administering an immunostimulatory nucleic acid to the subject on a periodic basis to prevent disease in said subject. The activated nucleic acid has a nucleotide sequence comprising SEQ ID NO: 1 (ODN 10102), SEQ ID NO: 19 (ODN 10103), SEQ ID NO: 45 (ODN 10104), SEQ ID NO: 118 (ODN 10105) or SEQ ID NO: 141 (ODN 10106) .

  In still another aspect, the present invention provides a method of inducing an innate immune response, the method comprising the steps of: administering an effective amount of an immunostimulatory nucleic acid to an innate immune response to a subject Said immunostimulatory nucleic acid comprises SEQ ID NO: 1 (ODN 10102), SEQ ID NO: 19 (ODN 10103), SEQ ID NO: 45 (ODN 10104), SEQ ID NO: 118 (ODN 10105) or SEQ ID NO: 141 ( It has a nucleotide sequence comprising ODN 10106).

  In yet another aspect, the invention provides a method of identifying an immunostimulatory nucleic acid, said method comprising: SEQ ID NO: 1 (ODN 10102), SEQ ID NO: 19 (ODN 10103), SEQ ID NO: 45 (ODN 10104) Measuring the activation control level of an immune cell population contacted with an immunostimulatory nucleic acid comprising the nucleotide sequence of SEQ ID NO: 118 (ODN 10105) or SEQ ID NO: 141 (ODN 10106), and immunity contacted with a test nucleic acid The steps of measuring the activation test level of the cell population and comparing the activation control level with the activation test level, wherein a test level equal to or exceeding the above control level is immunostimulatory Indicates nucleic acid.

  These and other aspects and embodiments of the invention are described in further detail herein.

(Detailed Description of the Invention)
That CpG-containing nucleic acids can be used to stimulate the immune system, thereby treating cancer, infections, allergies, asthma, and other disorders and protecting against infectious diseases after cancer chemotherapy. It was known in the prior art. The strong yet balanced cellular and humoral immune responses resulting from CpG stimulation reflect the body's own natural defense system against invading pathogenic microorganisms and cancer cells. CpG sequences relatively rare in human DNA are commonly found in DNA of infectious microorganisms such as bacteria. The human immune system elicits a rapid and strong immune response against invading pathogens, apparently to recognize CpG sequences as an early warning sign of infection, and without producing the side effects often seen with other immune stimulants. So, it evolved. Thus, CpG-containing nucleic acids that rely on this inactive immune defense mechanism can utilize unique and natural pathways for immunotherapy.

  The effect of CpG nucleic acids on immune modification was discovered by the inventors of the present patent application and has been extensively described in co-pending patent applications. Such co-pending applications include U.S. patent application Ser. No. 08 / 386,063 (filing date 02/07/95) (and related PCT US95 / 01570), 08 / 738,652 (filing date 10/30). No. 08 / 960,774 (filing date 10/30/97) (and related PCT applications / US 97/19791, WO 98/18810), 09 / 191,170 (filing date 11/13) No. 09 / 030,701 (filing date 02/25/98) (and related PC / US 98/03678, 09 / 082,649 (filing date 05/20/98) (and related PC /) US98 / 10408), 09 / 325,193 (filing date 06/03/99) (and related PC / US98 / 04703), 09 / 286,098 ( Application date 04/02/99) (and related PC / US99 / 07335), 09 / 306,281 (filing date 05/06/99) (and related PCT / US99 / 09863) are included. And the entire content of each of the patent applications is incorporated herein by reference.

The present invention is based, in part, on the unexpected discovery of several families of nucleic acids that are more immunostimulatory than previously reported CpG nucleic acids. Each family is represented by a particularly immunostimulatory nucleic acid. These nucleic acid families and their representative members are described in more detail below.
ODN10102 Family The nucleic acid family is composed of the nucleotide sequence having the formula of _{5'X 1 X 2 X 3 X 4} X 5 X 6 X 7 TT CGT CGT TTC GTC GTT3 '( SEQ ID NO: 3), wherein, _{X 1} , X ₂ , X ₃ , X ₄ , X ₅ , X ₆ and X ₇ are each independently selected residues, which are selected from the nucleotide group consisting of adenosine, guanosine, thymidine and cytosine It can be selected. In some embodiments, no flanking residues may be present. Such nucleic acids include the nucleotide sequence of 5 'TT CGT CGT TTC GTC GTT 3' (SEQ ID NO: 4).

In other embodiments, the nucleic acids are X ₁ ; X ₁ and X ₂ ; X ₁ , X ₂ , and X ₃ ; X ₁ , X ₂ , X ₃ and X ₄ ; or X ₁ , X ₂ , X ₃ , It may lack X ₄ and X ₅ or may lack X ₁ to X ₆ or may lack X ₁ to X ₇ .

In one embodiment, X ₁ is thymidine, and / or X ₂ is cytosine, and / or X ₃ is guanosine, and / or X ₄ is thymidine, and / or X ₅ is cytosine, and / or X ₆ is guanosine, And / or X ₁ is thymidine. One skilled in the art can determine the remaining nucleic acid sequences belonging to this family.

  The nucleic acids of this family are generally at least 17 nucleotides in length. In some embodiments, the nucleic acid is at least 19, at least 20, at least 21, at least 22, at least 23, and at least 24 nucleotides in length. In a preferred embodiment, the nucleic acid is 24 nucleotides in length. In still further embodiments, the nucleic acid is greater than 24 nucleotides in length. Examples include nucleic acids that are at least 50, at least 75, at least 100, at least 200, at least 500, at least 1,000 nucleotides in length, or longer. Preferably, the nucleic acid is 17 to 100, more preferably 24 to 100 nucleotides in length.

  All nucleic acids of this first family contain at least three CpG motifs. These nucleic acids may contain four or more CpG motifs. The CpG motifs may be adjacent to each other, or alternatively, they may be spaced apart from each other by a fixed or arbitrary distance from each other.

  Nucleic acids of this family also include overexpression of thymidine nucleotides. These nucleic acids may contain more than 60%, or less than 60%, or less than 55% thymidine.

The invention further assumes, in part, that one has unexpectedly discovered another nucleic acid family that is more immunostimulatory than previously reported CpG nucleic acids. The nucleic acid family is composed of a nucleotide sequence having the formula of Formula _{5'TCG TCG TTT CGT CGT TTC X 1} X 2 X 3 X 4 X 5 X 6 3 '( SEQ ID NO: 5), In the _{formula, X} 1, X ₂ , X ₃ , X ₄ , X ₅ and X ₆ are independently selected residues, which can be selected from the nucleotide group consisting of adenosine, guanosine, thymidine and cytosine . In some embodiments, no flanking residues may be present. Such nucleic acids include the nucleotide sequence of 5 'TCG TCG TTT CGT CGT TTC 3' (SEQ ID NO: 6).

In other embodiments, the nucleic acid, the absence of _{X 6,} lack of _{X 6} and _{X 5,} or lack of _X 6, _{X 5,} and _{X 4,} the lack of the _{X 6} to _{X 3,} from _{X 6} to _{X 2} Or a lack of X ₆ to X ₁ may have occurred. .

In one embodiment, X ₁ is cytosine. In another embodiment, X ₂ is guanosine. In another embodiment, X ₃ is thymidine. In another embodiment, X ₄ is thymidine. One skilled in the art can determine the remaining nucleic acid sequences belonging to this family.

  The nucleic acids of this latter family are generally at least 18 nucleotides in length. In some embodiments, the nucleic acid is at least 20, at least 22, at least 23, and at least 24 nucleotides in length. In a preferred embodiment, the nucleic acid is 24 nucleotides in length. In still further embodiments, the nucleic acid is greater than 24 nucleotides in length. Examples include nucleic acids at least 50, at least 75, at least 100, at least 200, at least 500, at least 1,000 nucleotides in length, or longer. Preferably, the nucleic acid is 18 to 100, more preferably 24 to 100 nucleotides in length.

  All nucleic acids of this second family contain at least four CpG motifs. These nucleic acids may, depending on the embodiment, comprise more than four CpG motifs. The CpG motifs may be adjacent to each other, or alternatively, they may be spaced apart from each other by a fixed or arbitrary distance from each other.

  Nucleic acids of this family also include overexpression of thymidine nucleotides. These nucleic acids may contain more than 60%, or less than 60%, or less than 55% thymidine.

  In another aspect, the invention provides a nucleic acid comprising the nucleotide sequence of TCG TCG TTT CGT CGT TTC GTC GTT (SEQ ID NO: 1) (ODN 10102). As described in more detail in the Examples, this nucleic acid was identified only after screening a large number of nucleic acids for nucleic acids having immunostimulatory activity similar to or greater than previously identified immunostimulatory nucleic acids. More specifically, this nucleic acid was compared to a nucleic acid having the nucleotide sequence of TCG TCG TTT TGT CGT TTT GTC GTT (SEQ ID NO: 2) which has already been shown to be immunostimulatory. Nucleic acids comprising SEQ ID NO: 1 were identified only after screening about 165 nucleic acids for those with greater immunostimulatory potential than those of nucleic acids comprising SEQ ID NO: 2. The difference in activity is surprising. This is because there is only a minimal difference (ie, the difference between the two nucleotides) between SEQ ID NO: 1 and SEQ ID NO: 2. It was unexpected that a minimal change in such a sequence would result in a statistically significant increase in immune stimulation.

  In yet another aspect of the invention, a nucleic acid is provided having the following nucleotide sequence: That is, 5'TCG TCG TTT CGT CGT CGT TTC GTC GT3 '(SEQ ID NO: 7), 5' TCG TCG TTT CGT CGT CGT TTC GTC G 3 '(SEQ ID NO: 8), 5' TCG TCG TTT CGT CGT TTC GTC 3 '(SEQ ID NO: 9) ), 5 'TCG TCG TTT CGT CGT TTC GT3' (SEQ ID NO: 10), 5 'TCG TCG TTT CGT CGT TGT G3' (SEQ ID NO: 11), 5 'CG TCG TTT CGT CGT TTC GTC GTT 3' (SEQ ID NO: 2) , 5 'G TCG TTT CGT CGT CGT TTC GTC GTT3' (SEQ ID NO: 13), 5 'TCG TTT CGT CGT CGT TTC GTC GTT 3' (SEQ ID NO: 14), 5 'CG TTT CGT CGT CGT TTC GT GTT3 '(SEQ ID NO: 15), 5' GTTT CGT CGT CGT TTC GTC GTT 3 '(SEQ ID NO: 16), 5' TTT CGT CGT TTC GTC GTT 3 '(SEQ ID NO: 17), and 5' TT CGT CGT TTC GTC GTT 3 ' It is sequence number 18).

  The nucleic acid of the present invention has other immunostimulatory motifs such as poly T motif, poly G motif, TG motif, poly A motif, poly C motif etc, provided that the core sequences of SEQ ID NO: 4 and SEQ ID NO: 6 are present. It can further include. These immunostimulatory motifs are described below or as described in US Ser. No. 09 / 669,187 (filed on Sep. 25, 2000) and published PCT patent application PCT / US00 / 26383 (publication number WO 01/22972), It is described in more detail.

(ODN 10103 family)
The nucleic acid family is composed of a nucleotide sequence having the formula _{5'X 1 X 2 X 3 X 4} X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 GGT CGT TTT3 '( SEQ ID NO: 20), In the formula, X ₁ , X ₂ , X ₃ , X ₄ , X ₅ , X ₆ , X ₇ , X ₈ , X ₉ , X ₁₀ , X ₁₁ and X ₁₂ are independently selected residues And the residue can be selected from the group of nucleotides consisting of adenosine, guanosine, thymidine and cytosine. In some embodiments, no flanking residues may be present. Such nucleic acids include the nucleotide sequence of 5'GGT CGT TTT 3 '(SEQ ID NO: 21).

In other embodiments, the nucleic acid is absent X ₁ , absent X ₁ and X ₂ , absent X ₁ , X ₂ and X ₃ , absent X ₁ , X ₂ , X ₃ and X ₄ , or Lack of X ₁ , X ₂ , X ₃ , X ₄ and X ₅ , Lack of X ₁ to X ₆ , Lack of X ₁ to X ₇ , Lack of X ₁ to X ₈ , X ₁ to X _It may be with a loss of _9; a loss of X ₁ to X _10; a loss of X ₁ to X ₁₁ ; and a loss of X ₁ to X ₁₂ .

In various embodiments, X ₁ is thymidine, and / or X ₂ is cytosine, and / or X ₃ is guanosine, and / or X ₄ is thymidine, and / or X ₅ is cytosine, and / or X ₆ is guanosine. And / or X ₇ is thymidine, and / or X ₈ is thymidine, and / or X ₉ is thymidine, and / or X ₁₀ is thymidine, and / or X ₁₁ is thymidine, and / or X ₁₂ is cytosine. . One skilled in the art can determine the remaining nucleic acid sequences belonging to this family.

  The nucleic acids of this family are generally at least 9 nucleotides in length. In some embodiments, the nucleic acid is at least 10, at least 12, at least 15, at least 18, at least 20, and at least 21 nucleotides in length. In a preferred embodiment, the nucleic acid is 21 nucleotides in length. In yet another embodiment, the nucleic acid is greater than 21 nucleotides in length. Examples include nucleic acids of at least 50, at least 75, at least 100, at least 200, at least 500, at least 1,000 nucleotides in length, or longer. Preferably, the nucleic acid is 9 to 100, more preferably 21 to 100 nucleotides in length.

  All nucleic acids of this first family contain at least one CpG motif. These nucleic acids may contain two, three, four, or more CpG motifs. The CpG motifs may be adjacent to each other or they may be spaced apart from each other by a fixed or arbitrary distance.

  Nucleic acids of this family also include overexpression of thymidine nucleotides. These nucleic acids can comprise at least 60%, at least 55%, or at least 50% thymidine.

The invention further assumes, in part, that one has unexpectedly discovered another nucleic acid family that is immunostimulatory comparable to previously reported CpG nucleic acids. This nucleic acid family consists of a nucleotide sequence having the formula ₅ 'TCG TCG TTT TTC X ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ X ₈ X ₉ 3' (SEQ ID NO: 22), wherein X ₁ to X ₉ are each independently selected residues, which can be selected from the nucleotide group consisting of adenosine, guanosine, thymidine and cytosine. In some embodiments, no flanking residues may be present. By way of example, the nucleic acid may comprise the nucleotide sequence of 5 'TCG TCG TTT TTC 3' (SEQ ID NO: 23).

In other embodiments, the nucleic acid, the absence of _{X 9,} lack of _{X 9} and _{X 8,} lack of X 9, _{X 8,} and _{X 7,} lack from _{X 9} to _{X 6,} from _{X 9} in _{X 5} lack, lack from _{X 9} to _{X 4,} the lack of the _{X 9} to _{X 3,} it may be one with a lack of lack from _{X 9} to _{X 2,} and from _{X 9} to _{X 1.}

In various embodiments, X ₁ is guanosine, and / or X ₂ is guanosine, and / or X ₃ is thymidine, and / or X ₄ is cytosine, and / or X ₅ is guanosine, and / or X ₆ is thymidine. And / or X ₇ is thymidine, and / or X ₈ is thymidine, and / or X ₉ is thymidine. One skilled in the art can determine the remaining nucleic acid sequences belonging to this family.

  The nucleic acids of this family are generally at least 12 nucleotides in length. In some embodiments, the nucleic acid is at least 15, at least 18, at least 21 nucleotides in length. In a preferred embodiment, the nucleic acid is 21 nucleotides in length. In still further embodiments, the nucleic acid is greater than 21 nucleotides in length. Examples include nucleic acids that are at least 50, at least 75, at least 100, at least 200, at least 500, at least 1,000 nucleotides in length, or longer. Preferably, the nucleic acid is 12 to 100, more preferably 21 to 100 nucleotides in length.

  All nucleic acids of this second family contain at least two CpG motifs. These nucleic acids may contain 3 or 4 or more CpG motifs, depending on the embodiment. The CpG motifs may be adjacent to one another or they may be spaced apart from one another by a fixed or arbitrary distance from one another.

  Nucleic acids of this family also include overexpression of thymidine nucleotides. These nucleic acids can comprise at least 60%, at least 55%, or at least 50% thymidine.

  In another aspect, the invention provides a nucleic acid comprising the nucleotide sequence of TCG TCG TTT TTC GGT CGT TTT (SEQ ID NO: 19) (ODN 10103). As described in more detail in the Examples, this nucleic acid was identified only after screening a large number of nucleic acids for nucleic acid having an immunostimulatory activity similar to or greater than previously identified immunostimulatory nucleic acids. . More specifically, this nucleic acid was compared to a nucleic acid having the nucleotide sequence of TCG TCG TTT TGT CGT TTT GTC GTT (SEQ ID NO: 2) which has previously been shown to be immunostimulatory. A nucleic acid comprising SEQ ID NO: 19 was identified only after screening approximately 165 nucleic acids for one having an immunostimulatory capacity similar to or greater than that of the nucleic acid comprising SEQ ID NO: 2. The difference in activity is surprising. Because there is a minimal difference between SEQ ID NO: 19 and SEQ ID NO: 2 (ie, SEQ ID NO: 19 includes three additional internal nucleotides (ie, TCG) compared to SEQ ID NO: 2) That's because it's only a 'nucleotide deletion'. It was unexpected that such changes in the sequence resulted in increased immune stimulation.

  In yet another aspect of the invention, a nucleic acid is provided having the following nucleotide sequence: That is, 5 'TCG TCG TTT TTC GGT CGT TT3' (SEQ ID NO: 24), 5 'TCG TCG TTT TTC GGT CGT 3' (SEQ ID NO 25), 5 'TCG TCG TTT TTC GGT CGT 3' (SEQ ID NO 26), 5 'TCG TCG TTT TTC GGT CG3' (SEQ ID NO: 27), 5 'TCG TCG TTT TTC GGT C 3' (SEQ ID NO: 28), 5 'TCG TCG TTT TTC GGT 3' (SEQ ID NO 29), 5 'TCG TCG TTT TTC GG3 '(SEQ ID NO: 30), 5' TCG TCG TTT TTC G3 '(SEQ ID NO: 44), 5' TCG TCG TTT TTC 3 '(SEQ ID NO: 31), 5' TCG TCG TTT TTC GGT CGT TTT 3 '(SEQ ID NO: 32), 5 CG TCG TTT TTC GGT CGT TTT3 '(SEQ ID NO: 33), 5' GTCG TTT TTC GGT CGT TTT 3 '(SEQ ID NO: 34), 5' TCG TTT TTC GGT CGT TTT 3 '(SEQ ID NO 35), 5' CG TTT TTC GGT CGT TTT3 '(SEQ ID NO: 36) 5' G TTT TTC GGT CGT TTT 3 '(SEQ ID NO: 37), 5' TTT TTC GGT CGT TTT 3 '(SEQ ID NO: 38), 5' TT TTC GGT CGT TTT 3 '(SEQ ID NO: 39) ), 5'TTTCGTCGT TTT 3 '(SEQ ID NO: 40), 5'TTCGGT CGT TTT3' (SEQ ID NO: 41), 5 'TCGGT CGT TTT3' (SEQ ID NO: 42), 5 'CGGT CGT TTT3' (Sequence number 43), 5'GGT CGT TTT 3 '(SEQ ID NO: 21).

  These immunostimulatory nucleic acids have the ability to activate the innate immune system and enhance both humoral and cellular antigen specific responses when co-administered with an antigen such as hepatitis B surface antigen. Have the ability to The examples provided herein demonstrate that these nucleic acids can stimulate human immune cells in vitro, and mouse cells in vitro and in vivo. Indicates that it can stimulate. When compared to sequences known to be potent adjuvants, the nucleic acid of SEQ ID NO: 19 is at least 10-15% as a vaccine adjuvant.

  The nucleic acid of the present invention has other immunostimulatory motifs such as poly T motif, poly G motif, TG motif, poly A motif, poly C motif and the like, provided that the core sequences of SEQ ID NO: 21 and SEQ ID NO: 23 exist. It can further include. These immunostimulatory motifs are described in more detail below or as described in US Ser. No. 09 / 669,187, filed Sep. 25, 2000, and published PCT patent application PCT / US00 / 26383 (publication number WO 01/22972). It is described in.

(ODN 10104 family)
The nucleic acid family is composed of a nucleotide sequence having the formula _{5'X 1 X 2 X 3 X 4} X 5 X 6 TTT CGT CGT TTT GTC GTT3 '( SEQ ID NO: 46), In the _formula, X 1, _{X 2} , X ₃ , X ₄ , X ₅ and X ₆ are independently selected residues, which can be selected from the nucleotide group consisting of adenosine, guanosine, thymidine and cytosine. In some embodiments, no flanking residues may be present. Such nucleic acids include the nucleotide sequence of 5 'TTT CGT CGT TTT GTC GTT 3' (SEQ ID NO: 47).

In other embodiments, the nucleic acid is absent X ₁ , absent X ₁ and X ₂ , absent X ₁ , X ₂ and X ₃ , absent X ₁ , X ₂ , X ₃ and X ₄ , or It may be with the absence of X ₁ , X ₂ , X ₃ , X ₄ and X ₅ . Accordingly, the present invention is intended to encompass nucleic acids having the following nucleotide sequences: That is, 5 'X ₂ X ₃ X ₄ X ₅ X ₆ TTT CGT CGT TTT GTC GTT 3' (SEQ ID NO: 48), 5 'X ₃ X ₄ X ₅ X ₆ TTT CGT CGT TTT GTC GTT 3' (SEQ ID NO: 49), _{5 '} X ₄ X ₅ X ₆ TTT CGT CGT TTT GTC GTT3' (SEQ ID NO: 50), _{5 '} X ₅ X ₆ TTT CGT CGT TTT GTC GTT 3' (SEQ ID NO: 51), 5 'X ₆ TTT CGT CGT TTT GTC GTT3 '(SEQ ID NO: 52).

In one embodiment, X ₁ is thymidine. In another embodiment, X ₂ is cytosine. In another embodiment, X ₃ is guanosine. In another embodiment, X ₄ is thymidine. In yet another embodiment, X ₅ is cytosine. In yet another embodiment, X ₆ is guanosine. The present invention further encompasses the combination of multiple flanking residues as follows (blank cells are N residues, ie naturally occurring, as noted herein or known in the art) Or non-naturally occurring nucleotides).

  Table 1 represents only some of the possible nucleic acids that are members of the first nucleic acid family. One skilled in the art can determine the sequence of the remaining nucleic acids belonging to this family.

  The nucleic acids of this family are generally at least 18 nucleotides in length. In some embodiments, the nucleic acid is at least 19, at least 20, at least 21, at least 22, at least 23, and at least 24 nucleotides in length. In a preferred embodiment, the nucleic acid is 24 nucleotides in length. In yet further embodiments, the nucleic acid is greater than 24 nucleotides in length. Examples include nucleic acids at least 50, at least 75, at least 100, at least 200, at least 500, at least 1,000 nucleotides in length, or longer. Preferably, the nucleic acid is 18 to 100, more preferably 24 to 100 nucleotides in length.

  All nucleic acids of this first family contain at least three CpG motifs. These nucleic acids may contain 4 or 5 or more CpG motifs. The CpG motifs may be adjacent to each other or they may be spaced apart from each other by a fixed or arbitrary distance.

  Nucleic acids of this family also include overexpression of thymidine nucleotides. These nucleic acids can contain more than 60% or less than 60%, or less than 55% thymidine.

The invention further assumes, in part, that one has unexpectedly discovered another nucleic acid family that is more immunostimulatory than previously reported CpG nucleic acids. This nucleic acid family consists of a nucleotide sequence having the formula 5 'TCG TCG TTT CGT CGT TTT GT X ₁ X ₂ X ₃ X ₄ 3' (SEQ ID NO: 95), wherein X ₁ , X ₂ , X ₃ and X ₄ are independently selected residues, which can be selected from the nucleotide group consisting of adenosine, guanosine, thymidine and cytosine. In some embodiments, no flanking residues may be present. By way of example, the nucleic acid comprises the nucleotide sequence of 5 'TCG TCG TTT CGT, CGT, TTT GT3' (SEQ ID NO: 96).

In other embodiments, the nucleic acid, the absence of _{X 4,} the lack of _{X 4} and _{X 3,} the lack of _{X 4} and _{X 3,} or _X 4, _{X 3,} and lack of _{X 2} is even arose Good. Accordingly, the present invention is intended to encompass nucleic acids having the following nucleotide sequences: That is, 5 'TCG TCG TTT CGT CGT TTT GT X ₁ X ₂ X ₃ 3' (SEQ ID NO: 97), 5 'TCG TCG TTT CGT CGT TTT GT X ₁ X ₂ 3' (SEQ ID NO 98), and 5 'TCG TCG TTT CGT CGT TTT GT X ₁ 3 '(SEQ ID NO: 99).

In one embodiment, X ₁ is cytosine. In another embodiment, X ₂ is guanosine. In another embodiment, X ₃ is thymidine. In another embodiment, X ₄ is thymidine. The present invention further includes the combination of multiple flanking residues as follows (blank cells are N residues, ie, as described herein or otherwise known, naturally occurring or natural) Can be any of the nucleotides not present in

  Table 2 represents only some of the possible nucleic acids that are members of the second nucleic acid family. One skilled in the art can determine the sequence of the remaining nucleic acids belonging to this family.

  The nucleic acids of this latter family are generally at least 20 nucleotides in length. In some embodiments, the nucleic acid is at least 21, at least 22, at least 23, and at least 24 nucleotides in length. In a preferred embodiment, the nucleic acid is 24 nucleotides in length. In yet further embodiments, the nucleic acid is greater than 24 nucleotides in length. Examples include nucleic acids of at least 50, at least 75, at least 100, at least 200, at least 500, at least 1,000 nucleotides in length, or longer. Preferably, the nucleic acid is 20 to 100, more preferably 24 to 100 nucleotides in length.

  All nucleic acids of this second family contain at least four CpG motifs. These nucleic acids may comprise 5 or more CpG motifs, depending on the embodiment. The CpG motifs may be adjacent to each other or they may be spaced apart from each other by a fixed or arbitrary distance.

  Nucleic acids of this family also include overexpression of thymidine nucleotides. These nucleic acids can comprise more than 60% or less than 60%, or less than 55% thymidine.

  In another aspect, the invention provides a nucleic acid comprising the nucleotide sequence of TCG TCG TTT CGT CGT TTT GTC GTT (SEQ ID NO: 45) (ODN 10104). As described in more detail in the Examples, this nucleic acid was identified only after screening a large number of nucleic acids for nucleic acid having immunostimulatory activity similar to or greater than previously identified immunostimulatory nucleic acids. More specifically, this nucleic acid was compared to a nucleic acid having the nucleotide sequence of TCG TCG TTT TGT CGT TTT GTC GTT (SEQ ID NO: 2) which has previously been shown to be immunostimulatory. A nucleic acid comprising SEQ ID NO: 45 was identified only after screening approximately 165 nucleic acids for a nucleic acid having an immunostimulatory capacity greater than that of the nucleic acid comprising SEQ ID NO: 2. The difference in activity is surprising. This is because there is only minimal difference between SEQ ID NO: 45 and SEQ ID NO: 2 (ie, substitution of thymidine (SEQ ID NO: 2) with cytosine (SEQ ID NO: 45)). It was unexpected that a minimal change in such sequences would lead to a statistically significant increase in immunostimulatory properties.

  In yet another aspect of the invention, a nucleic acid is provided having the following nucleotide sequence: That is, 5 'CG TCG TTT CGT CGT CTT TTT GTC GTT3' (SEQ ID NO: 110), 5 'GTCG TTT CGT CGT CGT TTT GTC GTT 3' (SEQ ID NO: 111), 5 'TCG TTT CGT CGT TTT GTC GTT 3' (SEQ ID NO: 112) ), 5 'CG TTT CGT CGT CGT TTT GTC GTT3' (SEQ ID NO: 113), 5 'GTTT CGT CGT CGT TTT GTC GTT 3' (SEQ ID NO: 114), 5 'TTT CGT CGT TTT GTC GTT 3' (SEQ ID NO: 47) 'TCG TCG TTT CGT CGT TTT GTC GT 3' (SEQ ID NO: 115), 5 'TCG TCG TTT CGT CGT TGT GTC G 3' (SEQ ID NO: 116), 5 'TCG TCG TTT C T CGT TTT GTC3 a '(SEQ ID NO: 117), 5'TCG TCG TTT CGT CGT TTT GT3' (SEQ ID NO: 96).

  The nucleic acid of the present invention has other immunostimulatory motifs such as poly T motif, poly G motif, TG motif, poly A motif, poly C motif and the like, provided that the core sequences of SEQ ID NO: 47 and SEQ ID NO: 96 exist. It can further include. These immunostimulatory motifs are described below or as described in US Ser. No. 09 / 669,187 (filed on Sep. 25, 2000) and published PCT patent application PCT / US00 / 26383 (publication number WO 01/22972), It is described in more detail.

(ODN 10105 family)
The nucleic acid family, having the formula _{5'X 1 X 2 X 3 X 4} X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 X 15 TTT TTT CGA3 '( SEQ ID NO: 119) A nucleotide sequence, wherein X ₁ , X ₂ , X ₃ , X ₄ , X ₅ , X ₆ , X ₇ , X ₈ , X ₉ , X ₁₀ , X ₁₁ , X ₁₁ , X ₁₂ , X ₁₃ , X ₁₄ and X ₁₅ are independently selected residues, which can be selected from the nucleotide group consisting of adenosine, guanosine, thymidine and cytosine. In some embodiments, no flanking residues may be present. Such nucleic acids include the nucleotide sequence of 5 'TTT TTT CGA 3' (SEQ ID NO: 120).

In other embodiments, the nucleic acid is absent X ₁ , absent X ₁ and X ₂ , absent X ₁ , X ₂ and X ₃ , absent X ₁ , X ₂ , X ₃ and X ₄ , or Lack of X ₁ , X ₂ , X ₃ , X ₄ , and X ₅ , Lack of X ₁ to X ₆ , Lack of X ₁ to X ₇ , Lack of X ₁ to X ₈ , X ₁ to X ₉ Lack of up to, X ₁ to X ₁₀ , X ₁ to X ₁₁ , L ₁ to X ₁₂ , L ₁ to X ₁₃ , L ₁ to X ₁₄ , It may be with a lack of X ₁ to X ₁₅ .

In various embodiments, X ₁ is thymidine, and / or X ₂ is cytosine, and / or X ₃ is guanosine, and / or X ₄ is thymidine, and / or X ₅ is cytosine, and / or X ₆ is guanosine. And / or X ₇ is thymidine, and / or X ₈ is thymidine, and / or X ₉ is thymidine, and / or X ₁₀ is thymidine, and / or X ₁₁ is guanosine, and / or X ₁₂ is thymidine, and And / or X ₁₃ is cytosine, and / or X ₁₄ is guanosine, and / or X ₁₅ is thymidine. One skilled in the art can determine the remaining nucleic acid sequences belonging to this family.

  The nucleic acids of this family are generally at least 9 nucleotides in length. In some embodiments, the nucleic acid is at least 10, at least 12, at least 15, at least 18, at least 20, at least 22, and at least 24 nucleotides in length. In a preferred embodiment, the nucleic acid is 24 nucleotides in length. In still further embodiments, the nucleic acid is greater than 24 nucleotides in length. Examples include nucleic acids of at least 50, at least 75, at least 100, at least 200, at least 500, at least 1,000 nucleotides in length, or more. Preferably, the nucleic acid is 9 to 100, more preferably 24 to 100 nucleotides in length.

  All nucleic acids of this first family contain at least one CpG motif. These nucleic acids may contain two, three, four or more CpG motifs. The CpG motifs may be adjacent to each other or they may be spaced apart from each other by a fixed or arbitrary distance.

  Nucleic acids of this family also include overexpression of thymidine nucleotides. These nucleic acids can comprise at least 60%, at least 55%, or at least 50% thymidine.

The invention further assumes, in part, that one has unexpectedly discovered another family of nucleic acids that is as immunostimulatory as previously reported CpG nucleic acids. This nucleic acid family consists of a nucleotide sequence having the formula 5 'TCG TCG TTT TGT CGT TTT TX ₁ X ₂ X ₃ X ₄ X ₅ 3' (SEQ ID NO: 121), wherein X ₁ to X ₉ are And each independently selected residue, which can be selected from the nucleotide group consisting of adenosine, guanosine, thymidine and cytosine. In some embodiments, no flanking residues may be present. As an example, such a nucleic acid comprises the nucleotide sequence of 5 'TCG TCG TTT TGT CGT TTT 3' (SEQ ID NO: 122).

In other embodiments, the nucleic acid, the absence of _{X 5,} lack of _{X 5} and _{X 4,} the lack of X 5, _{X 4,} and _{X 3,} the lack of the _{X 5} to _{X 2,} and from _{X 5} to _{X 1} The absence of may have occurred.

In various embodiments, X ₁ is thymidine, and / or X ₂ is thymidine, and / or X ₃ is cytosine, and / or X ₄ is guanosine, and / or X ₅ is adenine It is. One skilled in the art can determine the remaining nucleic acid sequences belonging to this family.

  The nucleic acids of this family are generally at least 19 nucleotides in length. In some embodiments, the nucleic acid is at least 20, at least 22, and at least 24 nucleotides in length. In a preferred embodiment, the nucleic acid is 24 nucleotides in length. In still further embodiments, the nucleic acid is greater than 24 nucleotides in length. Examples include nucleic acids of at least 50, at least 75, at least 100, at least 200, at least 500, at least 1,000 nucleotides in length, or more. Preferably, the nucleic acid is 19 to 100, more preferably 24 to 100 nucleotides in length.

  All nucleic acids of this second family contain at least three CpG motifs. These nucleic acids may, depending on the embodiment, comprise four or more CpG motifs. The CpG motifs may be adjacent to each other or they may be spaced apart from each other by a fixed or arbitrary distance.

  Nucleic acids of this family also include overexpression of thymidine nucleotides. These nucleic acids can comprise at least 60%, at least 55%, or at least 50% thymidine.

  In another aspect, the invention provides a nucleic acid consisting of a nucleotide sequence having the formula TCG TCG TTT TGT CGT TTT TTT CGA (SEQ ID NO: 118) (ODN 10105). As described in more detail in the Examples, this nucleic acid was identified only after screening a large number of nucleic acids for nucleic acid having immunostimulatory activity similar or superior to the already identified immunostimulatory nucleic acid. More specifically, this nucleic acid was compared to a nucleic acid having the nucleotide sequence of TCG TCG TTT TGT CGT TTT GTC GTT (SEQ ID NO: 2) which has already been shown to be immunostimulatory. A nucleic acid comprising SEQ ID NO: 118 was identified only after screening approximately 165 nucleic acids for those with immunostimulatory potential similar to or greater than that of the nucleic acid comprising SEQ ID NO: 2. The difference in activity is surprising. This is because there is 79% identity between SEQ ID NO: 118 and SEQ ID NO: 2 (ie, the 5 'nucleotides on the 3' side differ between SEQ ID NO: 118 and SEQ ID NO: 2). It was unexpected that changes within the sequence would result in increased immune stimulation.

In yet another aspect of the invention, a nucleic acid having the following nucleotide sequence is obtained: That is,
5 'TCG TCG TTT TGT CGT TTT TTT CG 3' (SEQ ID NO: 123), 5 'TCG TCG TTT TGT CGT TTT TTT C 3' (SEQ ID NO: 124), 5 'TCG TCG TTT TGT CGT TTT TTT 3' (SEQ ID NO: 125), 5 'TCG TCG TTT TGT CGT TTT TT 3' (SEQ ID NO: 126), 5 'CG TCG TTT TGT CGT TTT TTT CGA 3' (SEQ ID NO: 127), 5 'G TCG TTT TGT CGT TTT TTT CGA 3' (SEQ ID NO: 128), 5 ′ TCG TTT TGT CGT TTT TTT CGA 3 ′ (SEQ ID NO: 129), 5 ′ CG TTT TGT CGT TTT TTT CGA 3 ′ (SEQ ID NO: 130), 5 ′ G TT TGT CGT TTT TTT CGA 3 '(SEQ ID NO: 131), 5' T T TGT CGT TTT TTT CGA 3 '(SEQ ID NO: 132), 5' TT TGT CGT TTT TTT CGA 3 '(SEQ ID NO: 133), 5' T GT CGT TTT TTT CGA 3 '(SEQ ID NO 134), 5' TGT CGT TTT TTT CGA 3 '(SEQ ID NO: 135), 5' GT CGT TTT TTT CGA 3 '(SEQ ID NO: 136), 5' T CGT TTT TTT CGA 3 '(SEQ ID NO: 137), 5' CGT TTT TTT CGA 3 '( SEQ ID NO: 138), 5 'GT TTT TTT CGA 3' (SEQ ID NO: 139), and 5 'TTTT TTT CGA 3' (SEQ ID NO: 140).

  These immunostimulatory nucleic acids have the ability to activate the innate immune system and enhance both humoral and cellular antigen specific responses when co-administered with an antigen such as hepatitis B surface antigen . In the examples provided herein, these nucleic acids can stimulate human immune cells in vitro, and also in vitro and in vivo mouse cells. Demonstrate that it can stimulate. When compared to sequences known to be potent adjuvants, it is found that the nucleic acid of SEQ ID NO: 118 acts equally or better as a vaccine adjuvant.

  The nucleic acid of the present invention has another immunostimulatory motif such as poly T motif, poly G motif, TG motif, poly A motif, poly C motif, etc., provided that the core sequences of SEQ ID NO: 120 and SEQ ID NO: 122 exist. It can further include. These immunostimulatory motifs are described in more detail below or as described in US Ser. No. 09 / 669,187, filed Sep. 25, 2000, and published PCT patent application PCT / US00 / 26383 (publication number WO 01/22972). It is described in.

(ODN 10106 family)
The nucleic acid comprises a nucleic acid sequence having the formula TCG TCG TTT TTC GTG CGT TTT T (SEQ ID NO: 141) (ODN 10106).

  The above sequence can be flanked by a number of separately selected nucleotide residues which can be selected from the group of nucleotides consisting of adenosine, guanosine, thymidine and cytosine.

  The nucleic acids of this family are at least 22 nucleic acids long. In a preferred embodiment, the nucleic acid is 22 nucleotides in length. In yet further embodiments, the nucleic acid is greater than 22 nucleotides in length. Examples include nucleic acids of at least 50, at least 75, at least 100, at least 200, at least 500, at least 1,000 nucleotides in length, or more. Preferably, the nucleic acid is 12-100.

  All nucleic acids of this first family contain at least four CpG motifs. These nucleic acids may contain five or more CpG motifs. The CpG motifs may be adjacent to each other or they may be spaced apart from each other by a fixed or arbitrary distance.

  Nucleic acids of this family also include overexpression of thymidine nucleotides. These nucleic acids can contain more than 60%, less than 60%, or less than 55% thymidine.

  In another aspect, the invention provides a nucleic acid consisting of the nucleotide sequence of TCG TCG TTT TTC GTG CGT TTT T (SEQ ID NO: 141). As described in more detail in the Examples, this nucleic acid was identified only after screening a large number of nucleic acids for nucleic acid having immunostimulatory activity that is similar to or greater than the already identified immunostimulatory nucleic acid. More specifically, this nucleic acid was compared to a nucleic acid having the nucleotide sequence of TCG TCG TTT TGT CGT TTT GTC GTT (SEQ ID NO: 2) which has already been shown to be immunostimulatory. A nucleic acid comprising SEQ ID NO: 141 was identified only after screening about 165 nucleic acids for those with greater immunostimulatory potential than those of the nucleic acid comprising SEQ ID NO: 2. The difference in activity is surprising. This is because there is only a minimal difference between the sequence code 141 and SEQ ID NO: 2. It was unexpected that such minimal changes in sequence would result in statistically significant increases in immune stimulation.

  The nucleic acid of the present invention may further comprise another immunostimulatory motif such as poly T motif, poly G motif, TG motif, poly A motif, poly C motif etc, provided that the core sequence of SEQ ID NO: 141 is present. it can. These immunostimulatory motifs are described in more detail below or as described in US Ser. No. 09 / 669,187, filed Sep. 25, 2000, and published PCT patent application PCT / US00 / 26383 (publication number WO 01/22972). It is described in.

  It is understood that any of the embodiments detailed herein is equally applicable to the nucleic acids provided herein. Thus, for example, if the embodiment refers to SEQ ID NO: 1, it is understood that it applies equally to SEQ ID NO: 19, SEQ ID NO: 45, SEQ ID NO: 118 and SEQ ID NO: 141.

  The CpG motifs of the nucleic acids described herein are preferably not methylated. An unmethylated CpG motif is an unmethylated cytosine-guanine dinucleotide sequence (i.e. an unmethylated 5 'cytosine followed by a 3' guanosine and linked by a phosphate bond). All of the nucleic acids described herein are immunostimulatory. In some embodiments of the invention, the CpG motif is methylated. The methylated CpG motif is a methylated cytosine-guanine dinucleotide sequence (i.e. a methylated 5 'cytosine linked by a phosphate bond, followed by a 3' guanosine).

The CpG nucleic acid has the formula:
5 'X ₁ X ₂ CGX ₃ X ₄ 3'
Wherein C is not methylated, and X ₁ X ₂ and X ₃ X ₄ are nucleotides. In related embodiments, the 5 'X ₁ X ₂ CGX ₃ X ₄ 3' sequence is a non-palindromic sequence. In certain embodiments, X ₁ X ₂ is a nucleotide selected from the group consisting of GpT, GpG, GpA, ApA, ApT, ApG, CpT, CpA, CpG, TpA, TpT, and TpG, and ₃ X ₄ is a nucleotide selected from the group consisting of TpT, CpT, ApT, TpG, ApG, CpG, TpC, ApC, CpC, TpA, ApA, and CpA. In a more specific embodiment, X ₁ X ₂ is a nucleotide selected from the group consisting of GpA and GpT, and X ₃ X ₄ is TpT. In yet another embodiment, X ₁ X ₂ are both purine and X ₃ X ₄ are both pyrimidines. In another embodiment, X ₂ is T and X ₃ is pyrimidine. Examples of CpG nucleic acids are described in US Ser. No. 09 / 669,187 (filed Sep. 25, 2000) and published PCT patent application PCT / US00 / 26383 (publication number WO 01/22972).

  A T-rich nucleic acid is a nucleic acid comprising at least one polyT sequence and / or having a nucleotide composition in which T nucleotide residues are greater than 25%. A nucleic acid having a poly T sequence is one which comprises at least 4 T in succession, for example 5'TTTT3 '. Preferably, the T-rich nucleic acid comprises one or more poly T sequences. In preferred embodiments, the T-rich nucleic acid may have two, three, four or other numbers of poly-T sequences. Other T-rich nucleic acids according to the invention have a nucleotide composition where the T nucleotide residue is greater than 25%, but it is not essential that the poly-T sequence is included. In these T-rich nucleic acids, T nucleotide residues are separated from one another by other types of nucleotide residues (ie, G, C, and A). In some embodiments, the T-rich nucleic acid comprises 35%, 40%, 50%, 60%, 70%, 80%, 90%, and 99% T nucleotide residues, and any integer% in between It has more than nucleotide composition. Preferably, the T-rich nucleic acid has at least one poly-T sequence and a nucleotide composition with greater than 25% T nucleotide residues.

The poly G nucleic acid is preferably a nucleic acid having the following formula: That is,
5 'X ₁ X ₂ GGGX ₃ X ₄ 3'
In the formula, X ₁ , X ₂ , X ₃ and X ₄ are nucleotides. In a preferred embodiment, at least one of X ₃ and X ₄ is G. In another embodiment, both X ₃ and X ₄ are G. In yet another embodiment, the preferred formula is 5 'GGGNGGG 3', or 5 'GGGNGGGNGGG 3', N represents a nucleotide between 0 and 20.

  C-rich nucleic acids are nucleic acid molecules having at least one or preferably at least two poly C regions, or alternatively consisting of at least 50% C nucleotides. The poly C region is at least four consecutive C residues. Thus, the poly C region is included in the equation 5 'CCCC 3'. In some embodiments, it is preferred that the poly C region has the formula 5'CCCCCCCC3 '. Other C-rich nucleic acids according to the invention have a nucleotide composition in which the C nucleotide residue is greater than 50%, but it is not essential that the poly C sequence is included. In these C-rich nucleic acids, C nucleotide residues can be separated from one another by other types of nucleotide residues (ie, G, T, and A). In some embodiments, the C-rich nucleic acid has a nucleotide composition that is greater than 60%, 70%, 80%, 90%, and 99% C nucleotide residues, and any integer% in between. Preferably, the C-rich nucleic acid has at least one poly C sequence and a nucleotide composition greater than 50% C nucleotide residues, and in some embodiments is also T-rich.

  The immunostimulatory nucleic acid is double stranded or single stranded. In general, double-stranded molecules are more stable in vivo, while single-stranded molecules have high immune activity. Thus, in some aspects of the invention it is preferred that the nucleic acid be single stranded, and in other aspects it is preferred that the nucleic acid be double stranded.

  The terms "nucleic acid" and "oligonucleotide" are used interchangeably herein to bind to a phosphate group and to substituted pyrimidines (eg, cytosine (C), thymidine (T), or uracil (U) Or a plurality of nucleotides (ie, molecules containing a sugar (eg, ribose or deoxyribose)) linked to an exchangeable organic base which is either a substituted purine (eg, adenine (A) or guanine (G)) means. As used herein, the term refers to oligodeoxyribonucleotides as well as oligodeoxyribonucleotides. The terms also encompass polynucleosides (eg, phosphate-depleted polynucleotides) and other organic base-containing polymers. Nucleic acid molecules can be obtained from existing nucleic acid sources (eg, genomic DNA or cDNA), but are preferably synthetic (eg, generated by nucleic acid synthesis).

  The immunostimulatory oligonucleotides of the present invention include various chemical modifications and substitutions as compared to native RNA and DNA, and include internucleoside crosslinking with phosphodiester, β-D-ribose units, and / or Or natural nucleoside bases (adenine, guanine, cytosine, thymine, uracil) are included. Examples of chemical modifications are known to the person skilled in the art, for example Uhlmann E et al. (1990) Chem Rev 90: 543; "Protocols for Oligonucleotides and Analogs" Synthesis and Properties & Synthesis and Analytical Techniques, S., et al. Agrawal (Editor) Humana Press, Totowa, USA 1993; Crooke ST et al. (1996) Annu Rev Pharmacol Toxicol 36: 107-129; and Hunziker J et al. (1995) Mod Synth Methods 7: 331-417. Oligonucleotides according to the present invention may have one or more modifications, each modification being a specific phosphodiester as compared to an oligonucleotide of the same sequence composed of natural DNA or RNA. • located at internucleoside bridges and / or β-D-ribose units and / or at certain natural nucleoside base positions.

For example, the oligonucleotide may comprise one or more modifications, each modification independently being
a) substitution of a phosphodiester internucleoside bridge located at the 3 'and / or 5' end of the nucleoside by a modified internucleoside bridge,
b) substitution of phosphodiester crosslinks located at the 3 'and / or 5' end of the nucleoside by dephosphorization crosslinks.

c) replacing the sugar phosphate unit by another unit from the sugar phosphate backbone,
d) substitution of specific β-D-ribose units by modified sugar units, and e) substitution of natural nucleoside bases by modified nucleoside bases,
It is selected from

  More detailed examples of chemical modification of oligonucleotides are as follows.

  Nucleic acids also include substituted purines and pyrimidines (eg, C-5 propyne pyrimidine and 7-deaza-7 substituted purine modified bases). Wagner RW et al. (1996) Nat Biotechnol 14: 840-4. Purines and pyrimidines include, but are not limited to, adenine, cytosine, guanine, thymidine, 5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine And other naturally occurring and non-naturally occurring nucleoside bases (nucleobase), substituted and unsubstituted aromatic moieties. Other such modifications are well known to those skilled in the art. In all of the above embodiments, the X residue is also a non-naturally occurring nucleoside or nucleotide analog as described herein.

Modified bases are any bases that are chemically different from naturally occurring bases such as T, C, G, A, and U, etc. that are usually found in DNA and RNA, but their naturally occurring bases and basic chemistry Share the structure. The modified nucleoside base is, for example, hypoxanthine, uracil, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5- (C ₁ -C ₆ ) -alkyluracil, 5- (C _2- ) C ₆ ) -Alkenyluracil, 5- (C ₂ -C ₆ ) -alkynyluracil, 5- (hydroxymethyl) uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5- (C) C ₁ -C ₆ ) -alkylcytosine, 5- (C ₂ -C ₆ ) -alkenylcytosine, 5- (C ₂ -C ₆ ) -alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine, 5-bromocytosine , ^{N 2} - dimethyl guanine, 2,4-diaminopurine, 8-azapurine, substituted 7-deazapurine, More preferably, 7-deaza-7-substituted purines and / or 7-deaza-8-substituted purines, 5-hydroxymethycytosine, N4-alkylcytosine, such as N4-ethylcytosine, 5-hydroxydeoxycytidine, 5-hydroxymethyl Deoxycytidine, N 4 -alkyl deoxytidines, eg N 4 -ethyl deoxytidine, 6-thiodeoxy guanosine, and deoxyribonucleosides of nitropyrrole, C 5-propynyl pyrimidine, and diaminopurines (eg 2, 6 diaminopurines), inosine, It can be selected from 5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine, hypoxanthine, or other modifications of the natural nucleoside base. This list is meant to be exemplary and should not be construed as limiting.

  The particular formulas described herein define a set of modified bases. For example, the letter Y is used to refer to cytosine or a nucleoside containing modified cytosine. Modified cytosine, as used herein, is a pyrimidine base analog of naturally occurring or non-naturally occurring cytosine that can be substituted for this base without compromising the immunostimulatory activity of the oligonucleotide. Modified cytosines include, but are not limited to, 5-substituted cytosines (eg, 5-methyl-cytosine, 5-fluoro-cytosine, 5-chloro-cytosine, 5-bromo-cytosine, 5-iodo-cytosine, 5-hydroxy-cytosine, 5-hydroxymethyl-cytosine, 5-difluoromethyl-cytosine, and unsubstituted or substituted 5-alkynyl-cytosine), 6-substituted cytosine, N4-substituted cytosine (for example, N4-ethyl-cytosine), 5-aza-cytosine, 2-mercapto-cytosine, isocytosine, pseudo-isocytosine, cytosine analogues having fused ring systems such as N, N'-propylene cytosine or phenoxazine, and uracil and its derivatives such as 5- Fluoro-uracil, 5-bromo-uracil, 5-bromo Cycloalkenyl - uracil, 4-thio - uracil, 5-hydroxy - uracil, 5-propynyl - uracil) and the like. Some of the preferred cytosines include 5-methyl-cytosine, 5-fluoro-cytosine, 5-hydroxy-cytosine, 5-hydroxymethyl-cytosine, and N4-ethyl-cytosine. In another embodiment of the present invention, the cytosine base is by a common base (eg 3-nitropyrrole, P-base), an aromatic ring system (eg fluorobenzene or difluorobenzene) or a hydrogen atom (dSpacer) , Will be replaced. The letter Z is used to refer to guanine or modified guanine bases. As used herein, modified guanine is a purine base analog of naturally occurring or non-naturally occurring guanine, which can be substituted without impairing the immunostimulatory activity of the oligonucleotide. Examples of modified guanine include, but are not limited to, 7-deazaguanine, 7-deaza-7-substituted guanine (eg, 7-deaza-7- (C2-C6) alkynyl guanine), 7-deaza-8-substituted guanine Hypoxanthine, N2-substituted guanine (N2-methyl-guanine), 5-amino-3-methyl-3H, 6H-thiazolo [4,5-d] pyrimidine-2,7-dione, 2,6-diaminopurine , 2-aminopurine, purine, indole, adenine, substituted adenine (eg, N6-methyl-adenine, 8-oxo-adenine), 8-substituted guanine (eg, 8-hydroxyguanine and 8-bromoguanine), and 6- Thioguanine is mentioned. In another embodiment of the invention, the guanine base is a common base (eg 4-methyl-indole, 5-nitroindole and K base), an aromatic ring system (eg benzimidazole or dichloro-benzimidazole) 1-methyl-1H- [1,2,4] triazole-3-carboxylic acid amide) or a hydrogen atom (dSpacer).

  Oligonucleotides may include modified internucleotide linkages, such as those described above in (a) or (b). These modified bonds are partially resistant to degradation (eg, stabilized). A "stabilized nucleic acid molecule" is relatively resistant to in vivo degradation (eg, exonuclease or endonuclease). Stabilization is a function of length or secondary structure. Nucleic acids that are tens to hundreds of kilobases in length are relatively resistant to in vivo degradation. For shorter nucleic acids, secondary structure can stabilize and increase the effect of the nucleic acid. For example, if the 3 'end of the nucleic acid has self-complementarity to the upstream region, it folds to form a kind of stem-loop structure and then the nucleic acid begins to stabilize, thereby exhibiting higher activity.

  Nucleic acid stabilization can also be achieved via phosphate backbone modifications. Oligonucleotides with phosphorothioate linkages, in some embodiments, can provide maximal activity and protect the oligonucleotide from degradation by intracellular exonucleases and endonucleases.

  It has been demonstrated that modification of the nucleic acid backbone enhances the activity of the nucleic acid when administered in vivo. Constructs with phosphorothioate linkages have maximum activity and protect the nucleic acid from degradation by intracellular exonucleases and endonucleases. Other modified nucleic acids include phosphodiester modified nucleic acids, combinations of phosphodiester and phosphorothioate nucleic acids, methyl phosphonates, methyl phosphorothioates, phosphorodithioates, p-ethoxy, and combinations thereof. These combinations and the particular effect of the combination on immune cells are described in PCT published patent applications PCT / US95 / 01570 (WO 96/022555) and PCT / US 97/19791 (WO 98/18810) and US Pat. No. 6,239,116 B1 (issued May 29, 2001), as discussed for CpG nucleic acids. The entire contents of these documents are incorporated herein by reference.

  These modified nucleic acids show more stimulatory activity due to enhanced nuclease resistance, increased cellular uptake, increased protein binding, increased protein binding, and / or altered intracellular localization. It is also considered good.

  Other stabilized nucleic acids include the following. Non-ionic DNA analogues such as alkyl and aryl phosphates (charged phosphonate enzymes are replaced by alkyl or aryl groups), phosphodiesters, and alkyl phosphotriesters where the charged oxygen moiety is alkylated. ester. Nucleic acids that are substantially resistant to nuclease digestion also include nucleic acids that include diols such as tetraethylene glycol or hexaethylene glycol at one end or the other end.

  The oligonucleotide may comprise one or two accessible 5 'ends. For example, by combining two oligonucleotides via a 3'-3 'linkage to produce an oligonucleotide with one or two accessible 5' ends, such It is possible to generate modified oligonucleotides with The 3'-3 'linkage may be a phosphodiester, phosphorothioate, or any other modified internucleoside bridge. Methods for achieving such conjugation are known in the art. For example, such bonds are described by Seliger, H. et al. et al. Oligonucleotide analogs with terminal 3'-3'- and 5'-5'-internucleotidic linkages as antisence inhibotors of viral gene expression. Nucleosides & Nucleotides (1991), 10 (1-3), 469-77 and Jiang, et al. Pseudo-cyclic oligonucleotides: in vitro and in vivo properties, described in Bioorganic & Medicinal Chemistry (1999), 7 (12), 2727-2735.

  In addition, 3'-3 'linked ODNs where the linkage between the 3' terminal nucleoside is not a phosphodiester, phosphorothioate, or other modified crosslinks, using additional spacers (eg, tri- or tetra-ethylene glycol phosphate moieties) (Durand, M. et al, Triple-helix formation by an oligonucleotide containing one (dA) 12 and two (dT) 12 sequences bridged by two hexaethylene glycol chains, Biochemistry (1992), 31 (38) ), 9197-204, U.S. Patent No. 5,658,738, and U.S. Patent No. 5,668,265)). Alternatively, the non-nucleotide linker can be from ethanediol, propanediol, or abasic deoxyribose (dSpacer) units (Fontanel, Marie Laurence et al., Sterical recognition by T4 polynucleotide, using standard phosphoramidite chemistry It is derived from kinase of non-nucleosidic moities 5'-attached to oligonucleotide nucleotides; Nucleic Acids Research (1994), 22 (11), 2022-7). The incorporation of non-nucleotide linkers can be performed one or more times, or in combination with one another, to allow any desired distance between the two ODN 3 'ends to be joined.

The phosphodiester-nucleoside bridge located at the 3 'and / or 5' end of the nucleoside can be replaced by a modified internucleoside bridge. Here, the modified internucleoside bridge is, for example, phosphorothioate, phosphorodithioate, NR ¹ R ² -phosphoramidite, boranophosphate, α-hydroxybenzyl phosphonate, phosphate- (C ₁ -C ₂₁ ) -O-alkyl ester, phosphate - _[(C 6 _{-C 12)} aryl _{- (C 1} -C 21) -O- alkyl] _{ester, (C} 1 -C ₈₎ alkylphosphonate and / or _(C 6 _{-C 12)} aryl phosphonate A bridge, selected from (C ₇ -C ₁₂ ) -α-hydroxymethyl-aryl (for example as disclosed in WO 95/01373), (C ₆ -C ₁₂ ) aryl, (C ₆ -C ₂₀ ) aryl, and (C ₆ -C ₁₄₎ aryl, by halogen, alkyl, alkoxy, nitro, cyano Optionally substituted, ^{R 1} and ^{R 2} independently of one another, _hydrogen, (C 1 _{-C 18) alkyl,} (C 6 _{-C 20) aryl,} (C 6 _{-C 14)} aryl - _{(C 1} - C ₈ ) alkyl, preferably hydrogen, (C ₁ -C ₈ ) alkyl, preferably (C ₁ -C ₄ ) alkyl, and / or methoxyethyl, or alternatively R ¹ and R ² together with the nitrogen atom carrying them Form a 5-6 membered heterocycle which may further contain additional heteroatoms from the groups O, S and N.

  Dephospho crosslinking (for dephospho crosslinking, see, for example, Uhlmann E and Peyman A in "Methods in Molecular Biology", Vol. 20, "Protocols for Oligonucleotides and Analogs", S. Agrawal, Ed., Humana Press, Totowa 1993, Chapter 16 Substitution of the phosphodiester located at the 3 'and / or 5' end of the nucleoside, as described in pp. 355 ff), for example, dephospho cross-linked formacetal (formacetal), 3'-thioform acetal, methyl hydroxyl Amine, oxime, methylene dimethyl hydrazo, dimethylene sulfone, and / Or is selected from silyl groups.

The compositions of the invention may optionally have a chimeric backbone. As used herein, a chimeric backbone is a backbone that contains two or more types of bonds. In one embodiment, a chimeric backbone the formula: 5'Y ₁ N ₁ ZN can be represented by ₂ Y ₂ 3 '. Y ₁ and Y ₂ are nucleic acid molecules having 1 to 10 nucleotides. Y ₁ and / or Y ₂ each comprise at least one modified internucleotide linkage. These nucleic acids are an example of one type of "stabilized immunostimulatory nucleic acid", as at least two nucleotides of the chimeric oligonucleotides contain a backbone modification.

For chimeric oligonucleotides, Y ₁ and Y ₂ are considered to be independent of one another. This means that each of Y ₁ and Y ₂ may or may not have different sequences and different backbone linkages in the same molecule. In some embodiments, Y ₁ and Y ₂ have 3-8 nucleotides. N ₁ and N ₂ are nucleic acid molecules having 0-5 nucleotides, as long as N ₁ ZN ₂ has at least 6 nucleotides in total. The N ₁ ZN ₂ nucleotides have a phosphodiester backbone and do not include nucleic acids with modified backbones. Z is an immunostimulatory nucleic acid motif, preferably selected from those set forth herein.

The central nucleotide (N ₁ ZN ₂ ) of the formula Y ₁ N ₁ ZN ₂ Y ₂ has a phosphodiester internucleotide linkage, and Y ₁ and Y ₂ have at least one modified internucleotide linkage, It may have more than one or all modified internucleotide linkages. In a preferred embodiment, Y ₁ and / or Y ₂ have at least 2 or 2 to 5 modified internucleotide linkages, Y ₁ has 2 modified internucleotide linkages, and Y ₂ is Either having 5 modified internucleotide linkages, or Y ₁ has 5 modified internucleotide linkages, and Y ₂ has 2 modified internucleotide linkages. The modified internucleotide linkage is, in some embodiments, a phosphorothioate modified linkage, a phosphorodithioate modified linkage, or a p-ethoxy modified linkage.

  The nucleic acid also includes a nucleic acid having a backbone sugar, which is covalently linked at a 2 'position to a low molecular weight organic group other than a hydroxyl group and at a 5' position to a low molecular weight organic group other than a phosphate group. Thus, the modified nucleic acid may comprise a 2'-O-alkylated ribose group. Furthermore, instead of ribose, the modified nucleic acid may comprise a sugar such as arabinose or 2'-fluoroarabinose. Thus, the nucleic acid may be heterologous in backbone composition, and therefore contains any possible combination of polymer units that bind to each other, such as peptide-nucleic acid (having an amino acid backbone with a nucleic acid base). In some embodiments, the nucleic acids are homologous in backbone composition. Other examples are described in more detail below.

  Sugar phosphate units derived from a sugar phosphate backbone (ie, the sugar phosphate backbone is composed of sugar phosphate units) (ie, β-D-ribose and phosphate diesters that together form a sugar phosphate unit) The internucleoside bridge can be replaced by another unit, wherein the other unit is, for example, a "morpholino-derivative" oligomer (eg Stirchak EP et al. (1989) Nucleic acid). Acids Res 17: 6129-41 (ie, for example, substitution with morpholino derivative units), or construct a polyamide nucleic acid ("PNA") (eg, Nielsen PE et al. (1994) Bioconj ug Chem 5: 3-7), ie suitable for substitution, eg with 2-aminoethylglycine, eg with PNA backbone units. Oligonucleotides can be modified with other carbohydrate backbone modifications and substitutions (eg, peptide nucleic acids with phosphate groups (PHONA), locked nucleic acids (LNA), and oligonucleotides with backbone moieties with some alkyl or amino linkers) You may have The alkyl linker may be branched or unbranched, may be substituted or unsubstituted, and may be chirally pure or a racemic mixture.

The β-ribose unit or β-D-2′-deoxyribose unit can be replaced by a modified sugar unit, wherein the modified sugar unit is, for example, β-D-ribose, α-D-2 '-Deoxyribose, L-2'-deoxyribose, 2'-F-2'-deoxyribose, 2'-F-arabinose, 2'-O- (C ₁ -C ₆ ) alkyl-ribose (preferably, _{2'-O- (C 1 -C 6} ) alkyl - ribose is 2'-O- methyl _{ribose), 2'-O- (C 2} -C 6) alkenyl - ribose, 2 '- [O- ( C ₁ -C ₆ ) alkyl-O- (C ₁ -C ₆ ) alkyl] -ribose, 2′-NH ₂ -2′-deoxyribose, β-D-xylo-furanose, α-arabinofuranose, 2, 4-Dideoxy-β-D-erythro-hexo-pyra North, as well as carbocyclic sugar analogues (for example described in Froehler J (1992) Am Chemn Soc 114: 8320) and / or acyclic sugar analogues (for example described in Vandendriessche et al. (1993) Tetrahedron 49: 7223) And / or bicyclo sugar analogues (for example as described in Tarkov M et al. (1993) Helv Chim Acta 76: 481).

  In some embodiments, the sugar is 2'-O-methyl ribose, in particular for one or both nucleotides linked by a phosphodiester or phosphodiester-like internucleoside linkage.

  For use in the present invention, the oligonucleotides of the present invention can be synthesized de novo using any of a number of methods well known in the art. For example, b-cyanoethyl phosphoramidite method (Beaucage, S. L., and Caruthers, M. H., Tet. Let. 22: 1859, 1981), nucleoside H-phosphonate method (Garegg et al., Tet. Let) 27: 4051-4054, 1986; Froehler et al., Nucl. Acid. Res. 14: 5399-5407, 1986, Gareget et al., Tet. Let. 27: 4055-4058, 1986, Gaffney et al. , Tet.Let. 29: 2619-2622, 1988). These chemistries can be performed by a variety of automated nucleic acid synthesizers that are commercially available. These oligonucleotides are referred to as synthetic oligonucleotides. Alternatively, T-rich and / or TG dinucleotides may be generated in large amounts in a plasmid (see Sambrook, T., et al., "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory Press, New York, 1989). However, it can be separated into smaller pieces or administered as a whole. Nucleic acids can be prepared from existing nucleic acid sequences (eg, genomic DNA or cDNA) using known techniques such as those using restriction enzymes, exonucleases, or endonucleases.

  Modified backbones such as phosphorothioates can be synthesized using automated techniques that utilize either phosphoramidite or H-phosphonate chemistry. For example, aryl-phosphonates and alkyl-phosphonates can be produced as described in US Pat. No. 4,469,863. Alkyl phosphotriesters (charged oxygen moieties are alkylated as described in US Pat. Nos. 5,023,243 and EP 092,574) by automated solid phase synthesis using commercially available reagents Can be prepared. Methods for generating other DNA backbone modifications and substitutions have been described (eg, Uhlmann, E. and Peyman, A., Chem. Rev. 90: 544, 1990; Goodchild, J., Bioconjugate Chem. 1: 1.65. , 1990).

  Nucleic acids prepared in this way are referred to as isolated nucleic acids. In general, "isolated nucleic acid" refers to a nucleic acid which has been separated from components with which it is naturally associated. As an example, the isolated nucleic acid may be isolated from cells, from nuclei, from mitochondria or from chromatin.

  When administering a nucleic acid in combination with an antigen encoded in the nucleic acid vector (as described herein), the backbone of the nucleic acid is a phosphodiester and phosphorothioate (or other phosphate ester modified) chimera Combination is preferred. The cells may have problems in incorporating the plasmid vector in the presence of complete phosphorothioate nucleic acid. Thus, when delivering both vector and nucleic acid to a subject, the nucleic acid preferably has a chimeric or phosphorothioate backbone, but the plasmid is associated with a carrier that delivers the plasmid directly to the cell. It is preferable to eliminate the need for cellular uptake. Such carriers are known in the art and include, for example, liposomes and gene guns.

  The present invention further encompasses the use of any of these nucleic acids described above in the methods recited herein, and all of the use of immunostimulatory nucleic acids as described above and known. Do.

  It has been discovered by the present invention that the immunostimulatory nucleic acid surprisingly increases the immunostimulatory effect. For example, it has been demonstrated that the nucleic acids described herein can provide protection against infection, presumably by generally stimulating the immune system. In an example, a mouse subject challenged with herpes simplex virus type 2 (HSV-2) of a nucleic acid having the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 19, SEQ ID NO: 45, SEQ ID NO: 118 or SEQ ID NO: 141 Illustrate the ability to protect. The nucleic acid can be administered prior to or at the same time as the viral challenge.

  The ability of these nucleic acids to be demonstrated to induce immune stimulation is such that the nucleic acids in human and other subjects are vaccinated, cancer immunotherapy, asthma immunotherapy, and full enhancement of immune function. Evidence of enhanced hematopoietic recovery after radiation or chemotherapy and other immunomodulatory treatments.

  The nucleic acids of the invention can be used as a stand-alone therapy. Stand-alone therapies are those that can achieve a prophylactically or therapeutically useful result from the administration of a single agent or composition. Thus, the nucleic acids described herein can be used alone in the prevention or treatment of infections, cancer, and asthma and allergies. The reason for this is that the nucleic acid can induce an immune response that is useful for the treatment outcome of these diseases. Some of the methods described herein relate to the use of nucleic acids as stand-alone therapies while other methods relate to the use of nucleic acids in combination with other therapeutic agents.

  When used in a vaccine, the nucleic acid is administered with an antigen. Preferably, the antigen is specific for a disorder that requires prevention or treatment. For example, where the disorder is an infectious disease, the antigen is preferably derived from an infectious organism (eg, bacteria, virus, parasite, fungus, etc.). Where the disorder is cancer, the antigen is preferably a cancer antigen.

  Immunostimulatory nucleic acids are useful in some aspects of the invention as a prophylactic vaccine for the prevention of infections (ie, infections), cancer, allergies, or asthma. Preferably, a subject who has not been diagnosed as suffering from one of these symptoms, more preferably a prophylactic vaccination of a subject who is considered to be at risk of developing one of these symptoms. Use For example, the subject may be at risk of developing an infection with an infectious organism, at risk of developing a cancer for which a specific cancer antigen has been identified, or may develop an allergy for which the allergen is known. The risk of developing asthma or the risk of developing asthma that is predisposed to having asthma is known.

  As used herein, a subject at risk is a subject having any risk of exposure to an infectious agent, a carcinogen, or an allergen. Subjects at risk also include subjects predisposed to developing such a disorder. Some predisposing factors may be hereditary (and thus may be identified by either genetic analysis or family history). Some predisposing factors are environmental (eg, pre-exposed to carcinogens, etc.). An example of a subject at risk of developing an infection is a subject that is detected or expected to travel in an area where a specific type of infectious agent is detected or detected, or an infectious organism. It can be a subject directly or indirectly exposed to an organism via lifestyle or medical treatment by contact with a potentially containing body fluid. Subjects at risk of developing an infection also include the general population recommended by a healthcare provider to vaccinate against a particular infectious organism.

  If the antigen is an allergen, and the subject develops an allergic response to that particular antigen, and the subject is likely to be exposed to the antigen (ie, in the pollen season), the subject is There is a risk of exposure. Subjects at risk of developing an allergy to asthma have been identified as having allergy or asthma, but who have no active disease during immunostimulatory nucleic acid treatment, either genetically or environmentally. And subjects that are considered to be at risk of developing these diseases due to factors.

  The immunostimulatory nucleic acid can also be given without antigen or allergen for shorter term protection against infections, allergies or cancer, in which case repeated doses allow longer term protection.

  A subject at risk of developing a cancer is one that is likely to develop cancer (eg, more likely than in the general population). These subjects include, for example, subjects having a genetic abnormality (the presence of the genetic abnormality has been demonstrated to correlate with a higher probability of developing cancer than in the general population) Subjects that have been exposed to cancer causing agents (ie, carcinogens), such as tobacco, asbestos, or other chemical toxins, or subjects who have previously treated cancer and who are in remission on the surface . When a subject at risk of developing a cancer is treated with an antigen specific for the type of cancer at risk of developing the subject and an immunostimulatory nucleic acid, the subject is a cancer cell. It may be able to kill the cancer cells as they develop. When a tumor begins to form in a subject, the subject generates a specific immune response against the tumor antigen.

  In addition to using immunostimulatory nucleic acids as a preventive measure, the invention also encompasses using immunostimulatory nucleic acids for the treatment of a subject suffering from an infection, allergy, asthma, or cancer.

  A subject suffering from an infectious disease is a subject that is exposed to an infectious agent and has acute or chronic detectable levels of the agent in the body or excrement. When used therapeutically, the immunostimulatory nucleic acid can be used as a stand-alone or in combination with another therapeutic agent. For example, the immunostimulatory nucleic acid can be used therapeutically with an antigen that reduces the level of infectious pathogens or that is capable of clearing infectious pathogens and that enhances antigen-specific systemic or mucosal immune responses.

  As used herein, an infectious disease is a disease that results from the presence of foreign microorganisms in the body. It is particularly important to develop effective vaccine strategies and treatments that protect the mucosal surface of the body, which is the main pathogen entry site.

  As used herein, the terms treat, treated, treated or treating when used in connection with an infection refers to a subject suffering from an infection with a pathogen (infection (A subject who is at risk of (a) a subject who is at risk for preventing or treating the infection with a prophylactic treatment that reduces the likelihood that the subject is infected with a pathogen) It refers to treatment after a subject (infected subject) has been infected to reduce or eliminate the infection or prevent it from getting worse.

  A subject suffering from an allergy is a subject that has an allergic reaction in response to an allergen or is at risk of developing an allergic reaction. Allergy refers to acquired hypersensitivity to a substance (allergen). Allergic conditions include, but are not limited to, eczema, allergic rhinitis or coryza, hay fever, conjunctivitis, bronchial asthma, urticaria (hives) and food allergies, and other atopic conditions.

  At present, allergic diseases are generally treated by injection of a small amount of antigen followed by increasing the dose of antigen. This method is believed to induce tolerization to allergens and to prevent further allergic reactions. However, these methods can take several years to become effective and are associated with the risk of side effects such as anaphylactic shock. The method of the present invention avoids these problems.

  Allergy is generally caused by IgE antibody production against harmless allergens. Cytokines induced by systemic or mucosal administration of immunostimulatory nucleic acids are for the most part a class of cytokines called Th1 (for example, there is IL-12 and IFN-γ), which are humoral and Induce both cellular immune responses. The types of antibodies associated with Th1 responses are generally more protective as they have high neutralizing and opsonizing capabilities. The other major type of immune response is associated with the production of IL-4, IL-5, and IL-10 cytokines, referred to as a Th2 immune response. Th2 responses are mostly associated with antibodies, have little protective effect against infection, and some Th2 isotypes (eg, IgE) are associated with allergies. In general, allergic diseases are mediated by Th2-type immune responses, while excessive Th1 responses are associated with autoimmune diseases, but Th1 responses appear to provide the best protection against infection. Based on the ability of the immunostimulatory nucleic acid to transfer the subject's immune response from Th2 (associated with IgE antibody and allergy generation) to a Th1 response (which is protective against allergic response) A subject can be administered a dosage effective to induce an immune response to treat or prevent allergy.

  Thus, immunostimulatory nucleic acids have significant therapeutic utility in the treatment of allergic and non-allergic conditions such as asthma. Th2 cytokines, particularly IL-4 and IL-5, are increased in the airways of asthmatic subjects. These cytokines promote important aspects of the asthmatic inflammatory response including IgE isotope switching, neutrophil migration and activation, and mast cell proliferation. Th1 cytokines, in particular IFN-γ and IL-12, can suppress the formation of Th2 clones and the production of Th2 cytokines. Asthma refers to a disorder of the respiratory system characterized by inflammation, narrowing of the airways, and increased responsiveness of the airways to inhaled factors. Asthma is often associated with, but not limited to, atopic or allergic symptoms.

  A subject suffering from cancer is a subject having detectable cancer cells. The cancer may be a malignant or non-malignant cancer. The cancer or tumor includes, but is not limited to, biliary cancer, brain cancer, breast cancer, cervical cancer, choroidal cancer, colon cancer, endometrial cancer, esophageal cancer, gastric cancer, carcinoma in situ, lymphoma, liver cancer, lung cancer (Eg small cells and non-small cells), melanoma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, sarcoma, skin cancer, testicular cancer, thyroid cancer, and renal cancer, etc. Cancer and sarcoma. In one embodiment, the cancer is hairy cell leukemia, chronic myelogenous leukemia, cutaneous T cell leukemia, multiple myeloma, follicular lymphoma, malignant melanoma, squamous cell carcinoma, renal cell carcinoma, prostate carcinoma, bladder Cell carcinoma or colon carcinoma.

  Some cancer cells can be targets of the immune system because they are antigenic. In one aspect, combined administration of an immunostimulatory nucleic acid and a cancer drug, particularly one classified as cancer immunotherapy, is useful for stimulating a specific immune response to a cancer antigen.

  The theory of immune surveillance is that the main function of the immune system is to detect and eliminate neoplastic cells prior to tumorigenesis. The basic principle of this theory is that cancer cells are antigenically different from normal cells and thus elicit an immune response similar to that which causes rejection of immunologically incompatible allografts. Studies have confirmed that tumor cells differ in their antigen expression qualitatively or quantitatively. Such antigens are interchangeably referred to as tumor antigens or cancer antigens. Then, some of these antigens may be tumor specific antigens or tumor associated antigens. A "tumor-specific antigen" is an antigen that is specifically present on tumor cells but not on normal cells. Examples of major specific antigens are viral antigens in tumors induced by DNA or RNA viruses. "Tumor-associated" antigens are present on both tumor cells and normal cells, but in tumor cells are present in different amounts or in different forms. Examples of such antigens are oncofetal antigens (e.g. oncofetal antigens), differentiation antigens (e.g. T and Tn antigens) and oncogene products (e.g. HER / neu).

  Various types of cells have been identified that can kill tumor targets in vitro and in vivo: natural killer cells (NK cells), cytolytic T lymphocytes (CTL), lymphokine activated killer cells (LAK) , And activated macrophages. NK cells can kill tumor cells without prior sensitization to specific antigens, the activity of which is a class I antigen encoded by major histocompatibility complex (MHC) on target cells Does not require the presence of NK cells are thought to be involved in the control of neoplasia and in the control of metastatic growth. In contrast to NK cells, CTL can only kill tumor cells after being sensitized to tumor antigens and only when the target antigen is expressed on tumor cells that also express MHC class I . CTLs are thought to be effector cells in rejection of transplanted tumors and tumors caused by DNA viruses. LAK cells are a subset of null lymphocytes distinct from the NK and CTL populations. Activated macrophages, once activated, can kill tumor cells in a manner that is not antigen-dependent and not limited to MCH. Activated macrophages are thought to reduce the growth rate of the tumors they infiltrate. In vitro assays have identified antibodies and complements as well as other immune mechanisms such as antibody dependent cell mediated cytotoxic responses and lysis. However, these immune effector mechanisms may not be as important in vivo as NK, CTL, LAK, and macrophage functions in vivo (for a review, see Piessens, WF, and David, J. , "Tumor Immunology", Scientific American Medicine, Volume 2, Scientific American Books, NY, page 1-13, 1996).

  The goal of immunotherapy is to increase the patient's immune response to established tumors. One method of immunotherapy involves the use of an adjuvant. Adjuvant materials derived from microorganisms such as Bacillus Calmette-Guerin enhance the immune response and enhance the resistance to tumors in animals.

  As used herein, an "antigen" is a molecule capable of eliciting an immune response. Antigens include, but are not limited to, cells, cell extracts, proteins, polypeptides, peptides, polysaccharides, polysaccharide conjugates, peptides and non-peptide mimetics of polysaccharides and other molecules, small molecules, lipids, glycolipids, sugars Included are quality, viruses and viral extracts, and multicellular organisms such as parasites and allergens. The term antigen broadly includes any type of molecule that is recognized as foreign to the host's immune system. Antigens include, but are not limited to, cancer antigens, microbial antigens, and allergens.

  As used herein, "microbial antigens" are antigens of microorganisms, including but not limited to viruses, bacteria, parasites, and fungi. Such antigens include intact microorganisms and their naturally occurring isolates and fragments or derivatives, which are identical or similar to naturally occurring microbial antigens and which elicit an immune response specific for that microorganism Synthetic compounds are also mentioned. A compound is similar to a naturally occurring microbial antigen if it induces an immune response (humoral and / or cellular) to the naturally occurring microbial antigen. Such antigens are used routinely in the art and are well known to those skilled in the art.

  As used herein, a "cancer antigen" is a compound such as a peptide or protein present in a tumor or cancer cell and is expressed on the surface of an antigen presenting cell in the context of an MHC molecule. When it is a compound capable of eliciting an immune response. By preparing crude extracts of cancer cells (eg, as described in Cohen et al., 1994, Cancer Research, 54: 1055), by partially purifying the antigen, by recombinant techniques, Alternatively, cancer antigens can be prepared from cancer cells by de novo synthesis of known antigens. Cancer antigens include, but are not limited to, recombinantly expressed antigens, immunogenic portions of tumors or cancers, or tumors or whole cancers. Such antigens can be isolated or prepared recombinantly, or by any other means known in the art.

  By differentially expressing cancer or tumor antigens by the cancer cells, the cancer or tumor antigens can be utilized to target the cancer cells. Some of these antigens are not necessarily expressed but are encoded by normal cells. Characterize these antigens as being normally silent (ie, not expressed) in normal cells, expressed only at specific stages of differentiation, and transiently expressed, such as embryonic and fetal antigens be able to. Other cancer antigens are encoded by mutant cellular genes such as oncogenes (eg, activated ras oncogene), suppressor genes (eg, mutant p53), fusion proteins resulting from internal deletions or chromosomal translocations. Still other cancer antigens can be encoded by viral genes such as those carried on RNA and DNA tumor viruses.

  In some aspects of the invention, a subject is "exposed to" an antigen. As used herein, the term "exposed to" refers to either an active step of contacting a subject with an antigen or passively exposing a subject to an antigen in vivo. Point to Methods for actively exposing a subject to an antigen are well known in the art. Generally, the antigen is directly administered to the subject by any means such as intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration. The antigen can be administered systemically or locally. Methods of administering antigens and immunostimulatory nucleic acids are described in more detail below. When the antigen is available for exposure to immune cells in the body, the subject is passively exposed to the antigen. For example, a subject can be passively exposed to an antigen by invading a foreign pathogen into the body or by generating tumor cells that express the foreign antigen on the cell surface.

  The method of passively exposing a subject to an antigen may be particularly dependent on the timing of administration of the immunostimulatory nucleic acid. For example, in a subject at risk of developing cancer or an infectious disease or an allergic or asthmatic response, when the risk is at a maximum, ie during the season of allergy or after being exposed to a cancer causing factor, The immunostimulatory nucleic acid can be administered periodically. Furthermore, it is possible to administer the immunostimulatory nucleic acid to the traveler prior to traveling abroad at risk for exposure to an infectious agent. Similarly, if immunostimulatory nucleic acid is administered to a soldier or citizen at risk for biological warfare, and the subject is exposed to biological warfare, or if it is, then systemic or mucosal to the antigen An immune response can be induced.

  In some preferred embodiments, administration is local, but nucleic acids and other therapeutic agents can be administered systemically. Topical administration may include topical application to mucosal surfaces, such as those of the mouth, vagina, anus, and penis. In embodiments where the administration is topical, in particular to the mucosal surfaces of the vagina, anus and mouth, it is preferred that the nucleic acid is other than a CpG nucleic acid.

  In a specific embodiment, the present invention uses HIV-1, HIV-2, HIV-3, HTLV-I, HTLV-II, HTLV-III, type A, with topical mucosal administration of unmethylated CpG nucleic acid. Hepatitis virus, Hepatitis B virus, Herpes simplex virus (HSV) types 1 and 2, Papilloma virus, Neisseria gonorrhoeae, Treponema pallidum, Campylobacter sp. It is intended to prevent or treat human sexually transmitted diseases (STD) caused by cytomegalovirus (CMV), Chlamydia trachomatis, and Candida albicans.

  As used herein, STD is an infection that is primarily, but not exclusively, transmitted through sexual intercourse. In addition to transmission through sexual contact with infected subjects, some STDs can also be transmitted through contact with the fluid of infected subjects. As used herein, "bodily fluid" includes blood, saliva, semen, vaginal fluid, urine, feces, and tears. STD is most commonly transmitted via blood, saliva, seminal fluid, and vaginal fluid. As an example, blood and blood products transfusions are a common mode of transmission among many sexually transmitted disease pathogens, including HIV and hepatitis virus.

  Sexually transmitted pathogens are generally bacterial, viral, parasitic or fungal in nature. Organisms that cause STD include bacteria such as Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum, Haemophilus ducreyi, Conyloma acuminata, Calymmatobacterium granulomatatis, and Ureaplasma urealyticum, and human immunodeficiency virus (HIV-1 and HIV-2). Viruses such as lymphotropic virus type I (HTLV-I), herpes simplex virus type 2 (HSV-2), human papilloma virus (plural types), hepatitis B virus, cytomegalovirus, and infectious molluscoma virus , Trichomonas vaginalis and Pht There may be mentioned parasites such as hirus pubis, and fungi such as Candida albicans.

  Other infectious diseases are known to be transmitted sexually, even if sexual transmission is not their primary mode of transmission. This latter category includes bacteria such as Mycoplasma hominis, Gardnerella vaginalis, and group B streptococcus, human T lymphotropic virus type II (HTLV-II), hepatitis C and D viruses, herpes simplex virus type I (HSV-1) and viruses such as Epstein-Barr virus (EBV) and parasites such as Sarcoptes scabiei.

  The present invention is also intended to encompass STDs transmitted by sexual contact, including oral and fecal exposure. These STDs are described in Shigella spp. And Campylobacter spp. And the like, viruses such as hepatitis A, and parasites such as Giardia lamblia and Entamoeba histolytica.

  The "subject in need thereof" may be a subject at risk of developing STD or one suffering from STD (ie a subject suffering from STD).

  Nucleic acids are useful in some aspects as a preventative measure to prevent STD in a subject at risk of developing STD. As used herein, a "subject at risk of developing an STD" refers to a body fluid derived from contact with an infected subject or from an infected subject. A subject who has some risk of developing STD due to contact with For example, a subject at risk is one who has or will have a sexual partner who is infected with an STD-causing pathogen. As at-risk subjects, it is also possible to use the condom (ie male or female condom) without using the Or those involved in unprotected sexual activity such as having sexual intercourse in either of the vagina. Subjects at risk with multiple sexual partners (eg, prostitutes or frequent prostitution) or even subjects with one sexual partner with multiple sexual partners it is conceivable that. Other subjects at risk for developing STD are those involved in other forms of high risk transmission practices such as sharing a hypodermic needle. Subjects receiving blood products may also be considered at risk, particularly if the blood supply system is not closely monitored. An example of this latter category of subjects is a subject in a sub-Saharan African country that has a blood supply system partially or completely contaminated with STD-causing pathogens (eg, HIV). Subjects at risk are those for whom one or more STD-causing pathogens are planning to travel to a common area (in particular, it is known that such a pathogen is present in the area's blood supply system) Can also be Another subject at risk is one who has a profession that includes potential contact with another person's body fluid. Examples of this latter category include, but are not limited to, nurses, doctors, dentists, and paramedics (eg, ambulance personnel, paramedics, firefighters, and police officers). Subjects at risk also include fetuses and neonates born to mothers infected with STD-causing pathogens.

  All of the aforementioned activities associated with transmission of STD-causing pathogens are also referred to as "high risk activity". The nucleic acid and other potential prophylactic or therapeutic agents used in combination may be administered before, during, or after the subject's involvement in the high-risk activity. Subjects who receive the nucleic acid prior to participating in sexual activity may, for example, be at least one month, at least one week, at least 48 hours, at least 24 hours, at least 12 hours, at least 6 hours, at least 4 hours before having sexual intercourse. The nucleic acid can be received for at least 2 hours (or any time between the times explicitly listed herein). Preferably, the time of administration prior to engaging in high-risk activity is sufficient to activate the immune system to be active while the infectious agent is present in the subject's body. Subjects who receive nucleic acid after participating in high-risk activities are within 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours after participating in high-risk activities The nucleic acid can be received within 3, 4, 5, 6, 7, 14, 28 days or longer (or any time between the times explicitly recited herein for time).

  Preferably, the subject is a non-rodent subject. Non-rodent subjects include, but are not limited to, dogs, cats, horses, cows, pigs, sheep, goats, chickens, primates (e.g. monkeys), fish (aquaculture species such as salmon), And especially humans or vertebrates excluding rodents such as rats and mice.

  Antigens can be derived from a variety of sources including tumors, non-tumor cancers, allergens, infectious agents. Each of the lists listed herein is not intended to be limiting.

  Examples of viruses found in humans include, but are not limited to, Retroviridae (eg, human immunodeficiency virus such as HIV-1 (HTLV-III, LAV, or HTLV-III / LAV, or HIV-III also mentioned) And other isolates such as HIV-LP, Picornaviridae (eg, polio virus, hepatitis A virus, enterovirus, human coxsackie virus, rhinovirus, echovirus), Calciviridae (eg, gastroenteritis) (A strain causing causative agent), Togaviridae (eg, equine encephalitis virus, rubella virus), Flaviridae (eg, dengue virus, encephalitis virus, yellow fever virus), and Coronoviridae (eg, corona) Iris), Rhabdoviradae (eg, Vesicular Stomatitis Virus, Rabies Virus), Coronaviridae (eg, Coronavirus), Rhabdoviradae (eg, Vesicular Stomatitis Virus, Rabies Virus), Filoviridae (eg, Ebola Virus) , Paramyxoviridae (eg, parainfluenza virus, mumps virus, measles virus, respiratory rash virus), Orthomyxoviridae (eg, influenza virus), Bunyaviridae (eg, Hantan virus, Bunya virus, flavovirus) , And the virus, such as Arena viridae (hemorrhagic fever virus), and Reoviridae (for example, , Reovirus, Orbivirus, and Rotavirus, Birnaviridae, Hepadnaviridae (Hepatitis B Virus), Parvovirida (Parvoi Urus), Papovaviridae (Papiloma Virus, Polyoma Virus), Adenoviridae (mostly) Adenovirus), Herpesviridae (Herpes simplex (HSV) types 1 and 2, Varicella zoster virus, Cytomegalovirus (CMV), Herpes virus) and Poxviridae (痘 瘡 virus, vaccinia virus, pox virus) , Iridoviridae (eg, African swine fever virus), and unclassified viruses (eg, causative agent of cavernous encephalopathy, cause of hepatitis delta) (Considered to be a defective satellite of hepatitis B), factors of non-A and non-B hepatitis (class 1 is internally transmitted, class 2 is parenterally transmitted (ie hepatitis C)) Norwalk and related viruses, and astroviruses.

  Although many of the microbial antigens are described herein in connection with human disease, the invention is also useful for treating other non-human diseases. Non-human vertebrates can also develop infections that can be prevented or treated with the immunostimulatory nucleic acids described herein. For example, in addition to the treatment of infectious human disease, the methods of the invention are useful for treating infections in animals.

  Both gram negative and gram positive bacteria function as antigens in vertebrates. Such gram positive bacteria include, but are not limited to, Pasteurella species, Staphylococci species, and Streptococcus species. Gram negative bacteria include, but are not limited to, Escherichia coli, Pseudomonas species, and Salmonella species. Specific examples of infectious bacteria include, but are not limited to, Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia, Mycobacteria sps (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria ruonocytogenes, Streptococcus pyogenes (group A streptococcus), Streptococcus agalactiae (group B streptococcus), Streptococcus cus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic species), Streptococcus pneumoniae, pathogenic Campylobacter sp. , Enterococcus sp. , Haemophilus influenzae, Bacillus anthracis, corynebacterium diphtheriae, corynebacterium sp. , Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp. And Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, Rickettsia, and Actinomyces israelli.

  Bacterial pathogen polypeptides include, but are not limited to, iron-regulated outer membrane protein (IROMP), outer membrane protein (OMP), and Aeromonis salmonicida A protein, which cause flunculosis, and bacterial kidney disease (BKD) P57 protein of Renibacterium, major surface associated antigen (major surface associated antigen (msa)) of Yersinosis, surface expressed cytotoxin (surface expressed cytotoxin (mpr)), surface expressed hemolysin (surface expressed hemolysin (mpr)) and flagellar antigens, and extracellular protein (ECP) of pasclerosis, iron-regulated outer membrane protein (IROMP), and structure With proteins, Vibrosis anguillarum and V. v. ordalii OMP and flagellar proteins, Edwardsiellosis ictaluri and E. coli. The flagellar proteins of tarda, OMP proteins, aroA and purA, surface antigens of Ichthyophthirius, structural and regulatory proteins of Cytophaga columnari, and structural and regulatory proteins of Rickettsia can be mentioned.

  Parasitic pathogen polypeptides include, but are not limited to, surface antigens of Ichthyophthirius.

  Examples of fungi include Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, and Candida albicans. Other infectious organisms (ie, protozoa) include Plasmodium species such as Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax, and Toxoplasma gondii. As blood-derived and / or tissue parasites, Plasmodium species, Babesia microti, Babesia divergens, Leishmania tropica, Leishmania species, Leishmania braziliensis, Leishmania donovani, Trypanosoma gambiense, and Trypanosoma rhodesiense (African sleeping sickness), Trypanosoma cruzi (Clypus disease) And Toxoplasma gondii. Other medically relevant microorganisms are extensively described in the literature. For example, C.I. G. A. Thomas, Medical Microbiology, Bailliere Tindall, Great Britain 1983, the entire content of which is incorporated herein by reference.

  For many vaccines for the treatment of non-human vertebrates, see Bennett, K. et al. Compendium of Veterinary Products, 3rd ed. North American Compendiums, Inc. , 1995. As mentioned above, antigens include viruses such as viruses, parasites, bacteria, and fungi, derived from natural sources or synthesized, and fragments thereof. Infectious viruses of human and non-human vertebrates include retroviruses, RNA viruses, and DNA viruses. This group of retroviruses includes both single retroviruses and complex viruses. Single retroviruses include subgroups of B-type retroviruses, C-type retroviruses, and D-type retroviruses. An example of a type B retrovirus is mouse mammary tumor virus (MMTV). As C-type retroviruses, C-type A group (including rous sarcoma virus (RSV), avian leukemia virus (ALV), and avian myeloblastosis virus (AMV)), C-type B group (feline leukemia virus (feline leukemia virus) Subgroups with FeLV), Gibbon monkey leukemia (GALV), spleen necrosis virus (SNV), reticuloendotheliosis virus (RV), and simian sarcoma virus (SSV). Type D retroviruses include Mason-Pfizer monkey virus (MPMV) and simian retrovirus type 1 (SRV-1). Complex retroviruses include lentivirus, T cell leukemia virus, and subgroups of foamy viruses. Lentiviruses include HIV-1, but also include HIV-2, SIV, Visna virus, feline immunodeficiency virus (FIV), and equine infectious anemia virus (EIAV). T cell leukemia viruses include HTLV-1, HTLV-II, monkey T cell leukemia virus (STLV), and bovine leukemia virus (BLV). Foamy viruses include human foamy virus (HFV), monkey foamy virus (SFV), and bovine foamy virus (BFV).

  Examples of other RNA viruses that are antigens in vertebrates include, but are not limited to, Orthoreovirus (multiple serotypes of both mammalian and avian retroviruses), Orbivirus (Blue tongue virus, Eugenangee virus, Kemerovo)・ Reoviridae family including viruses, African equine virus, and Colorado tick fever virus), Rotavirus genus (Human rotavirus, Nebraska calf diarrhea virus, simian rotavirus, bovine or ovine rotavirus, avian rotavirus) Member of the genus Enterovirus (Polio virus, Coxsackie virus type A and B, enterocyte necrotic human orphan (ECHO) virus, hepatitis A virus, simian enterovirus Mouse encephalomyelitis (ME) virus, Poliovirus muris (Poliovirus muris), bovine enterovirus, porcine enterovirus), Cardiovirus genus (encephalomyocarditis virus (EMC), Mengovirus), Rhinovirus genus (at least 113 sub) The human rhinoviruses, including other types, the Picornaviridae family, including the Apthovirus genus (foot-and-mouth disease (FMDV)), and the porcine small vesicle rash virus, San Miguel seal virus, feline picorna virus, and Norwalk・ Calciviridae containing viruses, and Alphavirus (Eastern equine encephalitis virus, Semliki forest virus, Sindbis virus, Chikungunya virus , Onyonnon virus, Ross River virus, Venezuelan equine encephalitis virus, equine encephalitis virus, Flavirius (mosquito-borne yellow fever virus, dengue virus, Japanese encephalitis virus, St. Louis encephalitis virus, Murray Valley encephalitis virus, West Nile virus, Kunjin virus, Central European tick vector virus, Far East tick vector virus, Chiassanua forest virus, Jump disease virus, Powassan virus, Omsk hemorrhagic fever virus), Rubivirus genus (rubella virus), Pestivirus genus (mucosa Togaviridae family including disease virus, swine cholera virus, border disease virus, and Bunyavirus genus (Buniyanbera and related viruses, California encephalitis swarm) And Phlebovirus spp. (Sicillia Sashfly virus, Rift Valley fever virus), Nairovirus spp. Crimea-Congo hemorrhagic fever virus, Nairobi sheep disease virus, and Uukuvirus sp. (Uulcuniemi) and related viruses Bunyaviridae family, Influenza virus genus (influenza virus type A, many human subtypes), swine influenza virus, avian influenza virus, and equine influenza virus, influenza B type (many human sub Type), and the Orthomyxoviridae family including influenza C (possibly another genus), and the genus Paramyxovirus Parainfluenza virus type 1, Sendai virus, erythrocyte adsorption virus, parainfluenza virus types 2 to 5, Newcastle disease virus, mumps virus), Morbillivirus (measles virus, subacute sclerosing whole encephalitis virus, distemper virus , The paramyxoviridae family including Phymovirus spp., Pneumovirus spp. (Respiratory syncytial virus (RSV), bovine respiratory syncytial virus, and pneumovirus); and Vesiculovirus spp. (VSV) (viral encephalitis (Chandipura) virus, Flanders Heart Park virus (Flanders-Hart Park virus), Lyssavirus genus (rabies virus), fish Rhabdovi The Rhabdoviridae family, including us and two possible Rhabdoviruses (Marburg virus and Ebola virus), and the Arenaviridae family, including lymphocytic choriomeningitis virus (LCM), Tacaribe virus complex, and Lassa fever virus And the Coronoaviridae family including infectious bronchitis virus (IBV), hepatitis virus, human enteric corona virus, and feline infectious peritonitis (feline coronavirus). Exemplary DNA viruses that are antigens in vertebrates include, but are not limited to, Orthopoxvirus (大 痘 瘡, 小 痘 瘡, 痘 瘡, 痘 瘡, buffalo, buffalo, rabbit 痘, Ectromelia), Leporipoxvirus (myxoma, Fibroma), Avipoxvirus genus (fowlpox, other avian poxvirus), Capripoxvirus genus (sheeppox, goatpox), Suipoxvirus genus (porkpox), Parapoxvirus genus (contagious pustular dermatitis virus, pseudocapsular cow, bovine papules) Poxviridae family (including Stomatitis virus), Iridoviridae family (African swine fever virus, frog viruses 2 and 3 types, fish lymphocystoma virus), and alpha-Ierpes virus s (herpes simplex 1 and 2, varicella zoster, equine abortion virus, equine herpes virus 2 and 3, pseudorabies virus, bovine infectious keratoconjunctivitis virus, bovine infectious rhinotracheitis virus, feline nose Tracheitis virus, Infectious laryngeal tracheitis virus), Beta-herpesviruses (human cytomegalovirus and cytomegalovirus of pig and monkey), gamma-herpesviruses (Epstein-Barr virus (EBV), Marek's disease virus, squirrel monkey, Herpesviridae family including herpes, spider monkey / herpes virus, cotton rabbit / herpes virus, guinea pig / herpes virus, leuke cancer virus, and Mastadenovirus sp. (A, B, C, D, E, and unclassified, simian adenovirus (at least 23 serotypes), infectious canine hepatitis, and adenoviruses of cattle, pigs, sheep, frogs and many other species) And the Adenoviridae family, including the Aviadenovirus genus (Avian adenovirus), and non-culturable adenoviruses, and the Papillomavirus (human papilloma virus, bovine papilloma virus, rabbit chop papilloma virus, and various other species. Pathogenic papilloma virus, Polyomavirus (polyoma virus, simian vacuolar pathogen (SV-40), rabbit vacuolar pathogen (RKV), K virus, BK virus, JC virus, and lymphoproliferative papilloma virus etc. of Papoviridae family including other primate polyoma virus) and Parvoviridae family including Adeno-associated viruses, genus Parvovirus (feline panleukopenia virus, bovine parvovirus, canine parvovirus, aleutian Mink disease virus, etc.) Can be mentioned. Finally, DNA viruses may include viruses that are not compatible with the above families such as Kuru and Creutzfeldt-Jakob disease viruses, and chronic infectious neuropathic factors (CHINA virus).

  The immunostimulatory nucleic acid can also be used to induce an immune response, such as an antigen-specific immune response in birds such as hens, chickens, turkeys, ducks, geese, quails and pheasants. Birds are the main targets for many types of infections.

  Hatched birds are exposed to pathogenic microorganisms shortly after birth. These birds are initially protected against pathogens by antibodies from the mother, but this protection is temporary, and the bird's own immune system must begin to protect the birds against pathogens. It is desirable to protect the infection in young birds most susceptible to infection. When the birds live in a closed area and the disease spreads rapidly, it is desirable to prevent infection even with older birds. Therefore, it is desirable to administer the immunostimulatory nucleic acids and non-nucleic acid adjuvants of the invention to birds to enhance antigen-specific immune responses in the presence of antigen.

  An example of a common infection in chickens is chicken infectious anemia virus (CIAV). CIAV was first isolated in Japan in 1979 during the interruption of the Marek's disease vaccination survey (Yuasa et al., 1979, Avian Dis. 23: 366-385). Since that time, CIAV has been detected in commercial poultry in all the major countries producing poultry (van Bulow et al., 1991, pp. 690-699), Diseases of Poultry, 9th edition , Iowa State University Press).

  CIAV infection causes clinical disease characterized by anemia, hemorrhage, and immunosuppression in young susceptible chickens. Thymic and bone marrow atrophy and consistent lesions of CIAV infected chickens are also characteristic of CIAV infection. Lymphocyte depletion in the thymus and often in the bursa of Fabricius results in immunosuppression and increased susceptibility to secondary viral, bacterial or fungal infections, which then exacerbate the course of the disease. Immunosuppression may cause disease deterioration after infection with one or more of Marek's disease virus (MDV), infectious bursal disease virus, reticuloendotheliosis virus, adenovirus, or reovirus. The pathogenesis of MDV has been reported to be enhanced by CIAV (DeBoer et al., 1989, p. 28 In Proceedings of the 38th Western Poultry Diseases Conference, Tempe, Ariz.). Furthermore, CIAV has been reported to aggravate the signs of infectious Bulza disease (Rosenberger et al., 1989, Avian Dis. 33: 707-713). Chickens develop age tolerance to diseases experimentally induced by CAA. This is essentially complete by two weeks of age, but older birds are still susceptible (Yuasa, N. et al., 1979 (above); Yuasa, N. et. al., Arian Diseases 24, 202-209, 1980). However, when chickens are dually infected with CAA and immunosuppressants (IBDV, MDV, etc.), resistance to age is delayed (Yuasa, N. et al., 1979 and 1980 (supra); Bulow). von V. et al., J. Veterinary Medicine 33, 93-116, 1986). Properties of CIAV that can enhance disease transmission include environmental inactivation and high resistance to some common microbicides. The economic impact of CIAV infection on the poultry industry is evident from the fact that 10% to 30% of infected birds die from disease onset.

  As with other vertebrates, vaccination of birds can also occur at any age. Vaccination is usually performed by 12 weeks of age for viable organisms and 14-18 weeks of age for inactivated microorganisms or other types of vaccines. For in ovo vaccination, vaccination is performed in the last quarter of embryo development. The vaccine can be administered subcutaneously, by spray, orally, intraocularly, intratracheally, nasally, or by other mucosal delivery methods described herein. Thus, the normal vaccination schedule can be used to administer the immunostimulatory nucleic acids of the invention to birds and other non-human vertebrates, and after an appropriate period of time, as described herein. Can be administered.

  Cattle and livestock are also susceptible. The diseases which these animals develop, especially in cattle, cause serious economic losses. The methods of the invention can be used to prevent infections in livestock such as cattle, horses, pigs, sheep and goats.

  Cattle can be infected with bovine viruses. Bovine viral diarrhea virus (BVDV) is a small enveloped positive-strand RNA virus, which, along with porcine cholera virus (HOCV) and sheep border disease virus (BDV), is classified into the genus Pestivirus. Pestiviruses were previously classified into the Togaviridae family, but several studies have suggested a reclassification within the Flaviviridae family along with the Flavivirus and Hepatitis C virus (HCV) groups (Franki, et al., 1991).

  Based on cell culture analysis, BVDV, a serious bovine pathogen, can be distinguished into cytopathogenic (CP) and non-cytopathic (NCP) cell types. NCP cell types are more widespread, but both cell types are found in cattle. If a pregnant cow is infected with the NCP strain, it will persistently infect and give rise to a specifically tolerable calf, which will spread the virus in its lifetime. Persistently infected cattle die from mucosal disease, after which both biotypes can be isolated from the animal. Clinical signs can include abortion, teratogenesis, and respiratory disease, mucosal disease, and mild diarrhea. In addition, thrombocytopenia associated with infectious disease outbreaks that can lead to animal death has been described, and strains associated with this disease appear to be more virulent than conventional BVDV.

  The equine herpesvirus (EHV) comprises a group of antigenically distinct biological agents that cause a variety of infections in horses ranging from asymptomatic disease to fatal disease. These include the equine herpesvirus type 1 (EHV-1), a ubiquitous pathogen in horses. EHV-1 is associated with abortion, respiratory disease, and infectious diseases of central nervous system disorders. Primary infection of the upper respiratory tract in young horses results in a febrile illness lasting 8 to 10 days. Since immunologically experienced mares can re-infect via the respiratory tract before the disease can be identified, abortions usually occur without aura. Neurological syndromes are associated with respiratory disease or miscarriage, affecting animals of either sex at any age, leading to ataxia, weakness, and paralysis of the buttocks (Telford, EA R. et. al., Virology 189, 304-316, 1992). Other EHVs include EHV-2, or equine cytomegalovirus, previously classified as subtype 2 of EHV-1, EHV-3, equine herpesvirus, and EHV-4.

  Sheep and goats can be infected with various dangerous microbes, including Maedy visna.

  Primates such as monkeys, apes and macaques can be infected with simian immunodeficiency virus. Inactivated cell virus and a cell-free whole monkey immunodeficiency vaccine have been reported to allow protection with macaque (Stott et al. (1990) Lancet 36: 1538-1541; Desrosiers et al. PNAS USA (1989) 86: 6353-6357; Murphey-Corb et al. (1989) (1989) Science 246: 1293-1297; and Carlson et al. (1990) AIDS Res. Human Retroviruses 6: 1239-1246). Recombinant HIV gpl20 vaccine has been reported to enable protection with chimpanzees (Berman et al. (1990) Nature 345: 622-625).

  Cats (both pet cats and wild cats) are susceptible to various microorganisms. For example, feline infectious peritonitis is a disease that occurs in both domestic and wild cats (eg, lions, leopards, cheetahs, and jaguars). When it is desired to prevent infection of this and other types of pathogenic organisms in cats, the methods of the invention can be used to vaccinate cats and protect them from infection.

  Domestic cats are infected with multiple retroviruses, including but not limited to feline leukemia virus (FeLV), feline sarcoma virus (FeSV), endogenous Concornavirus (RD-114), feline syncytial virus (FeSFV) sell. Of these, FeLV is the most significant pathogen and is diverse, including lymphoret and myeloid neoplasia, anemia, immune-mediated disorders, and immunodeficiency similar to human acquired immunodeficiency syndrome (AIDS). Cause various symptoms. In recent years, a particular replication deficient FeLV variant designated FeLV-AIDS has been particularly relevant to the immunosuppressive properties.

  The discovery of feline T-lymphotropic lentivirus (also referred to as feline immunodeficiency) was first reported by Pedersen et al. (1987) Science 235: 790-793. For the characteristics of FIV, see Yamamoto et al. (1988) Leukemia, December Supplement 2: 2045-2155; Yamamoto et al. (1988) Am. J. Vet. Res. 49: 1246-1258; and Ackley et al. (1990) J.J. Virol. 64: 5652-5655. For FIV cloning and sequence analysis, see Olmsted et al. (1989) Proc. Natl. Acad. Sci. USA 86: 2448-2452 and 86: 4355-4360.

  Feline infectious peritonitis (FIP) is a sporadic disease that occurs unpredictably in domestic and wild felines. While FIP is primarily a disease of domestic cats, it has been diagnosed with lions, American lions, leopards, cheetahs, and jaguars. Smaller wild cats suffering from FIP include lynx and caracal, sun cats, and manul cats. In domestic cats, the disease mainly occurs in young animals, but cats of all ages are susceptible. Peak incidence occurs at 6 to 12 months of age. A decrease in incidence has been shown at 5 to 13 years of age, followed by an increase in incidence in cats of 14 to 15 years of age.

  Viral, bacterial and parasitic diseases in fish, crustaceans or other aquatic forms present a serious problem in the aquaculture industry. Due to the high density of animals in hatchery tanks or enclosed marine life aquaculture areas, infections may, for example, eradicate large stocks of fish, crustaceans, or other aquatic life form facilities There is. Once the disease has occurred, prevention of the disease is a more desirable response than intervention. Vaccination of fish is the only prophylaxis that can provide long-term protection via immunity. Nucleic acid based vaccination is described in US Pat. No. 5,780,448 issued to Davis.

  The fish immune system has many features similar to the mammalian immune system, such as the presence of B cells, T cells, lymphokines, complement, and immunoglobulins. Fish have a lymphocyte subclass with a role that appears to be similar in many ways to that of mammalian B and T cells. The vaccine can be administered by immersion or orally.

  Aquaculture species include, but are not limited to, fish, crustaceans, and other aquatic animals. Fish include all spinal fish and may be bony fish or cartilaginous fish (eg salmonids, carp, catfish, yellowtail, lyrea, and grouper). The salmonid family is a family of fish including trout (including rainbow trout), salmon, and alpine chars. Examples of crustaceans include but are not limited to clams, lobsters, shrimps, crabs, and oysters. Other cultured aquatic animals include, but are not limited to, eel, squid and octopus.

  Examples of polypeptides of viral aquaculture pathogens include, but are not limited to, glycoproteins (G) or nuclear proteins (N) of viral hemorrhagic septic virus (VHSV) and G of infectious hematopoietic necrosis virus (IHNV) Or N protein and infectious pancreatic necrosis virus (IPNV) VP1, VP2, VP3 or N structural protein, carp spring viremia (SVC) G protein and buty catfish virus (CCV) membrane association The proteins include tegmin or capsid proteins or glycoproteins.

  Typical parasites which infect horses are Gasterophilus species, Eimeria leuckarti, Giardia species, Tritrichomonas equi, Babesia species (RBC), Theileria equi, Trypanosoma species, Klossiella equi and Sarcocystis species.

  Typical parasites that infect pigs include Eimeria bebliecki, Eimeria scabra, Isospora suis, Giardia species, Balantidium coli, Entamoeba histolytica, Toxoplasma gondii and Sarcocystis species, and Trichinella spiralis.

  The main parasites of dairy cows and beef cattle include Eimeria species, Cryptosporidium sp. , Giardia species, Toxoplasma gondii, Babesia bovis (RBC), Babesia bigemina (RBC), Trypanosoma species (plasma), Theileria species (RBC), Theileria parva (lymphocytes), Tritrichomonas foetus and Sarcocytis species It can be mentioned.

  The main parasites of birds of prey are Trichomonas gallinae, Coccidia (Eimeria sp.), Plasmodium relictum, Leucocytozoon danilewskyi (Owls), Haemoproteus sp., Trypanosoma sp., Histoponos sp. , Toxoplasma and the like.

  Typical parasites that infect sheep and goats include Eimeria species, Cryptosporidium sp. Giardia sp. And Toxoplasma gondii, Babesia species (RBC), Trypanosoma species (plasma), Theileria species (RBC), and Sarcocystis species.

  Typical parasitic infections in poultry include Eimeria acervulina, E. coli. necatrix, E.I. These include coccidiosis caused by tenella, Isospora species, and Eimeria truncata, histomonosis caused by Histomonas meleagridis and Histomonas gallinarum, trichomoniasis caused by Trichomonas gallinae, and hexamitatia caused by Hexamita meleagridis. Poultry may also be infected with Emeria maxima, Emeria meleagridis, Eimeria adenoeides, Eimeria meleagrimitis, Cryptosporidium, Eimeria brunetti, Emeria adenoeides, Leucocytozoon species, Plasmodium species, Hemoproteus meleagridis, Toxoplasma gondicoc

  The methods of the invention can also be applied to the treatment and / or prevention of parasitic infections in dogs, cats, birds, fish and ferrets. As typical parasites of birds, Trichomonas gallinae, Eimeria species, Isospora species, Giardia, Cryptosporidium, Sarcocystis species, Toxoplasma gondii, Haemoproteus / Parahaemoproteus, Plasmodium species, Leucocytozoon / Akiba, and ATP Be Typical parasites which infect dogs are Trichinella spiralis, Isopora species, Sarcocystis species, Cryptosporidium species, Hammondia species, Hamardia species, Giardia duodenalis (canine) and Balantidium coli, Entamoeba histolytica, Hepatozoon canis and Toxoplasma gondii, These include Trypanosoma cruzi, Babesia canis, Leishmania amastigotes, and Neospora caninum.

  Typical parasites that infect feline species include Isospora species, Toxoplasma gondii, Sarcocystis species, Hammondia hammondi, Besnoitia species, Giardia species, Entamoeba histolytica, Hepatozoon canis, Cytauxzoon sp. , Cytauxzoon sp. , Cytauxzoon sp. (Red blood cells, RE cells).

  As typical parasites which infect fish, Hexamita species, Eimeria species, Cryptobia species, Cryptoma species, Nosema species, Myxosoma species, Myxosoma species, Chilodonella species, Trichodina species, Plistophora species, Myxosoma Henneguya, Costa species, Ichthyophithirius species, Oodinium species Can be mentioned.

  Typical parasites of wild mammals include Giardia species (carnivorous animals, herbivore animals), Isospora species (cariogenic animals), Eimeria species (cariogenic animals, herbivore animals), Theileria species (herbivore animals), Babesia species (carnivorous species) Animals, Herbivores), Trypanosoma species (carnivores, herbivores), Schistosoma species (herbivores), Fasciola hepatica (herbivores), Fascioloides magna (herbivores), Fasciola gigantica (herbivores), Trichinella spiralis (carnivores) Animals, herbivores).

  Parasitic infections at the zoo also present serious problems. Typical parasites of the bovine family (Bresbok, Antelope, Banten, Eland, Goua, Impala, Clip Springer, Kudu, Gazelle) include Eimeria species. Typical parasites of the Lepidoptera family (seals, sea lions) include Eimeria phocae. Typical parasites of the camelid family (camels, llamas) include Eimeria species. Typical parasites of the giraffe family (Giraffe) include Eimeria species. Typical parasites of the elephant family (African elephants and Asian elephants) include Fasciola species. Typical parasites of lower primates (chimpanzees, orangutans, apes, baboons, macaques, monkeys) include Giardia sp. And Balantidium coli, Entamoeba histolytica, Sarcocystis species, Toxoplasma gondii, Plasmodim species (RBC), Babesia species (RBC), Trypanosoma species (plasma), Leishmania species (macrophages) and the like.

  Cancer is one of the leading causes of death for companion animals (ie cats and dogs). Cancer usually attacks older animals than in household pets and is incorporated into their families. It is believed that 45% of dogs older than 10 years will die from the disease. The most common treatment options include surgery, chemotherapy and radiation therapy. Other treatment modalities used, with some success, are laser therapy, cryotherapy, hyperthermia and immunotherapy. The choice of treatment depends on the type of cancer and the degree of metastasis. Unless the malignancy is confined to isolated areas of the body, it is difficult to remove only the malignant tissue without affecting normal cells.

  Malignant diseases commonly diagnosed in dogs and cats include, but are not limited to, lymphosarcoma, osteosarcoma, breast tumor, mastocytoma, brain tumor, melanoma, adenosquamous cell carcinoma, carcinoid lung tumor, bronchial adenocarcinoma, fine cell Bronchial adenocarcinoma, fibroma, mycochondroma, lung sarcoma, neurosarcoma, osteoma, papilloma, retinoblastoma, Ewing sarcoma, Wilm's tumor, Burkitt's lymphoma, microglioma, neuroblastoma, It includes giant cell tumor of the bone, oral neoplasia, fibrosarcoma, osteosarcoma, and rhabdomyosarcoma. Other neoplasias in dogs include reproductive squamous cell carcinoma, tumors of sexually transmitted disease, testicular tumor, testicular tumor, seminome, Sertoli cell tumor, hemangiopericytoma, histiocytoma, green tumor (granulocytic sarcoma) These include corneal papilloma, squamous cell squamous cell carcinoma, hemangiosarcoma, pleural mesothelioma, basal cell carcinoma, thymoma, gastric tumor, adrenal cancer, oral papillomatosis, hemangioendothelioma, and cystadenoma. Additional malignancies diagnosed in cats include follicular lymphoma, intestinal lymphosarcoma, fibrosarcoma, and lung squamous cell carcinoma. Ferret, a previously popular household pet, is known to develop islet cell adenomas, lymphomas, sarcomas, neuromas, pancreatic islets cell tumors, gastric MALT lymphomas, and gastric adenocarcinoma.

  As neoplasia affecting agricultural livestock, leukemia, hemangiopericytoma, and bovine eye neoplasia (bovine), foreskin fibrosarcoma, ulcerative squamous cell carcinoma, foreskin cancer, connective tissue neoplasia, and mastocytoma ( Equine), liver cancer (pig), lymphoma and pulmonary adenomatous polyposis (ovine), lung sarcoma, lymphoma, rous sarcoma, reticuloendotheliosis, fibrosarcoma, nephroblastoma, B-cell lymphoma, and lymphoid leukaemia ( Avian species), retinoblastoma, hepatic neoplasia, lymphosarcoma (lymphoblastic lymphoma), plasmacytoid leukemia, and sarcoma sarcoma (fish), caseous lymphadenitis (CLA), bacterial sheep pseudo These include chronic, contagious, contagious diseases of sheep and goats caused by M. tuberculosis, and sheep's contagious lung tumors caused by ovine lung adenosis.

  An allergen refers to a substance (antigen) that can induce an allergic or asthmatic response in a susceptible subject. The list of allergens is huge and can include pollen, insect poisons, animal dander dust, fungal spores, and drugs (eg, penicillin). Examples of natural animal and plant allergens include, but are not limited to, proteins specific to the following genera: Thus, Canine (Canis familiaris), Dermatophagoides (e.g. Dermatophagoides farinae), Felis (Felis domesticus), Ambrosia (Ambrosia artemiisfolia), Lolium (e.g. Lolium perenne or Lolium multiflorum), Cryptromelia maria maria maria Rica , Alder, Alnus (Alnus gultinoasa), Betula (Betula verrucosa), Quercus (Quercus alba), Olea (Olea e ropa), Artemisia (Artemisia vulgaris), Plantago (for example Plantago lanceolata), Parietaria (for example Parietaria officinalis or Parietaria judaica), Blattalla (for example Blattalla germanica), Apis (for example Apis multiflorum), Cupressus (for example Cupressus sempervirens) , Cupressus arizonica, and Cupressus macrocarpa), Juniperus (eg, Juniperus sabinoides, Juniperus virginiana, Junipe us communis, and Juniperus ashei), Thuya (eg, Thuya orientalis), Chamaecyparis (eg, Chamaecyparis obtusa), Periplaneta (eg, Periplaneta americana), Agropyron (eg, Agropyron repens), Secale (eg, Secale cereale), Triticum For example, Triticum aestivum), Dactylis (eg Dactylis glomerata), Festuca (eg Festuca elatior), Poa (eg Poa pratensis or Poa compressa) Avena For example, Avena sativa), Holcus (eg Holcus lanatus), Anthoxanthum (eg Anthoxanthum odoratum), Arrhenatherum (eg Arrhenatherum elatius), Agrostis (eg Agrostis alba) Phleum (eg Phleum pratense) These include Phalaris arundinacea), Paspalum (eg, Paspalum notatum), Sorghum (eg, Sorghum halepensis), and Bromus (eg, Bromus inermis).

  The antigen may be a nucleic acid encoded by a nucleic acid vector or may not be encoded in a nucleic acid vector. In the former case, the nucleic acid vector is administered to a subject to express the antigen in vivo. In the latter case, the antigen can be administered directly to the subject. As used herein, an antigen not encoded in a nucleic acid vector refers to any type of antigen that is not a nucleic acid. For example, in some aspects of the invention, the antigen not encoded in the nucleic acid vector is a polypeptide. Minor modifications of the primary amino acid sequence of the polypeptide antigen may also result in a polypeptide having substantially equivalent antigenic activity as compared to the unmodified equivalent polypeptide. Such modifications may be deliberate as site-directed mutagenesis or naturally occurring. All of the polypeptides produced by these modifications are encompassed herein as long as antigenicity still exists. The polypeptide can be, for example, a viral polypeptide.

  As used herein, the term "substantially purified" refers to substantially other proteins, lipids, carbohydrates, or other substances with which the polypeptide is naturally associated. Not refers to a polypeptide. One skilled in the art can purify viral or bacterial polypeptides using standard techniques for protein purification. Substantially pure polypeptides will often produce a single major band on a non-reducing polyacrylamide gel. In the case of partially glycosylated polypeptides or those with multiple initiation codons, there may be multiple bands on non-reducing polyacrylamide gels, which form a unique pattern for the polypeptide. The purity of viral or bacterial polypeptides can also be determined by amino terminal amino acid sequence analysis. Other types of antigens that are not encoded in nucleic acid vectors, such as polysaccharides, small molecules, mimetics, etc. are described above and are encompassed within the present invention.

  The present invention also uses a polynucleotide encoding an antigenic polypeptide. It is envisioned that the antigen can be delivered to the subject in a nucleic acid molecule encoding the antigen such that the antigen is expressed in vivo. Such an antigen delivered to a subject in a nucleic acid vector is referred to as an antigen encoded by the nucleic acid vector. A nucleic acid encoding an antigen is operably linked to a gene expression sequence that directs expression of the antigen nucleic acid in a eukaryotic cell. The gene expression sequence is any regulatory nucleotide sequence, such as a promoter sequence or a promoter-enhancer combination, which facilitates efficient transcription and translation of the antigen nucleic acid to which the gene expression sequence is operably linked. The gene expression sequence may be, for example, a mammalian or viral promoter such as a constitutive or inducible promoter. Constitutive mammalian promoters include, but are not limited to, promoters for the following genes: Hypoxanthine phosphoribosyltransferase (HPTR), adenosine deaminase, pyruvate kinase, b-actin promoter, and other constitutive promoters. Exemplary viral promoters that function constitutively in eukaryotic cells include, for example, cytomegalovirus (CMV), simian virus (eg, SV40), papilloma virus, adenovirus, human immunodeficiency virus (HIV), rous sarcoma Included are the long terminal repeats (LTRs) of viruses, cytomegalovirus, Moloney leukemia virus and other retroviruses, and promoters derived from the thymidine kinase promoter of herpes simplex virus. Other constitutive promoters are known to those skilled in the art. Promoters useful as gene expression sequences of the invention also include inducible promoters. Inducible promoters are expressed in the presence of an inducing agent. For example, the metallothionein promoter is induced to promote transcription and translation in the presence of certain metal ions. Other inducible promoters are known to those skilled in the art.

  In general, gene expression sequences optionally include 5 'non-transcribed and 5' non-translated sequences involved in the initiation of transcription and translation, such as TATA box, capping sequence, CAAT sequences, respectively. In particular, such 5 'non-transcribed sequences include a promoter region comprising a promoter sequence for transcriptional control of the operably linked antigen nucleic acid. Gene expression sequences optionally include enhancer sequences or upstream activator sequences, as desired.

  The antigenic nucleic acid is operably linked to the gene expression sequence. As used herein, when an antigen nucleic acid sequence and a gene expression sequence are covalently linked such that expression or transcription and / or translation of the antigen coding sequence is under the influence or control of the gene expression sequence, said antigen nucleic acid The sequences and gene expression sequences are said to be operably linked. If transcription of the antigen sequence is caused by the promoter in the 5 'gene expression sequence, and if the nature of the bond between the two DNA sequences causes (1) no induction of frameshift mutations, (2) the antigen sequence of the promoter region The two DNA sequences are said to be operably linked if they do not interfere with their ability to direct transcription or (3) do not interfere with the ability of the corresponding RNA transcript to be translated into protein. Thus, where the gene expression sequence is capable of transcribing the antigen nucleic acid sequence such that the resulting transcript is translated into the desired protein or polypeptide, the gene expression sequence is operably linked to the antigen nucleic acid sequence. Do.

  The antigenic nucleic acid of the invention can be delivered to the immune system alone or in combination with a vector. In a broad sense, a vector is any carrier that can facilitate delivery of an antigen nucleic acid to cells of the immune system such that the antigen can be expressed and presented on the surface of the immune cell. The vector generally transports the nucleic acid to immune cells that have reduced degradation based on the degree of degradation that results in the absence of the vector. The vector optionally contains the gene expression sequence described above to enhance expression of the antigen nucleic acid in immune cells. In general, vectors useful in the present invention include, but are not limited to, plasmids, phagemids, viruses, or other carriers derived from viruses or bacterial sources engineered by insertion or incorporation of antigenic nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to, the following virus-derived nucleic acid sequences. Specifically, retroviruses such as Moloney murine leukemia virus, Harvey murine sarcoma virus, murine mammary tumor virus, and Rous sarcoma virus, adenovirus, adeno-associated virus, SV40 virus, polyoma virus, Epstein-Barr virus, papilloma Viruses, herpes viruses, vaccinia viruses, polio viruses, and RNA viruses such as retroviruses. Other vectors not described but known in the art can readily be used.

  Preferred viral vectors are non-cytopathic eukaryotic viruses, in which the non-essential gene has been replaced by the gene of interest. Non-cytopathic viruses include retroviruses, the life cycle of which involves reverse transcription of genomic viral RNA into DNA, followed by proviral incorporation into cellular DNA of the host. Retroviruses have been approved for human gene therapy trials. Most useful are replication defective (ie, capable of directing the synthesis of the desired protein but not the production of infectious particles) retroviruses. Such recombinant retroviral expression vectors are versatile for high efficiency transduction of genes in vivo. Standard protocols for producing replication defective retroviruses (incorporating exogenous genetic material into a plasmid, transfecting a packaging cell line with said plasmid, and producing a recombinant retrovirus by said packaging cell line (I.e., collecting the virus particles from the tissue culture medium, and infecting the target cells with the virus particles), as described in Kriegler, M. et al. , Gene Transfer and Expression, A Laboratory Manual W. H. Freeman C. O. , New York (1990) and Molecular Biology, vol. 7, Humana Press, Inc. Murry, E., in: Clifflon, New Jersey (1991). J. Method and is provided.

  The preferred virus for a particular application is the adeno-associated virus, which is a double stranded DNA virus. Adeno-associated viruses can be engineered into replication defective viruses, which can infect a wide range of cell types and species. The adeno-associated virus also allows multiple consecutive transductions due to heat and lipid solvent stability, high transduction frequency in cells of diverse lineages including hematopoietic cells, and lack of superinfection inhibition. It has the same advantages as having it. Reportedly, adeno-associated virus can be integrated into human cell DNA in a site-directed manner, thus minimizing the possibility of insertion mutations and variability of inserted gene expression characteristic of retroviral infection Make it In addition, wild-type adeno-associated virus infection is followed by more than 100 passages in tissue culture in the absence of selective pressure, suggesting that the adeno-associated virus genome integration is a relatively stable event. Adeno-associated virus can also function in an extrachromosomal manner.

  Other vectors include plasmid vectors. Plasmid vectors are widely described in the art and are well known to those skilled in the art. See, eg, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. In recent years, plasmid vectors have been particularly advantageous for delivering genes to cells in vivo due to their ability to replicate in and integrate into the host genome. There was found. However, those plasmids having a promoter compatible with the host cell can express peptides from genes operably encoded in the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC / CMV, SV40, and pBlueScript. Other plasmids are well known to those skilled in the art. In addition, restriction enzymes and ligation reactions can be used to custom design plasmids by removing and adding specific fragments of DNA.

  It has recently been discovered that bacteria can be used to deliver plasmids carrying genes to the immune system. Modified forms of bacteria, such as Salmonella, can be transfected with the plasmid and used as a delivery vehicle. A bacterial delivery carrier can be administered to the host subject orally or by other means of administration. The bacteria deliver the plasmid to the immune system (eg, B cells, dendritic cells), presumably by crossing the gut wall. Using this methodology, high levels of immune protection have been established. Such delivery methods are useful in aspects of the invention that employ systemic delivery of antigens, immunostimulatory nucleic acids, and / or other therapeutic agents.

  Thus, in addition to being suitable as stand-alone agents, immunostimulatory nucleic acids are particularly useful as vaccine adjuvants. It has previously been established that CpG oligonucleotides are excellent vaccine adjuvants. In order to identify the best immunostimulatory nucleic acids for use as vaccine adjuvants in humans and other non-rodent animals, in vivo screening of different nucleic acids for this purpose was performed. Several in vitro assays were evaluated in mice for their predictive value of adjuvant activity in vivo. During the course of this study, in vitro tests were identified that would be predictive of in vivo efficacy. Rather, it has surprisingly been found that activation of both B cells and NK cells is particularly correlated with the ability of the immunostimulatory nucleic acid to enhance the in vivo immune response to the antigen.

  The nucleic acids are also useful for improving dendritic cell survival, differentiation, activation, and maturation. Immunostimulatory nucleic acids have a unique ability to promote cell survival, differentiation, activation, and maturation of dendritic cells. Dendritic progenitor cells isolated from blood by immunomagnetic cell sorting generate morphological and functional characteristics of dendritic cells during a two day incubation with GM-CSF. Without GM-CSF, these cells undergo apoptosis. Immunostimulatory nucleic acids are superior to GM-CSF in promoting dendritic cell survival and differentiation (MHC II expression, cell size, particle size). Immunostimulatory nucleic acids also induce maturation of dendritic cells. Dendritic cells form a link between the innate and acquired immune systems by presenting antigens and through the expression of pattern recognition receptors that detect bacterial molecules such as LPS in their local environment Thus, the ability to activate dendritic cells with immunostimulatory nucleic acids is a function of their immunity to in vivo and ex-vivo immunotherapy against disorders such as cancer and allergic diseases or infections. Support the use of stimulatory nucleic acid based strategies. Immunostimulatory nucleic acids are also useful for activating and inducing dendritic cell maturation.

  Immunostimulatory nucleic acids also increase natural killer cell lytic activity and antibody-dependent cellular cytotoxicity (ADCC). ADCC can be performed using immunostimulatory nucleic acids in combination with antibodies specific for cellular targets such as cancer cells. When an immunostimulatory nucleic acid is administered to a subject with an antibody, the subject's immune system is induced to kill tumor cells. Antibodies useful in this ADCC procedure include antibodies that interact with cells in the body. Antibodies specific for many such cellular targets are described in the art and many are commercially available. Examples of these antibodies are listed below in the list of cancer immunotherapies.

  The nucleic acids are also useful for re-emerging an immune response from a Th2 immune response to a Th1 immune response. Re- eliciting an immune response from a Th2 immune response to a Th1 immune response can be assessed by measuring the levels of cytokines produced in response to this nucleic acid (eg, inducing monocytic cells and other cells To generate Th1 cytokines including IL-12, IFN-γ and GM-CSF). Reaging or rebalancing the immune response from a Th2 to a Th1 response is particularly useful for the treatment or prevention of asthma. For example, an effective amount for treating asthma may be that amount effective to re-emit a Th2-type immune response associated with asthma to a Th1-type response. Th2 cytokines, particularly IL-4 and IL-5, are elevated in the airways of asthmatic subjects. These cytokines promote key aspects of the asthmatic inflammatory response, including IgE isotype switching, eosinophil chemotaxis and activation, and mast cell growth. Th1 cytokines, in particular IFN-γ and IL-12, can suppress the formation of Th2 clones and the production of Th2 cytokines. The immunostimulatory nucleic acids of the present invention cause an increase in Th1 cytokines that help rebalance the immune system, mainly preventing or reducing the side effects associated with Th2 immune responses.

  The invention also includes methods for inducing broad spectrum resistance to non-specific antigen innate immune activation and infectious challenges using immunostimulatory nucleic acids. As used herein, the term antigen non-specific innate immune activation refers to activation of immune cells other than B cells, and for example, can respond in an NK cell, T cell or antigen independent manner. It may involve the activation of one or more immune cells or some combination of these cells. Broad spectrum resistance to infectious challenge is induced because immune cells are in active form and are primed to respond to any invasive compound or microorganism. These cells do not have to be specifically primed to a particular antigen. This is particularly useful in bacterial warfare, and other situations described above, such as travelers.

  The nucleic acids of the invention may be used in combination with other therapeutic agents, including antimicrobials, adjuvants, cytokines, anti-cancer treatments, allergy medicines, asthma medicines and the like.

  The nucleic acids of the invention can be administered to a subject with an anti-microbial agent. As used herein, an antimicrobial agent refers to a naturally occurring or synthetic compound that can kill or inhibit an infectious microorganism. The type of antimicrobial agent useful according to the invention depends on the type of microorganism at which the subject is infected or at risk of becoming infected. Antimicrobial agents include, but are not limited to, antibacterial agents, antiviral agents, antifungal agents and antiparasitic agents. Terms such as “anti-infective agent”, “anti-bacterial agent”, “anti-viral agent”, “anti-fungal agent”, “anti-parasitic agent” and “parasitic agent” are well established to those skilled in the art It has meaning and is defined in standard medical textbooks. In summary, anti-bacterial agents kill or inhibit bacteria, and include antibiotics and other synthetic or natural compounds with similar functions.

  Antibiotics are low molecular weight molecules produced as secondary metabolites by cells such as microorganisms. In general, antibiotics interfere with one or more bacterial functions or structures which are specific for the microorganism and which are not present in the host cell. Antiviral agents can be isolated or synthesized from natural sources and are useful for killing or inhibiting the virus. Antifungal agents are used to treat superficial fungal infections and opportunistic and primary systemic fungal infections. Antiparasitic agents kill or inhibit parasites.

  Antibacterial agents inhibit bacterial killing or bacterial growth or function. A large class of antibacterials are antibiotics. Antibiotics that are effective to kill or inhibit a wide range of bacteria are referred to as broad spectrum antibiotics. Other types of antibiotics are preferentially effective against gram positive or gram negative bacteria. These types of antibiotics are referred to as narrow spectrum antibiotics.

  Other antibiotics that are effective against a single microorganism or disease and not against other types of bacteria are referred to as limited spectrum antibiotics. Antibacterial agents are often classified based on their major mode of action. In general, antibacterial agents are cell wall synthesis inhibitors, cell membrane inhibitors, protein synthesis inhibitors, nucleic acid synthesis inhibitors or functional inhibitors, and competition inhibitors.

  Antibacterial agents useful in the present invention include, but are not limited to: Natural penicillin, semisynthetic penicillin, clavulanic acid, cephalosporin, bacitracin, ampicillin, carbenicillin, oxacillin, azlocillin, mezlocillin, mezlocillin, piperacillin, methicillin, dicloxacillin, nafcillin, cephalotin, cephalexin, cefamdol, cefaclor, cefazolin, Cefloxin, cefoxitin, cefotaxime, cefsoxadin, cefetamet, cefixime, ceftriaxone, ceftriaxone, cefoperazone, ceftazidine, moxalactam, carbapenem, imipenem, monobactem, ioiztreonam, polymyxin, amphotericin B, nystatin, imidazole, and , Flu Cona , Rifampin, ethambutol, tetracycline, chloramphenicol, macrolide, aminoglycoside, streptomycin, kanamycin, tobramycin, amikacin, gentamycin, tetracycline, minocycline, doxycycline, chlorotetracycline, erythromycin, roxithromycin, clarithromycin, oleandromycin, Azithromycin, chloramphenicol, quinolones, cotrimoxazole, norfloxacin, ciprofloxacin, enoxacin, nalidixic acid, temafloxacin, sulfonamides, gantricine, and trimethoprim; acedapson; sodium acetosulfone; alamesin; alexidine; amdinocillin; Amdinocilin Pipoxyl; Amisa Amifloxacin; amifloxacin mesylate; amikacin sulfate; amikacin sulfate; aminosalicylic acid; aminosalicylate sodium; amoxicillin; amphomycin; ampicillin; ampicillin sodium; aplacillin sodium; apramycin; aspartocin; astromicin sulfate; ; Avoparcin; azithromycin; azlocillin; azlocillin sodium; bacampicillin hydrochloride; bacitracin; bacitracin methylene disalicylate; bacitracin zinc; lambda vermycin; benzoylpath calcium; beristhromycin; benthamicin sulfate; biapenem; viniramycin; Pyrithione magsulfex; butycasin; butyrosine sulfate; Carbenicillin disodium; carbenicillin indanyl sodium; carbenicillin phenyl sodium; carbenicillin potassium; carmonam sodium; cefaclol; cefadroxil; cefamandol; cefamandol nafate; cefamandol sodium; Cefatorhidine; Cefazafural Sodium; Cefazolin Sodium; Cefbuperazone; Cefbuperazone; Cefdinir; Cephepimum; Cefepime Hydrochloride; Sodium; Cefotetan Disodium; Cefotiam Hydrochloride; Cefoxitin Sodium; Cefupimizole; Cefupimizole; Cefupimizole Sodium; Cefpyramide Sodium; Cefpyramide Sodium; Cefpiromusulphate; Cefpodoximuproxetyl; Cefprozil; Ceftazidime; ceftizidom; ceftizoxime sodium; ceftriaxone sodium; cefuroxime; cefloxime oxetyl; cefuroxime pivoxetyl; cefuroxime sodium; cefacetryl sodium; cephalexin; cephalexin hydrochloric acid; Cepharothin sodium; Cephapirin sodium; Cephalazine; cetocycline hydrochloride; cetophenicol; chloramphenicol Chloramphenicol palmitate; chloramphenicol pantothenate complex; chloramphenicol sodium succinate; chlorhexidine phosphine; chlorxylenol; chlortetracycline bisulphate; chlortetracycline hydrochloride; sinoxacin; Ciprofloxacin hydrochloride; cilolemycin; clarithromycin; clinafloxacin hydrochloride; clidamycin; clidamycin hydrochloride; clidamycin palmitate hydrochloride; clindamycin phosphate; clofazimine; cloxacillin benzathine; Xacillin sodium; croxiquine; choristimetate sodium; colistin sulfate; coumermycin; coumermycin sodium; cyclacillin; cycloserine; Dapson; Daptomycin; Demecrocyclin Hydrochloride; Demecyclin Hydrochloride; Demecyclin; Eryxamycin sodium; enoxacin hydrochloride; erythromycin assist erythromycin; erythromycin escrite; erythromycin ethyl succinate; erythromycin ethyl succinate; erythromycin single septate; erythromycin lactobionate; erythromycin propionate; Etambutol Hydrochloride; Ethionamide; Freloxacin; Fluxacillin; Fludalanin; Flumekin; Phosfomycin; Phosfomycin Tromethamine; Fumoxicillin; Furazolium Chloride; Furazolium Tartrate; Fusidium Sodium: Fusidic Acid; Gentamycin Sulfate Huloximein; hepacillin; hecacillin potassium; hexedin; ibafloxacin; imipenem; isoconazole; Hydrochloric acid; Lomefloxacin; Lomefloxacin hydrochloric acid; Lomefuroki Melocycline sulfosalicylic acid; megalomycin potassium phosphate; mekidox; meropenem; metacycline; methacycline hydrochloride; methenamine; methenamine hippuric acid; methenamine mandelic acid; methicillin sodium; Hydrochloric acid; metronidazole phosphoric acid; mezlocillin; mezulocillin sodium; minocycline; minocycline hydrochloric acid; myrinocamycin hydrochloride; monensin sodium; monensin sodium; nafcillin sodium; nalidixate sodium; nalidixate acid; natamycin; nebramycin; Neomycin sulfate; Neomycin undecylenate; Netylmicin sulfate; Neutra Nifuradene; nifuratoride; nifuratoril; nifludadil; niflupirol; nifluquinaazole; nifluthiazole; nitrocycline; nitrofurantoin; Oxyphosphoric acid; Oxytetracycline; Oxytetracycline calcium; Oxytetracycline hydrochloride; Pardimycin hydrochloride; Parachloromycin; paulomycin; Pefloxacin; Pefloxacin mesylate; Penamecillin G: Benzatine; Penicillin G potassium; Penicillin G procaine; Penicillin G sodium; V; Penicillin V Benza Penicillin V Hydrabamine; Penicillin V Potassium; Penyrididone Sodium; Phenylaminosalicylic Acid; Piperacillin Sodium; Pirvenicillin Sodium; Pyricillin Sodium; Pilrimycin Hydrochloride; Pivampicillin Hydrochloric Acid; Benton, polymyxin B sulfate, porphyromycin, propicamycin, pyrazine amide, pyrithione zinc, quindecamine acetate, quinupristin, lacephenicol, ramoplanin, ranimomycin, lepromycin, repromicin, rifabutin, rifamexin, rifamid, rifapentine, rifapentin, rifaximine; Loritetracycline; Loritetracycline nitrate; rosalamicin; rosalamicin butyric acid; rosalami Rosalamicin sodium phosphate; rosalamicin stearic acid; rosoxalin; roxisulomycin; sancycline; sanfetrinem sodium; salmoxicillin; salpicillin; scopafungin; sisomicin; Spectinomycin Hydrochloride; Spiramycin; Stalinomycin Hydrochloride; Stefinomycin; Streptomycin Sulfate; Strepticonidzide; Sulfabenz; Sulfabenzamide; Sulfacetamide; Sulfacetamide; Sodium Sulfatamide; Sulfaxene; sulfamelazine; sulfamethol; sulfamethazine; sulfamethizole; sulfa Sulfoxone; Sulfamonomethmethoxine; Sulfamoxol; Zinc Sulfanilate; Sulfanitrale; Sulfasalazine; Sulfasomeizole; Sulfathiazole; Sulfazamet; Sulfisoxazole; Sulfisoxazole Acetyl; Soxazole Diolamine; sulfomyxin; sultamem; sultamicillin; sunampicin sodium; thalampicin hydrochloride; teicoplanin; temafloxacin hydrochloride; temocillin; tetracycline; tetracycline hydrochloride; tetracycline hydrochloride complex; tetroxoprim; tiamphenicol; Ticarcillin cresyl sodium; ticarcillin disodium; ticarcillin monosodium; cyclaton; thiodonium chloride; Emissions; Tobramycin sulfate; tosufloxacin; Trimethoprim; trimetrexate Primus sulfates; Tris Alpha pyrimidine; troleandomycin; Toro spectinomycin sulfate; Chirosurishin; Bankomashiin; Bankomashiin hydrochloride; Bill Guinea clarithromycin; and resolver clarithromycin.

  Antiviral agents are compounds that prevent the infection of cells by viruses or the replication of viruses within cells. There are many antivirals but less than antibacterials. The process of viral replication is closely linked to DNA replication in host cells, as non-specific antiviral agents are often toxic to the host. There are several processes within the process of viral infection that can be blocked or inhibited by antiviral agents. These stages include attachment of the virus to the host cell (immunoglobulin or binding peptide), decoating of the virus (for example amantadine), synthesis or translation of viral mRNA (for example interferon), replication of viral RNA or DNA (for example For example, nucleoside analogs), maturation of novel viral proteins (eg, protease inhibitors), and budding and release of the virus.

  Nucleotide analogs are synthetic compounds that are similar to nucleotides, but have an incomplete or abnormal deoxyribose or ribose group. Once the nucleotide analogues are intracellular, they are phosphorylated to form the formed triphosphate which competes with normal nucleotides for incorporation into viral DNA or RNA. Once the triphosphate form of the nucleotide analog is incorporated into the elongated nucleic acid strand, it irreversibly associates with the viral polymerase and thus causes chain termination. Nucleotide analogues, without limitation, acyclovir (used for the treatment of herpes simplex virus and varicella-zoster virus), ganciclovir (useful for the treatment of cytomegalovirus), idoxuridine, treatment of ribavirin (RS virus) Useful), dideoxyinosine, dideoxycytidine, and zidovudine (azidothymidine). Interferons are cytokines that are secreted by virus-infected cells and immune cells. Interferons function by binding to specific receptors on cells adjacent to infected cells, causing changes in the cells that protect it from viral infection. Alpha-interferon and beta-interferon also induce the expression of class I and class II MHC molecules on the surface of infected cells, causing increased antigen presentation for immune cell recognition. Alpha-interferon and beta-interferon are available in recombinant form and are used for the treatment of chronic hepatitis B and C infections. At dosages that are effective for antiviral treatment, interferon has serious side effects such as fever, fatigue and weight loss.

  Immunoglobulin therapy is used for the prevention of viral infections. Immunoglobulin therapy for viral infections is different from bacterial infections. Because, rather than being antigen specific, this immunoglobulin therapy functions by binding to extracellular virions and preventing them from attaching to and entering cells susceptible to viral infection. This treatment is useful to prevent viral infection during the presence of the antibody in the host. In general, there are two types of immunoglobulin therapies, normal immunoglobulin therapies and hyperimmunoglobulin therapies. Normal immunoglobulin therapy utilizes antibody products prepared and pooled from normal blood donor serum. This pooled product contains low titers of antibodies against a wide range of human viruses such as hepatitis A, parvovirus, enterovirus (especially in newborns). Hyperimmunoglobulin therapy utilizes antibodies prepared from the sera of individuals having high titers of antibodies to a particular virus. These antibodies are then used against specific viruses. Examples of hyperimmunoglobulins are herpes zoster immunoglobulin (useful for the prevention of varicella in tolerized infants and neonates), human rabies immunoglobulin (useful for the prevention after exposure of subjects bitten by violent animals), hepatitis B Immunoglobulins (prevention of hepatitis B virus, particularly useful in subjects exposed to this virus), and RSV immunoglobulin (useful in the treatment of RS viral infection).

  Another type of immunoglobulin therapy is active immunization. This involves the administration of antibodies or antibody fragments to viral surface proteins. Two types of vaccines that can be used for active immunization of hepatitis B include serum-derived hepatitis B antibodies and recombinant hepatitis B antibodies. Both are prepared from HBsAg. These antibodies are administered at three doses to subjects at high risk of infection with hepatitis B virus, such as health care workers, sexual partners of chronic carriers, and infants.

  Thus, high viral agents useful in the present invention include, but are not limited to, immunoglobulins, amantadine, interferons, nucleoside analogs, and protease inhibitors. Detailed examples of antiviral agents include, but are not limited to: acemannan; acyclovir; acyclovir sodium; adefovir; alobudine; arvirceptos dotox; anantadine hydrochloride; alanotine; Ciparabine hydrochloride; delavidin mesylate; desciclovir; didanosine; dixoxalyl; edoxudin; embiloden; enviroxime; famciclovir; famothioline hydrochloride; Sodium; idox uridine; ketoxal; lamivuudin; lobcavir; memotine hydrochloride; methisazone; Penciclovir; pirodavir; ribavirin; rimantadine hydrochloride; saquinavir mesylate; somantadine hydrochloride; And ginviroxime.

  Antifungal agents are useful for the treatment and prevention of infectious fungi. Anti-fungal agents are sometimes classified by their mechanism of action. Some antifungal agents function as cell wall inhibitors by inhibiting glucose synthesis. These include, but are not limited to, basilyungin / ECB. Other antifungal agents function by destabilizing the integrity of the membrane. These include clotrimazole, sertaconzole, fluconazole, itraconazole, ketoconazole, miconazole, and imidazole such as voriconazole, as well as FK463, amphotericin B, BAY 38-9502, MK 991, pradimicin, UK 292, butenafin, and terbinafine. (But not limited to this). Other antifungal agents function by degrading chitin (eg, chitinase) or by immunosuppression (501 cream). Some examples of commercially available drugs are shown in Table 3.

  Therefore, as an antifungal agent useful in the present invention, imidazole, FK463, amphotericin B, BAY 38-9502, MK 991, pradimicin, UK 292, butenafin, chitinase, 501 cream, acrisolcin; ambroticin; amorolfin, amphotericin B; azaconazole; azaserine Bisulfangin, bifonaZole, biphenamin hydrochloride, bispyrithione magsulfexium nitrate, butoconazole nitrate, calcium undecylenate, candicidin, fuchsin chlorate, chlordantoin, ciclopirox, ciclopirox olamine, silyofungin, cisconazole, crotrimazole Denofungin, dipyrithione, deconazole, econazole, econazole nitrate, enilconazole, Gulsioflavin; glyseoflavin; hamycin; isoconazole; carafungin; ketoconazole; ketoconazole; lomofungin; lymimycin; Niphratel; nifluralon hydrochloride; nistatin; octanoic acid; olconazole nitrate; oxiconazole nitrate; oxyfungin hydrochloride; Scopafungin; Selenium sulfide; Sinefungin; Terbinafine; Terconazole; Thiram; Thicraton; Thioconazole; Tolucyclate; Torinate; Tolaphnate; Triacetin; Triafungin; Tridecungin; Undecylenic Acid; Bilidoflugin; Zinc Undecylenic Acid; and Dinoconazole Hydrochloride.

  Examples of antiparasitic agents, also called parasiticides, which are useful for human administration, include albendazole, amphotericin B, benznidazole, bithionol, chloroquine hydrochloride, chloroquine phosphate, clindamycin, dehydroemetin, diethylcarba Madin, diloxanide furoate (furoate), eflornithine, ephemerin, glucocorticoid, halofantrine, iodophanol, iodoquinol, mevalvazole, mefloquine, meglumine antimonioate, melarsoprol, metrifonate, metronidazole, niclosamide, ninic Fluthymox, oxamniquin, paromomycin, pentamidine isethionate, piperazine, praziquantel, primaquine phosphate, proguanyl, pyrantel pamoate, pyrimethamine Rufonamide, pyrimethamine sulfadoxine, quinacrine hydrochloride, quinine sulfate, quinidine gluconate, spiramycin, stibogluconate sodium (sodium antimony gluconate), suramin, tetracycline, doxycycline, thiabendazole, tinidazole, trimethoprim (sulfomethokil) Sazol, and tripalsamide (some of which are used alone or in combination with others) include (but are not limited to).

  Pesticides used for non-human subjects include piperazine, diethylcarbamazine, thiabendazole, fenbendazole, albendazole, oxyfendazole, oxybendazole, febantel, levamisole, pyrantel tartrate, pyrantelpamoate , Dichlorvos, ivermectin, doramectic, milbemycin oxime, iprinomectin, moxidectin, N-butyl chloride, toluene, hygromycin B tiacetalsemide sodium, melarsamine, praziquantel, epsiprantel, benzimidazole (eg, phenbendazole, albendazole, oxy Fendazole, chlorthrone, albendazole, amprolium), deciconate, lasaloside, monensin, sulfadimethoxy , Sulfamethazine, sulfaquinoxaline down, metronidazole, and the like.

  Pesticides used in horses include mebendazole, oxyfendazole, febantel, pyrantel, dichlorvos, trichlorfon, ivermectin, piperazine; For westeri: ivermectin, benzimidazole (e.g. thiabendazole, cambendazole, oxybendazole, and fenbendazole). Useful parasiticides for dogs include milbemycin oxime (oxine), ivermectin, pyrantel pamoate, and a combination of ivermectin and pyrantel. Treatment of parasites in pigs may include the use of levamisole, piperazine, pyrantel, thiabendazole, dichlorvos, and fenbendazole. In sheep and goats, anthelmintics include levamisole or ivermectin. Capulsolate is the cat D. It shows some efficacy in the treatment of immitis.

  Immunostimulatory nucleic acids can also be administered in combination with anti-cancer treatments. Anti-cancer treatments include cancer medications, radiation, and surgical procedures. As used herein, "cancer therapeutic agent" refers to an agent that is administered to a subject for the purpose of treating cancer. As used herein, "treatment of cancer" includes preventing the development of cancer, reducing the symptoms of cancer, and / or inhibiting the growth of the cancer that has developed. In another aspect, a cancer therapeutic agent is administered to a subject who is at risk of developing a cancer for the purpose of reducing the risk of developing the cancer. Various medicaments for the treatment of cancer are described herein. For the purpose of this specification, cancer therapeutics are classified as chemotherapeutics, immunotherapeutics, cancer vaccines, hormonal treatments, and biological response modifiers.

  As used herein, "cancer therapeutic agent" refers to an agent that is administered to a subject for the purpose of treating cancer. As used herein, "treatment of cancer" includes preventing the development of cancer, reducing the symptoms of cancer, and / or inhibiting the growth of the generated cancer. In another aspect, a cancer therapeutic agent is administered to a subject who is at risk of developing a cancer for the purpose of reducing the risk of developing the cancer. Various medicaments for the treatment of cancer are described herein. For the purpose of this specification, cancer therapeutics are classified as chemotherapeutics, immunotherapeutics, cancer vaccines, hormonal treatments, and biological response modifiers. In addition, the methods of the invention are intended to include the use of more than one cancer therapeutic along with the immunostimulatory nucleic acid. As an example, where appropriate, an immunostimulatory nucleic acid can be administered with both a chemotherapeutic agent and an immunotherapeutic agent. Alternatively, an immunotherapeutic agent and a cancer vaccine, or a chemotherapeutic agent and a cancer vaccine, wherein the cancer therapeutic agent is all administered to one subject for the purpose of treating a subject having cancer or at risk of developing cancer. Or a chemotherapeutic agent, an immunotherapeutic agent, and a cancer vaccine.

  Cancer therapeutics can function in various ways. Some cancer therapeutics work by targeting a physiological mechanism specific to tumor cells. Examples of such genes that target specific genes that are mutated in cancer, and the products of those genes (ie, primarily proteins), include oncogenes (eg, Ras, Her2, bcl- 2) including, but not limited to, tumor suppressor genes (eg, EGF, p53, Rb), and cell cycle targets (eg, CDK4, p21, telomerase). Cancer therapeutics may alternatively target signal transduction pathways and molecular mechanisms that are altered in cancer cells. Targeting cancer cells through epitopes expressed on their cell surface is achieved through the use of monoclonal antibodies. This latter type of cancer therapeutic is generally referred to herein as immunotherapy.

  Other cancer therapeutics target cells other than cancer cells. For example, some drugs stimulate the immune system to attack tumor cells (ie, cancer vaccines). Still other medications, called angiogenesis inhibitors, function by attacking the blood supply of solid tumors. Because most malignant cancers can metastasize (ie, be present at the primary tumor site and disseminated to distant tissue to form a secondary tumor), drugs that interfere with this metastasis are also Useful for treatment. Specific members of basic FGF, VEGF, angiopoietin, angiostatin, endostatin, TNFα, TNP-470, thrombospondin-1, platelet factor 4, CAI, and integrin family proteins as angiogenesis mediators , Is mentioned. One class of this type of drug is metalloprotease inhibitors, which inhibit the enzymes used by cancer cells to be present at the primary tumor site and to extravasate into another tissue.

  An immunotherapeutic agent is a drug derived from an antibody or antibody fragment that specifically binds to or specifically recognizes a cancer antigen. As used herein, cancer antigens are broadly defined as antigens expressed by cancer cells. Preferably, the antigen is expressed on the cell surface of a cancer cell. Even more preferably, the antigen is an antigen that is not expressed in normal cells, or at least not at the same level as cancer cells. Antibody-based immunotherapy can function by binding to the cell surface of cancer cells, thereby stimulating the underlying immune system to attack cancer cells. Another way in which antibody-based immunotherapy functions is as a delivery system for specific targeting of toxic substances to cancer cells. The antibodies are usually toxins such as lysine (eg, derived from castor bean), calicheamicin and maytansinoid, radioactive isotopes such as iodine-131 and yttrium-90, (herein Conjugated with a chemotherapeutic agent (as described), or a biological response modifier. In this way, toxic substances can be concentrated in the cancerous area and nonspecific toxicity to normal cells can be minimized. In addition to the use of antibodies specific for cancer antigens, antibodies that bind to the vasculature (eg, antibodies that bind to endothelial cells) are also useful in the present invention. This is generally because solid tumors survive depending on newly formed blood vessels, so most tumors can reinforce and stimulate the growth of new blood vessels. As a result, one strategy of many cancer mechanisms is to attack the blood vessels that feed the tumor and / or attack the connective tissue (or stroma) that supports such blood vessels.

  The use of immunostimulatory nucleic acids in combination with immunotherapeutic agents (eg, monoclonal antibodies) has a number of mechanisms (discussed above) including significant enhancement of ADCC, natural killer (NK) cell activation, and Long-term survival can be prolonged through the increase of IFNα levels. The nucleic acid used in combination with the monoclonal antibody acts to reduce the dose of antibody required to achieve a biological result.

  Examples of cancer immunotherapies currently in use or under development are described in Table 4.

  Still other types of chemotherapeutic agents that may be used in accordance with the present invention include aminoglutethimide, asparaginase, busulfan, carboplatin, chloronbucil, cytarabine hydrochloride, dactinomycin, daunorubicin hydrochloride, estramustine phosphate sodium, etoposide (VP 16 -213), floxuridine, fluorouracil (5-FU), flutamide, hydroxyurea (hydroxycarbamide), ifosfamide, interferon alpha-2a, interferon alpha-2b, leuprolide acetate (LHRH releasing factor analogue), lomustine (CCNU), Mechlorethamine hydrochloride (nitrogen mustard), mercaptopurine, mesna, mitototen (o p'-DDD), mitozanthrone hydrochloride, octreotide, plicamycin, hydrochloric acid Procarbazine, Streptozocin, Tamoxifen Citrate, Thioguanine, Thiotepa, Vinblastine Sulfate, Ansacrine (m-AMSA), Azacytidine, Erythropoietin, Hexamethylmelamine (HMM), Interleukin 2, Mitoguazone (Methyl-GAG; Methylglyoxal Bis-) These include guanylhydrazone; MGBG), pentostatin (2 'deoxycoformycin), semustine (methyl-CCNU), teniposide (VM-26), and vindesine sulfate.

  Cancer vaccines are medicaments intended to stimulate the endogenous immune response against cancer cells. Vaccines currently produced mainly activate the humoral immune system (ie, antibody dependent immune response). Other vaccines currently under development focus on the activation of cell-mediated immune systems, including cytotoxic T cells that can kill tumor cells. Cancer vaccines generally enhance the presentation of cancer antigens to antigen presenting cells (eg, macrophages and dendritic cells) and / or other immune cells (eg, T cells, B cells, and NK cells).

  Cancer vaccines may take one of several forms, as discussed below, but their purpose is by APC and by the final presentation of antigen presentation on the cell surface related to MHC class I molecules. In order to facilitate the endogenous processing of such antigens, to deliver cancer antigens and / or cancer associated antigens to antigen presenting cells (APCs). One form of cancer vaccine is a whole cell vaccine, which is a preparation of cancer cells removed from a subject, treated ex vivo, and then reintroduced into the subject as whole cells. Lysates of tumor cells can also be used as cancer vaccines to elicit an immune response. Another form of cancer vaccine is a peptide vaccine, which activates T cells using a cancer specific small protein or a cancer associated small protein. Cancer-associated proteins are proteins that are not expressed exclusively by cancer cells (ie, other normal cells may also express these antigens). However, expression of cancer-associated antigens is generally consistently upregulated by certain types of cancer. Yet another form of cancer vaccine is a dendritic cell vaccine, which comprises whole dendritic cells exposed to a cancer antigen or a cancer associated antigen in vivo. Dendritic cell lysates or membrane fractions may also be used as cancer vaccines. Dendritic cell vaccines can activate antigen presenting cells directly. Other cancer vaccines include ganglioside vaccines, heat shock protein vaccines, viral and bacterial vaccines, and nucleic acid vaccines.

  The use of an immunostimulatory nucleic acid in combination with a cancer vaccine has resulted in an improved, antigen-specific humoral immune response in addition to activating NK cells and endogenous dendritic cells and increasing IFNα levels. And provide antigen-specific cell-mediated immune responses. This enhancement makes it possible to achieve the same effective effect with a vaccine containing reduced antigen dose. In some cases, cancer vaccines can be used with an adjuvant as described above.

  Other vaccines take the form of dendritic cells, which have been exposed to cancer antigens in vitro to treat the antigens, and on their cell surface, to present effective antigens to other immune system cells. To express cancer antigens associated with MHC molecules).

  Immunostimulatory nucleic acids are used in one aspect of the invention in combination with dendritic cell based cancer vaccines. Dendritic cells are professional antigen presenting cells. Dendritic cells display an association between the prior and acquired immune systems through the expression of pattern recognition receptors, by presenting antigens and detecting microbial molecules such as LPS in their local environment. Form. Dendritic cells efficiently internalize, process, and present soluble specific antigens to which they are exposed. The processes of internalization and antigen presentation involve rapid upregulation of major histocompatibility complex (MHC) and costimulatory molecule expression, cytokine production, and where they are thought to be involved in T cell activation. Causes a transition to a certain lymphatic organ.

  Table 5 lists various cancer vaccines currently being used or under development.

  As used herein, chemotherapeutic agents include immunotherapeutics and all other forms of cancer therapeutics that do not fall under the cancer vaccine classification. As used herein, chemotherapeutic agents include both chemical and biological agents. These agents function to inhibit the cellular activity that cancer cells rely on for continued survival. Categories of chemotherapeutic agents include alkylating agents, alkaloid agents, anti-metabolites, hormones or hormone analogs, and various antineoplastic agents. Most, if not all, of these agents are directly toxic to cancer cells and do not require an immune response. The combination of chemotherapy and immunostimulatory nucleic acid administration increases the maximum tolerated dose of chemotherapy.

  Chemotherapeutic agents currently under development or in clinical use are shown in Table 6.

  In one embodiment, the methods of the invention use immunostimulatory nucleic acids instead of using IFNα therapy in the treatment of cancer. Currently, some treatment protocols require the use of IFNα. Because IFNα is produced following administration of several immunostimulatory nucleic acids, these nucleic acids can be used to endogenously produce IFNα.

  In another embodiment, the asthma drug / allergy drug is a PDE-4 inhibitor, a bronchodilator / β-2 agonist, a K + channel opener, a VLA-4 antagonist, a neurokin antagonist, a TXA2 synthesis inhibitor, xanthanine, an arachidonic acid antagonist, 5 lipoxygenase inhibitors, thrombosin A2 receptor antagonists, thromboxane A2 antagonists, 5-lipox activating protein inhibitors, and protease inhibitors, which are drugs selected from (but not limited to). In some important embodiments, the asthma drug / allergy drug is a bronchodilator / β-2 agonist selected from the group consisting of salmeterol, salbutamol, terbutaline, D2522 / formoterol, fenoterol, and orciprenaline.

  In another embodiment, the asthma drug / allergy drug is a drug selected from the group consisting of antihistamines and prostaglandin inducers. In one embodiment, the antihistamine agent is loratidine, cetirizine, buclidine, ceterizine analogue, fexofenadine, terfenadine, desloratadine, norastemizole, epinastine, ebastine, ebastine, astemizole, levocabastine, azelastine, tranilast, terfenadine, mizolastine It is selected from the group consisting of betataxine, CS560, HSR609. In another embodiment, the prostaglandin inducer is S-5751.

  In yet another embodiment, the asthma medication / allergy medication is selected from the group consisting of steroids and immunomodulators. Immunomodulators include anti-inflammatory agents, leukotriene antagonists, IL-4 muteins, soluble IL-4 receptors, immunosuppressants, anti-IL-4 antibodies, IL-4 antagonists, anti-IL-5 antibodies, soluble IL-13 receptors- It may be selected from the group consisting of (but not limited to) Fc fusion proteins, anti-IL-9 antibodies, CCR3 antagonists, CCR5 antagonists, VLA-4 inhibitors, and downregulators of IgE. In one embodiment, the downregulator of IgE is anti-IgE.

  In another embodiment, the steroid is selected from the group consisting of beclomethasone, fluticasone, tolanquinolone, budesonide, and budesonide. In still further embodiments, the immunosuppressive agent is a tolerized peptide vaccine.

  In one embodiment, the immunostimulatory nucleic acid is coadministered with an asthma drug / allergy drug. In another embodiment, the subject is an immunocompromised subject.

  The immunostimulatory nucleic acid can be further combined with other therapeutic agents (eg, an adjuvant) to enhance the immune response. Immunostimulatory nucleic acids, and other therapeutic agents, can be administered simultaneously or sequentially. When other therapeutic agents are administered simultaneously, they may be administered as the same or separate formulations, but simultaneously. If the administration of the other therapeutic agent and the immunostimulatory nucleic acid is temporally separated, the other therapeutic agents are administered sequentially with each other and with the immunostimulatory nucleic acid. The separation of time between the administration of these compounds can be in minutes or longer. Other therapeutic agents include (but are not limited to) adjuvants, cytokines, antibodies, antigens, and the like.

  Compositions of the invention may also include non-nucleic acid adjuvants. A non-nucleic acid adjuvant is any molecule or compound that can stimulate humoral and / or cellular immune responses other than the immunostimulatory nucleic acids described herein. Non-nucleic acid adjuvants include, for example, adjuvants that cause retention, immunostimulatory adjuvants, and adjuvants that cause retention and stimulate the immune system.

  As used herein, an adjuvant that causes a retention effect is an adjuvant that causes the antigen to be released slowly into the body, thereby prolonging the exposure of immune cells to the antigen. Adjuvants of this class include alum (eg aluminum hydroxide, aluminum phosphate); or emulsion based formulations (mineral oil, non-mineral oil, water-in-oil emulsions or oil-in-water emulsions, Montanide Adjuvants, Seppic ISA series (for example, oil-in-water emulsions such as Montanide ISA 720, AirLiquide, Paris, France); MF-59 (Span 85 and Tween 80 stabilized, squalene-in-water emulsions; Chiron Corporation , Emeryville, CA); and PROVAX (oil-in-water emulsions comprising a stabilizing surfactant and a micelle forming agent; IDEC, Pharmaceuticals Corpora ion, San Diego, CA), and the like (but not limited to).

  Immunostimulatory adjuvants are adjuvants that cause activation of cells of the immune system. This causes, for example, immune cells to produce and secrete cytokines. Adjuvants of this class include Q.I. Saponins purified from the bark of saponaria trees (eg, QS21 (a glycolipid which elutes in the 21st peak in HPLC fractions; Aquila Biopharmaceuticals, Inc., Worcester, Mass.)); poly [di (carboxylatophenoxy) Phosphazene (PCPP polymer; Virus Research Institute, USA); derivatives of lipopolysaccharide (eg, monophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc., Hamilton, MT), muramyl dipeptide (MDP; Ribi), and Onyl muramyl dipeptide (t-MDP; Ribi); OM-174 (glucosamine disaccharide related to lipid A; OM Pha ma SA, Meyrin, Switzerland); and Liu Leishmania elongation factor (purified Leishmania protein; Corixa Corporation, Seattle, WA), and the like (but not limited to).

  Adjuvants that cause a depot effect and stimulate the immune system are compounds that have both of the functions identified above. Adjuvants of this type include ISCOMS (mixed saponin, lipid-containing, immunostimulatory complex that forms particles of virus size with pores capable of retaining antigens; CSL, Melbourne, Australia); SB- AS2 (SmithKline Beecham adjuvant system # 2 (in oil-in-water emulsion containing MPL and QS21): SmithKline Beecham Biologicals [SBB], Rixensart, Belgium); SB-AS4 (SmithKline Beechem adjuvant system # 4 (including alum and MPL) ): SBB, Belgium); nonionic block copolymers which form micelles (eg CRL 1005), which are It includes straight sandwiched hydrophobic polyoxypropylene a strand of styrene; Vaxcel, Inc. , Norcross, GA); and Syntex Adjuvant Formulations (in oil-in-water emulsions including SAF, Tween 80 and nonionic block copolymers; Syntex Chemicals, Inc., Boulder, CO), but is not limited thereto. .

  Immunostimulatory nucleic acids are themselves useful as adjuvants to induce humoral immune responses. Thus, they can be delivered to a subject exposed to an antigen, resulting in an enhanced immune response to the antigen.

  Immunostimulatory nucleic acids are useful as mucosal adjuvants. It has already been discovered that both systemic and mucosal immunity are induced by mucosal delivery of CpG nucleic acids. The systemic immunity induced in response to this CpG nucleic acid included both humoral and cell-mediated responses to specific antigens that failed to elicit systemic immunity when administered alone to the mucosa. . In addition, both CpG nucleic acid and cholera toxin (CT, a mucosal adjuvant that induces a Th2-like response) induced CTL. This was surprising, as with systemic immunization, the presence of Th2-like antibodies is usually associated with a deficiency of CTL (Schirmbeck et al., 1995). Based on the results presented herein, it is predicted that immunostimulatory nucleic acids function in a similar manner.

  In addition, the immunostimulatory nucleic acid induces a mucous response both at the local mucosal site (eg, lung) and at the remote mucosal site (eg, lower gastrointestinal tract). Significant levels of IgA antibodies are induced at distant mucosal sites by immunostimulatory nucleic acids. CT is generally considered to be a very effective mucosal adjuvant. As previously reported (Snider, 1995), CT mainly induces antibodies of the IgG1 isotype, which show a Th2-type response. On the contrary, the immunostimulatory nucleic acid is more Th1 mainly by the IgG2 antibody, in particular after the boost or when the two adjuvants are combined. TH1-type antibodies generally have higher neutralizing capacity, and furthermore, Th2 responses in the lung are highly undesirable as they are associated with asthma (Kay, 1996, Hogg, 1997). Thus, the use of immunostimulatory nucleic acids as mucosal adjuvants has the advantage that other mucosal adjuvants can not achieve. The immunostimulatory nucleic acids of the invention are also useful as mucosal adjuvants that induce both systemic and mucosal immune responses.

  Mucosal adjuvants, referred to as non-nucleic acid mucosal adjuvants, can also be administered with the immunostimulatory nucleic acid. As used herein, a non-nucleic acid mucosal adjuvant is an adjuvant other than an immunostimulatory nucleic acid that can induce a mucosal immune response in a subject when administered to a mucosal surface in combination with an antigen. As mucosal adjuvants, bacterial toxins (eg, cholera toxin (CT), CT derivatives (hereinafter: CT B subunit (CTB) (Wu et al., 1998, Tochikubo et al., 1998); CTD53 (Val → Asp) (Fontana et al., 1995); CTK 97 (Val → Lys) (Fontana et al., 1995); CTK 104 (Try → Lys) (Fontana et al., 1995); CTD 53 / K 63 (Val → Asp, Ser → Lys) (Fontana et al., 1995); Arg → His) (Fontana et al., 1995); CTN 107 (His → Asn) (Fontana et al., 1995); CTE 114 (Ser → Glu) (Fontana et al., 1995); CTE 112 K (Glu → Lys) (Yamamoto et al.) CTS 61 F (Ser → Phe) (Yamamoto et al., 1997 a, 1997 b); CTS 106 (Pro → Lys) (Douce et al., 1997, Fontana et al., 1995); CTK 63 (Ser → Lys) (Douce et al., 1997, Fontana et al. Et al., 1995)), zonation zone toxins, zot, E. coli heat labile enterotoxin, Labile Toxin (LT), LT derivatives (hereinafter: LT B subunit (LTB) (LTB) ( Verweij et al., 1998); LT7K (Arg → Lys) (Komas et al., 1998, Douce et al., 1995); LT61F (Ser → Phe) (Komase et al., 1998); LT112 K (Glu → Lys) (Komase et al., 1998) LT118 E (Gly → Glu) (Komase et al., 1998); LT146 E (Arg → Glu) (Komase et al., 1998); LT192 G (Arg → Gly) (Komase et al., 1998); LTK63 (Ser → Lys) (Marchetti et al., 1998, Douce et al., 1997, 1998, Di Tommaso et al., 1996); and LTR72 (Ala-> Arg) (including but not limited to Giuliani et al., 1998)), pertussis toxin (PT) (Lycke et al.) 1992, Spangler BD, 1992, Freytag and Clemments, 1999, Roberts et al., 1995, Wilson et al., 1995) (this is PT-9K / 129G (Roberts et al., 1995, Crop). ey et al., 1995)); toxin derivatives (see below) (Holmgren et al., 1993, Verweij et al., 1998, Rappuoli et al., 1995, Freytag and Clements, 1999); lipid A derivatives (eg monophosphoryl lipid A, MPL) (Sasaki et al., 1998, Vancott et al., 1998; Muranyl dipeptide (MDP) derivatives (Fukushima et al., 1996, Ogawa et al., 1989, Michalek et al., 1983, Morisaki et al., 1983); Bacterial outer membrane proteins (eg Borrelia burgdorferi). Outer membrane protein A (OspA) lipoprotein, Neisseria meningitidis outer membrane protein) (Marinaro et al., 1999, Van de Verg et al., 1996); Oil-type emulsions (e.g., MF59) (Barchfield et al., 1999, Verschoor et al., 1999, O'Hagan, 1998); aluminum salts (Isaka et al., 1998, 1999); and saponins (e.g., QS21) (Antigenics, Inc. , Woburn, MA) (Sasaki et al., 1998, MacNeal et al., 1998), ISCOMS, MF-59 (Squaren-in-water emulsion stabilized with Span 85 and Tween 80; Chiron Corporation, Emeryville, CA); Seppic ISA series of Montanide adjuvant (Eg, Montanide ISA 720; AirLiquide, Paris, France); PROVAX (oil-in-water emulsion containing stabilized surfactant and micelle forming agent; IDEC Pharmaceuticals Corporation, San Diego, CA); Syntex Adjuvant Formulation (SAF; Syntex Chemicals, Inc., Bou der, CO); poly [di (carboxymethyl Lato phenoxy) phosphazene (PCPP polymer; Virus Research Institute, USA) and Liu Leishmania elongation factor (Corixa Corporation, Seattle, WA), and the like (but not limited to).

  The immune response may also be a cytokine (Bueler & Mulligan, 1996; Chow et al., 1997; Geissler et al., 1997; Iwasaki et al., 1997; Kim et al., 1997) or a B-7 costimulatory molecule (Iwasaki et al., 1997; Tsuji et al., It can be induced or enhanced by co-administration or colinear expression with the immunostimulatory nucleic acid in 1997). The cytokine can be administered directly with the immunostimulatory nucleic acid, or can be administered in the form of a nucleic acid vector encoding the cytokine, such that the cytokine can be expressed in vivo. In one embodiment, the cytokine is administered in the form of a plasmid expression vector. The term cytokine acts as a humoral regulator at nanomolar or picomolar concentrations, and a variety of soluble proteins and peptides that modulate the functional activity of individual cells and tissues, under either normal or physiological conditions. Used as a general name for a group. These proteins also mediate interactions between cells directly and regulate processes that take place in the extracellular environment.

  Examples of cytokines include IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-15, IL-18, granulocyte macrophage colony Stimulatory factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), interferon-gamma (gamma-IFN), IFN-alpha, tumor necrosis factor (TNF), TGF-beta, FLT-3 ligand, and CD40 Ligands include, but are not limited to.

Cytokines play a role in directing T cell responses. Helper (CD4 +) T cells modulate the mammalian immune response through the production of soluble factors that act on other immune system cells, including other T cells. Most mature CD4 + T helper cells express one of two cytokine profiles (Th1 or Th2). Th1 subsets promote delayed type hypersensitivity, cell mediated immunity, and immunoglobulin class switching to IgG _2a . Th2 subsets induce humoral immunity by activating B cells, promoting antibody production, and inducing class switching to IgG1 and IgE. In some embodiments, the cytokine is preferably a Th1 cytokine.

  The immunostimulatory nucleic acid can be administered directly to the subject or can be administered with the nucleic acid delivery complex. A nucleic acid delivery complex is associated (eg, with a means for targeting (eg, a molecule that produces higher binding affinity to the target cell (eg, B cell surface and / or increased cellular uptake by the target cell)). By ionic or covalent bond; or encapsulated therein is meant a nucleic acid molecule. Examples of nucleic acid delivery complexes are sterols (eg cholesterol), lipids (eg cationic lipids, virosomes or liposomes) or target cell specific binding agents (eg ligands recognized by target cell specific receptors) Can be mentioned. Preferred complexes may be stable in vivo sufficiently to prevent significant uncoupling prior to internalization by the target cell. However, this complex may be cleavable under appropriate conditions in cells such that the nucleic acid is released in functional form.

  Delivery vehicles or devices for delivering antigens and nucleic acids to surfaces have been described. The immunostimulatory nucleic acid and / or antigen and / or other therapeutic agent can be administered alone (eg, in saline or in a buffer) or using any delivery vehicle known in the art . For example, the following delivery vehicles are described: Cochleates (Gould-Fogerite et al., 1994, 1996); Emulsomes (Vancott et al., 1998, Lowell et al., 1997); ISCOMs (Mowat et al., 1993). Carlsson et al., 1991, Hu et al., 1998, Morein et al., 1999; Liposomes (Childers et al., 1999, Michalek et al., 1989, 1992, de Haan 1995a, 1995b); live bacterial vectors (e.g., Salmonella, Escherichia coli, Bacillus) calmatte-guerin, Shigella, Lactobacillus) (Hone et al., 1996, ouwels et al., 1998, Chatfield et al., 1993, Stover et al., 1991, Nugent et al., 1998; live viral vectors (eg vaccinia, adenovirus, herpes simplex) (Gallichan et al., 1993, 1995, Moss et al., 1996, Nugent) Et al., 1998, Flexner et al., 1988, Morrow et al., 1999; Microspheres (Gupta et al., 1998, Jones et al., 1996, Maloy et al., 1994, Moore et al., 1995, O'Hagan et al., 1994, Eldridge et al., 1989). Nucleic acid vaccines (Fynan et al., 1993, Kuklin et al., 1997, Sasaki et al., 1998, Okada et al., 1997, Ishii et al., 1997); polymers (e.g. Ruboxymethylcellulose, chitosan) (Hamajima et al., 1998, Jabbal-Gill et al., 1998); polymer ring (Wyatt et al., 1998); Proteosomes (Vancott et al., 1998, Lowell et al., 1988, 1996, 1997); Hashi et al., 1998); transgenic plants (Tacket et al., 1998, Mason et al., 1998, Haq et al., 1995); virosomes (Gluck et al., 1992, Mengiardi et al., 1995, Cryz et al., 1998); virus-like particles (Jiang et al.). , 1999, Leibl et al., 1998). Other delivery vehicles are known in the art, and some additional examples are provided below in the discussion of vectors.

The stimulation index of a particular immunostimulatory nucleic acid can be tested in various immune cell assays. Preferably, the stimulation index of the immunostimulatory nucleic acid for B cell proliferation is at least about 5, preferably at least about 10, more preferably at least about 5, as determined by ³ H uridine uptake in mouse B cell cultures. At least about 15, and most preferably at least about 20. This mouse B cell culture is contacted with 20 μM nucleic acid at 37 ° C. for 20 hours and pulsed with 1 μCi of ³ H uridine; and collected and counted after 4 hours (this is, respectively, 1995) PCT Published Patent Application No. PCT / US95 / 01570, which claims priority to US application Ser. Nos. 08 / 386,063 and 08 / 960,774, filed Feb. 7, and October 30, 1997. WO 96/02555) and PCT / US97 / 19791 (WO 98/18810) in detail). For example, it is important that the immunostimulatory nucleic acid be able to effectively induce an immune response (eg, antibody production) for in vivo use.

  Immunostimulatory nucleic acids are effective in non-rodent vertebrates. Different immunostimulatory nucleic acids can cause optimal immune stimulation, depending on the type of subject and the sequence of the immunostimulatory nucleic acid. It has been found according to the invention that many vertebrates respond to the same class of immunostimulatory nucleic acids (sometimes referred to as human specific immunostimulatory nucleic acids). However, rodents respond to different nucleic acids. As provided herein, immunostimulatory nucleic acids that cause optimal stimulation in humans do not generally cause optimal stimulation in mice, and vice versa. However, immunostimulatory nucleic acids that cause optimal stimulation in humans cause optimal stimulation in other animals (eg, cows, horses, sheep, etc.). Those skilled in the art will use the guidance provided herein to identify the subject of interest using the conventional assays described herein and / or conventional assays known in the art. Useful optimal nucleic acid sequences can be identified for species of

  The term effective amount of immunostimulatory nucleic acid refers to the amount necessary or sufficient to achieve the desired biological effect. For example, an effective amount of an immunostimulatory nucleic acid for inducing mucous immunity is the amount necessary to cause the development of IgA in response to an antigen when exposed to the antigen, whereas it is systemic. The amount required to induce immunity is the amount necessary to cause the development of IgG in response to the antigen upon exposure to the antigen. Various active compounds are selected in combination with the techniques provided herein and factors such as efficacy, relative bioavailability, patient weight, severity of adverse side effects and preferred mode of administration) An effective treatment or therapeutic treatment regimen (which does not cause substantial toxicity and is generally effective in treating a particular subject) may be planned by comparing. The effective amount for any particular application depends on such factors as the disease or condition being treated, the particular immunostimulatory nucleic acid being administered, the antigen, the size of the subject, or the severity of the disease or condition. Can change. One of ordinary skill in the art may empirically determine the effective amount of a particular immunostimulatory nucleic acid and / or antigen and / or other therapeutic agent without requiring undue experimentation.

  The subject dose of the compounds described herein for mucosal or topical delivery is typically in the range of about 0.1 μg to 10 mg per dose, which is daily, weekly or monthly and It depends on the application that can be given in any other amount of time between them. More typically, the mucosal or topical dose is in the range of 10 μg to 5 mg, most typically about 100 μg to 1 mg per dose, with 2 to 4 doses separated by days or weeks. It is done. More typically, the immunostimulatory dose is daily or weekly administration in the range of 1 μg to 10 mg, most typically 10 μg to 1 mg per dose. A subject dose of the compound is described herein for parenteral delivery to induce an antigen-specific immune response, wherein the compound is delivered with the antigen, but another therapeutic agent is Typically, 5 to 10,000 times higher, more typically 10 to 1,000 times higher, most typically 20 to 10 times higher than the effective mucosal dose for vaccine adjuvant or immunostimulatory application. ~ 100 times higher. When an immunostimulatory nucleic acid is administered in combination with other therapeutic agents or in a specific delivery vehicle, to induce an innate immune response, to increase ADCC, or to induce an antigen-specific immune response Doses of the compounds described herein for parenteral delivery are typically in the range of about 0.1 μg to 10 mg per dose, which is daily, weekly, or monthly and their Depends on the application that can be given in any other amount of time between. More typically, parenteral doses for these purposes range from about 10 μg to 5 mg per dose, most typically from about 100 μg to 1 mg, with 2 to 4 doses per day, per week Interval is made. However, in certain embodiments, parenteral doses for these purposes may be used in the range of 5 to 10,000 times higher than the representative doses described above.

  For any compound described herein, the therapeutically effective amount can be initially determined from animal models. Therapeutically effective doses are also known for CpG oligonucleotides being tested in humans (human clinical trials have been initiated), and for compounds known to exhibit similar pharmacological activity (eg other mucosal adjuvants (eg For example, for LT and other antigens for vaccination purposes, it can be initially determined from human data for mucosal or topical administration, although higher doses are required for parenteral administration. May be adjusted based on the relative bioavailability and potency of the compound being administered Based on the methods described above and other methods well known in the art, adjusting the dose to achieve maximal efficacy That is well within the ability of one skilled in the art.

  The formulations of the present invention are administered in pharmaceutically acceptable solutions, which conventionally comprise salts, buffers, preservatives, compatible carriers, adjuvants, and necessary pharmaceutically acceptable concentrations. May contain other therapeutic ingredients.

  For use in therapy, an effective amount of an immunostimulatory nucleic acid can be administered to the subject by any mode that delivers the nucleic acid to the surface of the exhaustion (eg, mucosal, systemic). Administering the pharmaceutical composition of the present invention may be accomplished by any means known to one of ordinary skill in the art. Preferred routes of administration include, but are not limited to, oral, parenteral, intramuscular, intranasal, intrathecal, inhaled, ocular, vaginal and rectal.

  For oral administration, the compounds (ie, immunostimulatory nucleic acids, antigens and other therapeutic agents) can be readily formulated by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the present invention to be taken orally by the subject to be treated, such as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. To be prescribed. Pharmaceutical preparations for oral use, if necessary, grinding the resulting mixture and, if desired, adding suitable auxiliaries to obtain tablets or dragee cores, of the granulate The mixture can be processed to obtain a solid excipient. Suitable excipients are, in particular, fillers (for example sugar (including lactose, sucrose, mannitol or sorbitol); cellulose preparations (for example corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, Methylcellulose, hydroxypropyl methyl-cellulose, sodium carboxymethylcellulose and / or polyvinyl pyrrolidone (PVP) If desired, disintegrants such as cross-linked polyvinyl pyrrolidone, agar or alginic acid or salts thereof such as sodium alginate Optionally, oral formulations can also be formulated with saline or buffer to neutralize internal acid conditions, or can be administered without any carrier.

  Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally be gum arabic, talcum, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol and / or titanium dioxide, lacquer solutions, and suitable organics It may comprise a solvent or solvent mixture. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

  Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. Push-fit capsules are active mixed with fillers (eg lactose), binders (eg starch) and / or lubricants (eg talc or magnesium stearate) and optionally stabilizers It may contain ingredients. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. Microspheres formulated for oral administration may also be used. Such microspheres are well defined in the art. All formulations for oral administration should be in dosages suitable for such administration.

  For buccal administration, the compositions may take the form of tablets or lozenges formulated in a conventional manner.

  For administration by inhalation, the compounds for use according to the invention use a suitable propellant (for example dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). It can be delivered conventionally in the form of an aerosol spray presentation from a pressure pack or a nebulizer. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a measured amount. Capsules and cartridges of, eg, gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

  The compounds may be formulated for parenteral administration by injection (eg, by bolus injection or continuous infusion) if systemic delivery is desired. Formulations for injection can be presented in unit dose form (eg, in ampoules) or in multidose devices, with the addition of preservatives. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles. The composition may include formulation agents (eg, suspending agents, stabilizing agents, and / or dispersing agents).

  Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

  Alternatively, the active compound may be in powder form for constitution with a suitable vehicle, eg, sterile pyrogen-free water, before use.

  The compounds may also be formulated in rectal or vaginal compositions (eg, suppositories or retention enemas), eg, with conventional suppository bases (eg, cocoa butter or other glycerides). .

  In addition to the above formulations, the above compounds can also be formulated as a depot preparation. Such long acting formulations may use suitable polymeric or hydrophobic substances (eg as an emulsion in an acceptable oil) or ion exchange resins, or foam soluble derivatives (eg foam dissolve) Can be formulated as a sexual salt).

  The pharmaceutical composition may also comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers (eg, polyethylene glycol).

  Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous or saline solutions for inhalation, microencapsulated, curled or coated on microscopic gold particles It is contained in liposomes, sprayed, aerosolized, pellets for skin transplantation, or dried on sharp objects to be scratched into the skin. The above pharmaceutical compositions may also be in the form of granules, powders, tablets, coated, (micro) capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations which release the active compound over time. The excipients and additives and / or adjuvants (eg, disintegrants, binders, coatings, expanding agents, lubricants, flavoring agents, sweetening agents or solubilizers), and their preparation excipients and additives as described above Used customarily. The above pharmaceutical compositions are suitable for use in various drug delivery systems. For a brief overview of methods for drug delivery, see, Langer, Science 249: 1527-1533, 1990, which is incorporated herein by reference.

  The immunostimulatory nucleic acid and optionally other therapeutic agents and / or agents can be administered per se (neat) or in the form of a pharmaceutically acceptable salt. When used in medicine, their salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts are conventionally used to prepare pharmaceutically acceptable salts thereof. obtain. Such salts include, but are not limited to, salts prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, p-toluenesulfonic acid Tartaric acid, citric acid, methanesulfonic acid, formic acid, malonic acid, succinic acid, naphthalene-2-sulfonic acid, and benzenesulfonic acid. Also, such salts may be prepared as alkaline metal or alkaline earth metal salts, such as sodium, potassium or calcium salts of the carboxylic acid group.

  Suitable buffers include acetic acid and salts (1-2% w / v); citric acid and salts (1-3% w / v); boric acid and salts (0.5-2.5% w / v) And phosphoric acid and salts (0.8-2% w / v). Suitable preservatives include benzalkonium chloride (0.003-0.03% w / v); chlorobutanol (0.3-0.9% w / v); parabens (0.01-0.25) % W / v) and thimerosal (0.004 to 0.02% w / v).

  As described in more detail herein, the pharmaceutical composition of the present invention may optionally comprise an effective amount of an immunostimulatory nucleic acid and optionally an antigen and / or other therapeutic agent. Included in a carrier that is acceptably acceptable. The term "pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to humans or other vertebrates. means. The term "carrier" means an organic or inorganic ingredient (whether natural or synthetic), with which the active ingredient is mixed to facilitate the application. The components of the above pharmaceutical compositions are also miscible with the compounds of the present invention and miscible with one another in such a manner that there is no interaction which substantially impairs the desired pharmaceutical efficacy.

  Immunostimulatory nucleic acids useful in the present invention can be delivered in a mixture with further adjuvants, other therapeutic agents, or antigens. The mixture may consist of several adjuvants in addition to the immunostimulatory nucleic acid or several antigens or other therapeutic agents.

  Various administration routes are available. The particular mode chosen will, of course, be dependent on the particular adjuvant or antigen chosen, the particular condition being treated, and the dosage required for therapeutic efficacy. The methods of the invention can generally be practiced using any medically acceptable mode of administration. This manner means any manner that results in an effective immune response level without causing adverse effects that are not clinically acceptable. Preferred modes of administration are discussed above.

  The compositions may be conveniently presented in unit dosage form and may be prepared by any of the methods in the art of pharmacy. All methods include the step of bringing the compound into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately associating the compounds with liquid carriers, finely divided solid carriers, or both, and then, if necessary, shaping the product . Liquid dose units are vials or ampoules. Suitable dosage units are tablets, capsules and suppositories. For the treatment of the patient, depending on the activity of the compound, depending on the mode of administration, the purpose of the immunization (ie prophylactic or therapeutic), the nature and severity of the disorder, the age and weight of the patient, Various doses may be required. The administration of a given dose may be carried out both by single administration of individual dose units or by several smaller dose units.

  Other delivery systems include timed release, delayed release, or sustained release delivery systems. Such a system may avoid repeated administration of the compound, which may increase convenience to the subject and physician. Many types of release delivery systems are available and known to those skilled in the art. They include polymer based systems such as poly (lactide-glycolide), copolyoxalate, polycaprolactone, polyesteramides, polyorthoesters, polyhydroxybutyric acid and polyanhydrides.

  Microcapsules of the above polymers containing drugs are described, for example, in US Pat. No. 5,075,109. Delivery systems also include non-polymeric systems, including lipids such as sterols (eg, cholesterol, cholesterol esters), and fatty acids or natural fats (eg, monoglycerides, diglycerides, and triglycerides); hydrogel release Systems; silastic systems; peptide-based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants etc. As specific examples, (a) an erosion system (US Pat. Nos. 4,452,775, 4,675,189 and 5,736,5) in which the agent of the present invention is contained in a matrix form And (b) diffusion systems in which the active ingredient penetrates at a controlled rate from the polymer (US Pat. Nos. 3,854,480, 5,133,974 and No. 5,407,686, but is not limited thereto. In addition, pump based hardware delivery systems may be used, some of which are adapted for implantation.

  The invention is further illustrated by the following examples. The following examples should not be construed as further limiting in any way. The entire contents of all references cited throughout this specification (literature references, issued patents, published patent applications, and co-pending patent applications) are expressly incorporated herein by reference. Incorporated by reference.

Example 1 (ODN 10102)
(Summary)
The present report summarizes in vitro data using human cells. This data indicates that ODN 10102 (SEQ ID NO: 1) behaves similarly to ODN 7909 (SEQ ID NO: 2), and in some aspects in a better manner. In vitro and in vivo data in mice show that CpG ODN 10102 is for activation of the innate immune system and for enhancing HBsAg specific humoral and cellular responses in mice when co-administered with the above antigens Show similar properties to CpG ODN 7909, and sometimes better properties.

  The assays performed were receptor binding (TLR9), B cell activation (expression of cell surface activation markers and B cell proliferation) and cytokine secretion (IL-10, IP-10, IFN-α and TNF-α) The All assays showed that ODN 10102 had characteristics that were nearly identical if not better than ODN 7909.

  In vitro studies (ie, B cell proliferation assay, NK lytic activity, cytokine secretion profile) were performed using unstimulated BALB / c mouse splenocytes. In vivo comparative studies were performed by testing the ability of these two ODNs to enhance the antigen-specific immune response to hepatitis B antigen (HBsAg).

(Materials and methods)
(Human research)
Oligodeoxynucleotides All ODNs were provided by Coley Pharmaceutical GmbH (Langenfeld, Germany). Control ODN did not contain any stimulatory CpG motifs. The ODN was diluted in phosphate buffered saline and stored at -20 <0> C. All dilutions were performed using pyrogen free reagents.

  TL9 Assay The cells used for this assay expressed human TLR9 receptor and contained a reporter gene construct. The cells were incubated with the ODN for 16 hours. Each data point was done in triplicate. Cells were lysed and assayed for reporter gene activity. The stimulation index was calculated with reference to reporter gene activity in media without the addition of ODN.

  Cell Purification Peripheral blood buffy coat preparations from healthy human donors were obtained from German Red Cross (Rathingen, Germany), from which PBMCs were purified by centrifugation against Ficoll-Hypaque (Sigma, Germany). The purified PBMCs were used fresh or were stored in freezing medium and stored at -70 ° C. If necessary, aliquots of these cells are thawed, washed, and 10% (v / v) heat-inactivated FCS, 1.5 mM L-glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin And resuspended in RPMI 1640 culture medium supplemented with

Cytokine detection Thawed PBMCs or fresh PBMCs were resuspended at a concentration of 5 × 10 ⁶ / ml and added to 48-well flat bottom plates (1 ml / well). Nothing was added to this plate, or different concentrations of ODN had been added. The cells were cultured at 37 ° C. in a humidified incubator. Culture supernatants were collected after designated time points. If not used immediately, the supernatant was frozen at -20 <0> C until needed. Certain cytokines in the supernatant were evaluated using commercially available ELISA kits (IL-10; Diaclone, USA) or in-house ELISA (IP-19 and IFN-α), and commercially available antibodies. Color was developed using (Pharmingen, Germany or PBL, USA).

  Culture for flow cytometric analysis of B cell activation Monoclonal antibodies to CD19 and monoclonal antibodies to CD86 were purchased from Becton Dickinson (Germany). PBMCs were incubated for 48 hours with or without the addition of various concentrations of ODN. B cells were identified by expression of CD19 by flow cytometry. Flow cytometry data were obtained on a FACSCalibur (Becton Dickinson). Data were analyzed using the computer program CellQuest (Becton Dickinson). After culturing proliferating CD19-positive B cells with CFSE-labeled PBMC (CFSE is a fluorescent dye that binds to all cell surfaces), use CFSE, using flow cytometry methods (see above). It identified by the decrease of content.

(In vitro and in vivo studies in mice)
Oligodeoxynucleotides CpG ODN (GMP quality) can be obtained from Coley Pharmaceutical Inc. Powered by (Wellesley, MA). Resuspend all ODN in endotoxin free sterile TE (pH 8.0) (OmniPer®; EM Science, Gibbstown, NJ), store and handle under sterile conditions, microbial contamination And endotoxin contamination was also prevented. Dilutions of ODN for assay were performed in endotoxin free sterile PBS (pH 7.2) (Sigma Chemical Company, St. Louis, MO).

  Animals Female BALB / c mice (6-8 weeks old) were used for all experiments. The animals were purchased from Charles River Canada (Quebec, Canada) and kept in microisolators at the Animal Care Facility at Ottawa Hospital Research Institute, Civic Site.

Harvested Splenocytes and Cultures Splenocytes of unstimulated BALB / c mice were used for all in vitro assays. The animals were anesthetized with isoflurane and euthanized by cervical dislocation. The spleen was removed under sterile conditions and placed in PBS + 0.2% bovine serum albumin (Sigma Chemical Company). The spleen is then homogenized and the splenocytes are diluted with 2% normal mouse serum (final concentration 100 U / ml; Cedarlane Laboratories, Ontario, Canada), penicillin-streptomycin solution (final concentration 1 mg / ml; Sigma Chemical Company) and 5 x 10 Resuspend in RPMI 1640 (Life Technologies, Grand Island, NY) tissue culture medium supplemented with ⁻⁵ M β-mercaptoethanol (Sigma Chemical Company).

B cell proliferation assay: Spleen cell suspensions were prepared and adjusted to a final concentration of 5 × 10 ⁶ cells per ml in complete RPMI 1640. Splenocyte suspension was poured into 96 well U bottom tissue culture plates (100 μl / well) with 100 μl of stimulant and diluted to the appropriate concentration in complete RPMI 1640. The stimuli used were CpG ODN (at 1, 3, 10 μg / ml) 7909 and 10102, 10103, 10104, 10105 or 10106. Concanavalin A (10 μg / ml, Sigma Chemical Company) and LPS (10 μg / ml, Sigma Chemical Company) were used as positive controls, and cells cultured in medium alone were used as negative controls. Each splenocyte sample was plated in triplicate and cells were incubated at 37 ° C. for 96 hours in a humidified 5% CO ₂ incubator. At the end of the incubation period, cells were pulsed with ³ H-thymidine (20 μCi / ml) for 16 hours after 96 hours of incubation, harvested, and radioactivity was measured.

Cytokine secretion profile: Spleen cell suspensions were prepared and plated in 96 well U bottom tissue culture plates as described for B cell proliferation assay. Each splenocyte sample was plated in triplicate and cells were incubated at 37 ° C. for 6, 12, or 48 hours in a humidified 5% CO ₂ incubator. At the end of the incubation period, the 96 well plate was centrifuged at 1200 rpm for 5 minutes, and the culture suspension was collected and stored at -80 ° C until assayed. A commercially available assay kit (mouse OptEIA kit; PharMingen, Mississauga, ON) is used according to the manufacturer's instructions for 6 hours (TNF-α), 24 hours (IL-12) and 48 hours (IL-6 and IL-). The cytokine levels of the culture suspension removed in 10) were assayed.

NK Assay: Splenocyte suspensions were prepared as above and prepared to a final concentration of 3 × 10 ⁶ cells / ml in complete RPMI 1640. Splenocyte cell suspension (10 ml; 30 × 10 ⁶ cells), T-25 tissue culture flask with CpG ODN (at 1, 3, 10 μg / ml) with either 7909 and 10102, 10103, 10104, 10105 or 10106 Plated in (Fisher Scientific, Ottawa, ON). Splenocytes cultured in medium alone were used as a negative control. Each splenocyte culture was incubated at 37 ° C. for 24 hours in a humidified 5% CO ₂ incubator. At the end of the incubation period, cells were plated with different effectors: target ratio of 96 well U bottom tissue culture plates (100 μl / well) with 100 μl of ⁵¹ Cr labeled target cells at 5 × 10 ⁴ cells / ml. The NK sensitive mouse lymphocyte cell line YAC-1 (ATCC No. TIB-160, ATCC, Manassas, Va.) Was used as a target cell line. Each sample was plated in triplicate and cells were incubated for 4 hours at 37 ° C. in a humidified 5% CO ₂ incubator. Target cells were incubated with medium alone or medium containing 2N HCL to determine spontaneous and maximal release, respectively. At the end of the incubation period, the supernatant was harvested and the level of radioactivity was measured using a gamma counter. The% dissolution was determined using the following formula;
% Specific release = (experimental release−spontaneous release) / (maximum release−spontaneous release) × 100
.

  Immunization of mice: BALA / c mice (n = 10 / group) with either 1 μg HBsAg subtype ad (International Enzymes, CA) alone or with 10 μg CpG ODN 7909 or CpG ODN 10102, 10103, 10104, 10105 or 10106 Immunized in combination. Animals were bled and boosted 4 weeks after primary immunization. One week after the boost, 5 animals from each group were euthanized and spleens were removed for CTL assay.

  Determination of antibody response: Antibodies specific for HBsAg (anti-HBs) (all IgG, IgG1 and IgG2a) are detected and quantified by endpoint dilution ELISA assay, which is an individual animal Was done in triplicate for samples derived from The end point titer was defined as the highest plasma dilution resulting in an absorbance (OD 450) that is twice as high as the absorbance of non-immune plasma with a cutoff value of 0.05. These were reported as group mean titers ± SEM.

Statistical analysis: Statistical analysis was performed using the InStat program (Graph PAD Software, San Diego). Statistical differences between groups can be 1-followed by Student's t-test (for 2 groups) or Tukey's test (for 3 or more groups) on raw data or transformed data (log ₁₀ , for heterogeneous populations). Tested by factor ANOVA.

result:
TLR9 binding: A receptor for recognition of CpG sequences was recently identified and shown to be a member of the Toll-like receptor (TLR) family (Hemmi et al., 2000). This receptor, TLR9, is readily activated by ODNs containing optimal immunostimulatory CpG sequences. We incubated cell lines that stably express different concentrations of ODN 7909 and 10102 and control ODN with human TLR9 (FIG. 1).

  The results show that these ODNs 10102 activated TLR9 to the same or higher than 7909. Both ODNs showed dose response curves and reached maximal activity at the same concentration. The control ODN used did not induce TLR9 activity even at the highest concentration of 12 μg / ml.

  Human B cells: One feature of type B ODNs is their ability to activate B cells very efficiently (Krieg et al., 1995). B cells and plasmacytotoid DCs are currently the only immune cell types known to express TLR9 (Krug et al., 2001; Bauer et al., 2001). Therefore, we measured the direct activity of B cells induced by ODN 7909 and 10102 by: a. Upregulation of cell surface marker CD86 (Figure 2) and b. Measurement of B cell proliferation (Figure 3). For CD86 expression of human B cells, PBMC from healthy blood donors were incubated with different ODNs as described in Materials and Methods, and B cell activation was measured.

  Both results show that 10102 and 7909 are very important stimulators of human B cells. FIG. 2 shows that these CpG ODN can stimulate B cells only at a concentration of 0.4 μg / ml in vitro. The plateau was reached at about 1.6 μg / ml. Similar results were obtained for B cell proliferation incubation (Figure 3), where the stimulation index reached a maximum at about 1.6 μg / ml.

  Cytokine secretion: B-class ODNs lead to Th1 dominant immune responses in vivo as well as in vitro. It has been found that these can typically induce Th1 cytokines (eg, IFN- and IFN-) and Th1-related cytokines (eg, MCP-1 and IP-10). In addition, low secretion of the proinflammatory cytokines IL-6 and TNF- and secretion of the negative regulator IL-10 can be observed. Therefore, we measured the secretion of the Th1 cytokine IFN-, the chemokine IP-10, and the regulator cytokine IL-10 and the proinflammatory cytokine TNF-. FIG. 4 shows the results for experiments performed with different donors at 0.2, 0.4, 1.6 and 5 μg / ml to measure IFN-secretion in vitro.

  Both CpG ODNs (7909 and 10102) induced the highest high levels of IFN-α reached at 0.4 (7909) or 1.6 μg / ml (10102). However, the largest increase in IFN-α secretion was more pronounced after stimulation at 10102 compared to 7909. In contrast, control ODN induced low amounts of IFN-α starting only at 5.0 μg / ml. Furthermore, in contrast to conotrol ODN, ODNs 7909 and 10102 induced high amounts of the chemokine IP-10 as shown in FIG.

  Very similar experiments were performed for IL-10 secretion (Figure 6). Again, as shown above for IFN-α, both CpG ODNs 7909 and 10102 exhibit nearly identical properties, with 10102 working better in some cases in these assays. By comparison, control ODN induces IL-10 secretion only at the highest concentration.

  As shown in FIG. 7, both ODNs 7909 and 10102 and control ODN showed a weak secretion profile of the proinflammatory cytokine TNF-α as compared to LPS. Again, it was found that the two ODNs also have very similar properties in this assay.

  In the first set of experiments, induction of proliferation of mouse spleen cells was investigated. According to the data shown in FIG. 8, CpG ODN 10102 is equally potent as CpG ODN for inducing mouse B cell proliferation at all concentrations tested.

  In the next set of experiments, secretion of various cytokines was tested upon incubation of spleen cells with 101012, 7909 and control 2137. According to the data shown in FIG. 9, both CpG ODN 7909 and 10102 have essentially equal potency for increasing cytokine secretion by mouse splenocytes.

  According to the data in FIG. 10, both CpG ODNs 7909 and 10102 have essentially equal potency for increasing the lytic activity of NK cells in mouse splenocyte cultures.

  According to the results of this study (Figure 11), the use of either CpG ODN 7909 or 10102 significantly increased antibody titers to HBsAg compared to antigen alone (p <0.001) When ODN was used in combination with HBsAg, anti-HBs responses were not significantly increased (p = 0.86).

  The increase in total IgG levels is slightly but significantly increased when CpG ODN 7909 is used compared to when CpG ODN 10102 is used (p = 0.04).

  In the mouse IgG isotype, partitioning is widely used as an indicator of immune response properties where high IgG2a / IgG1 ratios show a Th1-biased immune response (Constant and Bottomly, 1997). In this study, the use of CpG ODN significantly increased IgG2a titer compared to when the antigen was used alone or in combination with control ODN 2137 (p << Ag vs. 7909 or 10102 or Ag vs. Ag + 2137). 0.001). However, the level of IgG2a response was similar when either CpG ODN 7909 or 10102 were used in combination with HBsAg (p> 0.05). Thus, both CpG ODNs 7909 and 10102 are equally potent in their ability to induce a Th1 biased immune response, as measured by increased levels of IgG2a over IgG1.

Conclusion:
In vitro data using human cells indicated that ODN 10102 behaves similar to or better than previously identified ODN 7909 in the various assays performed.

  According to the results of in vivo and in vitro studies in mice, when treated with CpG ODN 10102, both ODN 7909 and the ability to enhance antigen-specific response in vivo against inactive immune responses It has similar or better immune enhancing properties.

(Example 2 (ODN 10103))
Summary: The ability of ODN 10103 (SEQ ID NO: 19) to stimulate human PBMC in vitro was compared to that of ODN 7909 (SEQ ID NO: 2). Immune stimulation, receptor (ie, TLR9) binding, B cell activation (eg, expression of cell surface activation markers and B cell proliferation) and cytokine secretion (eg, IL-10, IP-10, IFN-α and TNF) (A secretion of α) was analyzed. All assays showed that ODN 10103 had properties similar to or better than those of ODN 7909.

  The ability of ODN 10103 to stimulate mouse immune cells in vitro and in vivo was compared to ODN 7909. In vitro studies (eg, B cell proliferation assay, NK lytic activity and cytokine secretion profile) were performed using naive BALB / c mouse splenocytes. In vivo studies are performed by testing the strength of these two ODNs to enhance antigen-specific immune responses to hepatitis B surface antigen (HBsAg), humoral (antibody) and cell-mediated immune response (CTL activity) Was analyzed. In addition, Th bias of the induced immune response was tested by determining the strength of the CTL response as well as the IgG2a / IgG1 ratio.

Materials and methods:
For human studies, see Example 1 for a description of cell cultures for oligodeoxynucleotide, TLR9 assay, human cell proliferation, cytokine detection and flow cytometric analysis of B cell activation.

  For in vitro and in vivo studies of mice, description of oligodeoxynucleotides, recovery and culture of animals, splenocytes, B cell proliferation assay, cytokine secretion profile, NK assay, immunization of mice, measurement of antibody responses and statistical analysis See Example 1 for.

(Mouse in vitro / in vivo studies :)
Assessment of CTL responses: CT1 assays were performed as previously described by Davis et al. Results are shown as% specific lysis at different effector: target (E: T) ratios.

(result:)
TLR9 Binding: We incubated cell lines stably expressing human TLR9 with different concentrations of ODN 7909 and 10103 and control ODN (FIG. 13). Both ODNs showed concentration dependent dose-response curves achieving their maximal activity at the same concentration. Control ODN did not induce TLR9 activation even at the highest concentration of 24 μg / ml. ODN 10103 shows higher stimulation ability at lower doses (eg 6 g / ml and 12 g / ml) than show ODN 7909, which is lower as ODN 10103 achieves a similar immune stimulation index It may be used at doses, suggesting that it reduces potential toxicity.

  One characteristic of human B cells: Type B ODNs is their ability to activate B cells very effectively (Krieg et al., 1995). B cells and plasmacytotoid DC are only immune cell types that are currently known to express TLR9 (Krug et al., 2001; Bauer et al., 2001). Therefore, we measured the direct activity of B cells induced by ODNs 7909 and 10103 by upregulation of cell surface marker CD86 (Figure 14) and measured the proliferation of B cells (Figure 15). ). For CD86 expression on human B cells, PBMC of healthy blood donors were incubated with different ODNs and B cell activity was measured as described in Materials and Methods.

  Both results indicate that 10103 is at least equivalent to 7909 as a stimulator of human B cells. FIG. 14 shows that these CpG ODN can stimulate B cells starting at in vitro concentration (only at 0.4 μg / ml). A plateau was achieved at about 1.6 μg / ml, and over 60% of B cells were found to upregulate CD86 as compared to the control, which is a very small effect at the same concentration. The results show that ODN 10103 stimulates CD86 expression to higher levels at lower doses (eg, 0.4 μg / ml) than the doses of ODN 7909 in all three donors tested. Again, this suggests that lower doses of ODN 10103 can be used to achieve the desired level of immune stimulation. Similar results were obtained for induction of B cell proliferation (FIG. 15).

  Cytokine secretion: B-class ODNs lead to Th1 dominant immune responses in vivo as well as in vitro. It has been found that these can typically induce Th1 cytokines (e.g. IFN-y and IFN-a) and chemokines (e.g. MCP-1 and IP-10). In addition, low secretion of the proinflammatory cytokine Il-6 and TFN-α and secretion of the negative regulator IL-10 were observed. Thus, we measured the secretion of the Th1 cytokines IFN-α, the chemokine IP-10 and the regulatory cytokine Il-10 and the proinflammatory cytokine TNF-α.

  FIG. 16 shows the results for experiments performed with 6 different donors at 0.2 μg / ml, 0.4 μg / ml and 1.6 μg / ml to measure IFN-α secretion in vitro Indicates Both CpG ODNs (7909 and 10103) induced significant amounts of IFN-α in different donors. In contrast, control ODN did not induce any IFN-α or induced a small amount of IFN-α in one donor. This data suggests that patient variability may exist and that some patients respond better to an ODN such as 10103 when compared to ODN 7909. This finding indicates that the ODN can be classified in terms of subjects likely to be high responders.

  In addition to IFN-α, ODNs 7909 and 10103 induced the chemokine IP-10 as shown in FIG. ODN 10103 induced IP-10 at the same level as ODN 7909 or higher than ODN 7909 at all doses tested. In particular, at a concentration of 0.4 μg / ml, ODN 10103 produced more IP-10 than did ODN 7909. At a concentration of 1.6 μg / ml, ODN 10103 induced approximately 25% more IP-10 than ODN 7909 induced.

  As shown in FIG. 18, CpG ODNs 7909 and 10103 exhibited almost the same IL-10 inducing ability.

  As shown in FIG. 19, both ODNs 7909 and 10103 and the control ODN showed a low secretion profile of the proinflammatory cytokine TNF-α at all concentrations tested, as compared to LPS. At the highest dose tested (ie 6 μg / ml), ODN 10103 stimulated the secretion of higher levels of IL-10 than stimulated by ODN 7909. The dose response is shown in FIG.

  In vitro mouse studies: data show that both CpG ODN 7909 and 10103 are equally potent in inducing mouse B cell proliferation and have essentially the same potency in enhancing cytokine secretion by mouse splenocytes And possess essentially the same potency in enhancing the lytic activity of NK cells in mouse splenocyte cultures (FIG. 22). ODN 10103 appears to have a higher ability to stimulate the secretion of IL-6 and TNF-, especially at the lower doses tested. Likewise, the lytic activity profile of these ODNs varies with concentration.

  In Vivo Mouse Studies: The results of this study show that the use of either CpG ODN 7909 or 10103 significantly enhanced antibody titers to HBsAg as compared to antigen alone (p <0.001 and p <0.001 respectively). 0.01), there was no significant increase in anti-HBs response when using control ODN in combination with HBsAg (p = 0.85). (See Figures 23 and 24).

  Furthermore, both CpG ODN 7909 and 10103 enhanced antibody response to HBsAg in that there were no significant differences in anti-HBs responses (p = 0.13) in mice immunized with HBsAg + CpG ODN 7909 and HBsAg + CpG ODN 10103 Equally powerful in doing things.

  In mouse IgG, isotype distribution is widely used as an indicator of the nature of the immune response, where high IgG2a / IgG1 ratios indicate a Th1-biased immune response (1). In this study, the use of CpG ODN significantly enhanced IgG2a titer compared to using antigen alone or in combination with control ODN 2137 (Ag vs. 7909 or Ag vs. 10103 For p <0.001 and Ag + 7909 vs Ag + 2137 p <0.01 and Ag + 10103 vs Ag + 2137 p <0.05). However, the level of IgG2a response was similar when either CpG ODN 7909 or 10103 was used in combination with HBsAg (p> 0.05). Thus, CpG ODNs 7909 and 10103 are both equally potent in their ability to induce a Th1-biased immune response, as measured by increased levels of IgG2a to IgG1.

  As shown in FIG. 25, the CTL response in animals immunized with HBsAg using ODN 10103 appears to be greater than the CTL response induced CpG ODN 7909.

  Conclusion: In vitro data in human PBMC demonstrate that the B-class molecules (7909 and 10103) behave similarly but are not identical in all the assays performed. Of particular importance is the observation that for some of the assays and correlations that were tested, ODN 10103 induced greater immune stimulation than did the ODN 7909 identified above. This difference suggests that CpG nucleotides can be tailored for use in subjects, and that lower levels of CpG ODN can achieve the desired therapeutic endpoint with potentially lower toxicity events.

Example 3 (ODN 10104):
(In vivo effect):
Overview:
Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG dinucleotides have been shown to induce strong innate immune responses to infectious agents and cause Th1-like immune activation. The ability of transmucosal delivery of CpG ODN applied to genital mucosa to protect against or treat intravaginal (IVAG) infection with herpes simplex virus type 2 (HSV-2) was tested.

Materials and methods:
All ODNs were provided by Coley Pharmaceutical Group (Langenfeld, Germany) and had undetectable endotoxin levels (<0.1 EU / ml) measured by the Limulus assay (Bio Whittaker, Verviers, Belgium). The ODN was suspended in sterile endotoxin free Tris-EDTA (Sigma, Deisenhofen, Germany) and stored and processed under sterile conditions to prevent both microbial and endotoxin contamination. All dilutions were performed using pyrogen-free phosphate buffered saline (Life Technologies, Eggenstein, Germany).

  CpG ODN and non-CpG ODN were applied to the vagina of female C57B1 / 6 mice prior to IVAG HSV-2 infection or at various time points after IVAG HSV-2 infection. Mice were monitored daily for genital pathology, survival and genital virus titer following challenge.

result:
Female mice treated with CpG ODN in the genital tract 24 hours prior to IVAG HSV-2 challenge survive infection, show minimal genital pathology, and vaginal wash substantially over the first 6 days post infection There was no virus in it. Importantly, transmucosal delivery of CpG ODN to the genital tract prior to infection provided superior protection against local IVAG challenge as compared to intramuscular delivery of CpG ODN. In contrast, mice pretreated with control ODN alone were not protected, showed severe pathology and had high titers of HSV-2 in vaginal lavage. Immediately after IVAG HSV-2 infection, CpG ODN treated mice are protected and have low vaginal virus titers, but after 24 and 72 hours of IVAG HSV-2 infection, CpG ODN treated mice are protected There was no (see Figures 26 and 27).

Conclusion:
These results show that local transmucosal delivery of CpG ODN to the genital tract before or after genital HSV-2 challenge was very effective at preventing infection by sexually transmitted virus, and CpG-induced innate immunity is suggested to be involved.

(In vitro effect):
Materials and methods:
Peripheral blood buffy coat preparations from healthy human donors were obtained from German Red Cross (Rathingen, Germany), from which PBMCs were purified by centrifugation against Ficoll-Hypaque (Sigma, Germany). Purified PBMC were either used as is or suspended in frozen media and stored at -70 ° C. When necessary, aliquots of these cells were thawed, washed and supplemented with 10% (v / v) heat inactivated FCS, 1.5 mM L-glutamine, 100 U / ml penicillin and 100 μg / ml streptomycin Resuspend in RPMI 1640 culture medium.

Thawed PBMCs or intact PBMCs were resuspended at a concentration of 5 × 10 ⁶ / ml and added to 48-well flat-bottomed plates which had not been given any previous or various concentrations of ODN (1 ml / Well). The cells were cultured at 37 ° C. in a humidified incubator. Culture supernatants were collected 48 hours later. If not used immediately, the supernatant was frozen at -20 <0> C until needed. A commercially available ELISA kit (IL-10; Diaclone, USA) or an in-house ELISA (IFN) in which the amount of cytokine in the supernatant was modified using a commercially available antibody (Pharmingen or PBL respectively; Germany or Germany) It evaluated using-(alpha).

result:
B-class ODNs lead to Th1 dominant immune responses in vivo as well as in vitro. They have found that they can induce representative Th1 cytokines such as IFN-α and IFN-γ and Th1 related chemokines such as MCP-1 and IP-10. In addition, low secretion of the proinflammatory cytokines IL-6 and TNF-α, and secretion of the negative regulator IL-10 can be observed. Thus, we measured the secretion of the Th1 cytokine IFN-α and the regulatory cytokine IL-10. FIG. 28 shows 0.02 μg / ml, 0.05 μg / ml, 0.1 μg / ml, 0.2 μg / ml, 0.5 μg / ml and 1.0 μg to measure IFN-α secretion in vitro Results are shown for experiments performed with 3 different donors at 10104 / ml or control nucleic acid.

  Nucleic acid 10104 induced high levels of IFN-α in a dose dependent manner with peak induction at 10 μl nucleic acid at 0.1 μg / ml. In contrast, the control nucleic acid induced low amounts of IFN-α, which was comparable to the amount of IFN-α induced in the presence of media alone (FIG. 28).

  Similar experiments were performed for IL-10 secretion (Figure 4). Again, as shown above for IFN-α, nucleic acid 10104 induced IL-10 in a dose dependent manner with peak induction at 10 μg nucleic acid at 0.2 μg / ml. Control nucleic acid showed a similar but lower induction profile but its peak shifted to 0.5 μg / ml control nucleic acid. In this case, control levels were greater than media levels (Figure 29).

(TLR9 involvement):
Materials and methods:
Stably transfected HEK 293 cells expressing human TLR9 have been previously described (Bauer et al .; PNAS; 2001). Briefly, HEK 293 cells were transfected by electroporation using a vector expressing human TLR9 and a 6x NF kappa B luciferase reporter plasmid. Stable transfectants (3 × 10 ⁴ cells / well) were incubated with ODN for 16 hours at 37 ° C. in a humidified incubator. Each data point was done in triplicate. Cells were lysed and assayed for luciferase gene activity (using the Brightlite kit from Perkin-Elmer (Ueberlingen, Germany)). The stimulation index was calculated with respect to reporter gene activity in media without the addition of ODN.

result:
Recently, receptors for recognition of CpG sequences have been identified and shown to be members of the Toll-like receptor (TLR) family (Hemmi et al., 2000). This receptor, TLR9, is readily activated by ODNs containing optimal immunostimulatory CpG sequences. We incubated cell lines stably expressing human TLR9 with various concentrations of 10104 and control ODN (Figure 30).

  This result demonstrates that 10104 ODN activated TLR9 in a dose dependent manner with maximal stimulation at 0.625 μg / ml. On the other hand, control ODN was only stimulated at 10 μg / ml, yet its stimulation was much lower than that observed with 10104 nucleic acid.

(CpG10104 effect unrelated to delivery vehicle):
Figures 61A and 61B show the effect of topical CpG delivery using a BEMA disc or the effect of topical CpG delivery in saline on the local pathology of mice after intravaginal challenge with HSV-2. Female C57 / B16 mice were injected SC with 2 mg of progesterone per mouse (4 days prior to virus challenge). CpG 10104 (1 mg, 10 mg or 100 mg) in saline or CpG 10104 (1 mg, 10 mg or 100 mg) impregnated onto a bio-erodible mucoadhesive disc (BEMA) 24 hours prior to viral challenge It was instilled into the vaginal cavity (IVAG). For virus challenge, mice are coated with IVAG using a cotton applicator, turned supine, and held in halothane for 10 minutes, 10 ml IVAG IVAG containing 104 PFU HSV-2 (strain 333) for 1 hour. Infected by injection. Genital pathology was monitored daily after HSV-2 challenge and scoring was performed blindly. Pathology was recorded on a five-point scale: 0, no clear infection; 1, slight redness of the external vagina; 2, redness and swelling of the external vagina; 3, severe redness and swelling of the external vagina and surrounding tissue 4, Genital ulceration with severe redness, swelling and hair loss of reproductive tissue and surrounding tissues; 5, severe genital ulceration covering surrounding tissues. The mice were sacrificed when reaching stage 5. The graph shows mean pathology values for the time (day) after infection for CpG in BEMA discs (FIG. 61A), or for the time (day) after infection for CpG in saline (FIG. 61B) Indicates

  The results show that IVAG delivery to mice of CpG 10104 in saline or CpG 10104 on BEMA disks can reduce vaginal pathology associated with subsequent IVAG HSV-2 infection.

  62A and 62B show the effect of topical CpG delivery using a BEMA disc, or the effect of topical CpG delivery in saline on the survival rate of mice after intravaginal challenge with HSV-2. The mice were treated essentially as described above. The mice were monitored daily and sacrificed if severe genital ulceration was observed that extends to surrounding tissues. The graph shows% survival (FIG. 62A) for time (day) after infection for CpG in BEMA discs, or% survival (FIG. 62B) for time (day) for CpG in saline .

  This result demonstrates that IVAG delivery to mice of CpG 10104 in saline or CpG 10104 on BEMA disks can enhance survival after subsequent IVAG HSV-2 infection.

(Parental administration of CpG 10104):
Figures 63 and 64 show the effect of parenteral CpG 10104 delivery on IP-10 and IFN-y levels in mouse plasma. Female BALB / c mice were injected SC with CpG 10104 in 100 nmol saline, CpG 7909 in saline or resiquimod (R-848). The mice are bled at various time points after injection (1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours), Plasma was collected and IP-10 levels were determined by ELISA.

  This result indicates that CpG 10104 can induce significant amounts of IP-10 and IFN-γ in plasma after SC injection, the level reached being greater than that at R-848.

(Mucosal administration of CpG 10104):
Figure 65 shows the effect of intravaginal CpG 10104 delivery on IP-10 levels in mouse plasma. Female BALB / c mice were given CpG 10104 in 100 nmol saline, CpG 7909 in saline or Requimomod (R-848) instilled into the vaginal cavity. The mice are bled at various time points after injection (1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours), Plasma was collected and IP-10 levels were determined by ELISA. The results show that CpG 10104 can induce significant amounts of IP-10 in plasma after IVAG instillation.

  Figure 69 shows the effect of intravaginal CpG 10104 delivery on IP-10 levels in mouse vaginal lavage. Female BALB / c mice were given CpG 10104 in 100 nmol saline, CpG 7909 in saline or Requimomod (R-848) instilled into the vaginal cavity. At various time points after injection (15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 9 hours, 10 hours, 11 hours, 12 hours) , The vaginal cavity of the mouse was washed 3 times with 75 ml PBS. IP-10 levels in vaginal lavage were determined by ELISA. The results show that CpG 10104 can induce significant local production of IP-10 in the vaginal cavity after IVAG instillation.

(Local administration of CpG 10104)
Figure 67 shows the effect of local CpG delivery on local pathology of mice after intravaginal exposure with HSV-2. Female C57 / B16 mice were injected with 2 mg of progesterone SC per mouse (4 days before virus exposure). Twenty-four hours prior to virus exposure, CpG 10104 (1, 10 or 100 mg) or Resiquimod (1, 10 or 100 mg) in saline was infused into the vaginal cavity (IVAG). For virus exposure, wipe the mouse's IVAG with a cotton applicator, hold the halothane anesthesia for 1 hour, turn the mouse on its back and use 10 ml of 104 PFU HSV-2 (strain 333) for 1 hour The mice were infected by IVAG injection over Reproductive organ pathology was monitored daily and scored blindly after HSV-2 exposure. Pathology below: 0, no infection seen; 1, minor redness of external vagina; 2, redness and swelling of external vagina; 3, severe redness and swelling of external vagina and surrounding tissue; 4, genital and surrounding tissue Were scored at five stages of genital ulceration with severe redness, swelling and hair loss; 5, severe genital ulceration extending to surrounding tissues. When reaching stage 5, the mice were sacrificed.

  This graph shows the mean pathology score against time after infection (days) for CpG ODN 10104 or Resiquimod in saline. The result is that IVAG delivery of CpG 10104 to mice can reduce vaginal pathology associated with subsequent IVAG HSV-2 infection, and CpG 10104 can be more potent than a 10-fold dose of R-848 Explain that.

  Figure 68 shows the effect of local CpG delivery on mouse survival following intravaginal exposure of HSV-2. Essentially, mice were treated as described above. The mice were monitored daily and were sacrificed when severe genital ulcerations extending to surrounding tissues were observed. The graph shows% survival versus time post infection (days) for CpG ODN 10104 and Resiquimod in saline.

  This result illustrates that IVAG delivery of CpG 10104 to mice can enhance survival after subsequent IVAG HSV-2 infection, and CpG 10104 can be more potent than a 10-fold dose of R-848. Do.

70A and 70B show the effect of local CpG delivery on survival and local pathology of mice after vaginal exposure of HSV-2. Essentially, mice were treated as described above. CpG 10104 (100 mg) in saline or oil-in-water cream was injected into the vaginal cavity (IVAG), either a single application 4 hours after viral infection or multiple applications once daily for 5 days .
Reproductive organ pathology was monitored daily and scored blindly after HSV-2 exposure. Pathology below: 0, no infection seen; 1, minor redness of external vagina; 2, redness and swelling of external vagina; 3, severe redness and swelling of external vagina and surrounding tissue; 4, genital and surrounding tissue Were scored at five stages of genital ulceration with severe redness, swelling and hair loss; 5, severe genital ulceration extending to surrounding tissues. When reaching stage 5, the mice were sacrificed. The graph shows% survival (FIG. 70A) and time to local pathology scores (FIG. 70B) versus time (days) post infection for CpG 10104 in saline.

  This result illustrates that IVAG delivery of CpG 10104 in saline or oil-in-water cream may reduce pathology and enhance survival of mice previously infected with lethal doses of HSV-2.

(Immunization of mice)
FIGS. 71A and 71B show that CpG 10104 is as good as CpG 7909 in promoting a humoral immune response to HBsAg in BALB / c mice. BALB / c mice were immunized by intramuscular injection into the left tibialis anterior muscle with 1 mg HBsAg alone or with 10 mg ODN and / or alum (25 mg AL3 +). The animals were boosted 4 weeks after the first immunization. Antibody titers were determined 2 weeks after boost using an endpoint ELISA. FIG. 71A shows an experiment performed without alum and FIG. 71B shows an experiment performed with alum.

  FIGS. 72A and 72B show that CpG 10104 is as good as CpG 7909 in promoting Th1-induced immune responses (as determined by high IgG2a compared to IgG1 titers) to HBsAg in BALB / c mice. Show that BALB / c mice were immunized by intramuscular injection into the left tibialis anterior muscle with 1 mg HBsAg alone or with 10 mg ODN and / or alum (25 mg AL3 +). Animals were boothed 4 weeks after the first immunization. IgG isotype levels were determined 2 weeks after boost using an endpoint ELISA.

(Example 4 (ODN 10105))
(Summary):
This report summarizes in vitro data with human cells and illustrates that ODN 10105 performs as well as or better than ODN 7909 in human cell assays. Furthermore, ODN 10105 shows similar or better performance to ODN 7909 in in vitro and in vivo data of mice, and CpG ODN 10105 stimulates the natural immune system as well as humorality of the antigen co-administered mice It is described that it is useful for enhancing HBsAg-specific and cellular responses.

  The assays performed were receptor binding (TLR9), B cell activation (expression of cell surface activation markers and B cell proliferation) and cytokine secretion (IL-10, IP-10 and IFN-α). All assays demonstrated that ODN 10105 has properties similar to or better than ODN 7909. In vitro studies (ie, B cell proliferation assay, NK lytic activity, cytokine secretion profile) were performed using na し て ve BALB / c mouse splenocytes. Comparative in vivo studies were performed by comparing the potential of these two ODNs to enhance the antigen-specific immune response to hepatitis B antigen (HBsAg).

(Materials and methods):
For human studies, see Example 1 for a description of oligodeoxynucleotides, TLR9 assay, human cell purification, cytokine detection and culture for flow cytometric analysis of B cell activation.

  For mice in vitro and in vivo studies, description of oligodeoxynucleotides, animals, collection and culture of splenocytes, B cell proliferation assay, cytokine secretion profile, NK assay, immunization of mice, determination of antibody response and statistical analysis Examples See 1.

(result):
(TLR9 Binding): Cell lines stably expressing human TLR9 were incubated with different concentrations of ODN 7909 and ODN 10105 and control ODN (FIG. 31). This result illustrates that there was no statistically significant difference between the two B class ODNs when TLR9 was activated. Both ODN show the same dose-response curve and reached maximal activation at the same concentration. The control ODN used did not induce TLR9 activation even at the highest concentration of 24 μg / ml.

  (Human B cells): One feature of B-type ODNs is their ability to activate B cells very effectively (Krieg et al., 1995). B cells and plasmacytotoid DCs are currently the only immune cell types known to express TLR9 (Krug et al., 2001; Bauer et al., 2001). Direct activity of B cells induced by ODN 7909 and ODN 10105 was measured by upregulation of cell surface marker CD86 (FIG. 32) and proliferation of B cells (FIG. 33). For CD86 expression of human B cells, PBMC from healthy blood donors were incubated with different ODNs and B cell activity was measured as described in Materials and Methods.

  This result illustrates that ODN 10105 as well as ODN 7909 are very potent human B cell stimulants. FIG. 32 shows that these CpG ODN were able to stimulate B cells only at a concentration of 0.4 μg / ml in vitro. A plateau was reached at about 1.6 μg / ml, and over 70% of B cells were found to be upregulated in contrast to the much less powerful control, CD86. ODN 10105 was able to induce B cell proliferation even at a maximum test dose of 6 μg / ml, whereas ODN 7909 reached a plateau at the 1.6-3.0 μg / ml dose Similar results were obtained for B cell proliferation induction (FIG. 33).

  Cytokine secretion: B class ODN induces Th1 dominant immune responses in vivo as well as in vitro. They were found to be able to induce representative Th1 cytokines (eg IFN-γ and IFN-α) and chemokines (eg MCP-1 and IP-10). In addition, the proinflammatory cytokine IL-6 and the low secretion of TNF-α and the negative regulator IL-10 can be observed. After administration of ODN 10105 and ODN 7909, the secretion of the Th1 cytokines IFN-α, the chemokine IP-10 and the regulatory cytokines IL-10 and the proinflammatory cytokines TNF-α were measured. FIG. 34 shows the results of experiments performed with six different donors at 0.2, 0.4 and 1.6 μg / ml to measure IFN-α secretion in vitro.

  Both CpG ODNs induced high levels of IFN-α which were maximal at 0.4-1.6 μg / ml. In contrast, control ODN induced low amounts of IFN-α which only began to be expressed at 5.0 μg / ml. Compared to ODN 7909, ODN 10105 induced higher levels than IFN-α at both 1.6 μg / ml and 5.0 μg / ml doses. In contrast to control ODN, ODN 7909 and ODN 10105 induced the chemokine IP-10 as shown in FIG. 35, again ODN 10105 induced higher levels at the 0.4 μg / ml dose.

  The time dependent effects on different cytokines were also analyzed. Therefore, PBMC from different donors were incubated for 8 hours, 24 hours, 36 hours and / or 48 hours to measure secretion of IL-10 or IFN-α. Figures 36 and 37 illustrate the results obtained for IFN-α when incubated for 8 and 24 hours (Figure 36) or 36 and 48 hours (Figure 37) using two different donors. . When incubated with CpG ODN, IFN-α is first secreted as early as 8 hours and reaches its maximum amount at 24 hours, does this amount remain at this level between 24 and 48 hours? Or even increased. LPS did not induce any IFN-α. For both donors, ODN 10105 stimulated higher levels of IFN-α at 8 hours, at a concentration of 1.6 μg / ml.

Very similar experiments were performed for IL-10 secretion (Figures 38 and 39). This cytokine showed similar characteristics to IFN-α, but the maximum amount was obtained at 48 hours. Again, as described above for IFN-α, CpG ODN
Both 7909 and ODN 10105 showed almost identical characteristics in these assays as well as all other assays performed.

  In Vitro Mouse Studies: As shown in FIG. 40, ODN 10105 is capable of stimulating higher levels of B cell proliferation than ODN 7909 at all tested concentrations. According to the data shown in FIG. 41, both CpG ODN 7909 and ODN 10105 are capable of stimulating IL-10, IL-12, IL-6 and TNF- secretion. For IL-12 and TNF- secretion, ODN 10105 induces more than 7909 factor secretion at all tested concentrations.

  CpG ODNs have essentially the same potency in enhancing lytic activity of NK cells in mouse splenocyte cultures (FIG. 42).

  As shown in FIG. 43, either CpG ODN 7909 or ODN 10105 significantly enhanced antibody titers to HBsAg as compared to antigen alone (p <0.0001) while control ODN There was no significant increase in anti-HBs response when used in combination with HBsAg (p = 0.85).

  As shown in FIG. 44, the increase in total IgG levels is similar to both CpG ODNs. In mice, IgG isotype distribution is widely used as an indicator of the nature of the immune response, where high levels of IgG2a / IgG1 show a Th1-biased immune response (Constant and Bottomly, 1997). In the current study, the use of CpG ODN significantly increased IgG2a titer compared to when antigen alone is used or in combination with control ODN 2137 (p vs. Ag vs. ODN 7909 or 10105) P <0.01 for <0.001 and Ag + 7909 vs Ag + 2137 p <0.05 for Ag + 10105 vs Ag + 2137). However, IgG2a response levels were similar (p> 0.05) when either CpG ODN 7909 or 10105 was used in combination with HBsAg. Therefore, both CpG ODNs 7909 and 10105 are equally potent in their ability to induce a Th1-biased immune response as measured by the increased levels of IgG2 to IgG1.

(Closed):
ODN 10105 shows in vitro data with human cells showing that it behaves like ODN 7909 and in some cases better. According to the results of mouse studies, CpG ODNs 7909 and 10105 are similar both in their effects on innate immune responses in vivo and their ability to enhance antigen specific responses in vivo when the antigen is co-administered. Have immune enhancing properties.

Example 5 (ODN 10106):
(HCV research):
(Summary):
This experiment was conducted comparing CpG ODN 10106 and CpG ODN 7909 for their immunoactive properties in PBMCs isolated from normal healthy adult subjects and adult subjects chronically infected with HCV. . The ability of ODN to stimulate B cell proliferation, cytokine secretion (IL-10 and TFN-α) and chemokine secretion (IP-10) was evaluated. All assays showed that ODN 10106 had almost the same or better properties as ODN 7909, and similar results were normal, healthy adult subjects and chronically infected with HCV It was observed using PBMCs isolated from adult subjects.

(Materials and methods):
(Human (HCV) Research):
Oligodeoxynucleotides: CpG ODN 7909, 10106 and control ODN 4010 were manufactured in contract with the Coley Pharmaceutical Group. All ODN, no sterile endotoxin ^{TE (pH8.0) (OmniPer R;} EM Science, Gibbstown, NJ) and resuspended in storage and operation under sterile conditions to prevent contamination of both microbial and endotoxin did. Control ODN 4010 does not contain a stimulatory CpG motif. RPMI 1640 complete medium containing 10% normal human AB serum (Wisent Inc, St. Bruno, QC) (heat inactivated) and 1% penicillin / streptomycin (Gibco BRL, Gland Island, NY) immediately before use for dilution of ODN I did it inside. The ODN sequences used are shown in the following table.

  Table 1: ODN sequences used in these experiments.

PBMC isolation: 200 mL whole blood from 10 normal healthy adult subjects and an adult subject chronically infected with HCV that failed the previous 6 months with 10 IFN-α based treatments, It was collected by venipuncture into a heparinized green top vacutainer. Peripheral blood mononuclear cells (PBMCs) were purified by centrifugation at Ficoll-Pacque at 400 × g for 35 minutes. The cells were resuspended at a concentration of 10 × 10 ⁶ / ml in RPMI complete medium containing 10% normal human AB serum (heat inactivated) and 1% penicillin / streptomycin.

B cell proliferation assay: ODN was diluted to the following concentrations: 2 μg / ml, 6 μg / ml and 12 μg / ml in RPMI medium with 10% normal human AB serum (heat inactivated) and 1% penicillin / streptomycin . 100 μL of diluted ODN was added to the wells of a 96 well round bottom plate. Freshly isolated PBMCs are resuspended to 1 × 10 ⁶ / ml in complete RPMI medium with 10% normal human AB serum (heat inactivated) and 1% penicillin / streptomycin and then cell suspension The solution was added to each well (100 μL / well) to a final ODN concentration of 1 μg / mL, 3 μg / mL and 6 μg / mL. Cells were cultured for 5 days and then pulsed with ³ H-thymidine (1 μCi / well) for 16 hours to 18 hours. Following incubation, cells were collected on filter paper and the amount of radioactivity was measured. Results were reported as stimulation index (SI) versus untreated media control.

Cytokine detection: ODN were diluted to the following concentrations: 2 μg / ml, 6 μg / ml and 12 μg / ml in RPMI medium with 10% normal human AB serum (heat inactivated) and 1% penicillin / streptomycin. 100 μL of diluted ODN was added to the wells of a 96 well flat bottom plate. Freshly isolated PBMC resuspended at a concentration of 10 × 10 ⁶ / ml were added to each well (100 μl / well) to a final ODN concentration of 1 μg / mL, 3 μg / mL and 6 μg / mL . The cells were incubated for 48 hours at 37 ° C. with 5% CO ₂ . Following incubation, cell supernatants were collected from each well and frozen at -80 <0> C until assayed.

  The levels of IFNα and IL-10 and IP-10 in the supernatants are measured using ELISA Kit (Catalog number 41105, D1000 and DIP 100 respectively), commercially available from R & D Systems, Minneapolis, MN, based on the manufacturer's instructions. did.

Statistical analysis: Statistical analysis was performed using InStat (Graph PAD Software, San Diego). Statistical differences between groups were determined by one-way analysis of variance (ANOVA) on raw or transformed data (log ₁₀ ) followed by Tukey-Kremer multiple comparison test. Bartlett's test (Kurskal-Wallis test) was used to indicate that after transformation of the data, the difference between the standard deviations is a significant non-parametric ANOVA.

(result)
B cell proliferation: One feature of type B ODNs is their ability to activate B cells very efficiently (Krieg et al., 1995). The ability of two B class ODNs (7909 and 10106) to stimulate B cell proliferation is shown in FIG. 45 below.

  When compared to CpG ODN 7909, 10106 was equally effective at stimulating B cell proliferation. Furthermore, there was no significant difference in their ability to stimulate PBMCs derived from either groups of normal healthy subjects or subjects chronically infected with HCV.

  Cytokine / chemokine secretion: B-class ODN produce Thl dominant immune responses in vivo and in vitro. It has been found that the B class ODN can induce representative Th1 cytokines such as IFNα and IFN-α, and chemokines such as MCP-1 and IP-10. In addition, low secretion of the proinflammatory cytokines IL-6 and TNF-α, and secretion of the negative regulator IL-10 can be observed. Figures 46, 47 and 48 illustrate the ability of B-class ODNs to stimulate the secretion of the Thl cytokines IFNα, chemokine IP-10 and the regulatory cytokine IL-10.

  B-class ODNs (7909 and 10106) induced secretion of equivalent concentrations of IFNα.

  After stimulation of PBMC with either 7909 or 10106 B class ODN, equivalent concentrations of IP-10 were secreted. A difference is seen in the ability of these ODNs to stimulate the secretion of IP-10 from PBMCs isolated from normal healthy subjects or from subjects chronically infected with HCV It was not. Maximum concentrations of IP-10 were obtained at an ODN concentration of 3 μg / ml for both 7909 and 10106. The same analysis was performed on the secretion of IL-10 (Figure 48).

  CpG ODN 7909 and 10106 were able to cause the secretion of equivalent concentrations of IL-10 from PBMCs isolated from both adult populations. Maximal IL-10 induction for both ODNs was observed at 6 μg / ml.

(Conclusion)
In vitro using human peripheral blood mononuclear cells isolated from two different adult populations: (1) normal healthy subjects and (2) chronic HCV-infected subjects who have previously failed IFN-a therapy The data show that B-class CpG ODNs 7909 and 10106 are equally capable of stimulating B cell proliferation and secretion of IFN-α, IL-10 and IP-10 within the same population, and further this effect Indicates that it was identical for the two populations.

(Non-HCV research)
(wrap up)
CpG ODN 10106 is a class B nucleic acid. These experiments compare the nature of the immunostimulatory properties of CpG ODN 10106 and the immunostimulatory properties of CpG ODN 7909. In vitro and in vivo immunological parameters were used for this evaluation.

  In vitro data were obtained by comparing ODN 10106 and ODN 7909 for human PBMC. The assays performed included receptor binding (TLR9), activation of B cells (expression of cell surface activation markers and proliferation of B cells) and secretion of cytokines (IL-10, IP-10, IFN-α and TNF- included α). All assays showed that ODN 10106 had properties that were almost identical to ODN 7909.

  In vitro studies (ie, B cell proliferation assay, NK lytic activity, cytokine secretion profile) were also performed by using splenocytes of naive BALB / c mice. In vivo comparative studies were performed by examining the ability of these two ODNs to boost antigen-specific immune responses to hepatitis B antigen (HBsAg). In in vivo comparative studies, improvements in both humoral responses (antibodies) and cell mediated immune responses (CTL activity) were investigated. In addition, the nature of the elicited immune response (ie, Th1 vs. Th2) was examined by determining the IgG2a / IgG1 ratio and the strength of the CTL response.

(Materials and methods)
For human studies, see Example 1 for a description of oligodeoxynucleotides, TLR9 assays, human cell purification, cytokine detection, and cultures for flow cytometric analysis of B cell activation.

  For in vitro and in vivo studies of mice, oligodeoxynucleotides, animals, collection and culture of splenocytes, B cell proliferation analysis, cytokine secretion profile, NK assay, immunization of mice, quantification of antibody responses, and statistical analysis See Example 1 for a description of.

(result)
(TLR9 binding)
We incubated cell lines stably expressing human TLR9 with different concentrations of ODN 7909 and ODN 10106, as well as control ODN (FIG. 49). The results show that there was no significant difference between the two B-class ODNs with respect to activating TLR9. Both ODNs showed the same dose response curve. The control ODN used did not induce TLR9 activity even at the highest concentration of 12 μg / ml.

(Activation of human B cells)
One feature of type B ODNs is their ability to activate B cells very efficiently (Krieg et al., 1995). At present, only B cells and plasmacytoid DCs are immune cell types known to express TLR9 (Krug et al., 2001; Bauer et al., 2001). Therefore, we measure direct activation of B cells induced by ODN 7909 and ODN 10106 by upregulation of the cell surface marker CD86 (Figure 50) and by proliferation of B cells (Figure 51). did. For expression of CD86 on human B cells, PBMC from healthy blood donors were incubated with separate ODNs and B cell activation was measured as described in Materials and Methods.

(B cell proliferation)
Both results show that 10106 and 7909 are very potent human B cell stimulators. FIG. 50 shows that these CpG ODN were able to stimulate B cells very strongly at in vitro concentrations of only 0.4 μg / ml. The plateau was reached at about 1.6 μg / ml. Similar results were obtained for induction of B cell proliferation (FIG. 51), where the stimulation index reached a maximum at about 0.8 μg / ml.

(Cytokine secretion)
B-class ODNs lead to Th1 dominant immune responses in vivo and in vitro. These can induce representative Th1 cytokines such as IFN-γ and IFN-α, as well as chemokines such as MCP-1 and IP-10. In addition, low secretion of the proinflammatory cytokines IL-6 and TNF-α, and secretion of the negative regulator IL-10 can be observed. We thus measured the secretion of the Th1 cytokine IFN-α, the chemokine IP-10, and the modulator IL-10 and the proinflammatory cytokine TNF-α.

  FIG. 52 is performed with three different donors at 0.2 μg / ml, 0.4 μg / ml, 1.6 μg / ml and 5 μg / ml to measure IFN-α secretion in vitro Shows the results of the experiment. Both CpG ODNs (7909 and 10106) induced high levels of IFN-α, reaching a maximum at 0.4 μg / ml (7909) or 1.6 μg / ml (10106). However, the maximal elevation of IFN-α secretion appeared approximately three times higher after stimulation of 10106 compared to 7909.

  In addition, ODN 7909 and ODN 10106, in contrast to the control ODN, induced higher amounts of the cytokine IP-10 as shown in FIG. In this experiment, the plateau was already reached at about 0.2 μg / ml.

  Very similar experiments were performed on IL-10 secretion (FIG. 54). Again, as described above for IFN-α, both CpG ODN 7909 and ODN 10106 exhibited nearly identical characteristics in this assay, as did all other assays performed. By comparison, control ODN induces IL-10 secretion only at the highest concentration.

  As shown in FIG. 55, both ODN 7909 and ODN 10106, as well as the control ODN, showed a lower secretion profile of the pro-inflammatory cytokine TNF-α at all tested concentrations compared to LPS. Again, similar features can be observed after stimulation with these two ODNs.

  According to this data, both CpG ODN 7909 and ODN 10106 have substantially equal efficacy for enhancing cytokine secretion by mouse splenocytes (FIG. 57).

(B cell proliferation)
The data show that CpG ODN 10106 is equally effective at inducing murine B cell proliferation at all concentrations tested, even if not above CpG ODN 7909 (Figure 56).

(NK assay)
According to the data, both CpG ODN 7909 and ODN 10106 have substantially equivalent potency in enhancing the lytic activity of NK cells in mouse splenocyte cultures (FIG. 58).

(Total IgG response)
According to the results of this study, the use of either CpG ODN 7909 or ODN 10106 significantly enhanced antibody titers to HBsAg compared to antigen alone (p <0.0001) while Ag + CpG ODN 7909 Alternatively, there was no significant difference in antibody titers in animals immunized with Ag + CpG ODN 10106 (p = 0.86). In addition, control ODN did not significantly enhance the anti-HB response when used in combination with HBsAg (p = 0.86) (FIG. 59). The increase in total IgG levels is slightly but significantly (p = 0.04) greater when CpG ODN 7909 is used compared to when CpG ODN 10606 is used.

(Property of humoral response (IgG1 to IgG2a ratio))
In mice, IgG isotype distribution is used extensively as an indicator of the nature of the immune response, where high IgG2a / IgG1 ratios show a Th1-biased immune response (Constant and Bottomly, 1997). In this study, the use of CpG ODN significantly enhanced IgG2a titer compared to when the antigen was used alone or in combination with the control ODN 2137 (for Ag vs. 7909) p <0.01, p <0.001 for Ag vs. 10106, and p <0.001 for Ag + 7909 vs. Ag + 2137, and p <0.01 for Ag + 10106 vs. Ag + 2137). However, the levels of IgG2a responses were similar when either CpG ODN 7909 or ODN 10106 were used in combination with HBsAg (p> 0.05). Thus, both CpG ODN 7909 and ODN 10106 are equally potent in their ability to induce a Th1-biased immune response as measured by increased levels of IgG2a over IgG1 (FIG. 60).

(Conclusion)
In vitro data with human peripheral mononuclear cells show that the two molecules of the B class (7909 and 10106) behave very similar, if not identical, in the various assays performed. In some assays, ODN 10106 performed better than ODN 7909.

  According to the results of the above mouse studies, CpG ODN 7909 and ODN 10106 have both their in vitro effects on innate immune responses and their ability to enhance antigen specific responses in vivo when co-administered with the antigen , Have similar immune enhancing properties.

(Equivalent)
The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention should not be limited in scope by the examples provided. Because this example is intended as merely illustrative of one aspect of the present invention, and other functionally equivalent embodiments are within the scope of the present invention. Various modifications of the invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects of the invention are not necessarily encompassed by each embodiment of the invention.

TLR9 engagement by ODN 7909 and 10102. As described in Example 1, TLR9 expressing cultured cell lines were incubated with the indicated concentrations of ODN. The mean stimulation index on the media control is illustrated. IL-1 was used as a positive control for the reporter gene. B cells upstream control activity marker CD86 by incubation of PBMC with CpG ODN. Human PBMC were incubated with ODN 7909 and 10102 for 48 hours at the indicated concentrations. The average proportion (measured by flow cytometry) of CD86 expressing CD19 positive B cells of three different donors is illustrated. B cell proliferation induced by CpG ODN 7909 and 10102. PBMC pre-incubated with the dye CFSE were cultured for 5 days in the presence or absence of the indicated ODN concentration. The cells were harvested and the reduction of CFSE strains on proliferating CD19 positive B cells was measured by flow cytometry on three different donors (see also Example 1). IFNα secretion induced by ODN 7909 and 10102. Human PBMC of three different donors were incubated for 48 hours with the indicated concentrations of ODN. The supernatant was collected and IFN-α was measured by ELISA (see Example 1). For each concentration, the average, minimum and maximum amounts of IFNα obtained from three different donors are illustrated. IP-10 secretion induced by ODN 7909 and 10102. Human PBMC of three different donors were incubated for 48 hours with the indicated concentrations of ODN. The supernatant was collected and IP-10 was measured by ELISA (see Example 1). For each concentration, the average, minimum and maximum amounts of IP-10 obtained from three different donors are illustrated. IL-10 secretion induced by ODN 7909 and 10102. PBMC of three different blood donors were incubated with the indicated concentrations of ODN 7907, 10102 or control ODN. The supernatant was collected and IL-10 was measured by ELISA. The average, minimum, and maximum amounts of I-10 obtained from three different donors are illustrated for each concentration. TNFα secretion in response to ODN 7909 and 10102. PBMC of three different blood donors were incubated for 24 hours with the indicated concentrations of ODN 7907, 10102 or control ODN. The supernatant was collected and TNFα was measured by ELISA. The mean, minimum and maximum amounts from three different donors are illustrated for each concentration. Untreated BALB / c mouse spleen cells (5 × ¹⁰ 6 / ml or 2.5 × ¹⁰ 6 / m1) of medium (negative control) or with different amounts of CpG-ODN 10102 and incubated. Cells were pulsed with ³ H thymidine (20 μCi / ml) for 16 hours after 96 hours of incubation, harvested, and radioactivity was measured. Each bar indicates the stimulation index (counts of incubated cells / min (CPM) / CMP of cells incubated with medium). Untreated BALB / c mouse splenocytes (5 × 10 ⁶ / ml) were incubated with medium (negative control) or different amounts of CpG ODN 7909, 10102, or control ODN 2137. Recovery of the supernatant was performed at 6 hours (TNFα, panel D), 24 hours (IL-12, panel B), or 48 hours (IL-6, panel C and IL-10, panel A). Untreated BALB / c mouse splenocytes (30 × 10 ⁶ / ml) were incubated with medium (negative control) or different amounts of CpG ODN 7909,10102. NK activity was measured using a ⁵¹ Cr release assay. Adult (6-8 weeks) BALB / c mice were immunized with 1 μg HBsAg alone, or in combination with CpG-ODN (10 μg) 10102, 7909, or control ODN (10 μg) 2137. Animals were bled 4 weeks post immunization and plasma was assayed for total IgG levels (anti-HBs) against HBsAg. Each bar represents the geometric mean (± SEM) of ELISA endpoint dilution titers for the whole group (n = 10). The titer was defined as the highest dilution which gave an extinction value of twice the absorbance of non-immune plasma at a fractional value of 0.05. Adult (6-8 weeks) BALB / c mice were immunized with 1 μg HBsAg alone or in combination with CpG-ODN (10 μg) 7909, 10102 or control ODN (10 μg) 2137. The animals were bled four weeks post immunization and plasma was assayed for IgG1 and IgG2a levels (antibodies HBs) to HBsAg. Each bar represents the geometric mean (± SEM) of ELISA endpoint dilution titers for the whole group of ELISA weekend point dilutions (n = 10). The titer was defined as the highest dilution which gave an extinction value of twice the absorbance of non-immune plasma at a fractional value of 0.05. TLR9 engagement by ODN 7909 and 10103. TLR-9 expressing cell lines were incubated with the indicated concentrations of ODN as described in Example 2. The mean stimulation index on media controls is shown for four independent experiments. IL-1 was used as a positive control for the reporter gene. B cells upstream control activity marker CD86 by incubation of PBMC with CpG ODN. Human PBMC were incubated with ODN 7909 and 10103 and control ODN for 48 hours at the indicated concentrations. The average proportion (measured by flow cytometry) of CD86 expressing CD19 positive B cells of three different donors is illustrated. B cell proliferation induced by CpG ODN 7909 and 10103. PBMC pre-incubated with the dye CFSE were cultured for 5 days in the presence or absence of the indicated ODN concentration. The cells were harvested and the reduction of CFSE strain on proliferating CD19 positive B cells was measured by flow cytometry (see also Example 2). INF-α secretion induced by ODN 7909 and 10103. Human PBMC of six different donors were incubated for 48 hours with the indicated concentrations of ODN. The supernatant was collected and IFN-α was measured by ELISA (see Example 2). For each concentration, the amount of IFNα obtained from six different donors is illustrated. IP-10 secretion induced by ODN 7909 and 10103. Human PBMC of three different donors were incubated for 48 hours with the indicated concentrations of ODN. The supernatant was collected and IP-10 was measured by ELISA (see Example 2). The average amount of IP-10 obtained from three different donors is illustrated for each concentration. IL-10 secretion to incubation with different concentrations of 7909, 10103, and control ODN. The means from three different donors obtained by incubation for 48 hours are illustrated. TNF-α Secretion: PBMC of 3 different blood donors were incubated with the indicated concentrations of ODN 7909, 10103 or controls for 48 hours. The supernatant was collected and TNF-α was measured by ELISA. Average values for three donors are shown. Naive BALB / c mouse spleen cells (5 × ¹⁰ 6 / ml or 2.5 × ¹⁰ 6 / m1) of medium (negative control) or different amounts of CpG ODNs 7909 (white bars), 10103 and (black bars) Incubated. Cells were pulsed with ³ H thymidine (20 μCi / ml) for 16 hours after 96 hours of incubation, harvested, and radioactivity was measured. Each bar indicates the stimulation index (counts of incubated cells / min (CPM) / CMP of cells incubated with medium). Naive BALB / c mouse splenocytes (5 × 10 ⁶ / ml) were incubated with medium (negative control) or different amounts of CpG ODN 7909, 10103, or control ODN 2137. Supernatant recovery is performed at 6 hours (TNF-α, panel D), 24 hours (IL-12, panel B), or 48 hours (IL-6, panel C and IL-10, panel A). The Naive BALB / c mouse splenocytes (30 × 10 ⁶ / ml) were incubated with medium (negative control) or different amounts of CpG-ODN 7909 and 10103. NK activity was measured using a ⁵¹ Cr release assay. Adult (6-8 weeks) BALB / c mice were immunized with 1 μg HBsAg alone, or in combination with CpG-ODN (10 μg) 10103, 7909, or control ODN (10 μg) 2137. The animals were bled 4 weeks post immunization and plasma was assayed for total IgG levels (anti-HBs) against HBsAg. Each bar represents the geometric mean (± SEM) of ELISA endpoint dilution titers for the whole group (n = 5). The titer was defined as the maximum dilution resulting in an absorbance value of twice the absorbance value of non-immune plasma at a fractional value of 0.05. Adult (6-8 weeks) BALB / c mice were immunized with 1 mg HBsAg alone or in combination with 10 mg CpG ODN 7909, 10103 or control ODN (10 μg) 2137. The animals were bled four weeks post immunization and plasma was assayed for IgG1 and IgG2a levels (antibodies HBs) to HBsAg. Each bar represents the geometric mean (± SEM) for the whole group of ELISA end point dilution titers (n = 5). The titer was defined as the maximum dilution resulting in an absorbance value of twice the absorbance value of non-immune plasma at a fractional value of 0.05. Adult (6-8 weeks) BALB / c mice were immunized with 1 mg HBsAg in combination with either CpG-ODN (10 μg) 7909 or 10103. Four weeks after immunization, spleens were taken and splenocytes were used for measurement of CTL activity by ⁵¹ Cr release assay. CTL activity is shown as mean% specific cell lysis (± SEM) against groups of animals (n = 5) at different effector: target ratios. FIG. 16 is a graph showing mean pathological scores as a function of days post infection in mice challenged with HSV-2 and administered nucleic acid 10104. FIG. FIG. 16 is a graph showing survival as a function of days post infection in mice challenged with HSV-2 and administered nucleic acid 10104. FIG. Figure 5 is a bar graph showing human IFN-a induction in human PBMC after 48 hours with nucleic acid 10104 or control. IFN-α was measured by ELISA and the results are shown as mean ± SEM from three blood donors. 16 is a bar graph showing human IL-10 induction in human PBMC after 48 hours with nucleic acid 10104 or control. IL-10 was measured by ELISA and the results are shown as mean ± SEM from three blood donors. 16 is a bar graph depicting human TLR9 mediated NFkB stimulation after 16 hours exposure to nucleic acid 10104 or control. Stimulation was measured using a reporter gene upstream assay. TLR9 engagement by ODN 7909 and 10105. TLR9 expressing cell lines were incubated with the indicated concentrations of ODN as described in Example 4. The mean stimulation index on media controls is shown for four independent experiments. IL-1 was used as a positive control for the reporter gene. During incubation of PBMC with CpG-ODN, the activation marker CD86 is upregulated by B cells. Human PBMCs were incubated with ODNs 7909 and 10105 as for the control ODN for 48 hours at the indicated concentrations. What is shown is the average percentage of CD86 expressing CD19 positive B cells (as measured by flow cytometry) of three different donors. B cell proliferation induced by CpG ODN 7909 and 10105. PBMC preincubated with the dye CFSE were cultured for 5 days in the presence or absence of the indicated ODN concentration. Cells were harvested and the reduction of CFSE strain on proliferating CD19 positive B cells was measured by flow cytometry (see also Example 4). IFN-α secretion induced by ODN 7909 and 10105. Human PBMC of three different donors were incubated for 48 hours with the indicated concentrations of ODN. The supernatant was collected and IFN-α was measured by ELISA (see Example 4). The average amount of IFN-α obtained is graphed for three different donors at each concentration. IP-10 secretion induced by ODN 7909 and 10105. Human PBMC of three different donors were incubated for 48 hours with the indicated concentrations of ODN. The supernatant was collected and IP-10 was measured by ELISA (see Example 4). The average amount of IP-10 obtained is graphed for three different donors at each concentration. Time kinetics of IFN-α secretion. PBMC of two different blood donors were incubated for 8 or 24 hours with the indicated concentrations of ODN 7909, 10105 or control ODN. The supernatant was collected and IFN-α was measured by ELISA. The individual IFN-α amounts obtained at different times are shown for two different donors. Time kinetics of IFN-α secretion. PBMC of two different blood donors were incubated with the indicated concentrations of ODN 7909, 10105, or control for 36 hours or 48 hours. The supernatant was collected and IFN-α was measured by ELISA. The individual IFN-α amounts obtained at different time points are shown for two donors. Time kinetics of IL-10 secretion. PBMCs of three different blood donors were incubated with indicated concentrations of ODN 7909, 10105 or control ODN for 8, 24 or 48 hours. The supernatant was collected and IL-10 was measured by ELISA. The individual IL-10 quantities obtained at different time points are shown for three different donors. Time kinetics of IL-10 secretion. The same experiment as in Example 8 is shown. The average amount of IL-10 at each concentration and time point between the three donors was calculated. Naive BALB / c mouse spleen cells (5 × ¹⁰ 6 / ml or 2.5 × ¹⁰ 6 / m1) of medium (negative control) or with different amounts of CpG-ODNs 7909 and 10105 and incubated. Cells were pulsed with ³ H thymidine (20 μCi / ml) for 16 hours after 96 hours of incubation, harvested, and radioactivity was measured. Each bar indicates the stimulation index (counts of incubated cells / minute (CMP) / CMP of cells incubated with medium). Naive BALB / c mouse splenocytes (5 × 10 ⁶ / ml) were incubated with medium (negative control) or different amounts of CpG-ODN 7909, 10105, or control ODN 2137. Supernatant recovery is performed at 6 hours (TNF-α, panel D), 24 hours (IL-12, panel B), or 48 hours (IL-6, panel C and IL-10, panel A). The Naive BALB / c mouse splenocytes (30 × 10 ⁶ / ml) were incubated with medium (negative control) or with different amounts of CpG-ODN 7909 and 10105. NK activity was measured using a ⁵¹ Cr release assay. Adult (6-8 weeks) BALB / c mice were immunized with 1 μg HBsAg alone, or in combination with CpG-ODN (10 μg) 10105, 7909, or control ODN (10 μg) 2137. The animals were bled 4 weeks post immunization and plasma was assayed for total IgG levels (anti-HBs) against HBsAg. Each bar represents the geometric mean (± SEM) of ELISA endpoint dilution titers for the whole group (n = 10). The titer was defined as the maximum dilution resulting in an absorbance value of twice the absorbance value of non-immune plasma at a fractional value of 0.05. Adult (6-8 weeks) BALB / c mice were immunized with 1 μg HBsAg alone or in combination with 10 μg CpG ODN 7909, 10105, or control ODN (10 μg) 2137. The animals were bled four weeks post immunization and plasma was assayed for IgG1 and IgG2a levels (antibodies HBs) to HBsAg. Each bar represents the geometric mean (± SEM) for the whole group of ELISA end point dilution titers (n = 10). The titer was defined as the maximum dilution giving an absorbance value twice the absorbance of non-immune plasma at a fractional value of 0.05. B cell proliferation induced by CpG ODN. PBMCs from normal and healthy subjects (n = 10) or subjects chronically infected with HCV (n = 10) at a concentration of 0.5 × 10 ⁶ / ml, or medium (negative control) or Incubate with increasing amounts of CpG ODN 7909 and 10106 or 4010 at 6 μg / mL. After 5 days of incubation, cells were pulsed with ³ H-thymidine (1 μCi / well) for 16-18 hours, harvested, and radioactivity was measured. Each bar represents the mean stimulation index (counts of cells incubated at ODN / min (CPM) / CPM of cells incubated at medium). Secretion of IFN-α induced by CpG ODN. Human PBMC from normal and healthy subjects and subjects chronically infected with HCV were incubated with control ODN 4010, 7909, or 10106 at a concentration in the range of 1 to 6 μg / mL. The supernatant was collected and IFN-α was measured by ELISA (see Example 5). The detection limit of this assay is 31.2 pg / mL and subjects with IFN-α results below the detection limit are not represented on the graph. The mean value shown as a straight line was determined by detectable IFN-α for these subjects. IP-10 secretion induced by CpG ODN. Human PBMCs from 10 normal and healthy subjects and 10 HCV chronically afflicted subjects were incubated with control ODN 4010, 7909 or 10106 in the concentration range 1 to 6 μg / mL. The supernatant was collected and IP-10 was measured by ELISA (detection limit 15.6 pg / mL) (see Example 5). IL-10 secretion induced by CpG ODN. Human PBMCs from 10 normal and healthy subjects and 10 HCV chronically afflicted subjects were incubated with control ODN 4010, 7909 or 10106 in the concentration range 1 to 6 μg / mL. The supernatant was collected and IL-10 was measured by ELISA (see Example 5). The detection limit for the ELISA assay is 23.4 pg / mL. If the treatment group has subjects with undetectable IL-10 concentrations, the number of subjects with detectable IL-10 is shown on the graph as a percentage of the total number of patients evaluated. The mean and standard deviation determined are for patients with detectable IL-10. TLR9 engagement by ODN 7909 and 10106. TLR9 expressing cell lines were incubated with the indicated concentrations of ODN as described in Example 5. The mean stimulation index on the media control is illustrated. IL-1 was used as a positive control for the reporter gene. B cells upregulate the activity marker CD86 upon incubation of PBMC with CpG-ODN. Human PBMC were incubated with ODN 7909 and 10106 for 48 hours at the indicated concentrations. The average proportion (measured by flow cytometry) of CD86 expressing CD19 positive B cells of three different donors is illustrated. B cell proliferation induced by CpG-ODN 7909 and 10106. PBMC preincubated with the dye CFSE were cultured for 5 days in the presence or absence of the indicated ODN concentration. The cells were harvested and the reduction of CFSE strains on proliferating CD19 positive B cells was measured by flow cytometry for three different donors (see also Example 5). INF-α secretion induced by ODN 7909 and 10106. Human PBMC of three different donors were incubated for 48 hours with the indicated concentrations of ODN. The supernatant was collected and IFN-α was measured by ELISA (see Example 5). The average, minimum and maximum amounts of IFN-α obtained are graphed for three different donors at each concentration. IP-10 secretion induced by ODN 7909 and 10106. Human PBMC of three different donors were incubated for 48 hours with the indicated concentrations of ODN. The supernatant was collected and IP-10 was measured by ELISA (see Materials and Methods). The average, minimum and maximum amounts of IP-10 obtained are plotted for three different donors at each concentration. IL-10 secretion. PBMC of three different blood donors were incubated with the indicated concentrations of ODN 7909, 10106 or controls. The supernatant was collected and IL-10 was measured by ELISA. The average, minimum and maximum amounts of IL-10 obtained from 3 donors are illustrated. TNF-α Secretion: PBMC of 3 different blood donors were incubated with the indicated concentrations of ODN 7909, 10106 or control for 16 hours. The supernatant was collected and TNF-α was measured by ELISA. The average, minimum and maximum amounts of three donors are illustrated. Naive BALB / c mouse spleen cells (5 × ¹⁰ 6 / ml or 2.5 × ¹⁰ 6 / m1) of medium (negative control) or with different amounts of CpG-ODNs 7909 and 10106 and incubated. Cells were pulsed with ³ H thymidine (20 μCi / ml) for 16 hours after 96 hours of incubation, harvested, and radioactivity was measured. Each bar indicates the stimulation index (counts of incubated cells / min (CPM) / CPM of cells incubated in medium). Naive BALB / c mouse splenocytes (5 × 10 ⁶ / ml) were incubated with medium (negative control) or different amounts of CpG-ODN 7909, 10106, or control ODN 2137. Supernatant recovery is performed at 6 hours (TNF-α, panel D), 24 hours (IL-12, panel B), or 48 hours (IL-6, panel C and IL-10, panel A). The Naive BALB / c mouse splenocytes (30 × 10 ⁶ / ml) were incubated with medium (negative control) or different amounts of CpG ODN 7909 and 10106. NK activity was measured using a ⁵¹ Cr release assay. Adult (6-8 weeks) BALB / c mice were immunized with 1 μg HBsAg alone, or in combination with CpG-ODN (10 μg) 10106, 7909, or control ODN (10 μg) 2137. The animals were bled 4 weeks post immunization and plasma was assayed for total IgG levels (anti-HBs) against HBsAg. Each bar represents the geometric mean (± SEM) of ELISA endpoint dilution titers for the whole group (n = 10). The titer was defined as the maximum dilution resulting in an absorbance value of twice the absorbance value of non-immune plasma at a fractional value of 0.05. Adult (6-8 weeks) BALB / c mice were immunized with 1 μg HBsAg alone or in combination with 10 μg of CpG ODN 7909, 10106 or control ODN (10 μg) 2137. The animals were bled four weeks post immunization and plasma was assayed for IgG1 and IgG2a levels (antibodies HBs) to HBsAg. Each bar represents the geometric mean (± SEM) for the whole group (n = 10) of ELISA endpoint dilution titers. The titer was defined as the maximum dilution giving an absorbance value twice the absorbance of non-immune plasma at a fractional value of 0.05. FIG. 61A shows the effect of local CpG delivery using BEMA discs on the local pathology of mice after intravaginal challenge with HSV-2. FIG. 61B shows the effect of local CpG delivery in saline on the local pathology of mice after vaginal challenge with HSV-2. FIG. 62A shows the effect of topical CpG delivery with BEMA discs on mouse survival following vaginal challenge with HSV-2. Figure 62B shows the effect of topical CpG delivery in saline on survival of mice after vaginal challenge with HSV-2. Figure 7 shows the effect of parenteral CpG 10104 delivery on IP-10 levels in mouse plasma. Figure 7 shows the effect of parenteral CpG 10104 delivery on IFN-g levels in mouse plasma. Figure 7 shows the effect of intravaginal CpG 10104 delivery on IP-10 levels in mouse plasma. Figure 7 shows the effect of local CpG delivery on mouse local pathology after vaginal challenge with HSV-2. Figure 7 shows the effect of local CpG delivery on mouse survival following vaginal challenge with HSV-2. Figure 7 shows the effect of intravaginal CpG 10104 delivery on IP-10 levels of mouse vaginal lavage. FIG. 69A shows the effect of local CpG delivery on mouse local pathology after vaginal challenge with HSV-2. FIG. 69B shows the effect of local CpG delivery on mouse survival following vaginal challenge with HSV-2. FIG. 70A shows that CpG 10104 is as good as CpG 7909 for increased humoral response to HBsAg in BALB / C mice in the absence of alum. FIG. 70B shows that CpG 10104 is as good as CpG 7909 for increased humoral response to HBsAg in BALB / C mice in the presence of alum. FIG. 71A shows that CpG 10104 is as good as CpG 7909 in promoting Th1 mediated immune responses to HBsAg in BALB / C mice (as measured by high IgG2a compared to IgG1 titer) in the absence of alum. FIG. 71B shows that CpG 10104 is as good as CpG 7909 in promoting Th1 mediated immune responses to HBsAg in BALB / C mice (as measured by high IgG2a compared to IgG1 titer) in the presence of alum.

【Sequence list】

Claims (98)

  A composition comprising an immunostimulatory nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 19, SEQ ID NO: 45, SEQ ID NO: 118 or SEQ ID NO: 141.   The composition according to claim 1, wherein said immunostimulatory nucleic acid molecule consists of the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 19, SEQ ID NO: 45, SEQ ID NO: 118 or SEQ ID NO: 141.   The composition of claim 1 further comprising an antigen.   The composition according to claim 3, wherein the antigen is selected from the group consisting of microbial antigens, cancer antigens, and allergens.   5. The composition according to claim 4, wherein the microbial antigen is selected from the group consisting of microbial antigens, viral antigens, fungal antigens and parasitic antigens.   The composition according to claim 3, wherein the antigen is encoded by a nucleic acid vector.   4. The composition of claim 3, wherein the nucleic acid vector is separated from the immunostimulatory nucleic acid.   The composition according to claim 3, wherein the antigen is a peptide antigen.   The composition according to claim 1, further comprising an adjuvant.   The composition according to claim 9, wherein the adjuvant is a mucosal adjuvant.   The composition of claim 1 further comprising a cytokine.   The composition according to claim 1, further comprising a therapeutic agent selected from the group consisting of anti-microbial agents, anti-cancer agents, and allergy / asthma medications.   The composition according to claim 12, wherein the antibacterial agent is selected from the group consisting of an anti-bacterial agent, an antiviral agent, an antifungal agent, and an antiparasitic agent.   13. The composition of claim 12, wherein the anti-cancer agent is selected from the group consisting of a chemotherapeutic agent, a cancer vaccine, and an immunotherapeutic agent.   The allergy / asthma drug includes PDE-4 inhibitors, bronchodilators / β-2 agonists, K + channel openers, VLA-4 antagonists, neurocin antagonists, TXA2 synthesis inhibitors, xantanine ( Xanthanine), arachidonic acid antagonist, 5 lipoxygenase inhibitors, thromboxin A2 receptor antagonists, thromboxane A2 antagonists, inhibitors of 5-lipox activation protein, and protease inhibitors The method according to claim 12, wherein Composition.   The composition according to claim 1, wherein the immunostimulatory nucleic acid has a nucleotide backbone comprising at least one backbone modification.   18. The composition of claim 17, wherein the backbone modification is a phosphorothioate modification.   18. The composition of claim 17, wherein the nucleotide backbone is a chimera.   18. The composition of claim 17, wherein the nucleotide backbone is fully modified.   The composition of claim 1 further comprising a pharmaceutically acceptable carrier.   The composition of claim 1, wherein the immunostimulatory nucleic acid does not comprise a methylated CpG dinucleotide.   The composition of claim 1, wherein the immunostimulatory nucleic acid comprises at least four CpG moieties.   The composition of claim 1, wherein the immunostimulatory nucleic acid is T-rich.   The composition of claim 1, wherein the immunostimulatory nucleic acid comprises a polyT sequence.   The composition of claim 1, wherein the immunostimulatory nucleic acid comprises a poly G sequence.   The composition of claim 1, wherein the immunostimulatory nucleic acid is formulated for oral administration.   The composition according to claim 1, wherein the immunostimulatory nucleic acid is formulated as a nutritional supplement.   29. The composition of claim 28, wherein the adjuvant is formulated as a capsule, a tablet, or a sublingual tablet.   The composition of claim 1, wherein the immunostimulatory nucleic acid is formulated for topical administration.   The composition of claim 1, wherein the immunostimulatory nucleic acid is formulated for parenteral administration.   The composition of claim 1, wherein the immunostimulatory nucleic acid is released in a sustained release device.   The composition of claim 1, wherein the immunostimulatory nucleic acid is formulated to be delivered to a mucosal surface.   The composition according to claim 1, wherein the mucosal surface is selected from the group consisting of the oral cavity, the nose, the rectum, the vagina, and the surface of the eye.   The composition of claim 1, wherein the immunostimulatory nucleic acid stimulates a mucosal immune response.   The composition of claim 1, wherein the immunostimulatory nucleic acid stimulates a systemic immune response.   The composition of claim 1, wherein the immunostimulatory nucleic acid is provided in an amount effective to stimulate a mucosal immune response.   The composition of claim 1, wherein the immunostimulatory nucleic acid is provided in an amount effective to stimulate a systemic immune response.   The composition of claim 1, wherein the immunostimulatory nucleic acid is provided in an amount effective to stimulate an innate immune response.   The composition according to claim 1, wherein the immunostimulatory nucleic acid is provided in an amount effective to treat or prevent an infectious disease.   The composition according to claim 1, wherein the immunostimulatory nucleic acid is provided in an amount effective to treat or prevent allergy.   The composition according to claim 1, wherein the immunostimulatory nucleic acid is provided in an amount effective to treat or prevent asthma.   The composition of claim 1, wherein the immunostimulatory nucleic acid is provided in an amount effective to treat or prevent cancer.   33. The composition of claim 32, wherein the sustained release device is a microparticle.   41. The composition of claim 40, wherein the infectious disease is herpes simplex virus infection.   A method of stimulating an immune response in a subject in need thereof to stimulate an immune response, said method comprising: SEQ ID NO: 1, SEQ ID NO: 19, SEQ ID NO: 1, in an amount effective to stimulate an immune response 45. A method comprising administering to a subject an immunostimulatory nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 118 or SEQ ID NO: 141.   47. The method of claim 46, wherein the subject has or is at risk of developing an infection.   48. The method of claim 47, wherein the infection is selected from the group consisting of bacterial infection, viral infection, fungal infection and parasitic infection.   Said virus infection is human immunodeficiency virus (HIV-1 and HIV-2), human T lymphotrophic virus type I (HTLV-I), human T lymphotrophic virus type II (HTLV-II), herpes simplex virus type I (HSV-1), Herpes simplex virus type II (HSV-2), human papilloma virus (multiplex type), hepatitis A virus, hepatitis B virus, hepatitis C virus and hepatitis D virus, EB virus (EBV), 49. The method of claim 48, wherein the method is selected from the group consisting of cytomegalovirus and infectious lichen tumor virus.   50. The method of claim 49, wherein the viral infection is herpes simplex virus infection.   47. The method of claim 46, wherein the subject has or is at risk of developing an allergy.   47. The method of claim 46, wherein the subject has or is at risk of developing asthma.   47. The method of claim 46, wherein the subject has or is at risk of developing cancer.   47. The method of claim 46, further comprising administering an antigen to the subject.   54. The method of claim 53, wherein the antigen is selected from the group consisting of microbial antigens, cancer antigens, autoantigens and allergens.   55. The method of claim 54, wherein the microbial antigen is selected from the group consisting of bacterial antigens, viral antigens, fungal antigens and parasitic antigens.   The antigen is, for example, herpesvirus, retrovirus, orthomyxo, Toxoplasma, Hemophilus, Campylobacter, Clostridium, E. coli. 56. The method of claim 55, wherein the method is derived from a microorganism selected from the group consisting of E. coli and staphylococci.   47. The method of claim 46, wherein the immune response is an antigen specific immune response.   54. The method of claim 53, wherein the antigen is encoded by a nucleic acid vector.   60. The method of claim 59, wherein the nucleic acid vector is separated from an immunostimulatory nucleic acid.   55. The method of claim 54, wherein the antigen is a peptide antigen.   47. The method of claim 46, further comprising administering an adjuvant to the subject.   63. The method of claim 62, wherein the adjuvant is a mucosal adjuvant.   47. The method of claim 46, further comprising administering a second therapeutic agent to the subject.   65. The method of claim 64, wherein the second therapeutic agent is an antimicrobial agent.   66. The method of claim 65, wherein the antibacterial agent is selected from the group consisting of antibacterial agents, antiviral agents, antifungal agents, and antiparasitic agents. 65. The method of claim 64, wherein the second therapeutic agent is an anti-cancer agent.   68. The method of claim 67, wherein the anti-cancer agent is selected from the group consisting of a chemotherapeutic agent, a cancer vaccine, and an immunotherapeutic agent. 65. The method of claim 64, wherein the second therapeutic agent is an allergy / asthma drug.   The allergy / asthma drugs include PDE-4 inhibitors, bronchodilators / β-2 agonists, K + channel openers, VLA-4 antagonists, neurocin antagonists, TXA2 synthesis inhibitors, zansanin, arachidonic acid antagonists, 5 lipoxygenase inhibitors, thrombosin A2 70. The method of claim 69, wherein the method is selected from the group consisting of receptor antagonists, thromboxane A2 antagonists, inhibitors of 5-lipox activation protein, and protease inhibitors.   47. The method of claim 46, wherein the immunostimulatory nucleic acid has a nucleotide backbone comprising at least one backbone modification.   72. The method of claim 71, wherein the backbone modification is a phosphorothioate modification.   72. The method of claim 71, wherein the nucleotide backbone is a chimera.   72. The method of claim 71, wherein the nucleotide backbone is fully modified.   47. The method of claim 46, wherein the immunostimulatory nucleic acid does not comprise methylated CpG dinucleotides.   47. The method of claim 46, wherein the immunostimulatory nucleic acid comprises a poly G sequence.   47. The method of claim 46, wherein the immunostimulatory nucleic acid is administered orally.   47. The method of claim 46, wherein the immunostimulatory nucleic acid is administered locally.   47. The method of claim 46, wherein the immunostimulatory nucleic acid is administered parenterally.   47. The method of claim 46, wherein the immunostimulatory nucleic acid is administered in a sustained release device.   47. The method of claim 46, wherein the immunostimulatory nucleic acid is administered to a mucosal surface.   47. The method of claim 46, wherein the immune response is a mucosal immune response.   47. The method of claim 46, wherein the immune response is a systemic immune response.   82. The method of claim 81, wherein the mucosal surface is selected from the group consisting of the oral cavity, the nose, the rectum, the vagina, and the surface of the eye. 47. The method of claim 46, wherein the method comprises:
Separating immune cells from the subject,
Contacting the immune cells with an amount of the immunostimulatory nucleic acid effective to activate the immune cells, and readministering the activated immune cells to the subject,
Method, including   86. The method of claim 85, wherein the immune cell is a white blood cell.   86. The method of claim 85, wherein the immune cell is a dendritic cell.   86. The method of claim 85, further comprising contacting the immune cell with the antigen.   47. The method of claim 46, wherein the subject is a human.   47. The method of claim 46, wherein the subject is selected from the group consisting of dogs, cats, horses, pigs, sheep, goats, birds, monkeys, and fish.   47. The method of claim 46, wherein the subject has or is at risk of developing an infectious disease, wherein the method is a method of treating or preventing the immunological disease. the method of.   47. The method of claim 46, wherein the subject has or is at risk of developing asthma, wherein the method is a method of treating or preventing the asthma in the subject.   47. The method of claim 46, wherein the subject has or is at risk of developing an allergy, wherein the method is a method of treating or preventing the allergy.   47. The method of claim 46, wherein the subject has or is at risk of developing a cancer, wherein the method is a method of treating or preventing the cancer.   95. The method according to claim 94, wherein said cancer is biliary cancer; osteosarcoma; cancer of brain and CNS; breast cancer; cervical cancer; choriocarcinoma; colon cancer; connective tissue cancer; endometrial cancer; Gastric cancer; Hodgkin's lymphoma; intraepithelial neoplasia; laryngeal cancer; lymphoma; liver cancer; lung cancer (eg, small and non-small cells); melanoma; neuroblastoma; oral cancer; Prostate cancer; rectal cancer; sarcoma; skin cancer; testicular cancer; thyroid cancer;   47. The method of claim 46, further comprising the step of administering an antibody specific for a cell surface antigen, wherein said immune response comprises antigen dependent cellular cytotoxicity (ADCC). How to cause)).   A method of preventing a disease in a subject comprising administering an immunostimulatory nucleic acid to the subject in a regular basis to prevent the disease in the subject. Wherein the immunostimulatory nucleic acid has the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 19, SEQ ID NO: 45, SEQ ID NO: 118 or SEQ ID NO: 141.   A method of inducing an innate immune response comprising administering to the subject an immunostimulatory nucleic acid in an amount effective to stimulate the innate immune response, wherein The method wherein the immunostimulatory nucleic acid has the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 19, SEQ ID NO: 45, SEQ ID NO: 118 or SEQ ID NO: 141. A method of identifying an immunostimulatory nucleic acid, said method comprising
Measuring the control level of activation of the immune cell population contacted with the immunostimulatory nucleic acid, wherein the immunostimulatory nucleic acid comprises SEQ ID NO: 1, SEQ ID NO: 19, SEQ ID NO: 45, SEQ ID NO: 118 or SEQ ID NO: A process comprising 141 nucleotide sequences
Measuring the test level of activation of the contacted immune cell population in contact with the test nucleic acid, and comparing the control level of activation to the test level of activation;
And wherein the test level above the control level is indicative of an immunostimulatory nucleic acid.

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