JP2003520568A - Advanced antigen presentation platform - Google Patents

Abstract

  (57) [Summary] The present invention provides particles for hapten presentation for the purpose of eliciting an immune response. The amino acid sequence of the monomers constituting the particles is derived from the core protein of duck hepatitis B virus. The particles can also deliver nucleic acids. The nucleic acid can be delivered to enhance an immune response or, for example, for gene therapy. In addition, the present invention relates to particles comprising a nucleocapsid protein monomer, wherein the primary sequence of the particle is from hepatitis B virus, where the monomer is not cross-reactive with antibodies to human HBcAg.

Description

Detailed Description of the Invention

BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to the development of vaccines, and in particular to improved methods for antigen presentation. The invention further relates to delivering nucleic acids to cells via particles.

BACKGROUND OF THE INVENTION The goal of all vaccinations is to induce specific immunity that prevents microbial invasion, eliminates microbes that have already invaded the host, or neutralizes microbial toxins. . Unfortunately, the development of effective protein subunit or peptide vaccines against intracellular pathogens has been largely hampered by technological and conceptual deficiencies.

[0003] One such drawback is the inability to elicit a strong TH1 immune response.
The immune system's response to antigens during vaccination is somewhat variable depending on the size and composition of the disease-specific antigens used to enhance the immune response, and especially the adjuvant. Potential responses include TH1 responses, TH2 responses or mixed (TH1 / TH2) responses.

Cytokine profiles determine T cell regulation and effector functions in the immune response. Cytokines play a role in directing T cell responses.
Helper (CD4 +) T cells regulate the immune response of mammals through the production of soluble factors that act on other immune system cells, including other T cells. Most mature C
D4 + T helper cells have two cytokine profiles (TH1 or TH
Express one of 2). TH1 cells contain IL-2, IL-3, IFN-γ, TN
Secretes F-β, GM-CSF and high levels of TNF-α. TH2 cells
IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13
, GM-CSF and low levels of TNF-α. The subset of TH1 is
Promotes delayed type hypersensitivity, cell-mediated immunity as well as conversion of immunoglobulin class to IgG2. TH2 subsets activate B cells, promote antibody production, and I
Induce humoral immunity by inducing class conversion to gG and IgE.

Several factors have been shown to influence the orientation of TH1 or TH2 profiles. The best characterized regulators are cytokines. IL-1
2 and IFN-γ are positive TH1 regulators and negative TH2 regulators.
IL-12 promotes the production of IFN-γ, and IFN-γ provides positive feedback for IL-12. IL-4 and IL-10 are TH2
It is likely necessary to establish a cytokine profile and downregulate TH1 cytokine production; the effects of IL-4, in some cases, IL-12.
Dominates the effect of. IL-13 has been shown to inhibit the expression of inflammatory cytokines (including IL-12 and TNF-α by LPS-induced monocytes) in a manner similar to IL-4. The IL-12 p40 homodimer binds to the IL-12 receptor and antagonizes the biological activity of IL-12; thus IL-
The 12 p40 homodimer blocks the proTH-1 effect of IL-12.

The TH1 response is characterized by the migration of macrophages, B cell antibody-producing cells, professional antigen presenting cells (APCs), and cytotoxic T lymphocytes (CTLs) for elimination by microbial phagocytes. To be Furthermore, TH1 response induces the expression of IFN-γ. IFN-γ promotes the conversion of B cells to isotypes such as IgG2, which fixes complement and promotes phagocytosis by macrophages. IFN-γ also activates the antibacterial function of macrophages. Thus, the TH1 immune response induces a phagocyte-dependent host response that is important for elimination of intracellular microbes. Therefore, a vaccine that elicits a strong TH1 response is highly desirable.

[0007] Several studies have reported that the cytokine IL-12 plays an important role in inducing antiviral effects in vivo by promoting a TH1-type immune response. IL-12 is produced primarily by activated antigen presenting cells (APCs), including macrophages, dendritic cells, and B cells, and is reported to increase antibody, CD4 ⁺ , and CTL responses. . IL-12 induces the maturation of type 1 TH cells into IFN-γ producing cells, which is a natural killer (N
K) Promotes activity and enhances CTL maturation. It is a heterodynamic consisting of two subunits, p35 and p40.
ic) It is a cytokine. The p35 subunit is constitutively (constit
units, whereas the p40 subunit is expressed only upon APC activation. Therefore, a vaccine that eliminates the production of IL-12 is highly desirable.

The protein subunit or peptide vaccines themselves are often not immunologically active and must be administered with an adjuvant. Aluminum hydroxide (Alum) is currently the only adjuvant approved for human use. A major drawback of alum is TH2 rather than TH1 type immune response.
Inducing an immune response, which can prevent the induction of CTLs. Indeed, in mice immunized with recombinant hepatitis B surface antigen (HBsAg), the addition of alum selectively blocks CD8 + CTL activation (S
chirmbeck et al., 1994). Although not essential for protective immunity to HBV, CTL may nevertheless play an important role.

The use of alum has been associated with TH2-type disorders. Very high asthma (TH2 type disease) prevalence in developed countries may be associated with high hygiene levels and rapid treatment of infectious diseases in early childhood (Cookson and Moffatt, 1997).
. Early exposure to bacterial DNA pushes the immune system away from the TH2-type response towards a TH1-type response, which in developing countries where there is a very high frequency of upper respiratory tract infections in infancy. It may explain the low incidence of asthma. It is advantageous to have pediatric vaccines available that can re-establish a TH1-type response, thereby reducing the incidence of asthma.

Potent TH1 due to antigen accumulated in extracellular fluid (for most vaccines)
Very high concentrations of antigen are required to elicit a response. This cannot be achieved in a safe and effective manner. However, one solution to this problem is to deliver the disease-specific antigen on the surface of the particles. Both macrophages and dendritic cells internalize particles within large vacuoles. Here, the foreign antigen is class I.
It can be transferred to both the presentation route and the class II presentation route. This exogenous class I presentation pathway is an important goal for vaccine development. Because it provides a means of inducing CTL immunity with antigens that accumulate in extracellular fluid.
It has been demonstrated that when foreign antigens are naturally particles, they are presented 1,000-10,000 fold more efficiently than soluble antigens in both class I and class II pathways (Harris et al. , 1992; Griffiths et al., 1993; Schodel et al., 1994; Schirmback et al., 1995.
And Raychaudhuri and Rock, 1998). One example of the use of particles that transport antigens is the example of human hepatitis B virus core antigen (HBcAg). It is possible that human HBcAg may serve as an effective carrier for foreign epitopes, either chemically bound to the protein sequence or engineered into the protein sequence at some selected sites. Shown (Millich,
1990; Schodel et al., 1992; Schodel et al., 1993; Sch.
odel et al., 1994; Milich et al., 1995). Also, for example, the following T
US Pat. Nos. 4,882,145 to Hornton et al .; 4,882,14.
5, and 5,143,726, which are incorporated herein by reference. These patents relate to the use of fusion proteins composed of the T cell stimulatory region of the human hepatitis B virus (HBV) nucleocapsid protein linked to a polypeptide immunogen. However, one important drawback to the use of human HBcAg as a vaccine carrier is that antibodies against HBcAg itself (anti-HBc) also develop in recipients of the vaccine.
This is HBV in humans and blood (donated for use in transfusions)
The detection of infection is problematic because it is by screening for anti-HBc in the blood. Thus, the widespread use of human HBcAg-based vaccines creates deficiencies in the hepatitis B screening systems currently used. The use of Hepatitis B-based particles from some other species (eg Woodchuck) also poses the same problem. Because they are cross-reactive with human HBcAg. It is estimated that about 500 million people worldwide are infected with HBV. In these individuals, administration of a human HBcAg-based vaccine has no effect. Because their immune system probably attacks and destroys the vaccine particles before they exert their immune effects. Therefore, it would be advantageous to have a particulate vaccine carrier available that does not interfere with current HBV screening procedures.

Another major drawback of current vaccines is the "quas" of invading bacteria.
is unable to produce a vehicle to deliver a hapten that is effective in stimulating a particular immune response against "ispiesies)". Similar species are the first microbial offspring to develop during infection. The genetic characteristics of similar species are altered by mutations, allowing these structurally similar microorganisms to escape clearance by the immune system and maintain a pathological condition. Therefore, it would also be highly desirable to develop hapten vehicles for use in vaccines. This hapten vehicle was able to elicit a protective response against a wide variety of structurally similar haptens, such as those exhibited by genetically hypervariable microbes (eg, viruses). Such hapten vehicles can stimulate an immune response against the target microorganism and similar species produced during infection.

SUMMARY OF THE INVENTION The present invention provides a composition composed of a plurality of nucleocapsid protein monomers, the primary sequence of the nucleocapsid protein monomers being derived from duck hepatitis B virus. Here, the plurality of monomers aggregate to form particles. The monomer may further include a first hapten and a second hapten, as well as multiple haptens, depending on the use of the particles. The particle may further include nucleic acid. These nucleic acids can have, but are not limited to, sequences corresponding to SEQ ID NOs: 3-19.

The hapten may be associated with a disease state caused by a factor selected from the group consisting of: single stranded DNA virus, double stranded DNA virus, single stranded RNA virus, double stranded RNA virus, Intracellular parasites, fungi, bacteria, and cancer.

The present invention further provides a nucleic acid to a subject in need thereof by administering a composition composed of a plurality of nucleocapsid protein monomers assembled in the form of particles, the particles containing the desired nucleic acid sequences. Methods of delivering

The present invention also provides a method for making particles, which method comprises the following steps: a plurality of nucleocapsid protein monomers, the primary sequence of which is derived from duck hepatitis B virus. Providing a composition (the plurality of monomers assembles to form particles), exposing the particles to a charged agent to degrade the particles, and nucleic acids or other nucleoside capsid monomers containing different haptens. To remove the charged factor (removal of the charged factor allows the monomers to reassemble). Further steps such as removing unwanted nucleic acids and adding desired nucleic acids can be included in the method. The charged factor can be a divalent cation selected from the group consisting of: Mg ⁺² , Zn ⁺² , Ba ⁺² , Sr ⁺² , Ca ⁺² and Pb ⁺² .

Detailed Description of the Invention Definitions: Antibody: A molecule that is a member of a family of glycosylated proteins called immunoglobulins (eg, IgG, IgE, etc.). This molecule can specifically bind to the antigen.

Antigen: A term historically used to describe an entity bound by an antibody, and also to indicate an entity that induces the production of antibodies. In more recent usage, the meaning of antigen is limited to the entity bound by the antibody. On the other hand, the term "immunogen" is used with respect to the entity that induces antibody production. When the entity discussed herein is both immunogenic and antigenic, reference to this entity as either an immunogen or an antigen is typically done according to its intended use. Be seen.

Antigenic determinant: The actual structural part of an antigen that is immunologically bound by an antibody that binds the antigen binding site or T cell receptor. The term is interchangeable with "epitope".

Cytokine: A protein hormone that promotes cell proliferation and differentiation. Cytokines are secreted by immune cells (T cells, B cells, macrophages, dendritic cells, etc.) in response to antigenic stimulation.

GM-CSF: Granulocyte monocyte colony-stimulating factor is a cytokine that generally acts on the bone marrow to increase the production of inflammatory leukocytes. It is also a macrophage activating factor and promotes the differentiation of Langerhans cells into dendritic cells.

Hapten: A disease-specific antigenic determinant identified by biochemical, genetic, or computational means.

HBcAg: A T cell stimulating protein or polypeptide having an amino acid residue sequence corresponding to the amino acid residue sequence encoded by the hepatitis B virus nucleocapsid protein gene.

IFN-γ: Interferon-γ is produced by activated CD4 + and CD8 + T cells, and NK cells. The production of IFN-γ is a direct result of antigen activation and is enhanced by the presence of the cytokines IL-2 and IL-12.

Monocytic cells: Monocytic cells are released from the bone marrow as incompletely differentiated cells and are referred to as "monocytes."

Nucleic acid (or oligonucleotide): A polymeric form of nucleotides of at least 5 bases in length. Nucleotides include deoxyribonucleotides, ribonucleotides,
Or any modified form thereof. They can be double-stranded or single-stranded.

Polypeptide or peptide: A linear chain of amino acids linked together by peptide bonds between the α-amino and carboxy groups of adjacent amino acids.
They may or may not include their neutral (uncharged) or salt forms and modifications such as glycosylation, side chain oxidation, phosphorylation and the like,
It can be of various lengths. Also included are proteins modified by additional substituents (eg, glycosyl units, lipids, or inorganic ions (eg, phosphates) and chemical transformations or chain-related modifications (eg, sulfhydryl group oxidation)). .

[0027]   Protein: A polypeptide having about 70 or more amino acids.

TH (also in TH1 and TH2): T helper cells. Unsensitized CD4 + cells can differentiate into a subset of THI and TH2. This differentiation is influenced by a cytokine environment that is prematurely produced in response to the microorganisms that elicit an immune response. The principle effector function of TH1 cells is phagocyte-mediated defense against infection. TH2 cells are primarily involved in antibody development and allergic immune responses.

The present invention presents haptens for inducing an immune response, for delivering nucleic acids for use in enhancing an immune response, for gene therapy, and for other uses. To provide a new type of vehicle for use in. The vehicle is a particle, and the structure of the particle is the duck hepatitis B virus core antigen (
Duck HBcAg) based in part. Applicants have shown that particles formed by assembling a recombinant form of duck HBcAg are highly immunogenic,
It has been found to elicit an H1-type immune response and that the particles can be disassembled and reassembled under relatively mild, non-denaturing conditions. This latter finding, combined with known recombinant DNA technology, opens up the prospect of producing particles carrying a wide variety of different haptens on a single particle. In addition, the reversible disassembly process may be immunostimulatory (to further enhance the immune response) or may be useful for some other purpose (eg, for gene therapy). Means are provided for producing particles comprising.

Native duck HBcAg particles are 32-34 nm particles composed of 240 identical subunit monomers and very similar to the structure of human HBcAg. However, Applicants have found that duck HbcAg does not cross-react with woodchuck or human HBcAg antibodies. This is an important finding for two reasons: First, the use of duck HBcAg particles as a hapten carrier does not produce human HBAg antibodies, as anti-human HbcAg antibodies are not produced.
Does not interfere with current protocols for detecting cAg. Second, anti-human HBcA
Individuals who already have g. antibody can still be immunized with duck HBcAg. Therefore,
This finding represents a significant advance in the development of particulate vaccine vehicles.

In a preferred embodiment of the invention, the major amino acid sequences of the monomers making up the particles of the invention, and the nucleic acid sequences encoding them, are derived from the duck HBcAg.
Be induced. The duck HBcAg monomer consists of 262 amino acids (SEQ ID NO: 1 shown in Figure 1), the assembly domain consists of residues 1-200,
The arginine-rich nucleic acid then binds to the domain consisting of residues 201-262. By "derived from" the invention is that the sequence of the recombinant monomer is identical both to the sequence of the native duck HbcAg and also to the amino acid and / or nucleic acid sequence of the duck HBcAg. It is meant to include particles that are also composed of recombinant monomers that have variability. For example, alternative cloning sites can be engineered into the nucleic acid sequence for inserting sequences into the vector, inserting haptens, and making amino acid substitutions, and so on. Any of the genetic engineering possibilities known to those skilled in the art can be applied, so long as the resulting monomers still retain the ability to function in the practice of the invention. In particular, duck B shown in FIG.
Hepatitis core protein can be assembled into particles as shown in FIG. The sequences of proteins and nucleic acids corresponding to duck HBcAg are deleted, inserted, if necessary, in order to carry out or optimize the practice of the present invention.
Alternatively, it may be modified by substitution, and all such modifications are intended to be within the scope of the invention. A modified protein derived from a duck hepatitis B core protein may comprise one or more haptens (or the shortened versions shown in Figures 17 and 19 or other shortened versions) inserted in SEQ ID NO: 1, It then has the ability to be assembled into the particles shown in FIG. Methods for making such genetic modifications are well known to those of skill in the art.

In a preferred embodiment of the invention, the monomer sequence is derived from a recombinant form of duck HBcAg known as BC-201D, the amino acid sequence of which is shown in FIG.
It is shown in (SEQ ID NO: 1). The nucleic acid sequence encoding BC-201D is shown in Figure 2 (SEQ ID NO: 2). The correspondence between the one-letter codes and amino acids or nucleobases is shown in Table 1.

[0033]

[Table 1] Recombinant duck HBcAg (eg, when made in Escherichia coli) spontaneously self-assembles into polymeric core particles. With respect to obtaining particles for use in carrying out the present invention, means for producing suitable amounts of particles and for producing them are well known to those of skill in the art. For example, Itskura U.S. Pat. No. 4,356,270 and Murray 4,563,423.
No., which are incorporated herein by reference.

In a preferred embodiment of the invention, the particles of the invention are E. E. coli recombinant system. However, the particles can be produced by the expression of monomers in a variety of other recombinant expression systems. For example, yeast system, insect cell system (for example, using baculovirus expression vector), plant cell system (for example, tobacco, potato, corn, etc.), transgenic animal system or mammalian cell culture system. Any suitable expression system that accurately produces the particles of the invention can be used in the particles of the invention. Such systems and their use for the production of recombinant proteins are well known to those skilled in the art.

In a preferred embodiment of the invention, the particles comprise one or more foreign (ie
It is composed of monomers that are commonly designed to include non-duck) haptens. This hapten is transported by the particles to the host during vaccination and is thus presented to the host's immune system. Examples of such foreign non-duck haptens include haptens associated with double-stranded DNA or RNA viruses, single-stranded DNA or RNA viruses, intracellular parasites, fungi, bacteria and cancer, It is not limited to these. Particularly important exemplary immunogens are derived from: Bacteria (eg, A. pertussis, S. parathyphoid A and S. parathyphoid B, C. diphtheriae, C. te.
tanus, C.I. botulinum, C.I. perifringens, A .; a
nthracis, A .; pestis, V.I. cholera, N .; gonorr
hea, H .; influenzae, T.W. palladium), virus (poliovirus, adenovirus, parainfluenza virus, measles, mumps, RS virus, influenza virus, equine encephalomyelitis, swine fever virus, Newcastle disease virus, poultry pox virus, rabies virus, feline distemper virus And canine distemper virus, foot-and-mouth disease virus, human and simian immunodeficiency virus, etc .; Rickettsia immunogens such as typhus and typhus and spot fever. Any hapten that can be inserted into the monomer subunits of the particles of the invention and in which it is desirable to elicit an immune response can be utilized in carrying out the invention. The sequence of duck HBcAg is known, and many suitable hapten sequences are recombinant DNA required for hapten insertion.
It is known to a person skilled in the art as a technique.

Several methods for confirming that the expressed particles contain the desired hapten are well known to those of skill in the art. For example, the sequence of the monomers can be determined by any of several well known methods of amino acid sequencing.

In a preferred embodiment of the invention, a single copy of one hapten, several copies of one hapten or several copies of different haptens is a single region or number of recombinant duck HBcAg monomers. Can be inserted into the area. Thus, the particles of the present invention are highly versatile vehicles for the presentation of haptens and offer broad flexibility in the design of immunogenic particles.

In a preferred embodiment of the invention, the region into which the hapten can be inserted is a region on the particle assembly that induces an immune response in the hapten. In a preferred embodiment of the invention, the hapten-containing amino acids are inserted in such a way that they replace an equal number of amino acids of the recombinant monomer. In other embodiments, fewer, more, or no recombinant monomeric amino acids are
It can be replaced by a hapten. In general, haptens of about 10-30 amino acids in length can be effectively inserted into recombinant monomers. However, longer or shorter haptens can also be inserted. The inserted hapten (or hapten when more than one copy is inserted) is the ultimate due to the amino-terminal and carboxy-terminal flanking sequences, which correspond to sequences for or derived from duck HBcAg protein. Can be adjacent to each other. Further, more than one copy of the hapten can be inserted at a single site or different sites on the same monomer, or different haptens can be inserted in tandem at a single site, or different haptens can be the same. It can be inserted at different sites of the monomer. Haptens of any length and combination can be inserted as long as the monomers produced can be assembled into particles that elicit an immune response.

The choice of region into which the hapten can be inserted depends on the relevant hepatitis core antigen molecule (eg
This can be done by comparison with the known primary sequence and the three-dimensional structure of human HBcAg). See, for example, the detailed discussion of human hepatitis virus core protein structure in Bringas (1997) and Wyne et al. (1999). One of skill in the art will appreciate that several methods can be used to find relevant proteins (eg, human HBcAg and duck HBcA).
It is recognized that it exists for the comparative alignment of the functional domains of g).

In another preferred embodiment of the invention, the particles of the invention are composed of monomers without non-duck haptens. These particles may or may not contain nucleic acids. For example, the shortened form of duck HBcAg (t-duck HB
cAg), from which the nucleic acid binding domain has been deleted, can be used by itself as a general immunostimulatory agent, such as an adjuvant. further,
Particles composed of non-duck hapten-free monomers may contain nucleic acids, either random nucleic acids from a recombinant host or defined nucleic acids added during reassembly of the particles, and It can serve as a delivery mechanism for nucleic acids. The defined nucleic acid is used for gene therapy or gene medicine (eg, antisense RN).
A, for delivery of DNA sequences (which encode therapeutic agents, etc.),
It can be delivered by this particle.

A “stimulation index” can be measured to determine whether a particle stimulates an immune response. This stimulus index is a measure of the ability of the particle to mount an immune response, and measures immune parameters (eg, antibody forming capacity, number of lymphocyte subpopulations, mixed leukocyte response, lymphocyte proliferation, etc.). Thus, it can be tested in various immune cell assays. Stimulation of the immune response can be measured in assays that determine resistance to infection or tumor growth. Methods of measuring stimulation indices are well known to those of skill in the art. For example, one assay is the incorporation of ³ H uridine in murine B cell cultures. The culture was contacted with 10 μg of particles at 37 ° C. for 20 hours, then pulsed with ³ H uridine, 4 hours later, cells were harvested and RNA incorporated ³ H uridine was measured. To do. Induction of secretion of specific cytokines can also be used to assess stimulation indicators. Although not meant to be bound by theory, for in vivo use (eg, to treat a subject at risk of exposure to a pathogenic agent), the particles are It is important to be able to effectively induce cytokine secretion by the activity of lymphocyte and / or natural killer (NK) cell lysis. In one method, the stimulation index of the particles on B cell proliferation is at least about 5, preferably at least about 10, more preferably about 15, and most preferably at least about 2.
On the other hand, although it is 0, it is recognized that there is a difference in stimulation index between individuals.

Due to the high level of variability, the particles of the invention can be of several types. First, they can be either "non-mosaic" or "mosaic" in nature. By "non-mosaic" is meant a particle containing a single type of hapten. The monomers that make up the non-mosaiced particles can have a single copy of the hapten or two or more copies of the same hapten. This hapten (s)
Can be inserted in a single region of the monomer or in more than one region.

Mosaic particles are particles that contain two or more different haptens. Mosaic particles can be either "endogenous" or "exogenous." Endogenous mosaic particles are particles composed of monomers in which two or more different haptens are present on the same monomer. Endogenous mosaic particles can be produced by genetically engineering two or more different haptens into the region (s) of a single monomer subunit.

Exogenous mosaic particles are particles that contain two or more different haptens, in which the different haptens are on different monomers, and the particles are formed from a mixture of such monomers. It One means for producing such exogenous mosaic particles is to produce two or more different non-mosaic particles (or two or more different mosaic and non-mosaic particles) which are converted into monomers. Disassembling, mixing the monomers, and reassembling them into particles. Each reassembled particle is composed of a mixture of each type of monomer and thus represents a mixture of haptens.

The development of the exogenous mosaic particles of the present invention was made possible by Applicants' discovery that recombinant duck HBcAg particles can be reversibly degraded under mild, non-denaturing conditions. This finding allows the relatively easy production of a wide variety of exogenous mosaic particles containing 1 to several hundred different haptens per particle for presentation to the immune system. In this way, the particles of the invention can be used in a single immunity to immunize against subspecies polycythemia. This is a significant improvement over the previous hapten transport.

The reason that the recombinant duck HBcAg particles can be degraded under mild conditions is that this monomer has only a single cysteine residue and is present in the free sulfhydryl form. In contrast, the human HBcAg monomers are contacted with each other in the particle by multiple disulfide bonds, requiring stringent conditions to degrade the particle.

The ability of the particles of the invention to decompose and assemble provides a method for carrying out another aspect of the invention. The recombinant duck HBCAg monomer has a nucleic acid binding domain (arginine-rich amino acid residues 201-262) as a result of which:
It has an endogenous ability to bind to a nucleic acid molecule. The native function of the nucleic acid binding domain allows the hepatitis B virus particles to bind and "package" their own genetic material, thus, during infection, from cell to cell and from host to host. Is to be transferred to. The particles of the invention (eg, when isolated from an E. coli recombinant expression system) have within them a short, heterogeneous population of random nucleic acids from a bacterial host. The particles of the invention can be degraded and the monomer is
It can be separated from the host nucleic acid and then the monomer can be reassembled in the presence of a select different nucleic acid molecule. The nucleic acid molecule of choice is bound by the monomer and "packaged" into the reassembled particles.

The particles of the invention can be assembled to accommodate any nucleic acid of choice. Examples of such nucleic acids include, but are not limited to, single-stranded DNA and / or RNA and / or double-stranded DNA and / or RNA (or DNA / RNA hybrid). This nucleic acid may be transcribed or translated into a therapeutic entity, for example, or to bind to any other nucleic acid already present in the body to enhance or prevent transcription or translation of those nucleic acids. It can be delivered for therapeutic purposes. Nucleic acids for use in the present invention can be de novo synthesized using any of a number of procedures well known in the art. For example, β-
Cyanoethyl phosphoramidite method (Beuage and Caruther
S., 1981); Nucleotide H-phosphonate method (Gareggg et al., 198).
6; Froelder et al., 1986; Garegg et al., 1986; and Gaf.
fney et al., 1988). These chemicals can be carried out by various commercially available oligonucleotide automated synthesizers. Alternatively, oligonucleotides may be prepared from the presence of nucleic acid sequences (eg genomic DNA or cDNA) using known techniques, such as those using restriction enzymes, exonucleases or endonucleases.

For in vivo use, the nucleic acid is preferably resistant to degradation (eg, via endonucleases and exonucleases). Secondary structures, such as stem loops, can stabilize nucleic acids against degradation. Alternatively, nucleic acid stabilization may be achieved through modification of the phosphate ester backbone. Preferred stabilized nucleic acids have a backbone that is at least partially modified with phosphorothionate. Phosphorothioates can be synthesized using automated techniques using either phosphoramidite or H-phosphonate chemistries. Aryl phosphonates and alkyl phosphonates can be made (eg, described in US Pat. No. 4,469,863); and alkyl phosphotriesters (
The charged oxygen sites therein are alkylated as described in US Pat. No. 5,023,243 and EP 092,574) by automated solid phase synthesis using commercially available reagents. You can Methods for making other DNA backbone variants and substitutions have been described (Uhlmann and Pey.
man, 1990; Goodchild, 1990).

Nucleic acids may include the use of phosphothioate or phosphodithioate end linkages, or methylphosphothioate end linkages rather than phosphodiesterase linkages within the intramolecular backbone (Kreig et al., 1996; Bogg et al., 1997). . Modification of the phosphate backbone can occur at the 5'end of nucleic acids (eg, the 5'ends of the first two nucleotides of a nucleic acid). This modification of the phosphate backbone can occur at the 3'end of the nucleic acid (eg, the 3'end of the first two nucleotides of the nucleic acid). Non-classical bases such as inosine and queosin, as well as acetyl, thio, and similarly modified forms of adenine, cytidine, guanine, thymidine, and uridine may also be included,
These cannot be easily recognized by endogenous endonucleases. Other stabilized nucleic acid molecules include nonionic DNA analogs such as alkylphosphonates and arylphosphonates, where the charged oxygen sites are alkylated. Also included are nucleic acid molecules that include a diol, such as tetrahylene glycol or hexaethylene glycol, at either or both termini. The term "oligonucleotide"
Includes both single-stranded and double-stranded DNA forms.

In a preferred embodiment of the invention, the particles of the invention are reassembled to include immunostimulatory nucleic acids. Such immunostimulatory nucleic acids have recently been described (Ca
rson and Raz, 1997; Davis et al., 1998; Kreig et al., 1
995; Lipford et al., 1997a; Lipford et al., 1997b; Pi
setsky, 1996; Superwasser et al., 1997; Spawas.
Ser et al., 1998; Stagey et al., 1996; Sun et al., 1998; and Zimmermann et al., 1998). DNA was first described as an immunostimulant when bacterial DNA was observed to cause regression of implantable tumors in vivo without direct tumor cytotoxicity. This tumor resistance induced by DNA resulted from proliferated natural killer (NK) cell activity. NK
Although activity can be stimulated by DNA from various bacterial sources, mammalian DN
It cannot be stimulated by A. This indicates that base modifications and / or nucleotide sequences, but not the DNA backbone, are critical mediators of NK stimulation. Studies evaluating the immunostimulatory activity of randomly selected oligonucleotides show that a defined motif (RACGTY, where R = A or G, and Y = C or T) causes potent induction of NK cells. Was shown. For mammalian DNA, this sequence is under-represented, and this sequence is
If found in A, it is masked by the modification. Experiments have determined that oligonucleotides with these modified sequences have neither NK activity nor other immunostimulatory activity. The mechanism of immune stimulation by these nucleic acids is still unknown. Cytokines IL-12, TNF-α, IL in antigen presenting cells (APC)
It is known that the production of -6 and IL-1 is stimulated. Initial studies indicate that individual levels of these cytokines can be regulated by alterations in the sequences surrounding defined immunostimulatory motifs.

The use of immunostimulatory nucleic acids in vaccines has been previously described. For example, H
Utcherson et al., US Pat. No. 5,723,335 and PCT International Application Publication WO 98/40100, both of which are incorporated herein by reference, are immunopotentiated to enhance the immune response to a vaccine. The use of nucleic acids is described. However, this delivery mechanism is simply injection of the nucleic acid with other vaccine components, a method that may not deliver sufficient amounts of the nucleic acid in an effective manner.

Bacterial DNA is a bulk phase of endocytosis or protocytosis (p
It appears to invade immune cells by rotocytosis), where they are then processed by antigen presenting cells. Antigen presenting cells appear to process the particles of the invention by either bulk phase endocytosis or receptor-mediated endocytosis. By including the immunostimulatory nucleic acid as an endogenous part of the particle, the immunostimulatory nucleic acid is transported to the host in concentrated form and processed directly by the antigen presenting cells, maximizing the effect of the immunostimulatory nucleic acid as an adjuvant. .

In a preferred embodiment of the invention, the immunostimulatory nucleic acid is a base sequence 5′-RACGTY-3 ′ (surrounded by up to 20 nucleotides on either side.
It is characterized by an oligonucleotide having SEQ ID NO: 20). Preferably, the immunostimulatory oligonucleotide ranges in base size from about 8-30. For use in the present invention, the nucleic acid may be composed of various modified bases as listed above, and, as listed above, using any of a number of procedures well known in the art, It can be newly synthesized. For example, "Immune st
imitation by phosphorothioate oligonucleotide
International patent application WO 95/26 entitled "Ucleotide analogs"
204 reports the non-sequence specific immunostimulatory effect of phosphorothioate modified oligonucleotides. Alternatively, CpG dinucleotides can be produced on a large scale in plasmids (Sambrook et al., 1989), which dinucleotides are degraded to oligonucleotides after administration to a subject. Oligonucleotides can be prepared by using known techniques (eg, techniques using restriction enzymes, exonucleases or endonucleases) to existing nucleic acid sequences (eg, genome D).
NA or cDNA).

The immunostimulatory nucleic acid may be a cytokine producing (eg, IL-6, IL-12, IFN
-Γ, TNF-α and GM-CSF). As an example array,
These include, but are not limited to:

[0056]

[Chemical 1] Immunostimulatory nucleic acids can stimulate natural killer cell (NK) lytic activity in a subject. Specific, but non-limiting examples of such sequences include:

[0057]

[Chemical 2] Immunostimulatory nucleic acids can stimulate B cell proliferation. Specific, but non-limiting examples of such sequences include:

[0058]

[Chemical 3] Any suitable immunostimulatory nucleic acid can be used in the practice of the invention.

The present invention provides a method for the degradation and reassembly of the particles of the present invention under relatively mild physiological conditions. Generally, this method comprises the steps of 1) providing a particle composed of a plurality of nucleocapsid protein monomers, a primary amino acid sequence derived from duck hepatitis B virus, wherein the plurality of monomers are Assembling and forming particles; 2) exposing the particles to a charged agent; and 3) removing the charged agent. Upon removal of the charged agent, this monomer spontaneously reassembles into particles. A preferred embodiment of the method is as follows: The particles can be isolated from the recombinant expression system by a simple 4-step method. For example, cells are pelleted by centrifugation and lysed by French press. Ammonium sulfate is added up to 277 g per liter. The precipitated material is collected by centrifugation, resuspended in phosphate buffer and dialyzed overnight against phosphate buffer. The resulting dialysate was applied to a hydroxyapatite column and washed with phosphate buffer until the optical density at 280 nm was less than 0.1, and 277 g ammonium sulfate per liter was added to the pooled fractions. , And the precipitate is collected by centrifugation. The resulting pellet is resuspended in minimal buffer and the Sepha
Apply to a rose-CL4B size exclusion chromatography column. Peak fractions representing this particle are determined by SDS-PAGE and pooled. The particles were treated with a neutral buffer (eg, 20 mM Tris HCl, pH 7.0) and a high concentration (0.1 to 1 M) of a divalent cation (eg, Mg ⁺² , Zn ⁺² , Ba ⁺² , Resuspend in Ca ⁺² , Pb ⁺² and Sr ⁺² ). The particles were dissociated into monomers (with gentle mixing) in about 1 hour and the progress of dissociation was determined by Fast P
Rotate Liquid Chromatography (FPLC) Se
It can be monitored by phase-CL4B size exclusion column chromatography or native agarose gel electrophoresis. Ribonuclease A may be added to remove nucleic acids (eg, recombinant host nucleic acids) that were originally contained within the particle when isolated. Reassembly is accomplished in the following manner: a truncated form of duck HBcAg lacking the nucleic acid binding domain (t-duck H
For BcAg), reassembly and particle formation was 100-200 mM N
Occurs on simple dialysis against the appropriate buffer plus aCl. Duck HBc
The non-truncated form of Ag reassembles only in the presence of nucleic acid. Thus, this monomer can be mixed with selected nucleic acids and dialyzed against a suitable buffer to form dialysis reassembly particles containing the nucleic acids. In a preferred embodiment of the invention, the molar ratio of nucleic acid to monomer is about 1: 1. Exogenous DNA that has not been incorporated into the reassembled particles can be removed by size exclusion column chromatography. Those skilled in the art will appreciate that in practicing the methods of the invention, many insignificant modifications can be made without significantly altering the results of the methods. For example, a procedure can be "scaled up" or "scaled down" for production purposes. In addition, some modifications (eg, modifications in ionic strength, pH, exact ratio of nucleic acid to monomer, etc.) can be beneficial to optimize the procedure for a particular monomer type. All such modifications and optimizations are included within the scope of the inventive method.

The dissociation / reassembly process is illustrated in FIG. In this figure, the particles 10 are
For example, the duck HBcAg monomer 11 containing a pertussis-specific hapten, and the particles 20 are, for example, a duck HBc containing a polio-specific hapten.
It is composed of Ag monomer 21. Particles 10 and 20 are exposed to a charged agent (eg, a divalent cation) and exposed particles 10 and 20 are dissociated into monomers 11 and 21. Unwanted nucleic acids contained inside the particles can be removed, for example, by the addition of nucleases. After removal of the nuclease, the specific nucleic acid 30
It can be added to the mixture of monomers 11 and 21. When removing charged agents,
Monomers 11 and 21 reassemble to form exogenous mosaic particles 40, which contain nucleic acids 30.

In another aspect of the invention, the exogenous non-duck hapten can be attached to particles rather than genetically engineered to attach to monomer sequences. Individual polypeptide haptens (eg, polypeptides, carbohydrates, etc.) operatively linked to the particle through amino acid residue side chains and immunogenic conjugates (ie, branched chain polypeptides and / or polypeptides / carbohydrates) Polymers). Such particles are considered to be "derived" from the duck HBcAg as described above and are intended for use within the practice of the present invention.

The present invention provides particles for use in eliciting an immune response in a host. In a preferred embodiment of the invention, the particles have a classical meaning (ie, to prophylactically evoke the host's immune system prior to exposure to the disease causing entity).
Can be used as a vaccine in. In yet another preferred embodiment of the invention, the particles of the invention are therapeutically (ie to boost a subject's immune response against a pathogen that has already infected the subject), eg in the case of HIV infection. Can be used.

The present invention also provides a composition for use in eliciting an immune response comprising the particles of the invention. The preparation of such compositions for use as a vaccine is well known to those of skill in the art. Typically, such compositions are prepared either as liquid solutions or suspensions, but solid forms such as tablets, pills, powders and the like are also contemplated. Solid forms suitable for solution in, or suspension in, liquid prior to administration can also be prepared. The preparation can also be emulsified. The active ingredient may be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients include, for example, water, saline, dextrose, glycerol,
Examples include ethanol and the like, or a combination thereof. Further, this composition
Small amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like can be included. In addition, the composition may include other adjuvants. If administration of the composition in oral form is desired, various thickeners, seasonings, diluents, emulsifiers, dispersing aids or binders and the like may be added. The compositions of the present invention may include any such additional ingredients in order to provide the composition in a form suitable for administration. The final amount of active ingredient in the formulation may vary. However, in general, the amount of active ingredient (particles) in the formulation is 1-99%.

The invention also provides a method for eliciting an immune response in a subject, the method comprising administering to the subject an effective amount of a composition comprising the particles of the invention. . An effective amount refers to whether the subject prevents, treats, or treats a condition in the subject.
Or, the amount of the composition of the present invention required to at least partially prevent. The subject can be any vertebrate, such as any mammalian species (eg, human) or avian species. Precise amounts of the compound to be administered will vary from subject to subject and may depend on, for example, weight, sex, age, general physical condition and the like.
Such variability is well understood and best evaluated by those of skill in the art.

The compositions of the present invention may be administered in any of a variety of ways well known to those of skill in the art and include, but are not limited to, parenteral (eg, subcutaneous or intradermal injection or intramuscular injection). By oral injection), oral, optamically (opth
Almally), vagina, rectum, intranasal, percutaneous, etc. Any suitable method of administration can be used in the practice of the invention so long as the composition is delivered in an effective manner.

The following examples are provided to illustrate some aspects of the present invention and should not be construed in any way to limit the application of the present invention.

(Example) (Material) (Duck HBcAg particles) The duck HBcAg particles were mixed with E. E. coli recombinant expression system. Duck HBc
Ag particles have the amino acid sequence shown in Figure 1 (SEQ ID NO: 1) and may include a heterogeneous population of host-derived nucleic acids. The duck HBcAg (1-239) particles have the amino acid sequence shown in Figure 17 (SEQ ID NO: 21) and the nucleic acid sequence shown in Figure 18 (SEQ ID NO: 22). The t-duck HBcAg particles have the amino acid sequence shown in Figure 19 (SEQ ID NO: 23) and Figure 20 (SEQ ID NO: 2).
4) having the nucleic acid sequence shown. t-Duck HBcAg (“truncated” duck HBcAg) particles are composed of monomeric subunits whose nucleic acid binding domain has been genetically engineered to be deleted.

(Method) (Quantification of Specific RNA) J774A. 1 specific RNA produced by macrophage cells, different types of stimulation: 1) duck HBcAg particles containing nucleic acid or 2) t-free nucleic acid.
Quantification after exposure to duck HBcAg particles). Cells are treated with 10 μg of duck HB
Treated with either cAg particles or t-duck HBcAg particles, and total RNA was isolated from duplicate samples at various time points. Total RNA was reverse transcribed with random hexamers and then PCR was performed in the linear region with gene specific primers. PCR products were analyzed by gel electrophoresis and quantified by densitometry. Prior to use, recombinant protein should be treated with Triton
Treatment with X-114 was performed 3 times to remove lipopolysaccharide contaminants.

IL-12 Quantification A capture-ELISA was used to measure IL-12 p70 protein in cell culture medium and cell extracts using an R & D ELISA kit. IL-12
The biologically active form of is a heterodimeric disulfide-bonded glycoprotein consisting of p35 and p40 subunits. IL-12 is produced by activated antigen presenting cells including macrophages, dendritic cells, and B cells. After activation, p40 expression is induced and translated. The resulting p40 protein binds to the constitutively expressed p30 subunit and is secreted to perform various biological functions. Increasing amounts of duck HB using capture ELISA
After treatment with cAg, it is secreted by dendritic cells and J774A. The concentration of IL-12 secreted in one macrophage and located intracellularly was measured. Microplates coated with antibody to capture IL-12 p70 (8 wells x 12 rows / plate) were exposed to duck HBcAg for 18 hours with clarified cell culture medium or cell extracts. Incubated. This mixture was allowed to incubate for 2 hours. After incubation, the wells were washed 5 times with wash buffer. After washing, a buffer containing anti-p70 antibody conjugated to horseradish peroxidase that binds to a distinct binding motif other than the capture antibody is added and incubated for 2 hours at room temperature. After the incubation, the wells were washed again 5 times. A colorimetric substrate that reacts with horseradish peroxidase is added for 30 minutes at room temperature in the dark. After 30 minutes, the acidic solution is added to stop the reaction. The absorbance of each sample was then measured at 450 nm and corrected at 540 nm. The results were compared to a standard reaction carried out under the same conditions at the same time and were obtained in units of pg / ml.

Flow Cytometry Cellular markers (CD4 +, CD8 +, Ly-6A / E and B7-2) were monitored by flow cytometry in the following manner: Cell Culture: J774A. One cell was administered 500,000 per well 1 day before the experiment.
Divided into 0 cells. On the day of the experiment, LPS duck HBcAg was added directly to the cell culture medium to the appropriate concentration and allowed to incubate with the cells for 18 hours. At 18 hours, cells were removed by pipetting and 10 at 500 xg.
Centrifuge for minutes and then wash three times in phosphate buffered saline. Then F
To block the c receptor, cells were incubated with 10 μg / ml mouse IgG in phosphate buffered saline containing 1% bovine serum albumin (BSA). Mouse B7.2 (CD86), H-2k b (MHC-I), I
Fluorescein isocyanate (FITC) conjugated with a primary antibody against -A ^b (MHC-II); Fluorescein isocyanate conjugated with primary antibodies against Ly6A / E and Ly6 / C (FITC) was added, respectively, And it incubated at 4 degreeC for 30 minutes. A non-specific antibody with fluorescein isotype matching was used as a negative control. At least 10 ⁵ cells were collected for data analysis. Cells were pelleted by centrifugation and unbound antibody was washed off with three washes with PBS containing 1% BSA. The cells were then resuspended in FACS buffer and cell surface markers were quantified by Beck-Dickson FACscanner. Antibody against mouse CD8-RPE and CD4-RPE (R-phycoerythrin)
A similar method was used on splenocytes except that it also included an antibody against.

Quantification of Ig titers, both total and isotype Mice were treated with lipopolysaccharide duck HBcAg particles (0 μg,
Immunization was carried out with 1 μg or 10 μg). Blood was collected on day 10, 24 and then 14 days post secondary immunization and tested for duck HBcAg antibody. Approximately 100 μl of blood is retro-orbital (retro-orbital).
) Collected by puncture. After this blood collection, the sample was coagulated. The clot was removed by centrifugation. The resulting serum then contained the IgG fraction. Serum samples were serially diluted to 10 (1) -10 (6).

ELISA coated with duck HBcAg protein (particulate form)
Plates were prepared by incubating a commercial ELISA plate overnight at room temperature with duck HBcAg protein in bicarbonate buffer. Plates were washed 5 times with wash buffer, then bovine serum albumin (BSA)
Was used to block non-specific binding for 2 hours at room temperature. Plates were washed 5 times with wash buffer and stored at 4 ° C before use.

100 μl of diluted serum sample was incubated with the precoated plate for 2 hours at room temperature. After incubation, the wells were washed 5 times with wash buffer. After washing, buffer containing rat anti-mouse IgG antibody conjugated with horseradish peroxidase is added and incubated for 2 hours at room temperature. After incubation, wash again 5 times. A colorimetric substrate that reacts with horseradish peroxidase is added for 30 minutes at room temperature in the dark. After 30 minutes, an acidic stop solution is added and the reaction is stopped. The absorbance at 450 nm of each sample is then measured at 450 nm and corrected at 540 nm. The results are compared to a standard reaction run at the same time under the same conditions and are given in units of relative titer.

For isotype-specific assays, rat anti-mouse antibody specific for antibody isotype was used in place of the generic rat anti-mouse antibody. All other conditions remained the same.

(Duck HBcAg, duck HBcAg (1-239) and t-duck HB
Method for disassembling and reassembling cAg) Duck HBcAg particles, duck HBcAg (1-239) particles and t-duck HBcAg particles can be reversibly assembled between monomeric and particulate forms. It's been found. This process uses neutral buffers (eg Tris HC
1 pH 7.0) and high concentrations (eg 0.1-1 M) of divalent cations (eg Mg ⁺² , Zn ⁺² , Ba ⁺² , Ca ⁺² , Pb ⁺² and Sr ⁺² ). It can be performed under non-denaturing conditions. Particles as isolated from a recombinant expression system can be dissociated into monomers for approximately 1 hour (by gentle mixing), and the progress of dissociation is determined by FPLC (Pharmaica) Sepharose-CL4B size exclusion column chromatography. Or by native agarose gel electrophoresis. Ribonuclease A (eg, 7.5 μg / mL)
Is added to remove the recombinant host nucleic acid contained in the particles. Because, it results in isolated reassembly in the following manner: Non-nucleic acid bound t-duck HBcAg monomer can be reassembled by simple dialysis in neutral buffer containing 100-200 mM NaCl. . The duck HBcAg monomer and duck HBcAg (1-239) monomer are dependent on the presence of nucleic acid for proper reassembly. These monomers are mixed with the depleted nucleic acid and dialyzed against neutral buffer. During this dialysis, reassembled particles containing the nucleic acid are formed. A preferred monomer to oligonucleotide molar ratio is 1: 1.
Is. Exogenous DNA that is not incorporated into the reassembled particles can be removed by size exclusion column chromatography.

Example 1. In vitro induction of cytokines Which cytokines, if any, were added to recombinant duck HBcAg and t-duck HBcAg in J774A. To determine if induced by exposure of one macrophage cell, experiments were performed as described in Methods. The results are given in Figure 4. As can be seen, TH1 type cytokines IL-1α, IL-
Increased transcription of 6 and IL-12 was demonstrated by duck HBcAg and t-duck H.
Induced by both BcAg. This indicated that the core protein is responsible for inducing the TH1 cytokine profile.

Example 2. Induction of IL-12 p70 protein production J774A. 1 Duck HBcAg and t-duck H to macrophage cells
Experiments were performed in vitro to determine whether the addition of BcAg, as well as the addition of duck HBcAg to mouse myeloid dendritic cells, induced the production of IL-12 p70 protein. IL-1 in cell culture medium and cell extracts
Analysis of the amount of 2 p70 is performed as described in the method and the results are given in Figures 5A, 5B and 6. As can be seen, exposure to duck HBcAg and t-duck HBcAg for 72 hours was performed in culture medium (FIG. 5A) and in J774A. In the cells themselves of one macrophage cell (FIG. 5B), IL-
12 concentrations were increased. Exposure of mouse myeloid dendritic cells from both BALB / C and B10 mice to duck HBcAg for 24 hours resulted in an increase in IL-12 in the culture medium (FIG. 6). This response is consistent with the induction of a TH1-type immune response.

Example 3. Analysis of cell markers on J774A.1 macrophage cells J774A. 1 Duck HBcAg and t-duck H to macrophage cells
BcAg was added to the cell markers (MHC-I, MHC-II, Ly-6A / E).
And to determine if it could induce the presence of B7-2). These cell surface markers are directly involved in the TH1-type immune response. The presence of cell markers was determined by flow cytometry as described in the method. The results are given in Figure 6. As can be seen, this data indicated that each type of cell surface antigen was upregulated after 18 hours treatment with duck HBcAg. This means that macrophages
It has been shown that it can be stimulated by duck HBcAg and t-duck HBcAg.

Example 4 Immunogenicity of Ducks HBcAg The immunogenicity of ducks HBcAg was tested by determining the total IgG titer for ducks HBcAg in the following manner: two strains of mice. (B
10 and BALB / C) were immunized as described in the method. The results are given in FIG. As can be seen, both B10 (FIG. 7A) and BALB / C mice (FIG. 7B) produced antibody titers greater than 10 ⁵ . This indicated that duck HBcAg is highly immunogenic. Further, the isotypes of the IgG fractions shown in FIG. 8 are IgG2a, IgG
Deflection of TH1 through production of 2b and IgG3. Even though their genetic background is usually biased towards the TH2 immune response, BALB / C mice have TH
The fact of showing a deviation of 1 is particularly important.

Example 5. Analysis of cell markers in spleen cells After death blood sampling, spleen cells were obtained from B10 mice and cultured with 1 μg duck HBcAg for 24 hours. Cell markers (CD4 +, CD8 +, Ly-6) were analyzed using flow cytometry (as described in Methods).
A / E and B7-2) were monitored. The result is shown in FIG. As can be seen, both 1 μg and 10 μg immunized mice had a substantial increase in the presence of these cellular markers over control cells from non-immunized mice. This showed a strong T cell response.

Example 6. Demonstration of lack of cross-reactivity of duck HBcAg with human and woodchuck HBcAg As described in the methods to compare crossreactivity of duck, woodchuck and human HBcAg. Experiments were conducted. As can be seen in FIG. 11A, the Woodchuck antibody exhibits extensive cross-reactivity with human HBcAg and no duck HBcAg. Similarly, human HBcA
The g antibody shows extensive cross-reactivity with woodchuck HBcAg and no tolerance reactivity with duck HBcAg (FIG. 11A).

Example 7. Deassembly and Reassembly of Duck HBcAg Particles Experiments were performed to show that duck HBcAg particles can be successfully disassembled and reassembled. Duck HBcAg (1 mg / mL)
Incubated at 37 ° C. with 0.3 M MgCl ₂ , 7.4 μg / mL Rnase A, or both. After 30 minutes, each sample was dialyzed against Tris-buffered saline (TBS) for 4 hours and analyzed by native agarose electrophoresis. Treatment with magnesium and subsequent dialysis was performed with duck H
This resulted in the reassembly of the treated protein into particles (FIG. 12, lane 2) that co-migrated with the BcAg standard (FIG. 12, lane 1). Treatment with Rnase A alone did not alter the electrophoretic mobility of duck HBcAg (Figure 12, lane 4), which has no apparent effect on duck HBcAg particles with Rnase A alone. I suggested that. However, treatment with both Rnase A and MgCl ₂ resulted in the quantitative removal of co-migrating bands with the duck HBcAg standard (FIG. 12, lane 3). This is due to the presence of Rn in the presence of divalent cations.
It is suggested that case A can catalyze the hydrolysis of ribonucleic acid in the particles (ie the particles disassemble) and that the nucleic acid is required for reassembly of the separated duck HBcAg. A similar experiment was performed in which the Rnase A / MgCl ₂ treated duck HBcAg was supplemented with endogenous DNA and tRNA during the dialysis step. Reassembled particles were not detected by these methods.

Example 8. Deassembly and Reassembly of Duck HBcAg (1-239) Particles Duck HBcAg (1-239) was analyzed by the same method as described in Example 7. Identical results were obtained. And this is given in FIG. Treatment with magnesium and subsequent dialysis reassembles the processed protein into particles that co-migrated with the duck HBcAg (1-239) standard (FIG. 13, lane 1) (FIG. 13, lane 2). Occurs. Treatment with Rnase A alone did not alter the electrophoretic mobility of duck HBcAg (1-239) (Figure 13, lane 4), which indicates that Rnase A alone does not duck HBcAg (1-239).
It suggested that it had no obvious effect on the particles. However, Rnase A
Treatment with both MgCl ₂ and MgCl ₂ results in quantitative removal of bands that co-migrate with the duck HBcAg (1-239) standard (FIG. 13, lane 3). This suggests that in the presence of divalent cations, Rnase A can catalyze the hydrolysis of ribonucleic acid in particles (ie, disassemble the particles) and that nucleic acid is required for reassembly. .

However, in contrast to the results obtained with the full length duck HBcAg, Rnase
Addition of endogenous DNA to A / MgCl ₂ -treated duck HBcAg (1-239) favors reassembly of separated monomers into a nucleic acid-containing core structure. This is illustrated in FIGS. 14 and 15. In FIG. 14, lane 1 is Rnase.
Duck HBcAg (1-239) treated with both A and MgCl ₂ and dialyzed against TBS. Figure 14, lane 2, dialyzed against treated with both Rnase A and MgCl ₂ and TBS, and the exogenous oligonucleotides were added before dialysis, showing a duck HBcAg (1-239). As can be seen, a migrating band can be observed at the position of the control duck HbcAg (1-239) (lane 3), indicating reassembly of the particles in the presence of exogenous oligonucleotide. These results are further illustrated in FIG. Lane 1 of FIG. 15 shows control duck HbcAg (1
-239). Lane 2 of FIG. 15 shows duck HbcAg (1-239) treated with both Rnase A and MgCl ₂ and dialyzed against TBS and exogenous oligonucleotide added prior to dialysis. As can be seen, a migrating band can be observed at the position of the control duck HbcAg (1-239) (lane 1), indicating reassembly of the particles in the presence of the exogenous oligonucleotide.

Example 9 Reassembly of duck HBcAg (1-239) with exogenous nucleic acid was performed using Se
Confirmed by chromatography on pharose 4B. The results are shown in FIG. 16, where peak 2 of 16A is duck HBcAg (1-2
39) native core peak (Peak 1 is a breakthrough material that represents bacterial aggregates of high molecular weight). Peak 3 of 16B corresponds to the separated core monomer (after exposure to divalent cations) and peak 4 is exogenous DNA. 16C peak 5 shows the reassembled duck HBcAg after removal of divalent cations and addition of exogenous DNA.
(1-239) corresponding to the core particle; peak 6 is the unincorporated exogenous DN
Represents A.

[0086]

[Table 2]

[Brief description of drawings]

[Figure 1]   FIG. 1 is the amino acid sequence of duck HbcAg.

[Fig. 2]   FIG. 2 is the nucleic acid sequence of duck HbcAg.

[Figure 3]   FIG. 3 is an illustration of exogenous mosaic recombinant duck HbcAg particles.

Figures 4A and 4B show J774A.D after exposure to recombinant duck HBcAg particles (4A) or recombinant t-duck HBcAg particles (4B). Analysis of specific RNA in one macrophage cell. RNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR) and visualized by agarose gel electrophoresis.

FIG. 5A shows J774A.J after treatment with recombinant duck HBcAg particles or recombinant t-duck HBcAg particles for 72 hours. 1 Macrophage cell culture medium (5A
) Is the concentration of IL-12.

FIG. 5B shows J774A.B after treatment with recombinant duck HBcAg particles or recombinant t-duck HBcAg particles for 72 hours. 1 Macrophage cell extract (5
It is the concentration of IL-12 in B).

FIG. 6 IL-12 in culture medium of bone marrow-derived dendritic cells from BALB / C and B10 mice after treatment with recombinant duck HBcAg particles for 24 hours.
Is the concentration of.

FIGS. 7A, 7B, 7C, 7D and 7E show that J774A.JE after treatment with recombinant duck HBcAg particles. Flow cytometric analysis of cell surface antigen expression on 1 macrophage cells. 7A: CD86; 7B: MHC I; 7C: MHC
II; 7D: Ly-6A / E; 7E: Ly-6C. Arrows: 1, shaded area: isotype control; 2, solid line: untreated control; 3, dotted line: treated with 10 μg of recombinant duck HBcAg particles.

8A and 8B are total IgG titers of duck HBcAg in vivo.
8A: B10 mouse. 8B: BALB / C mouse.

FIG. 9. Duck HBcAg in B10 and BALB / C mice.
Is the IgG isotype titer.

10A, 10B, 10C and 10D are flow cytometric analyzes of cell surface antigen expression in spleen cells after restimulation with 1 μg duck HBcAg in vitro for 24 hours. 10A: CD86; 10B: Ly-6C; 10C
: CD4 +; 10D: CD8 +. Arrows: Immunized with 1.0 μg of duck HBcAg. 2, immunized with 1 μg of duck HBcAg. 3, 10 μg of duck HBcA
Immunized with g.

11A and 11B are a comparison of woodchuck cross-reactivity with duck and human antibody (11A) and duck and woodchuck antibody (11B).

FIG. 12: Duck HBcAg particles with nucleic acid and duck HB without nucleic acid.
Decomposition / reassembly of cAg particles. Lane 1: duck HBcAg; Lane 2: M
Duck HBcAg treated with gCl ₂ and dialyzed against TBS; lane 3
: Duck HB treated with both MgCl ₂ and Rnase A and dialyzed
cAg; Lane 4: Duck HBcAg treated with Rnase A and dialyzed.
.

FIG. 13: Degradation / reassembly of duck HBcAg (1-239) particles with nucleic acid and duck HBcAg (1-239) particles without nucleic acid. Lane 1:
Duck HBcAg (1-239); Lane 2: treated with MgCl ₂ and TB
Duck HBcAg (1-239) dialyzed against S; Lane 3: Duck HBcAg (1-239) treated with MgCl ₂ but not dialyzed; Lane 4: Rnas.
dA HBcAg (1-239) treated with eA and dialyzed; lane 5:
Duck HBc treated with both MgCl ₂ and Rnase A and dialyzed
Ag (1-239).

FIG. 14   Figure 14 shows duck HBcAg (1-239) particles with nucleic acid and nucleic acid.
No Decomposition / reassembly of duck HBcAg (1-239) particles. Lane 1:
Rnase A and MgCl₂Treated with both and dialyzed against TBS
Duck HBcAg (1-239). Lane 2: Rnase A and MgCl ₂ Duck HBcAg (1-23 treated with both
9); Exogenous oligonucleotide was added before dialysis; Lane 3: duck HBc.
Ag (1-239).

FIG. 15: Degradation / reassembly of duck HBcAg (1-239) particles with nucleic acid and duck HBcAg (1-239) particles without nucleic acid. Lane 1:
Duck HBcAg (1-239); Lane 2: Rnase A and MgCl ₂
Duck HBcAg (1-239) treated with both
); Exogenous oligonucleotide was added prior to dialysis.

16A, 16B and 16C show duck HBcAg (1 with exogenous nucleic acid.
-239) Degradation / reassembly of particles: confirmation by chromatography. 16A
: Peak 1: Breakthrough material shows high molecular weight bacterial aggregates; Peak 2: Duck HBcAg native core peak. 16B: Peak 3: dissociated core monomer; Peak 4: exogenous DNA. 16C: Peak 5: Reassembled duck HB
cAg core particles; peak 6: exogenous DNA not incorporated.

FIG. 17   FIG. 17 is the amino acid sequence of duck HBcAg (1-239).

FIG. 18   FIG. 18 is the nucleic acid sequence of duck HBcAg (1-239).

FIG. 19   FIG. 19 is the amino acid sequence of t-duck HBcAg.

FIG. 20   FIG. 20 is the nucleic acid sequence of t-duck HBcAg.

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, SD, SL, SZ, TZ, UG, ZW ), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, C R, CU, CZ, DE, DK, DM, EE, ES, FI , GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, K Z, LC, LK, LR, LS, LT, LU, LV, MA , MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, S K, SL, TJ, TM, TR, TT, TZ, UA, UG , UZ, VN, YU, ZA, ZW (72) Inventor Peterson, Darrell El.             United States Virginia 23832,             Chesterfield, Round Hill             Drive 4345 F term (reference) 4B024 AA01 BA33 CA02 GA11 HA17                 4C085 AA03 BA89 BB01 BB11 CC08                       EE03 EE06 FF02                 4H045 AA11 AA20 AA30 BA10 CA02                       DA86 EA29 FA74

Claims (34)

[Claims] 1. A particle comprising a nucleocapsid protein monomer, comprising:
The primary sequence of the particle is from hepatitis B virus, wherein the monomer is human HB
Particles that do not cross-react with antibodies to cAg. 2. The particle of claim 1, wherein a portion of the nucleocapsid protein monomer comprises a first hapten. 3. The particle of claim 2, wherein the second portion of the nucleocapsid protein monomer comprises a second hapten that is different from the first hapten. 4. The particle of claim 1, wherein the particle further comprises nucleic acid bound to the monomer. 5. The particle according to claim 1, wherein the hepatitis B virus is a duck hepatitis B virus. 6. The particle of claim 2, further comprising a nucleic acid that binds to the particle.
Particles according to. 7. The particle of claim 3, further comprising a nucleic acid that binds to the particle.
Particles according to. 8. The nucleic acid comprises SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, sequence. The particle according to claim 4, which is selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19. 9. The nucleic acid comprises SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, sequence. The particle according to claim 6, which is selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19. 10. The nucleic acid comprises SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,
The particle according to claim 7, which is selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19. 11. The particle according to claim 2, wherein the first hapten is a single-stranded DNA virus, a double-stranded DNA virus, a single-stranded RNA virus,
Particles associated with a disease state caused by a factor selected from the group consisting of double-stranded RNA viruses, intracellular parasites, fungi, bacteria and cancer. 12. The particle according to claim 3, wherein the second hapten is a single-stranded DNA virus, a double-stranded DNA virus, a single-stranded RNA virus,
Particles associated with a disease state caused by a factor selected from the group consisting of double-stranded RNA viruses, intracellular parasites, fungi, bacteria and cancer. 13. The particle of claim 1, further comprising first and second haptens, wherein the particle is assembled as an exogenous mosaic. 14. The particle of claim 1, further comprising first and second haptens, wherein the particle is assembled as an intrinsic mosaic. 15. A method of delivering a nucleic acid to a subject, the method comprising administering to the subject a particle comprising a nucleocapsid protein monomer and a nucleic acid, wherein the first sequence of the monomer is The method, which is derived from hepatitis B virus, wherein the monomer does not cross-react with antibodies to human HBcAg. 16. The method of claim 15, wherein the first portion of the nucleocapsid protein monomer comprises a first hapten. 17. The method of claim 15, wherein the second portion of the nucleocapsid protein monomer comprises a second hapten that is different from the first hapten. 18. The method of claim 15, wherein the hepatitis B virus is duck hepatitis B virus. 19. A method of making nucleocapsid protein monomer particles, the method comprising the steps of: providing a composition comprising a plurality of nucleocapsid protein monomers, wherein the primary sequence of the monomers is in hepatitis B virus. From which the monomer does not react with antibodies to human HBcAg, and wherein a plurality of said monomers are assembled to form particles; exposing said particles to a charge factor to render said monomer in non-particulate form Producing a mixture of :, and assembling particles from the nucleocapsid protein monomer in the mixture by removing the charge factor from the mixture. 20. The method of claim 19, further comprising removing unwanted nucleic acids from the mixture produced in the exposing step. 21. The method of claim 19, wherein the method comprises the steps of:
(A) step of adding endogenous mosaic particles; (b) step of adding exogenous mosaic particles; (c) step of adding nucleic acid; (d) in the step of assembling (
The method further comprising the step of adding any combination of a), (b) or (c). 22. The method according to claim 19, wherein the exposing step is performed.
The charge factor used in⁺², Zn⁺², Ba⁺², Sr⁺², Ca ⁺² , And Pb⁺²The method is a divalent cation selected from the group consisting of: 23. A method for eliciting an immunogenic response in a subject, the method comprising administering to the subject an effective amount of a composition comprising particles comprising nucleocapsid monomers. And the primary sequence of the monomer is from hepatitis B virus, and wherein the monomer does not cross-react with antibodies to human HBcAg. 24. The hepatitis B virus is duck hepatitis B.
The method according to 3. 25. The method of claim 23, wherein the composition further comprises a nucleic acid that binds to the particle. 26. The method of claim 23, wherein the composition further comprises one or more haptens associated with at least a portion of a plurality of the nucleocapsid monomers. 27. A vaccine comprising particles assembled from a plurality of nucleocapsid protein monomers, the primary sequence of said monomers being from hepatitis B virus, wherein said monomers are cross-linked with antibodies to human HBcAg. Not reacting, vaccine. 28. The primary sequence further comprises one hapten.
Vaccine according to. 29. The vaccine of claim 27, wherein the primary sequence further comprises one hapten from a double stranded DNA virus. 30. The vaccine of claim 27, wherein the primary sequence further comprises a second hapten from a double stranded DNA virus. 31. The vaccine of claim 28, wherein the hapten is from HIV. 32. The vaccine of claim 28, wherein the hapten is derived from cancer cells. 33. A method of eliciting a TH1-type immune response in a subject, said method comprising administering to said subject an effective amount of a composition comprising particles comprising nucleocapsid monomers. The method, wherein the primary sequence of the monomer is from hepatitis B virus, wherein the monomer comprises at least one hapten that does not cross-react with antibodies to human HBcAg and is associated with disease states. 34. The method of claim 33, wherein the disease state is cancer or AIDS.