WO2011028963A2 - Immunogenic hiv composition - Google Patents

Abstract

Novel compositions useful as HIV immunogens are provided. The compositions enable a host response to sites normally not recognized by a host. Also provided are antibodies raised to such immunogens. Also provided are complexes of antibodies or antigen-binding portions thereof and HIV antigens.

Description

IMMUNOGENIC HIV COMPOSITION

Peter L. Nara, Greg Tobin & George Lin

[0001] The instant application claims benefit to U.S. Ser. No. 61/239,757 filed 3 September 2009, the contents of which are incorporated herein by reference in entirety.

BACKGROUND

[0002] The current stable of licensed vaccines is generally successful against what are termed "Class One pathogens." Class One pathogens (such as measles, mumps and rubella viruses) are those pathogens, which, in general: (1) infect or cause the most serious disease in children/young adults; (2) carry a relatively stable microbial genome; (3) have a natural history of disease which results in spontaneous recovery; and (4) induce durable memory, associated with polyclonal and

multi-epitope antigen recognition.

[0003] In contrast, Class Two pathogens, such as, viral influenza, HIV-1, malaria, tuberculosis, trypanosomes, schistosomes, leishmania, anaplasma, enterovirus, astrovirus, rhinovirus, Norwalk viruses, toxigenic/pathogenic E. coli, Neisseria, Streptomyces, nontypeable Haemophilus influenza, hepatitis C, streptococcus, staphylococcus, cancer cells etc. are characterized by quite opposite features. For example, Class Two pathogens: (1) tend to infect and are transmitted in a significantly extended host age range, with infections occurring and reoccurring from childhood through the geriatric period; (2) exhibit genetic instability in defined

02a31b28 1 regions of their genome (a hallmark of the successful evolution of such pathogens); (3) in some cases, spontaneous recovery of disease frequently leaves the host vulnerable to multiple repeated annual infections and/or the establishment of either a chronic/active or chronic/latent infectious state; (4) induce oligoclonal, early immune responses that are directed to a very limited set of immunodominant epitopes which provide either narrow strain-specific protection, no protection and/or enhanced infection, in many cases, induced immune responses that only provide strain-specific immunity and thus do not provide broader protective immunity that would be desirable for many pathogens that undergo mutation; and (5) cause immune dysregulation following infection or vaccination, e.g. epitope blocking antibody, atypical primary immune response Ig subclasses, anamnestic cross reactive recall and inappropriate T_Hi and/or T_H2 cytokine metabolism. Thus, an immunodominant decoy, dysregulating or immune steering/biasing epitope or its equivalent ligand-receptor interaction of any of the innate host defense system molecules that someway alters the hosts acquired/adaptive immune response to the pathogen is considered to be the main focus and target of technologies designed to modify, alter or eliminate them from the molecule so as to result in the host immune system responding in an alternate manner resulting in an either/and or earlier and more broadly protective immune response following active immunization of the host.

[0004] At the immune level, very different etiologic agents can yield diverse pathogenesis and disease outcome as observed, for example, with human

immunodeficiency virus type 1 (HIV-1) as compared to human rhinovirus. Highly successful immune evading strategies, such as, Deceptive Imprinting, have evolved

02a31b28 2 and are selected and maintained across host and microbial taxa. Thus, the operational failures of the vertebrate immune system, for example, by Deceptive Imprinting, are fundamentally the same whether infected with HIV-1 or with a common cold virus for an average of 2-6 times a year for 60 years.

[0005] Although some advances in antigen delivery and expression have improved the immunogenicity of some Class Two microbial pathogens, current vaccine technologies have not readily translated into new, broadly effective and safe licensed vaccines. That may be due, in large part, to a poor understanding of the fundamental laws governing the vertebrate host defense system origin, repertoire development, maintenance, activation, senescence and co-evolution in similar and dissimilar environments.

[0006] At the heart of the problem in the annual global virus tracking programs and subsequent "reactionary" vaccine production that ensues, especially notable in the field of influenza, is the issue of antigenic variation. Antigenic variation is an evolved mechanism to ensure rapid sequence variation of specific pathogen gene(s) encoding homologues of an individual protein antigen, usually involving multiple, related gene copies, resulting in a change in the structure of an antigen on the surface of the pathogen. Thus, the host immune system during infection or re-infection is less capable of recognizing the pathogen and must make new antibodies to recognize the changed antigens before the host can continue to combat the disease. As a result, the host cannot stay completely immune to the viral disease. That phenomenon stands as one of the more, if not, most formidable problem challenging modern vaccine development today.

02a31b28 3 [0007] Original antigenic sin, first described in 1953 by Francis (Ann. Int. Med., 1953, 399:203) is a primary immune response, that when boosted not by the homologous, but by a cross reacting vaccine or incoming viral subtype/strain, results in the newly formed antibodies reacting better with the previous virus antigen challenge than with the incoming virus, and with varying levels of responses to other antigens of other viruses.

[0008] Original antigenic sin was not considered pathogenic and a means to avoid the immune system until 1990 (Nara et al., J. Virol. 62:2622-2628, 1988) when it was uncovered that original antigenic sin was a means, for example, for NA viruses such as HIV to avoid the host immune system. The immune avoidance strategy was observed to be implemented by a number of microbiological and possibly eukaryotic organisms.

[0009] Thus, pathogens often express epitopes which are preferentially recognized by a host immune system, so called immunodominant epitopes or determinants. However, those epitopes may not serve an important function for pathogen viability or function, for example. Thus, such immunodominant epitopes are essentially decoys. That observation indicates that immunodominant epitopes can be modified practicing methods known in the art which then will enable a host to raise an immune response to other epitopes displayed by the pathogen.

[0010] The loss of immune specificity directed by this aleatory recall poses a real problem for the host immune system to mount equal and potent immune responses to the changing virus both during an infection and between reinfections with various strains of the same pathogen. Thus, it is not surprising that natural

02a31b28 4 infection and vaccination fail to yield a more functional cross-reactive primary and anamnestic immunity as the repertoire development against those less immunogenic epitopes which may be more conserved and capable of generating cross-strain immunity, are lower on the hierarchy of immune system reactivity. The immunologic phenomenon whereby immunodominant epitopes misdirect the immune response away from more conserved and less immunogenic regions on an antigen was initially termed "clonal dominance" (Kohler et al., J Acquir Immune Defic Syndr 1992;

5: 1158-68), which later was renamed as "Deceptive Imprinting" (Kohler et al., Immunol Today 1994 (10):475-8).

[0011] The immunologic mechanisms for immunodominance are not fully understood, and no one mechanism yet fully explains how or why certain epitopes have evolved to be immunoregulatory and immunodominant. The range of immune responses observed in the phenomena include the induction of highly strain-specific or isolate-specific neutralizing antibody capable of inducing passive protection in experimental animal model-viral challenge systems all the way to the induction of a binding non-protective/non-neutralizing, blocking and even pathogen-enhancing antibody that in some cases prevents the host immune system from recognizing nearby adjacent epitopes, to interfering with CD₄ T-cell help. The same decoying of the immune response through immunodominance resulting in a more narrowly focused set of epitopes is observed with T cells of the host (helper and cytotoxic cell-mediated immunity. (Gzyl et al., Virology 2004; 318(2):493-506; Kiszka et al., J Virol. 2002 76(9):4222-32; and Goulder et al, J Virol. 2000; 74(12):5679-90).

02a31b28 5 [0012] The immune response to HIV is composed of an initial cell-mediated immune response followed by the subsequent development of neutralizing antibodies. Within weeks of infection, virus titers in the blood fall coincident with the induction of anti-HIV cellular and humoral immune responses. The fall in viremia correlates with the appearance of anti-HIV major histocompatibility complex (MHC) class I-restricted CD₈ ⁺ cytotoxic T cells. Recent evidence has shown a strong correlation of anti-HIV CD₄ ⁺ T cell responses and reduced viral loads. Therefore, the presentation of HIV antigens in the context of MHC class II molecules to CD₄ ⁺ T cells may be a key aspect of the control of HIV infection.

[0013] Due to previous successes in preventing viral diseases using subunit, live-attenuated viral, and inactivated viral vaccines, the scientific community was initially optimistic that a vaccine would be developed to prevent the spread of HIV. However, early optimism soon diminished because of repeated failures.

[0014] Vaccines based on subunit immunogens, although extremely safe, are limited in the breadth of antigens that are presented to the immune system because only one or a few of the viral proteins are utilized as immunogens and choice of subunit antigens may limit the likelihood of cross protection, for example, among clades of HIV. Also, the production of vaccines based on subunit immunogens requires the molecular manipulation of the viral proteins into cloning or expression vectors, perhaps leading to increased production time and cost.

[0015] The genetic diversity of HIV is due to the extremely high replication rate of the virus in infected individuals, the high rate of mutation caused by the error prone reverse transcriptase, the substantial viral load, and selection within infected

02a31b28 6 individuals. Diversity is so great that the presence of closely related but not identical strains of HIV, known as quasispecies, commonly appear in a single, infected individual. The quasispecies may diverge increasingly over time and changes tend to be within the env gene, particularly the five variable regions (VI -V5). Although changes also may occur in the gag, pol and accessory genes, those differences tend to be less substantial.

[0016] When significant changes accumulate and are seen in a large group of individuals, the strain is commonly considered a new family or new clade of HIV. Phylogenetic studies of HIV have shown that there are two major families of HIV, HIV-1 and HIV-2.

[0017] The strains of HIV-1 can be classified into four groups: the "major" group M, the "outlier" group O, and two new groups, N and P (Hemelaar et al., AIDS 20(16)W 13-23, 2006). Those four groups may represent four separate introductions of simian immunodeficiency virus into humans. Group O appears to be restricted to west-central Africa and group N, a strain discovered in 1998 in Cameroon, is extremely rare. In 2009, a new strain closely relating to gorilla simian

immunodeficiency virus was discovered in a woman from Cameroon. It was designated HIV-1 group P. More than 90% of HIV-1 infections belong to HIV-1 group M. Within group M, there are known to be at least nine genetically distinct subtypes (or clades), A, B, C, D, F, G, H, J and K.

[0018] Subtype B is prevalent in the Western Hemisphere, while subtypes A, C and D commonly occur in Africa. In Asia, the most frequently found subtypes are

02a31b28 7 E, C and B, with the subtype E having a high prevalence in Southeast Asia. In India, the prevalent subtype is C.

[0019] Accordingly, the most effective vaccines for invoking a strong and complete immune response (such as a modified live virus vaccine) may also carry the most risk of harming the individual while the safer alternatives (subunit) may induce as complete a set of immunity as a whole virus vaccine mentioned above but require a properly chosen antigen from the virus that is immune refocused and delivered in a licensed and safe adjuvant.

SUMMARY OF THE INVENTION

[0020] The invention relates, in part, to novel HIV antigens with enhanced or novel immunogenicity. An HIV composition of interest can serve as an improved vaccine, resulting from modifications providing the virus or viral antigen with a different array of and/or newly recognizable epitopes.

[0021] The more efficient and rapid use of recombinant technology coupled to a novel immune refocusing or modifying technology resulting in antigenic subunit compositions, attenuated virus or inactivated virus or virions carrying a modified epitope of interest greatly change the current practice of vaccine development by generating an HIV vaccine with improved effectiveness and an enhanced ability to stimulate increased cross-protective immune responses.

[0022] Additional features and advantages are described herein, and will be apparent from, the following Figures and Detailed Description.

02a31b28 8 BRIEF DESCRIPTION OF THE FIGURES

[0023] Figure 1 provides a portion of Table 1 listing certain mutations.

[0024] Figure 2 is a continuation of Table 1.

[0025] Figure 3 provides a portion of Table 2 providing primers.

[0026] Figure 4 is a continuation of Table 2.

[0027] Figure 5 is a continuation of Table 2.

[0028] Figure 6 provides the conclusion of Table 2.

DETAILED DESCRIPTION OF THE INVENTION

[0029] "Wild type" refers to a naturally occurring organism. The term also relates to forms of nucleic acids and of proteins found in a natural occurring organism of a naturally occurring population arising from natural processes, such as seen in polymorphisms arising from natural mutation and maintained by genetic drift, natural selection and so on, and does not include a nucleic acid or protein with a sequence obtained by, for example, purposeful modifications of the sequences through either a biologic or chemical selective process, or through molecular manipulations, such as a mutagenesis method. A wild type allele or antigen usually is but need not be the most prevalent form in a population. Also, herein, wild type is used as an equivalent of the parental form. Thus, if allele A is mutated to form allele A*, then A is the parental or wildtype form in that relations. Allele A itself can be a rare mutation in a population.

[0030] "Immunogen" and "antigen" are used interchangeably herein as a molecule that elicits a specific immune response, for example, containing an antibody that binds to that molecule or eliciting T cells, such as CD₄ ⁺ or CD₈ ⁺ cells, capable of

02a31b28 9 destroying an infected cell. That molecule can contain one or more sites to which a specific antibody binds. As known in the art, such sites are known as epitopes or determinants. An antigen can be polypeptide, polynucleotide, polysaccharide, a lipid and so on, as well as a combination thereof, such as a glycoprotein or a lipoprotein. An immunogenic compound or product, or an antigenic compound or product is one which elicits a specific immune response, which can be a humoral, cellular or both.

[0031] A vaccine is an immunogen or antigen used to generate an

immunoprotective response, that is, for example, the antibody reduces the negative impact of the immunogen or antigen which can be found on an infectious virus or entity expressing same, in a host. The dosage is derived, extrapolated and/or determined from preclinical and clinical studies, as known in the art. Multiple doses can be administered as known in the art, and as needed to ensure a prolonged prophylactic or anamnestic (memory) state. The successful endpoint of the utility of a vaccine for the purpose of this invention is the resulting presence of an induced immune response (e.g. humoral and/or cell-mediated) resulting, for example, in the production of serum antibody or antibody made by the host in any tissue or organ, that binds the antigen or immunogen of interest, or in a cellular response that recognizes the intended or cognate antigen. In some embodiments, the induced antibody in some way combines with a compound, molecule and the like carrying the cognate antigen or immunogen or directs the host to neutralize, reduce or prevent and/or eliminate a viral pathogen from infecting and causing serious clinical disease. Successful measurement of vaccine outcome may include, but is not exclusively confined to, immunity that either protects against infection or which reduces disease or inactivity

02a31b28 10 on infection. Immunoprotection for the purposes of the instant invention is another endpoint and is a marker of inducing presence of such circulating anti-pathogen antibody that binds the immunogen or the pathogen, or cells that express an antigen of the pathogen. That can be determined using, for example, any known immunoassay, such as an ELISA. Alternatively, one can use a viral neutralization assay to ascertain presence of circulating anti-viral antibody. For the purposes of the instant invention, observing immunoprotection, that is, presence of circulating antibody or presence of immune cells reactive with virus or virus-infected cells, is evidence of efficacy of a vaccine of interest. Although not relevant for determining efficacy of a vaccine of interest, the period of immunoprotection in a host can be at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 8 days, at least 10 days, at least 14 days, at least 15 days, at least 20 days, at least 21 days, at least 25 days, at least 28 days, at least 30 days, at least 35 days, at least 40 days, at least 42 days, at least 45 days, at least 49 days, at least 56 days, at least 60 days, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 1 year, at least 2 years or longer. Preferably the immunoprotection is observed in outbred populations, different geographic populations, clades and so on. In some

circumstances, an antigen or epitope can be a toleragen. It is known that tolerance can be obtained, for example, at low dose or at high does. In other circumstances, situations known as passive immunity or passive immunization when antibody is administered to a host not to act as an antigen but as an antibody, sometimes is called a passive vaccine or passive vaccination.

02a31b28 11 [0032] "Immunodominant epitope" is an epitope that selectively provokes an immune response in a host organism to the effective or functional exclusion, which may be partial or complete, of other epitopes on the same or another antigen of the pathogen. An immunodominant epitope may be specific for a strain, a clade, a group, a family, a species, a genus and so on. Alternatively, an immunodominant epitope that is modified in one strain, clade and so on, may reveal an epitope that is generic to a much wider range of pathogen. Hence, for example, in the context of HIV, an immunodominant epitope may reside in a particular site of a structural protein, for example, the envelope protein, such as the V3 loop, and modifying a V3 loop immunodominant epitope may reveal epitopes in regions aside from V3, such as those associated with the VI loop, V2 loop, V4 loop, V5 loop, A loop, B loop, E loop, an a helix region and so on (Kwong et al., Nature 393:648-659, 1998).

[0033] "To immunodampen an epitope," "to immunocloak an epitope," "to resurface an antigen," "to immunosmith an antigen," "to immunorefocus an antigen," "to immunodisable an antigen," 'to immunorefocus," "to eliminate antigenic regions," "to immunomodify an epitope," "to modify an epitope," "to epitope modify" and other equivalent phrases is to modify an epitope to substantially prevent the immune system of the host organism from producing antibodies, helper or cytotoxic T cells against that antigen or epitope, which terms are used interchangeably herein.

However, immunodampen, and equivalent terms, do not necessarily have to result in the complete removal of said epitope. Also, immunodampening can exert an influence on epitopes distal from the modified site. It may be necessary or beneficial to modify plural sites of an epitope or plural epitopes of a pathogen. Hence, an

02a31b28 12 epitope that is immunomodified is one which is not as or is no longer

immunodominant as compared to the original parental, wild type epitope prior to modification.

[0034] Immunodampening of an immunodominant epitope of an antigen can result in the production in a host organism of high titer antibodies or T cell responses against non-dominant epitopes on that antigen and/or new titers of antibodies or T cell responses to otherwise relatively immune silent epitopes or those which previously were poorly immunogenic. Such immunodampened antigens can serve as effective vaccines against organisms that have an antigen with a moderately or highly variable and/or conserved immunodominant epitope.

[0035] Thus, the instant invention also relates to an antibody or a cell that is reactive to an epitope previously not recognized or poorly recognized by a host because of one or more immunodominant epitopes. Hence, the antibody or cell is one which binds a non-immunodominant epitope or binds to an antigen which does not comprises or is devoid of an immunodominant epitope. Said antibody, or binding portion thereof, or cell can be derived, for example, as a monoclonal antibody, and used for diagnostic or therapeutic purposes, such as passive immunization using a human or humanized antibody as known in the art, see for example, W02009032661. The invention also relates to complexes of an antibody and an HIV antigen. The HIV antigen may comprise an immunodominant epitope, an immunodampened

immunodominant epitope or does not comprise an immunodominant epitope.

[0036] An immunodominant epitope can be identified by any of a variety of means and methods, for example, computationally, serologically, cellularly, using a

02a31b28 13 T cell-based peptide assay, structural analyses and so on. Multiple approaches can be used. Often, patient samples prior to infection are used as a control. Thus, serum antibodies or T cell reactivity of a host organism before and once infected with the pathogenic organism can be tested. The serum is evaluated for content of antibodies that bind to the identified antigens, usually either as pre-existing antibodies (naive human or animal) or occurring within a short amount of time after exposure or immunization that are likely to cause an immune response in a host organism. If an immunodominant epitope is present, substantially many antibodies in the serum will bind to and/or T cells will recognize the immunodominant epitope(s), with reduced to no binding/recognition to other epitopes present in the antigen. Regions other than immunodominant B and T cell epitopes may be targets for immune refocusing. Other domains that engage various host cell receptors (receptotopes) involved in host defense of the pathogens protein(s) may have evolved to stimulate a dysregulatory response (engaging host defense pathways that are inappropriate, e.g. those early signals and pathways that determine which antibody or cell-mediated response should be activated, thus sending the early immune response in the wrong direction).

Dysregulating epitopes may cause, for example, stimulation of deleterious or suboptimal expression and release of various cytokines and chemokines which turn on and off various immune cells needed for activation of a pathway.

[0037] After an immunodominant, decoy, dysregulating, immune steering, immune biasing and so on epitope has been identified and mapped, the

immunodominant epitope is modified as taught herein using the materials and methods taught herein and as known in the art as a design choice. The process of

02a31b28 14 immunodampening can be performed through any of a variety of methods, including, but not limited to, site-specific mutagenesis, antibody-induced evolution,

amplification, immune or drug selection or other in vitro or in vivo selection methods.

[0038] For example, a sequence that leads to post-translational glycosylation can be introduced or eliminated to dampen an immunodominant epitope. Thus, sequences that encode, for example, N-linked glycosylation sequons (NXT/S) can be altered or added in or near immunodominant epitopes. Methods for altering nucleic acids of sequons are taught herein.

[0039] The presence of N-linked carbohydrate (CHO) can be determined by the primary amino acid sequence of the polypeptide. A triplet amino acid sequence consisting of asparagine, followed by any amino acid, and ending with a serine or threonine (N-X-S/T), where X is any amino acid other than proline or aspartic acid is a target for N-linked CHO addition for dampening an epitope or antigen from immune system recognition. Hence, a glycosylation site can be introduced in the vicinity of an epitope or determinant for removal or immunodampening.

[0040] A particular amino acid of the immunodominant epitope can be replaced, substituted or deleted to dampen immunogenicity, for example, by substituting, removing or adding amino acids, modifying amino acids, by adding or removing a lipidation or glycosylation site, such as an O-glycosylation site or an N-glycosylation site, and so on. As known in the art, lipid groups can be added to polypeptides, sometimes referred to as prenylation (Young, J Lipid Res 46:2529-30, 2005) although other hydrophobic groups can be added to a polypeptide, such as, myristoyl and palmitoyl groups (Magee, J Cell Sci 97:581-584, 1990). Thus, a

02a31b28 15 polypeptide can be altered by adding, deleting or substituting one or more molecules, groups, compounds and the like at a target site or at an epitope, or in or at an adjacent or distal site which alters the target site or epitope. Hence, a particular amino acid can be derivatized or functional groups thereon can be removed, modified or added, for example, chemically, to effect such a change, such as, adding a saccharide group thereto, such as a glycol, such as a polyglycol, such as polyethylene glycol.

Immunodampening can occur by replacing, substituting or eliminating one amino acid, two amino acids, three amino acids or more of the immunodominant epitope, for example, by site-directed mutagenesis of the nucleic acid encoding the antigen to express another amino acid which is less immunogenic or which changes the pattern or hierarchy of immunogenicity. Methods for altering nucleic acids and/or polypeptides are provided herein, and are known in the art.

[0041] For example, a recombinant gpl20 of HIV B clade that displays a molecularly introduced N-linked sequon (NXT/S), which resulted in the addition of a supernumerary N-linked glycan in the immunodominant V3 domain, exhibited novel antigenic properties, such as the inability to bind antibodies that recognize wildtype V3 epitopes while inducing antibody responses to other previously silent or less immunogenic epitopes. Presence of the supernumerary carbohydrate moiety did not compromise the infectious viability of the HIV-1 recombinant virus. Test animals immunized with the recombinant glycoprotein showed moderate to high titers of antibodies that neutralize infection to both homologous and heterologous wildtype HIV-1 in vitro. Thus, immunodampening of the immunodominant epitope within the V3 domain of gp 120/ 160 caused the immune response to refocus on other neutralizing

02a31b28 16 epitopes that are located on the same antigen, see U.S. Pat. Nos. 5,585,250 and 5,853,724.

[0042] Suitable sites of HIV for immune modification include, but are not limited to the VI loop, V2 loop, V3 loop, V4 loop, V5 loop, CI region, C2 region, C3 region, superantigen regions and so on of gpl20; and the a helix, N terminus, C terminus and so on of gp 41. Suitable other sites of HIV for epitope modification include non-env proteins, gag, protease, pol, vif, tat, T, nef, rev, and other viral proteins. Suitable sites of immune modification also include RNA domains such as TAR and RRE. In some embodiments, a plurality of modifications can occur in more than one of the above regions. In other embodiments, the one or more modifications to the above-noted regions are coupled to a modification in the V3 loop.

[0043] The phrases and terms, as well as combinations thereof, "functional fragment, portion, variant, derivative or analog" and the like, as well as forms thereof, of a pathogen, antigen, component, subunit and the like thereof, such as, gpl20, gpl60, gag, pol and the like relate to an element having qualitative biological activity in common with the wild-type or parental element from which the variant, derivative, analog and the like was derived. For example, a functional portion, fragment or analog of HIV is one which stimulates an immune response as does native HIV, although the response may be to different epitopes on the HIV or a derived molecule.

[0044] Thus, included within the scope of the invention are functional equivalents of a virus, or portion or derivative thereof, of interest. The term

"functional equivalents" includes the virus and portions thereof with the ability to stimulate an immune response to HIV, although modified.

02a31b28 17 [0045] Parts of a pathogen, such as, HIV of interest, such as a membrane preparation carrying, for example, gpl20, gpl40 or gpl60, as well as preparations of any other HIV antigens, such as gag or pol, can be obtained practicing methods known in the art. When one or more epitopes are removed, cloaked, disabled or dampened, for example, by intramolecular modifications (e.g. deletions, charge changes, adding one or more N-linked sequons and so on) and given as an antigen to a naive animal, the altered epitope can induce a new hierarchy of immune responses at either or both the B and T cell levels (Garrity et al, J Immunol. (1997) 159( 0:279-89) against subdominant or previously silent epitopes. That technology as described herein is known as "Immune efocusing," which is synonymous with other equivalent terms used herein, such as immunosmithing or immunomodifying.

[0046] Thus, for example, a recombinant gpl20 (rgpl20), gp41 and or gpl60 subunit protein vaccine that is immunomodified as taught herein can be sufficient to protect against challenge from plural strains of HIV.

[0047] As provided herein, immunodampening can be implemented by any of a variety of techniques such as, altering or deleting specific amino acids of the epitope, or adding, for example, a glycosylation site at or near the epitope. The changes can occur at the level of the polypeptide or at the level of the polynucleotide, practicing methods known in the art.

[0048] Once a change is made, one then determines whether the change in the antigen, the mutein or the mutated antigen alters, such as, reduces the reactivity of the immunodominant epitope now modified, the "dampened or disabled epitope, antigen and so on." That can be tested in vitro by determining the reactivity of the dampened

02a31b28 18 antigen with defined antisera known to react with the immunodominant epitope, such as, by an ELISA or Western blot, for example. Candidates demonstrating reduced reactivity with those defined antisera are chosen for testing in vivo to determine whether those dampened antigens are immunogenic and the host generates an immune response thereto. Hence, for example, a mouse or other animal is immunized with the dampened or disabled antigen as known in the art, serum is obtained and is tested in an in vitro assay for reactivity therewith. That antiserum then can be tested on wildtype virus to determine if the antibody still recognizes the wild type epitope or the wild type antigen. That can be done, for example, in an ELISA or a Western blot. The latter can be informative, revealing whether the particular immunodominant epitope is bound, and if the antiserum remains reactive with the original pathogen, the size and possibly, the identity of the molecule carrying the epitope reactive with the antiserum.

[0049] Those candidate immunodampened or immunodisabled antigens less or no longer reactive with known antisera that bind to or exhibit cell-mediated immunoreactivity to the parent immunodominant antigen, yet remain immunogenic in hosts, are selected as candidate vaccines for further testing. For example, either the immune antiserum or immune cells can be tested for reactivity with a number of pathogen strains or clades in standardized assays to determine how generic that antibody or reactive cell is, that is, whether the newly recognized epitopes on the dampened antigen are generic to a wide range of pathogen strains, clades, variants, varieties and so on, and if the antibody or cell has antipathogen activity.

02a31b28 19 [0050] Many techniques are available to one of ordinary skill in the art to permit manipulation of immunogenic structures. The techniques can involve substitution of various amino acid residues at a site of interest, followed by a screening analysis of binding of the mutein to defined, known antibody that binds to one or more immunodominant epitopes of HIV. For example, a polypeptide can be synthesized to contain one or more changes to the primary amino acid sequence of the immunodominant epitope. Alternatively, the nucleic acid sequence of the

immunodominant epitope can be modified to express an immunodampened epitope. Hence, the nucleic acid sequence can be modified by, for example, site-directed mutagenesis to express amino acid substitutions, insertions, deletions and the like, some of which may introduce further modification at or near the immunodominant epitope, such as, introducing a glycosylation site, such as, mutations which cause N-glycosylation or O-glycosylation at or near the immunodominant epitope and so on.

[0051] One procedure for obtaining epitope mutants or muteins (a mutant epitope that varies from wildtype) and the like is "alanine scanning mutagenesis" (Cunningham & Wells, Science 244: 1081-1085 (1989); and Cunningham & Wells, Proc Nat. Acad Sci USA 84:6434-6437 (1991)). One or more residues are replaced by alanine or polyalanine residue(s). Those residues demonstrating functional sensitivity to the substitutions then can be refined by introducing further or other mutations at or for the sites of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. Similar substitutions can be attempted with other amino acids, depending on the desired property of the scanned residues.

02a31b28 20 [0052] A more systematic method for identifying amino acid residues to modify comprises identifying residues involved in immune system stimulation or immunodominant antibody recognition and those residues with little or no

involvement with immune system stimulation or immunodominant antibody recognition. An alanine scan of the involved residues is performed, with each Ala mutant tested for reducing immune system stimulation to an immunodominant epitope or immunodominant antibody recognition. In another embodiment, those residues with little or no involvement in immune system stimulation are selected to be modified. Modification can involve deletion (removal) or substitution of a residue or an amino acid, or insertion (addition) of one or more residues or amino acids adjacent to a residue or amino acid of interest. However, normally the modification involves substitution of the residue by another amino acid. A conservative substitution can be a first substitution. If such a substitution results in reduction of immune system stimulation or reduced reactivity with known immunodominant antibody, then another conservative substitution can be made to determine if more substantial changes are obtained.

[0053] Even more substantial modification in the ability to alter the immune system response away from the immunodominant epitope can be accomplished by selecting an amino acid that differs more substantially in properties from that normally resident at a site. Thus, such a substitution can be made while maintaining: (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation; (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.

02a31b28 21 [0054] For example, the naturally occurring amino acids can be divided into groups based on common side chain properties:

[0055] (1) hydrophobic: methionine (M or met), alanine (A or ala), valine (V or val), leucine (L or leu) and isoleucine (I or ile);

[0056] (2) neutral, hydrophilic: cysteine (C or cys), serine (S or ser), threonine (T or thr), asparagine (N or asn) and glutamine (Q or gin);

[0057] (3) acidic: aspartic acid (D or asp) and glutamic acid (E or glu);

[0058] (4) basic: histidine (H or his), lysine (K or lys) and arginine ( or arg);

[0059] (5) residues that influence chain orientation: glycine (G or gly) and proline (P or pro), and

[0060] (6) aromatic: tryptophan (W or trp), tyrosine (Y or tyr) and

phenylalanine (F or phe).

[0061] Some amino acids can fit more than one of the above categories, which is not intended to be the only means to classify amino acids.

[0062] Non-conservative substitutions can entail exchanging an amino acid with an amino acid from another group. Conservative substitutions can entail exchange of one amino acid for another from within a group.

[0063] Preferred amino acid substitutions are those which dampen or disable an immunodominant epitope, but can also include those which, for example:

(1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter immune system stimulating activity and/or (4) confer or modify other

physico-chemical or functional properties of such analogs. Analogs can include various muteins of a sequence other than the naturally occurring peptide sequence.

02a31b28 22 For example, single or multiple amino acid substitutions (such as conservative amino acid substitutions) may be made in the naturally occurring sequence. A conservative amino acid substitution generally should not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or to disrupt other types of secondary structure that characterizes the parent sequence) unless of a change in the bulk or conformation of the R group or side chain (Proteins, Structures and Molecular Principles (Creighton, ed., W. H. Freeman and Company, New York (1984);

Introduction to Protein Structure, Branden & Tooze, eds., Garland Publishing, New York, NY (1991)); and Thornton et al. Nature 354: 105 (1991)).

[0064] Ordinarily, the epitope mutant with altered biological and/or structural properties will have an amino acid sequence having at least 75% amino acid sequence identity or similarity with the amino acid sequence of the parent molecule, at least 80%, at least 85%, at least 90% and often at least 95% identity. Identity or similarity with respect to parent amino acid sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical (i.e., same residue) or similar (i.e., amino acid residue from the same group based on common side chain properties, supra) with the parent molecule residues, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Nevertheless, a sequence identity of less than 75% is not impossible so long as the altered epitope is immunodisabled as taught and determined herein and retains function, if present the parent molecule. Hence, sequence identity can be less than 75%o, less than 70%, less than 65%, less than 60%>, less than 55%, less than 50%, less

02a31b28 23 than 45%, less than 40%, and so on so long as immunodisabling of the epitope is achieved without loss of function.

[0065] Covalent modifications of the molecules of interest are included within the scope of the invention. Such may be made by chemical synthesis or by enzymatic or chemical cleavage of the molecule, if applicable. Other types of covalent modifications of the molecule can be introduced into the molecule by reacting targeted amino acid residues of the molecule with an organic derivatizing agent that is capable of reacting with selected side chains or with the N-terminal or C-terminal residue.

[0066] In cases where the immunodominant epitope to be immune modified is unable to be precisely mapped, the use of an antibody that is naturally derived from infection or by immunization to the immunodominant epitopes as either a polyclonal or monoclonal antibody can be used.

[0067] For all embodiments disclosed herein relating to an antibody or an immunoglobulin, an Ab or an Ig molecule can be one from any of the Ig family of molecules, IgG, IgA, IgM, IgD or IgE, or equivalents from other animals including birds, camels or invertebrates (any living organism capable of making an antibody, an antibody-like molecule or an antigen-binding molecule), or minimal fragments (F_ab₂, F_ab, or mini body light or heavy chain pieces etc.) of the epitope-paratope antibody binding site can be used. The antibody or epitope-binding fragments thereof can be derived naturally or experimentally from the whole Ig, enzymatic cleavage or synthetically using either peptide chemistry or recombinant protein expression technology. Hence, the antibody is made as known in the art, such as by immunizing

02a31b28 24 an animal, recombinant expression, phage display and so on, and modified, such as, truncated, as known in the art.

[0068] Also, various organic chemistry methods and materials can be practiced to modify a component of an epitope. For example, WO05/35726 teaches various methods for introducing, modifying, changing, replacing and so on substituents found on biomolecules.

[0069] Cysteinyl residues can be reacted with a-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to yield carboxylmethyl or carboxyamidomethyl derivatives. Cysteinyl residues also can be derivatized by reaction with bromotrifiuoroacetone, a-bromo-P-(5- imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2- pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate,

2-chloromercura-4-nitrophenol or chloro-7-nitrobenzo-2-oxa-l,3-diazole, for example.

[0070] Histidyl residues can be derivatized by reaction with

diethylpyrocarbonate at pH 5.5-7.0. p-Bromophenacyl bromide also can be used, the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.

[0071] Lysinyl and a terminal residues can be reacted with succinic or other carboxylic acid anhydrides to reverse the charge of the residues. Other suitable reagents for derivatizing a amino-containing residues include imidoesters, such as, methyl picolinimidate, pyridoxal phosphate, pyridoxal chloroborohydride, trmitrobenzenesulfonic acid, O-methylisourea and 2,4-pentanedione, and the amino acid can be transaminase-catalyzed with glyoxylate.

02a31b28 25 [0072] Arginyl residues can be modified by reaction with one or several conventional reagents, such as, phenylglyoxal, 2,3-butanedione,

1 ,2-cyclohexanedione and ninhydrin. Derivatization of arginine residues often requires alkaline reaction conditions. Furthermore, the reagents may react with lysine as well as the arginine ε-amino group.

[0073] The specific modification of tyrosyl residues can be made with aromatic diazonium compounds or tetranitromethane. For example,

N-acetylimidizole and tetranitromethane can be used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosyl residues can be iodinated using methods known in the art.

[0074] Carboxyl side groups (for example, of aspartyl or glutamyl residues) can be modified by reaction with carbodiimides (R-N=C=C-R'), where R and R' can be different alkyl groups, such as l-cyclohexyl-3-(2-morpholinyl-4-ethyl)

carbodiimide or l-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore, aspartyl and glutamyl residues can be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

[0075] Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues, respectively, under neutral or basic conditions. The deamidated form of those residues falls within the scope of the instant invention.

[0076] Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of serinyl or threonyl residues, methylation of the a amino groups of lysine, arginine, and histidine (Creighton, Proteins: Structure and

02a31b28 26 Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)), and acetylation of the N-terminal amine and amidation of any C-terminal carboxyl group.

[0077] Another type of covalent modification involves chemically or enzymatically coupling glycosides to the molecules of interest. Depending on the coupling mode used, the sugar(s) may be attached to: (a) arginine and histidine; (b) free carboxyl groups; (c) free sulfhydryl groups, such as those of cysteine; (d) free hydroxyl groups, such as those of serine, threonine or hydroxyproline; (e) aromatic residues such as those of phenylalanine, tyrosine or tryptophan; or (f) the amide group of glutamine. Such methods are described in WO 87/05330 and in Aplin & Wriston, C C Crit Rev Biochem, pp. 259-306 (1981). For example, a polyglycol can be appended to an amino acid.

[0078] Removal of any carbohydrate moieties present on the molecule of interest may be accomplished chemically or enzymatically. Chemical

deglycosylation, for example, can require exposure of the molecule to the compound, trifluoromethanesulfonic acid, or an equivalent compound, resulting in cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or

N-acetylgalactosamine), while leaving the remainder of the molecule intact.

Chemical deglycosylation is described, for example, in Hakimuddin et al., Arch Biochem Biophys 259:52 (1987) and in Edge et al, Anal Biochem 118: 131 (1981). Enzymatic cleavage of carbohydrate moieties on molecules can be achieved by any of a variety of endoglycosidases and exoglycosidases as described, for example, in Thotakura et al., Meth Enzymol 138:350(1987). Thus, a mannosidase, a fucosidase, a glucosaminosidase, a galactosidase and so on can be used.

02a31b28 27 [0079] RNA or DNA encoding the g l20, gp 160 and the like of HIV is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to the relevant genes, Innis et al. in PCR Protocols. A Guide to Methods and Applications, Academic (1990), and Sanger et al, Proc Natl Acad Sci 74:5463 (1977)). Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells, such as, E. coli cells, NS0 cells, insect cells, prokaryotes, COS cells, Chinese hamster ovary (CHO) cells or myeloma cells, to obtain synthesis of the protein of interest in the recombinant host cells. The RNA or DNA also may be modified, for example, by substituting bases to optimize for codon usage in a particular host or by covalently joining to the coding sequence of a heterologous polypeptide. Such an approach could be the basis for developing a subunit vaccine.

[0080] Thus, in one embodiment, the gpl20 of HIV was selected as a target for refocusing the host immune response to other nondominant sites on gpl20 as novel targets for an immunoprotective response, preferably one of broad scope and spectrum active on a wide variety of strains, clades and so on.

[0081] As taught herein, the above alterations to immunodominant decoy epitopes or dysregulatory sites (receptotopes) can be obtained by manipulating nucleic acids encoding an immunodominant epitope, such as, by cloning, site-directed mutagenesis, amplification and so on as known in the art. Similarly RNA, proteins, structures and domains can be altered by site-specific mutation to nucleotides that participate in the formation of the structure or domain. In some cases, mutations,

02a31b28 28 such as nucleotide base substitutions, can be made without changing the sequence of the encoded protein, (e.g silent mutation-synonymous), essentially recoding.

[0082] To obtain approval from regulatory agencies, such as the US Food and Drug Administration or European Medicines Agency for human products and the US Department of Agriculture for veterinary products, biological pharmaceutics must meet purity, safety and potency standards defined by the pertinent regulatory agency. To produce a vaccine that meets those standards, the recombinant organisms should be maintained in culture media that is, for example, certified free of transmissible spongiform encephalopathies (herein referred to as "TSE").

[0083] Preferably, plasmids harboring the vaccine-encoding sequence carry a non-antibiotic selection marker, since it is not always ideal to use antibiotic resistance markers for selection and maintenance of plasmids in hosts. In one embodiment, therefore, the present invention provides a selection strategy in which, for example, a catabolic enzyme is utilized as a selection marker by enabling the growth of bacteria in medium containing a substrate of said catabolic enzyme as a carbon source. An example of such a catabolic enzyme includes, but is not restricted to, lacYZ encoding lactose uptake and β-galactosidase (Genbank Nos. J01636, J01637, K01483 or K01793). Other selection markers that provide a metabolic advantage in a defined medium include, but are not restricted to, galTK (GenBank No. X02306) for galactose utilization, sacPA (GenBank No. J03006) for sucrose utilization, trePA (GenBank No. Z54245) for trehalose utilization, xylAB (GenBank No. CAB 13644 and

AAB41094) for xylose utilization etc. Alternatively, the selection can involve the use

02a31b28 29 of antisense mRNA to inhibit a toxic allele, such as the sacB allele (GenBank No. NP_391325).

[0084] In some embodiments, the administered product is a nucleic acid encoding a modified epitope of interest. In other embodiments, the administered product is a nucleic acid vector carrying the coding sequence of a modified epitope of interest. Hence, once administered, the nucleic acid vector then expresses the modified epitope. A suitable vector can be an adenoviral vector, an influenza vector, another viral vector, a bacterial vector, a yeast vector, a mammalian cell vector and so on. In some embodiments, a modified epitope of interest is produced in an edible product for ingestion by a target host to be immunized, such as a plant, a food and so on, such as a vegetable, a fruit, a milk, a milk product, a grain, a grain product and so on.

[0085] The modified antigen or epitope of interest then is manipulated using materials and methods known in the art. Often, the materials and methods relate to recombinant methods. The antigen of interest can be secreted or excreted in a culture medium, can be cell bound or can be contained within a cell. Standard procedures are practiced to isolate the product of interest.

[0086] The immunogen of the present invention may be used to treat a mammal. The immunogen of interest can be administered to a nonhuman mammal for the purpose of obtaining preclinical data or for treatment, for example. Exemplary nonhuman mammals include nonhuman primates, dogs, cats, livestock, such as, cattle, goat, sheep, rabbits and so on, poultry, such as, domesticated fowl, turkey and duck, rodents and other mammals. Such mammals may be established animal models for a

02a31b28 30 disease or may be used to study toxicity of the immunogen of interest. In each of those embodiments, dose escalation studies may be performed in the mammal. A product of the invention of interest can be used to treat same.

[0087] The specific method used to formulate the novel vaccines and formulations described herein is not critical to the present invention and can be selected from materials and methods known in the art. Hence, a formulation of interest can include a physiological buffer (U.S. Pat. No. 5,589,466) or other suitable carrier.

[0088] It may be desirable to further provide an adjuvant. Such molecules suitably are present in combination in amounts that are effective for the purpose intended. The adjuvant can be administered sequentially, before or after antigen administration or simultaneously therewith. Examples include aluminum phosphate or aluminum hydroxyphosphate (e.g. Ulmer et al., Vaccine, 18: 18 (2000)), monophosphoryl-lipid A (also referred to as MPL or MPLA; Schneerson et al., J. Immunol, 147:2136-2140 (1991); e.g. see Sasaki et al, Inf. Immunol, 65:3520-3528

(1997) ; and Lodmell et al, Vaccine, 18: 1059-1066 (2000)), QS-21 saponin (e.g.

Sasaki et al, J. Virol, 72:4931 (1998)); dexamethasone (e.g., Malone et al, J. Biol. Chem. 269:29903 (1994)); CpG DNA sequences (Davis et al, J. Immunol, 15:870

(1998) ); interferon-a (Mohanty et al, J. Chemother. 14(2): 194-197, (2002)), lipopolysaccharide (LPS) (Hone et al, J. Human Virol, 1 : 251-256 (1998)) and so on.

[0089] The formulation herein also may contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely impact each other. Hence, a

02a31b28 31 compound of interest can be administered with another antiviral compound. In some embodiments, a compound of interest is administered with an antibody or

antigen-binding portion thereof that specifically binds said compound of interest. In some embodiments a compound of interest and antibody or antigen-binding portion thereof are administered as a complex wherein said antibody is bound to said compound of interest.

[0090] Thus, the immunogen of interest can be used with a second

component, such as a therapeutic moiety conjugated to or mixed with same, administered as a conjugate, separately in combination, mixed prior to use and so on as a therapeutic, see, for example, Levine et al., eds., New Generation Vaccines. 2^nd Marcel Dekker, Inc., New York, NY, 1997). The therapeutic moiety can be any drug, vaccine and the like used for an intended purpose. Thus, the therapeutic moiety can be a biological, a small molecule and so on. The immunogen of interest can be administered concurrently or sequentially with a second HIV immunogenic composition, immunodampened or not, for example. Thus, an immunodampened antigen of interest can be combined with an existing vaccine, although that approach would minimize the use thereof if the existing vaccine, for example, is made in eggs, or an administered formulation can comprise two or more immunodampened epitopes.

[0091] The term "small molecule" and analogous terms include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogues,

polynucleotides, polynucleotide analogues, carbohydrates, lipids, nucleotides, nucleotide analogues, organic or inorganic compounds (i.e., including heterorganic

02a31b28 32 and/organometallic compounds), which can have a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds, which can have a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds, which can have a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds, which can have a molecular weight less than about 500 grams per mole, and salts, esters, combinations thereof and other pharmaceutically acceptable forms of such compounds which stimulate an immune response or are immunogenic, or have a desired pharmacologic activity.

[0092] Thus, the immunogen of the invention may be administered alone or in combination with other types of treatments, including a second immunogen or a treatment for the disease being treated. The second component can be an

immunostimulant.

[0093] In addition, the immunogen of the instant invention may be conjugated to various effector molecules such as heterologous polypeptides, drugs,

radionucleotides and so on, see, e.g., WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EPO 396,387. An immunogen may be conjugated to a therapeutic moiety such as an antibiotic (e.g., a therapeutic agent or a radioactive metal ion (e.g., a emitters such as, for example, ²¹³Bi)), an adjuvant or a targeting molecule.

[0094] Therapeutic compounds of the invention alleviate at least one symptom associated with a disease, disorder, or condition associated with HIV amenable for treatment with an immunogen of interest. The products of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as

02a31b28 33 described herein. The terms "physiologically acceptable," "pharmacologically acceptable" and so on mean approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized

Pharmacopeia for use in animals and more particularly in humans.

[0095] The products of interest can be administered in any acceptable manner. Methods of introduction include, but are not limited to, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intranasal, epidural, inhalation and oral routes, and if desired for immunosuppressive treatment, intralesional administration. Parenteral infusions include intramuscular, intradermal, intravenous, intraarterial or

intraperitoneal administration. The products or compositions may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal, genital and intestinal mucosa etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the therapeutic products or compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an

intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. In addition, the product can be suitably administered by pulse infusion, particularly with declining doses of the products of interest. The dosing can be given by injection, such as, intravenous or subcutaneous injections, depending, in part, on whether the administration is brief or chronic.

02a31b28 34 [0096] Various other delivery systems are known and can be used to administer a product of the present invention, including, e.g., encapsulation in liposomes, microparticles, microcapsules (see Langer, Science 249: 1527 (1990); Liposomes in the Therapy of Infectious Disease and Cancer, Lopez -Berestein et al., eds., (1989)), cochleates or other synthetic carrier structure or compound.

[0097] The active ingredients may be entrapped in a microcapsule prepared, for example, by coascervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsule and

poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, A. Osal, Ed. (1980).

[0098] Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. The composition of interest may also be administered into the lungs of a patient in the form of a dry powder composition, see e.g., U.S. Pat. No. 6,514,496.

[0099] It may be desirable to administer the therapeutic products or compositions of the invention locally to the area in need of treatment; that may be achieved by, for example, and not by way of limitation, local infusion, topical application, by injection, by means of a catheter, by means of a suppository or by means of an implant, said implant being of a porous, non-porous or gelatinous material, including hydrogels or membranes, such as silastic membranes or fibers.

02a31b28 35 Preferably, when administering a product of the invention, care is taken to use materials to which the protein does not absorb or adsorb.

[00100] The compositions can take the form of solutions, suspensions, an emulsion, tablets, pills, capsules, powders, sustained release formulations, depots and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate etc. Examples of suitable carriers are described in "Remington's Pharmaceutical Sciences," Martin. Such compositions will contain an effective amount of the immunogen preferably in purified form, together with a suitable amount of carrier so as to provide the proper and desired form for administration to the patient. As known in the art, the formulation will be constructed to suit the mode of administration.

[00101] In yet another embodiment, the product can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, Science 249: 1527 (1990); Sefton, CRC Crit Ref Biomed Eng 14:201 (1987);

Buchwald et al, Surgery 88:507 (1980); and Saudek et al, N Engl J Med 321 :574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer et al., eds., CRC Press (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen et al., eds., Wiley (1984); Ranger et al, J Macromol Sci Rev Macromol Chem 23:61 (1983); see also Levy et al, Science 228: 190 (1985); During et al, Ann Neurol 25:351 (1989);

02a31b28 36 and Howard et al., J Neurosurg 71 :105 (1989)). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target.

[00102] Sustained release preparations may be prepared for use with the products of interest. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the immunogen, which matrices are in the form of shaped articles, e.g., films or matrices. Suitable examples of such sustained release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethylmethacrylate), poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (such as injectable microspheres composed of lactic acid-glycolic acid copolymer) and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release cells, proteins and products for and during shorter time periods. Rational strategies can be devised for stabilization depending on the mechanism involved. Thus, in some embodiments, an amino acid substitution or other change can stabilize an epitope, particularly if there is another change therein which might disrupt the normal secondary, tertiary or quaternary structure of the protein carrying said epitope, said epitope or regions adjacent to said epitope.

[00103] Therapeutic formulations of the product may be prepared for storage as lyophilized formulations or aqueous solutions by mixing the product having the desired degree of purity with optional pharmaceutically acceptable carriers, diluents, excipients or stabilizers typically employed in the art, i.e., buffering

02a31b28 37 agents, stabilizing agents, preservatives, isotonifiers, nonionic detergents, antioxidants and other miscellaneous additives, see Remington's Pharmaceutical Sciences, 16th ed., Osol, ed. (1980). Such additives are generally nontoxic to the recipients at the dosages and concentrations employed, hence, the excipients, diluents, carriers and so on are pharmaceutically acceptable.

[00104] An immune refocused polypeptide (which includes an antigen, a portion thereof, an epitope, a determinant and so on) which can be produced as a subunit substantially free of contaminating proteins, including other pathogen proteins, in combination with other viral or non-viral polypeptides; as an

immunodisabled polypeptide of interest which can be expressed or produced in recombinant viruses, VLP's or in combination with one or more proteins of virus or cell origin; as an immunodisabled polypeptide which can be expressed or produced as an isolated molecule and then combined with one or more proteins of virus or cell origin; and so on); can be obtained or made in substantially pure form. Alternatively, the epitope of interest can be expressed on a structure and used as is, for example, on a virion, a virus-like particle, a liposome, other carrier molecule, for example, constructed of saccharides or polypeptides, or combinations thereof and so on.

Hence, an epitope of interest can be incorporated into any known delivery means.

[00105] An "isolated" or "purified" immunogen is substantially free of contaminating proteins from the medium from which the immunogen is obtained, or substantially free of chemical precursors or other chemicals in the medium used which contains components that are chemically synthesized. The language

"substantially free of subcellular material" includes preparations of a cell in which the

02a31b28 38 cell is disrupted to form a component mixture which can be separated from

subcellular components of the cells, including dead cells, and portions of cells, such as cell membranes, ghosts and the like, from which the immunogen is isolated or recombinantly produced. Thus, an immunogen that is substantially free of subcellular material includes preparations of the immunogen having less than about 30%, 20%, 25%o, 20%o, 10%), 5%o, 2.5%. or 1%>, (by dry weight) of subcellular contaminants or any other element that differs from the product of interest.

[00106] As used herein, the terms "stability" and "stable" in the context of a liquid formulation comprising an immunogen refer to the resistance of the immunogen in a formulation to, for example, thermal and chemical aggregation, degradation, fragmentation or other change which degrades an intended function or functions under given manufacture, preparation, transportation and storage conditions, such as, for one week, for two weeks, for three weeks, for one month, for two months, for three months, for four months, for five months, for six months or more. The "stable" formulations of the invention retain biological activity equal to or more than 80%, 85%, 90%, 95%, 98%, 99% or 99.5% of the original measured property under given manufacture, preparation, transportation and storage conditions over the above-noted time periods. The stability of said immunogen preparation can be assessed by degrees of aggregation, degradation or fragmentation by methods known to those skilled in the art, including, but not limited to, physical observation, such as, with a microscope, particle size and count determination and so on, compared to a reference. The metric can be a biologic property or activity.

02a31b28 39 [00107] The instant invention encompasses formulations, such as, liquid formulations that are maintained or stored at temperatures found in a commercial refrigerator and freezer found in the office of a physician or laboratory, such as from about -20° C to about 5° C, said stability assessed, for example, by microscopic analysis, for storage purposes, such as for about 7 days, 10 days, 14 days, 20 days, 30 days, 45 days, 60 days, for about 120 days, for about 180 days, for about a year, for about 2 years or more. The liquid formulation of the present invention also exhibit stability, as assessed, for example, by particle analysis, at room temperatures, for at least a few hours, such as one hour, two hours or about three hours prior to use.

Stability can also be measured using a biologic or functional property.

[00108] The term, "carrier," refers to a diluent, adjuvant, excipient or vehicle with which the therapeutic is administered. Such physiological carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a suitable carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions also can be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

02a31b28 40 [00109] Any carrier, diluent or excipient, while being pharmaceutically acceptable, also can be further treated, for example, to be hypoallergenic.

[00110] Buffers are preferably present at a concentration ranging from about 0.1 mM to about 50 mM. Suitable buffering agents for use with the instant invention include both organic and inorganic acids, and salts thereof, such as citrate buffers (e.g., monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-monosodium citrate mixture etc.), succinate buffers (e.g., succinic acid-monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid- sodium hydroxide mixture etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture, fumaric acid-disodium fumarate mixture, monosodium fumarate- disodium fumarate mixture etc.), gluconate buffers (e.g., gluconic acid-sodium glyconate mixture, gluconic acid-sodium hydroxide mixture, gluconic acid-potassium gluconate mixture etc.), oxalate buffers (e.g., oxalic acid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture etc.), lactate buffers (e.g., lactic acid-sodium lactate mixture, lactic acid-sodium hydroxide mixture, lactic acid-potassium lactate mixture etc.) and acetate buffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide mixture etc.). Phosphate buffers, carbonate buffers, histidine buffers, trimethylamine salts, such as Tris, HEPES and other such known buffers can be used.

[00111] The present invention can provide liquid formulations of an immunogen having a pH ranging from about 5.0 to about 7.0; about 5.5 to about 6.5;

02a31b28 41 about 5.8 to about 6.2; about 6.0; about 6.0 to about 7.5; about 6.5 to about 7.0; and so on.

[00112] Preservatives may be added to retard microbial growth, and may be added in amounts ranging from 0.1%-1% (w/v). Suitable preservatives for use with the present invention include phenol, benzyl alcohol, m-cresol,

octadecyldimethylbenzyl ammonium chloride, benzylconium halides (e.g., chloride, bromide and iodide), hexamethonium chloride, alkyl parabens, such as, methyl or propyl paraben, catechol, resorcinol, cyclohexanol and 3-pentanol.

[00113] Isotonicifiers are present to ensure physiological isotonicity of liquid compositions of the instant invention and include polyhydric sugar alcohols, such as, trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol. Polyhydric alcohols can be present in an amount of between about 0.1% to about 25%, by weight, about 1% to about 5%>, and so on, taking into account the relative amounts of the other ingredients.

[00114] Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which solubilizes the therapeutic agent or helps to prevent denaturation or adherence to the container wall. Typical stabilizers can be polyhydric sugar alcohols; amino acids, such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine,

2-phenylalanine, glutamic acid, threonine etc.; organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, arabitol, erythritol, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol and the like, including cyclitols such as inositol; polyethylene glycol; amino acid polymers; sulfur-containing reducing agents, such as

02a31b28 42 urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol,

a-monothioglycerol and sodium thiosulfate; low molecular weight polypeptides (i.e., <10 residues); proteins, such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone, saccharides, monosaccharides, such as xylose, mannose, fructose or glucose;

disaccharides, such as lactose, maltose and sucrose; trisaccharides, such as raffinose; polysaccharides, such as, dextran and so on. Stabilizers can be present in the range from 0.1 to 10,000 w/w per part of immunogen.

[00115] Additional miscellaneous excipients include bulking agents,

(e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine or vitamin E) and cosolvents.

[00116] As used herein, the term "surfactant" refers to organic substances having amphipathic structures, namely, are composed of groups of opposing solubility tendencies, typically an oil-soluble hydrocarbon chain and a water-soluble ionic group. Surfactants can be classified, depending on the charge of the surface-active moiety, into anionic, cationic and nonionic surfactants. Surfactants often are used as wetting, emulsifying, solubilizing and dispersing agents in pharmaceutical compositions and preparations of biological materials.

[00117] Nonionic surfactants or detergents (also known as "wetting agents") may be added to help solubilize the therapeutic agent, as well as to protect the therapeutic protein against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stresses without causing denaturation of the protein. Suitable nonionic surfactants include polysorbates (20, 80 etc.),

02a31b28 43 polyoxamers (184, 188 etc.), Pluronic polyols and polyoxyethylene sorbitan monoethers (TWEEN-20^®, TWEEN-80^® etc.). Nonionic surfactants may be present in a range of about 0.05 mg/ml to about 1.0 mg/ml, about 0.07 mg/ml to about 0.2 mg/ml, and so on.

[00118] As used herein, the term, "inorganic salt," refers to any compound, containing no carbon, that results from replacement of part or all of the acid hydrogen or an acid by a metal or a group acting like a metal, and often is used as a tonicity adjusting compound in pharmaceutical compositions and preparations of biological materials. Common inorganic salts are NaCl, KC1, NaH₂P0₄ etc.

[00119] Examples of diluents include a phosphate-buffered saline, buffer for buffering against gastric acid in the bladder, such as citrate buffer (pH 7.4) containing sucrose, bicarbonate buffer (pH 7.4) alone, or bicarbonate buffer (pH 7.4) containing ascorbic acid, lactose or aspartame. Examples of carriers include proteins, e.g., as found in skim milk, sugars, e.g., sucrose, or polyvinylpyrrolidone. Typically these carriers would be used at a concentration of about 0.1-90% (w/v) but also at a range of 1-10% (w/v).

[00120] The formulations to be used for in vivo administration must be sterile. That can be accomplished, for example, by filtration through sterilization or filtration membranes. For example, the subcellular formulations of the present invention may be sterilized by filtration.

[00121] The immunogen composition will be formulated, dosed and administered in a manner consistent with good medical practice. Factors for consideration include the particular disorder being treated, the particular mammal

02a31b28 44 being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to practitioners.

[00122] The amount of antigen is not critical to the present invention but is typically an amount sufficient to induce the desired humoral and/or

cell-mediated immune response in the target host. The amount of immunogen of the present invention to be administered will vary depending on the species and the physical properties and characteristics of the subject, as well as the disease or condition that is being treated. Generally, the dosage employed can be about

10-1500 μg/dose.

[00123] As used herein, the term "effective amount" refers to the amount of a therapy (e.g., a prophylactic or therapeutic agent), which is sufficient to reduce the severity and/or duration of a targeted disease, ameliorate one or more symptoms thereof, prevent the advancement of a targeted disease or cause regression of a targeted disease, or which is sufficient to result in the prevention of the development, recurrence, onset, or progression of a targeted disease or one or more symptoms thereof. For example, a treatment of interest can increase survivability of the host, based on baseline or a normal level, by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%. In another embodiment, an effective amount of a therapeutic or a prophylactic agent reduces the symptoms of a targeted disease, such as a symptom of HIV by at least 5%, preferably at least 10%,

02a31b28 45 at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.

[00124] Also used herein as an equivalent is the term, "therapeutically effective amount." In the context of the instant invention, an effective amount is one which generates in an in vitro system a specific immune response, whether humoral or cellular. Thus, if an immunodisabled peptide reacts with a specific HIV antibody or a cell reacts to or with HIV-infected cells (for example, as evidenced by DNA synthesis), then an effective amount is that which provides a response above background. Similarly, and independently, an effective amount is that which generates in vivo, in a host of design choice, a specific immune response to the disabled peptide, be that response humoral or cellular.

[00125] Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine or other "caine" anesthetic to ease pain at the site of the injection.

[00126] Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or concentrate in a sealed container, such as an ampule or sachet indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampule of sterile water for injection or saline can be provided, for example, in a kit, so that the ingredients may be mixed prior to administration.

02a31b28 46 [00127] An article of manufacture containing materials useful for the treatment of the disorder described above is provided. The article of manufacture can comprise a container and a label. Suitable containers include, for example, bottles, vials, syringes and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is effective for preventing or treating a targeted condition or disease and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The label on or associated with the container indicates that the composition is used for treating the condition of choice. The article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as, water, phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes and package inserts with instructions for use.

[00128] The instant invention also includes kits, e.g., comprising an immunogenic composition of interest, homolog, derivative thereof and so on, for use, for example, as a vaccine, and instructions for the use of same and so on. The instructions may include directions for using the composition, derivative and so on. The composition can be in liquid form or presented as a solid form, for example, desiccated or lyophilized. The kit can contain suitable other reagents, such as a buffer, a reconstituting solution and other necessary ingredients for the intended use. A packaged combination of reagents in predetermined amounts with instructions for

02a31b28 47 use thereof, such as for a therapeutic use is contemplated. In addition, other additives may be included, such as, stabilizers, buffers and the like. The relative amounts of the various reagents may be varied to provide for concentrates of a solution of a reagent, which provides user flexibility, economy of space, economy of reagents and so on.

[00129] Citation of any of the references discussed hereinabove shall not be construed as an admission that any such reference is prior art to the present invention. All references cited herein are herein incorporated by reference in entirety.

[00130] The invention now will be exemplified in the following non-limiting examples.

Example 1

[00131] Serologic data from CM235 isotype (Genbank accession number L03698) immunized baboons are used to identify immunodominant epitopes within gpl20, especially those within linear domains. Analysis using 118 12mer CM235 envelope peptides overlapping by 8 amino acids show 4 immunodominant epitopes including the V2 and V3 domains, V2 being the most immunodominant. In this example, both V2 and V3 domains are targeted for immunodampening. In other examples, additional domains of the gpl60 glycoprotein are targeted for immunodampening.

[00132] The V2 loop in CM235 is delineated by two cysteines:

161 171 181 191 201

CSFNMTTEL DKKQKVHALF YKLDIVPIED NKTSSEYRLI NC (V2) (SEQ ID NO: l)

02a31b28 48 [00133] Baboons immunized with E-g l20 (E clade) are immunoreactive with V2 peptides beginning at amino acids 166, 170 and 174 of SEQ ID NO: 1. Of those, peptide 170 is the most immunoreactive. The data suggest an immunodominant epitope within the 12 amino acids of peptide 170. peptide 166 TTELRDKKQKVH (residues 6-17 of SEQ ID NO: 1) peptide 170 RDKKQKVHALFY (residues 10-21 of SEQ ID NO: 1) peptide 174 QKVHALFYKLDI (residues 14-25 of SEQ ID NO: 1)

[00134] Immune dampening strategies are developed to completely dampen the VI /V2 immunodominant domain. Examples of immune dampened mutations of this region are provided in Table 1 in Figures 1 and 2.

[00135] The V3 domain of CM235 is:

302 05 10 15 20 25 30 35

CTR PSNNTR TSIPI GPGQA FYRTG DIIGD IRKAYC

(SEQ ID NO:2)

[00136] Immunodominant V3 peptides identified by the CM235 immune sera from baboons include: peptide 306 SNNTRTSIPIGP (residues 5-16 of SEQ ID NO: 2) peptide 310 RTSIPIGPGQAF (residues 9-20 of SEQ ID NO: 2) peptide 314 PIGPGQAFYRTG (residues 13-24 of SEQ ID

NO: 2 [00137] Immune dampening strategies are developed to completely dampen the V3 immunodominant domain. Examples of immune dampened mutations of V3 are depicted in Table 2 in Figures 3-6.

Example 2

[00138] Genes and gene segments encoding unmodified, wildtype gpl20, gpl40, and gpl60 Clade E envelope genes are cloned into the pC II cloning vector for ease of manipulation. Negative sense oligonucleotide primers containing artificial translational stop codons are used in PCR cloning amplifications to synthesize gpl60 gene fragments encoding the gpl20 subunit and the gpl40 domain. The wild type constructs and gene products are used as controls for studies in which immune refocused antigens were assessed for stimulation of broadened immune responses.

[00139] Translationally silent mutations containing unique restriction sites are introduced at nucleotides 238 (BamHI), 620 (Sphl), and 1010 (Kpnl) to facilitate insertion of subgenic fragments in which immune refocused mutations were engineered.

[00140] After immune refocusing mutations are introduced into the gpl20, gpl40, and gpl60 genes, they are subcloned into the pSC65 vaccinia virus transfer vector. Vaccinia virus expression is used as a convenient method for the production of preclinical antigens. The pSC65 vector permits identification of recombinant virus via coexpression of the LacZ gene product and subsequent staining

02a31b28 50 with X-gal. In addition, the pSC65 vector recombines into the TK gene of vaccinia leading to dual selection (TK^" and blue staining plaques) in cell lines lacking thymidine kinase activity.

[00141] To derive recombinant vaccinia virus, HeLa cells are transfected with the pSC65 transfer vector containing the gpl20, gpl40 and gpl60 genes, and then infected with the WR strain of vaccinia virus. After three days, the crude lysates of the cultures are prepared, diluted and used to infect TK^" cell monolayers in the presence of BrdU. Once the infection proceeded, the culture fluids are replaced with agarose overlays containing X-gal and blue recombinant plaques are picked. Plaques are screened for expression of Env proteins. Positive plaques are purified to homogeneity through multiple rounds of plaque purification.

[00142] A series of deletion and multiple point mutations are designed to dampen the immunodominant variable domains of gpl20E. Mutations are designed to introduce codons to direct the addition of glycosylation residues into immunodominant epitopes, replace charged amino acids with uncharged residues, and delete peptides thought to dysregulate immune responses. The deletions are designed to retain some of the proposed structural elements of the glycoproteins. Examples of immune refocused mutations are presented in Table 1. Oligonucleotides used to engineer the mutations are shown in Table 2.

[00143] The mutated gp 120/ 140/ 160 fragments are cloned into the vaccinia transfer vector pSC65 such that a series of gpl20, gpl40, and gpl60 genes are derived. Recombinant vaccinia viruses are engineered by transfecting the pSC65 vectors into HeLa cells as described above.

02a31b28 51 [00144] Recombinant g l60 glycoproteins are expressed as

membrane-bound proteins and are recovered from HeLa cells infected with recombinant vaccinia virus by extraction using buffers containing nonionic detergents such as 20 mM Tris-HCl, pH 8.0 with 0.1% Tween-20. Recombinant gpl20 and gpl40 glycoproteins are expressed as secreted proteins and are recovered from the conditioned culture supernatant.

[00145] Recombinant gpl20, gpl40, and gpl60 glycoproteins are purified using lectin affinity chromatography with lentil lectin sepharose and galanthus nivalis lectin agarose. The glycoproteins are analyzed by immunoblotting, SDS-PAGE and total protein composition.

Example 3

[00146] In addition to mutations shown in the VI, V2, and V3 domains of Example 2, other sites of the gpl60 molecule are targets of immune re focusing. These sites may include amino acids in or near contact points for antibodies that have strain-specific or nonprotective activities, cell receptors, or coreceptors and sites that may contribute to deceptive imprinting.

[00147] For example, a domain of the gp41 region may be a target of immune refocusing by the two mutations, M14 (CSGKIICT (SEQ ID NO: 3) -» CSNGTICT) (SEQ ID NO: 48) and M15 (GLWGCSGKIIC (SEQ ID NO: 4) -> GLAGCSGAIIC) (SEQ ID NO: 49). M14 introduces an N-linked glycosylation site into the C-C loop and Ml 5 introduces nonconservative amino acid substitutions into the same loop. Additional domains of the gp41 region may also be targeted by immune refocusing including an immune refocusing including an immunodominant epitope in the following section of the Clade B sequence: VQQQSNLLRAIEAQQHLLQLTVWGIKQLQARVLAVE RYLKDQKFLGLWGCSGKIICTT (SEQ ID NO:5). Further, an alpha helix structure of gp41 may also be targeted by immune refocusing. The Clade B sequence, SNLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYLK (SEQ ID NO:6) can be immune refocused by changing charged or other residues in either the faces of the helix or the sequences N-terminal or C-terminal to the helix. M22 (QARVLAV ERYLK (SEQ ID NO: 7)-> QAGVLAVQGYLK) (SEQ ID NO: 50) and M23 (LRAIEA (SEQ ID NO: 8) -> LGAIOA) (SEQ ID NO: 51) introduce charge changes to the alpha helix while M24 (LLQLTVWGIKQLQARVL (SEQ ID NO: 9) -> LGQLTVWGIKQLQARGL) (SEQ ID NO: 52) and M25 (LLRAIEAQQ

HLLQLTVWGIKQLQ (SEQ ID NO: 10) -> LGRAIEAQGHLLQLTGWGIKQGQ) (SEQ ID NO: 53) introduce changes to the face of the helix.

[00148] In a similar manner, other regions that have been identified as containing immunodysregulatory or deceptive imprinting sites such as superantigens can be targeted for immune refocusing. A superantigen site in gpl20 of Clade B can be immune refocused with mutations such as Ml 8 (PCKIKQIINM (SEQ ID NO: 11) PCAIVQIINM) (SEQ ID NO: 54).

[00149] The intervening peptide sequence between epitopes thought to convey broadened immunity may be immune dampened. The gpl20 sequence between epitopes of monoclonal antibodies 3b and 2F5 may be immune dampened with modifications such as those of Ml 9

RSYEEIWNNMTWIEWERENYTNQIYEILTESQNQQDRNEKD (SEQIDNO: 12)-» RSYnvtW NMTWIEWqvqNYTNOIYEILTESONOODRNvtD (SEQ ID NO: 55) and M20 (YEEIWNNMTWIEWERENYTNQIYEILTESQNQQDRNEKDLLEL (SEP ID NO: 13) ->

YnvtWNNMTWIqWqvqNYTNOIYglLTqSONOODRNvtDLLgL) (SEQ ID NO: 56).

Example 4

[00150] In addition to separate mutations, immune refocused immunogens may be composed of combinations of mutations at different sites. Thus, Ml 3 may consist of a V1/V2 mutation in the presence of a V3 mutation while Ml 6 may consist of the M13 mutation in the presence of M14 or M15.

Example 5

[00151] The immune refocused Env antigens may be used to raise antibodies that have novel and broadened therapeutic, diagnostic or investigatory properties. Animals, such as, but not restricted to mice, may be immunized with purified gpl20, gpl40, and/or gpl60 glycoproteins by using either single immune refocused antigens or a combination of immune-refocused antigens. Polyclonal or monoclonal antibodies may be derived using methods known to the art. In addition, novel and broadly reactive antibodies, antibody fragments, or their derivatives (such as, minibodies) may be derived from animal or humans that have been exposed, infected or naive to a pathogen, immune refocused or antibody immune refocused or other altered antigens. Thus, TNA or DNA from the immune cells (e.g. B cells, B cell precursors or pluripotential stem cells, for example, is isolated. Relevant genes or gene fragments encoding an immunoglobulin or portions thereof are cloned directly from the nucleic acid or cloned into an expression library for screening with the same or wild-type antigens or other structural mimetic probes.

[00152] The antibodies may be tested for antiviral activities against a panel of HIV- 1 isolates. Antibodies that crossneutralize or otherwise inactivate strains of virus different from the strain from which the antigen had been derived are considered to contain broadened crossreactive activities. The antibodies may be further characterized by antiviral testing against multiple strains with increasing evolutionary distance from the parental strain. Broadly reactive antibodies may be manufactured for use as therapeutics, preventatives, diagnostics, or experimental reagents.

Example 6

[00153] Antibodies may be used to immune refocus antigens by binding to decoy epitopes, receptotopes, immunodominant epitopes or other domains of the antigen. Antibodies raised from either unmodified or immune refocused antigens, or parts thereof, may be used to form antibody-antigen complexes for the purpose of producing a vaccine or deriving an antibody. As described above, immunization with gpl20, gpl40, or gpl60 antigens or infection with virus can stimulate the induction of strain-specific antibodies to the immunodominant epitopes. Thus, antibodies prepared in an immunization or infection process can be purified and bound to the antigen to form an immune stimulatory complex. Immunization with the complex may result in the stimulation of broadened crossprotective immunity. The antibody in the complex function to reduce the accessibility or to coat the immunodominant, strain-specific

02a31b28 55 epitopes so as to refocus the immune system to sites not covered by the bound antibody. The sites not covered may be more highly conserved and elicit broadened immunity.

[00154] Antibody fractions may be used to immune refocus antigens by binding to decoy epitopes. Antibody molecules (e.g., IgG) may be digested with papain or trypsin to yield F_ab or F(_ab)₂ fragments which, due to their smaller size relative to whole IgG, penetrate and bind the strain-specific decoy epitope more efficiently than whole IgG. F_ab or F(_ab)₂ fragments can be complexed with gpl20, gpl40, or gpl60 antigens to form an immune stimulatory complex which may stimulate broadened crossprotective immunity or stimulate the production of antibodies with broadened therapeutic activities.

[00155] Recombinant and single chain antibodies and antibody fragments may be used to immune refocus antigens by binding to decoy epitopes using a similar technique.

Example 7

[00156] The safety, toxicity and potency of recombinant immunogens are evaluated according to the guidelines in 21 CFR 610, which include: (i) general safety test; (ii) stringent safety test in immunocompetent mice; (iii) guinea pig safety test; and (iv) acute and chronic toxicity tests, as described below.

[00157] Groups of eight BALB/c mice are inoculated intraperitoneally with 100 μΐ of immunogen containing 300 μg of the immunogen of interest. Suitable negative and positive controls are used.

02a31b28 56 [00158] The animals are monitored for general health and body weight for 14 days post infection. Similar to animals that receive placebo, animals that receive the immunogen remain healthy, and do not lose weight or display overt signs of disease during the observation period.

[00159] For the more stringent safety test, groups of 15 healthy BALB/c mice are injected with 300 μg of the immunogen.

[00160] One day after inoculation, 3 mice in each group are sacrificed and the spleen, lung and liver homogenates are analyzed for immunogen. At week 4, 8, 12, and 16 post infection, 3 mice in each group are sacrificed and spleen, live and lung homogenates are obtained and analyzed to assess presence of the immunogen.

[00161] The safety of immunogen is also assessed in the guinea pig model. First, the effect of the immunogen on the general health status of the animals is examined, including weight gain.

[00162] Groups of 8 guinea pigs are inoculated intramuscularly with

300 μg of the immunogen.

[00163] The general health and body weight of the animals are monitored for six weeks post inoculation. If any animals are sacrificed before the six- week period concludes due to serious adverse affects, each sacrificed animal will be subjected to a detailed post-mortem examination. All animals are sacrificed at the end of six weeks post-inoculation and gross pathology is performed. The immunogen is deemed safe if no adverse health effects are observed and the animals gain weight at the normal rate compared to animals inoculated with placebo as an internal control.

02a31b28 57 [00164] To evaluate the acute and chronic toxicity of an immunogen, groups of 16 guinea pigs are inoculated intradermally with 300 μg of the immunogen at graded doses or saline.

[00165] Three days postinoculation, 8 animals in each group are sacrificed to access the acute effects of the immunogen on the animals. At 28 days post-inoculation, the remaining 8 animals in each group are sacrificed to evaluate any chronic effects on the animals. At both time points, the body weight of each animal is obtained. In addition, the gross pathology and appearance of the injection sites are examined. Blood is taken for blood chemistry, and the histopathology of the internal organs and injection sites are performed at each time point.

[0100] The mice are given a total of 3 doses of vaccine at 0, 14 and 60 days and the immune response to HIV is measured by ELISA using sera collected from the tail vein of individual mice at 10 day intervals, as described. The neutralization of HIV is measured in the collected 80 days after the first vaccination. The results of the study show that the vaccine of interest has the capacity to substantially increase the magnitude and potency of the humoral response to HIV and therefore possesses useful adjuvant properties.

[0101] All references cited herein are herein incorporated by reference in entirety.

[0102] It should be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present subject matter and without diminishing its intended

02a31b28 58 advantages. It is therefore intended that such changes and modifications be embraced by the appended claims.

References

Thomas Francis, Jr. in Proceedings of the American Philosophical Society, Vol. 104, No. 6 (Dec. 15, 1960), pp. 572-578, according to The Swine Flu Episode and the Fog of Epidemics by Richard Krause in DCD's Emerging Infectious Diseases Journal Vol. 12, No. 1 January 2006 published December 20, 2005.

Garrity, R.R., G. Rimmelzwaan, A. Minassian, W.P. Tsai, G. Lin, J.J. de Johg, J. Goudsmit, and P. L. Nara. 1997. Refocusing neutralizing antibody response by targeted dampening of an immunodominant epitope. J. Immunol 159:279-89.

Kohler H, Goudsmit, J. Nara P. Clonal dominance: cause for a limited and failing immune response to HIV-1 infection and vaccination. J. Acquir Immune Defic Syndr 1992; 5(l l): 1158-68.

Andreansky, S.S., John Stambas, Paul G. Thomas, Weidong Xie, Richard J. Webby, and Peter C. Doherty Consequences of immunodominant epitope deletion for minor influenza virus-specific CD8⁺ T-cell responses. J Virol 2005 Apr;79(7):4329-39.

Nara, P.L., Smit, L., Dunlop, N., Hatch, W., Merges, M., Waters, D., Kelliher, J., Gallo, R.C., Fischinger, P.J. and Goudsmit, J.: Emergence of viruses resistant to neutralization by V3 -specific antibodies in experimental human immunodeficiency virus type 1 IIIB infection of chimpanzees. J Virol 64: 3779-3791, 1990.

Nara, P. L., and R. Garrity. 1998. Deceptive imprinting: a cosmopolitan strategy for complicating vaccination. Vaccine 16: 1780-7.

Nara, P.L., R.R. Garrity, and J. Goudsmit. 1991. Neutralization of HIV-1 : a paradox of humoral proportions. FASEB J. 5:2437-55.

Nara, P. L., and G. Lin. 2005. HIV-1 : the confounding variables of virus

neutralization. Curr Drug Targets Infect Disord 5: 157-70.

Trujiollo, J.D., N.M. Kumpula-McWhirter, K. J. Hotzel, M. Gonzalez, and W.P. Cheevers. 2004. Glycosylation of immunodominant linear epitopes in the carboxy- terminal region of the caprine arthritis-encephalitis virus surface envelope enhances vaccine-induced type-specific and cross-reactive neutralizing antibody responses. J Virol 78:9190-202.

02a31b28 59

Claims

CLAIMS The invention is claimed as follows:

1. An isolated composition comprising an epitope of HIV that is not immunodominant as in a wild-type HIV, wherein said epitope comprises a VI loop, a V2 loop, a V4 loop, a V5 loop, a CI region, a C2 region or a C3 region of an env polypeptide, a gp41 polypeptide, a gag polypeptide, a nef polypeptide, a pol polypeptide, a tat polypeptide, a rev polypeptide, a T polypeptide or a protease polypeptide, or portion thereof.

2. The composition of claim 1 wherein said epitope comprises addition or removal of a glycosylation site.

3. The composition of claim 1, wherein said epitope comprises an amino acid addition, substitution or deletion.

4. The composition of claim 1, further comprising a pharmaceutically acceptable carrier, diluent or excipient.

5. The composition of claim 3, wherein said substitution stabilizes said epitope.

6. The composition of claim 1, wherein said epitope comprises a modified amino acid.

7. The composition of claim 8, wherein said modified amino acid comprises a lipid or a saccharide.

8. The composition of claim 1, comprising a virus-like particle or a virion.

02a31b28 60

9. The composition of claim 1, wherein said epitope is recombinant.

10. The composition of claim 1, further comprising an antibody, or antigen-binding portion thereof.

11. The composition of claim 10, wherein said antibody does not specifically bind to an HIV immunodominant epitope.

12. The composition of claim 10, wherein said antibody specifically binds an HIV immunodominant epitope.

13. The composition of claim 1, further comprising a second epitope of HIV that is not immunodominant as in a wild-type HIV.

14. The composition of claim 13, further comprising an epitope of a V3 loop.

15. The composition of claim 10, wherein said HIV is devoid of an immunodominant epitope.

16. An antibody which specifically binds to a non- immunodominant epitope of HIV.

02a31b28 61