WO2014039775A1 - Hiv-1 antigens with discrete conformational forms of the v1/v2 domain and methods of use thereof - Google Patents

Description

HIV-1 ANTIGENS WITH DISCRETE CONFORMATIONAL FORMS OF THE V1 V2

DOMAIN AND METHODS OF USE THEREOF

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims fee benefit of U.S. Provisional Patent Application Serial No. 61/697,979, filed September 7, 2012. The contents of the foregoing application is

incorporated by reference in its entirety.

GOVERNMENT INTERESTS

This invention was made with government support under grant numbers R01 AI-46383, R01 AI102718-01 and P01 AI-0888610 from the National Institutes of Health. The United States government has certain rights to this invention.

FIELD OF THE INVENTION

The present invention relates to a novel composition of recombinant HIY-1 Env proteins that contain structurally and immunologically distinct V1/V2 domains.

BACKGROUND OF THE INVENTION

Recent studies have shown a role for the V1/V2 domain of the HlV-1 ENV protein as a target in HLV-1 vaccines. A series of potent and broadly neutralizing monoclonal antibodies (MAbs) targeting a class of quaternary epitopes mat were dependent on several positions in the V2 region were isolated in one study (Walker et al., 2009, Science 326:285-289; Moore et al., 2011, Journal of Virology 85:3128-3141). These antibodies react preferentially with native trimeric Env complexes. Further studies have shown that a small conserved sequence in the V2 doinain interacted with «4β7 integrin, the mucosal homing receptor for activated T cells, and that this interaction strongly enhanced infection of those cells (Nawaz et al.. 2011, PLoS Pathog 7:el001301; Cicala et al, 2009, Proc Natl Acad Sci USA 106:20877-20882). A third finding came from the analysis of correlates of protection in the recently concluded RV144 vaccine trial conducted in Thailand, the first HIV vaccine trial that resulted in some protection against infection (Rerks-Ngarm et al., 2009, N Engl J Med 361 :2209-2220). Protection in this trial was shown to correlate with an increased titer of antibodies that bound to a VI. / 2 fusion protein , but not to any other factor analyzed, including avidity and titers of Env-specific IgG and IgA, viral neutralizing activity or levels of Env-specifk CD4+ T cells (Haynes et al, 2012, The New England Journal of Medicine 366:1275.-1286).

SUMMARY OF THE INVENTION

The present invention relates in part to isolated and purified polypeptides comprising the

VI V2 domain of a. HIV envelope (Env) protein having a. structural configuration of a."C" form, as at least shown in Figure 6 and Figure 7. As used herein, "HIV" is meant to represent either HIV-1 or HIV-2, and any and all subtypes thereof. Upon review of the specification, it will be understood that any such polypeptide is encompassed within the scope of invention which possesses the ability present and maintain the conformational integrity of the "C" form of the V1/V2 domain. It is disclosed herein that this "C" form presents one of several structural conformations based on an alternative form of disulfide bonding within this 1/V2 domain, which dramatically impacts the mununoreactivity of these proteins. To this end, an embodiment of the present invention further relates to a polypeptide which presents and maintains the conformational, antigenic, and immunoreactive integrity of the alternative "C" form of the V1/V2 domain, including but not limited to wherein the polypeptide is native gpI20, or wherein the polypeptide native is gpl40₅ or wherein the polypeptide is a recombinant form of gpl20, or wherein the polypeptide is a recombinant form of g l40. Thus, another embodiment of the present invention further relates to such a polypeptide maintaimiig the structural integrity as discussed herein where the recombinant gpl20-based form comprises a portion of the epl20 coding region, again, such that the antigenicity immunoreactivity of the I ! structural configuration of the "C" form is maintained. A specific, but non-limiting embodiment of the present invention relates to such a presented polypeptide wherein the amino-terniinal sequence represents a portion of MuLV gp70, including but not limited to a polypeptide presenting the "C" form which has the amino acid sequence as set forth in Figure 14 (SEQ∑D NO; 2). An additional embodiment of the present invention relates to nucleic acid molecules encoding a polypeptide with at least 90% amino acid sequence identity to a polypeptide which presents and maintains the conformational antigenic, and immunoreactive integrit of the alternative "C" form of the V1. V2 domain, as well as related recombinant DMA molecules housing such a nucleic acid molecule, and any cell line transfected or transformed with any such recombinant DNA molecule. The present invention also relates to isolated and purified polypeptides comprising the VI /V2 domain of a HIV envelope (Env) protein having a structural configuration of yet another form, identified herein as a "B" form, as at least shown in Figure 6 and Figure 7, or in the alternative, presented as both a "B" form and an "A" form (""B A" form"). Again, upon review of the specification, it will be understood that any such polypeptide is encompassed within the scope of invention which possesses the ability to present and maintain the conformational integrity of the "B" form of the VI V2 domain, or in the alternative a "B"/"A" combined form. Either form/forms as disclosed herein will again present one of several structural conformations based on an alternative form of disulfide bonding within this V1/V2 domain, dramatically impacting the immunoreactivity of these proteins. As disclosed immediately above in regard to the "C forms, the present invention fiirther relates to a pohpeptide which presents and maintains the conformational, antigenic, and immunoreactive integrity of the alternative "B" form (or in the alternative a "B'V'A" combined form) of the VI V2 domain, including but not limited to wherein the polypeptide is native gpl20, native gpl40, recombinant gpl20, recombinant form gpl40, a construction based upon a portion of the gpl20 or gp 140 coding region, again, such that the antigenicity and immunoreactivity of the V1 V2 structural configuration of the "B" form (or in the alternative a "B'V'A" combined form) are maintained. An additional, but non-limiting embodiment of the present invention relates to such a presented polypeptide wherein the ammo-tenninal sequence represents a portion of MuLV gp70, including but not limited to a polypeptide presenting the "B" form (or a "B'V'A" combined form) which has the amino acid sequence as set forth in Figure 14 (SEQ ID NG:2). An additional embodiment of the present invention relates to nucleic acid molecules encoding a polypeptide with at least 90% amino acid sequence identity to a polypeptide which presents and maintains the conformational, antigenic, and immunoreactive integrity of the alternative "B" form of the V1/V2 domain, or in the alternative a combined "B'V'A" form) as well as related recombinant DNA molecules housing such a nucleic acid molecule, and any cell line transfected or transformed with any such recombinant DNA molecule. It will be evident upon review of this specification that combinations of an "A" form, a "B" form and/or a "C" form are also contemplated herein as relating to the present invention.

The present invention also relates to utilizing the polypeptides of the present invention, including but not limited to polypeptides which maintain the conformational integrity disclosed herein as a "C" form, a "B" form and or a combined "B/A" forai in methods of treating a. HIV positive subject wliich involves adii iistering to such a subject a pharmaceutical coiiipositioii comprising any such polypeptide disclosed herein, or combination thereof, with such a pharmaceutical composition optionally containing a pharmaceutically active carrier. It will be the goal of any suc treatment^' to result in^' a reduction in the HIV viral load of a subject

The present invention further relates to utilizing the polypeptides of the present invention, including but not limited to polypeptides which maintain the conformational integrity disclosed herein as a "C" form, a "B" form and/or a combined "B/A" form in methods of vaccinating a human subject against infection or progression of HIV which comprises administration of a pharmaceutical composition comprising any, or combination thereof, of such polypeptides disclosed herein, with the pharmaceutical composition optionally containing a pharmaceutically active carrier, wherein administration of the pharmaceutical composition results in generation of an immune response against HTV infection.

To this end, the present invention relates to any such methodology utilized to induce an enhanced immunological response against a HTV antigen in a mammalian host, wherein the mammalian host is inoculated with a polypeptide disclosed herein as a "C^* form, a "B" form and/or a combined "B/A" form and, optionally, a pharmaceutically acceptable carrier. Thus, the present invention also relates to any such phannaceutical composition which comprises an effective amount of such a polypeptide and, optionally, a pharmaceutically acceptable carrier.

Another embodiment rela tes to methods of identifying an HIV antiviral compound, which comprises the steps of: (a) combining component (i)— a test compound, component (ii)— a "C" form-specific antibody, or a "B" form-specific antibody and/or an antibody which specifically recognizes a combination of the "B" and "A" form, and component (iii)— a polypeptide of "C" form, a "B" form and/or a combined "B/A" form; (b) measuring the effect the test compound has on the affinity of component (ii) for component (iii) of step (a); and. (c) comparing the effect the test compound has on the affinity of component (ii) for component (iii) versus the affinity of component (ii) for component (iii) in the absence of the test compound. Such methods may utilize test compounds selected from the group consisting of a peptide, a protein, a non- proteinaceous organic or inorganic molecule, DNA and UNA. BRIEF DESCRIPTION OF THE FIGURES

Figure L Evidence of stnictiitai inmiunological heterogeneity of the V1/V2 domain was seen for several rg l20s upon seqiientiai radioinmiimoprecipitations. (a) Analyses by sequential iimnunoprecipitations revealed that a substantial fraction (-25%) of three rgp! 20s tested were non-reactive with 697D, directed against a conformational V2 epitope, (b) ClOSg is a mAb isolated from an HIV-infected chimp, that recognizes a glycan -dependent V1 V2 epitope overlapping with the PG9/PG16 site and possesses potent neutralizing activity against a limited set of viruses. C108g also recognized only a fraction of BaL rg l20 containing the correctly- folded V2 domain.

Figure 2. To facilitate the structural analysis of the native V1 V2 doma in, this region of a primary c ade B Env (CaseA2) sequence was expressed as a fusion protein, joined to an N- temiinal fragment of the MuLV Env protein, gp70. This fusion protein system allowed the expression of the VI V2 domain as a native glycosylated and disiilfide-boiided protein. Antibody reactivity with this V1/V2 fusion protein was the only variable shown to have a significant inverse correlation with risk of infection in the Thailand RV144 vaccine.

Figure 3. Separation of the V1/V2 domain from the gp70 domain by cleavage at a proteolytic site engineered between the two domains shows that these regions form distinct and independent folded domains.

Figure 4. The deglycosylated gp70-Vl V2caseA2 protein is resolved as a doublet by SDS- PAGE under non-reducing conditions, indicating distinct conformational forms.

Figure 5. The two VLV2 conformational forms possess distinct immimoreactiviti.es with Vl/Y2-specifie mAbs. Mabs isolated from infected humans and mice immunized with rgp!20 all specifically recognize the V1/V2 lower band form. Mabs from rats immunized with V1 V2 fusion protein recognize pper band or both bands.

Figure 6. Three V1/V2 conformers can exist, which differ in their disulfide bond patterns. Structure A was determined for the H XB2 strain of HTV g l20 by Leonard et aL, 1990, J. Biol. Chem. 265:10373-10382,

Figure 7. Homogeneous conformational forms of the VI /V2 fusion protein were isolated by immnnoaffmity chromatography and analyzed by mass spectrometry. The two forms were fractionated by immunoaffmity on columns containing lower band-specific mab 238. The flow- through contained uppe band forms, while the column eluate contained the lower band form. The upper band form contained peptides at mass of 1573 and 2634 predicted for the C form, while the lower band form contained the complex peptide with mass of 4206 characteristic of forms A and B,

Figure 8. Analysis of alternate V1/V2 confomieric forms of the CaseA2 and BaL V1 V2 fusion proteins^'. For the CaseA2 protein, rat Mab Ί0ΑΊ 1 (directed, against a site in the gp70 carrier domain) polls down both forms, mouse Mab SC-258 recognizes only lower form and human Mab 8.22.2 (from immunized Xenomouse, which contained only human Ig genes) recognized predominantly upper band form. The two forms were also seen for BaL V1/V2 fusion protein, although they were nor resolved as well Again, K10A11 recognized both bands, rat Mab 10/76b, directed against a linear Y2 epitope, recognized both upper and lower band equally well while chimp Mab C108g recognizes only the lower band form. The C108g epitope was not well-expressed in this protein, perhaps because of its glycan-dependence.

Figure 9. Analysis of reactivity of human vaccinee sera with coiiformers of the gp70- Vl V2_CaseA2 protein.

Figure 10. Titrations of HTV-1 -positive human immune sera show different patterns against specific V1 V2 confonners.

Figure 11. Alignment of V1 V2 region of sequences analyzed in Fig. 12. Indicated are plasmid mmibers and corresponding names of Envs these sequences were obtained from.

Sequences are divided to indicate different regions of the VI /V2 domain; the conserved flanking regions, the VI hypervariable region, the semi-conserved V2 region, the V2 hypervariable region and the V2 flank. The sequences are arranged by decreasing size of the V I hypervariable region, and this order is reflected in the gel shown in Fig. 12.

Figure 12. Analysis of conformational heterogeneity in VI V2 domain demonstrated by

SDS-PAGE analysis of deglycosylated gp70-fusion proteins (+PngaseF, Non-Reduced). The plasmid nmnber and sequence name are indicated above each sample. Samples are arranged according to the list in Fig. 11, according to size of the VI hypervariable region. The CaseA2 sequence (p3020) is repeated in each gel to serve as a standard. Protein markers are also included as size standards. All samples were deglycosylated by treatment with PNGaseF before analysis. The data presented in this Fig. 12 represents gels run in unreduced form, while the bottom gels. Figure 13. Analysis of conformational heterogeneity in V1/V2 domain demonstrated by SDS-PAGE analysis of deglycosylated gp70-fusion proteins (+PngaseF, Reduced). See also. Fig. 12 legend. The data presented in this Fig. 13 represents the same samples analyzed as per Fig. 12, but after reduction of disulfide bonds by treatment with DTT.

Figure 14. The CaseA2 gp^'70-Vl /V2 sequence.

Figure 15, The sequence of P565 plasinkt

Figure 16. The gp70-Vl/V2 sequence of P565.

DETAILED DESCRIPTION OF INVENTION

The present invention is based on the discovery thai the V1/Y2 domain of HIV Env- based antigens possesses conformational heterogeneity resulting from alternative disulfide bonding, wliich dramatically impacts the iimnunoreactivity of these proteins. In accordance with the present invention, the conformationaliy heterogenous antigens can be resolved to provide HIV Env antigens with homogeneous conformational forms of the V1/V2 domain.

HIV envelope (Env) glycoproteins, including for example g l20 and pi 40, are known in the art and contain five constant and five variable domains. The V1/V2 domain comprises about 90 amino acids, including four cysteine residues designated herein as CI, C2, C3, and C4„ with CI being the most N-terminal and C4 being the most C -terminal (Figs. 2 6, and 7). The HIV Env antigens with homogeneous confomiational forms of the VI/V2 domain provided herein are designated Form A, Form B, and Form C. In Form A, there is a disulfide bond between CI and C4, and a disulfide bond between C2 and C3. hi Form B, there is a disulfide bond between CI and C3, and a disulfide bond between C2 and C4. hi Form C, there is a disulfide bond between CI and ( ^"2. and a disulfide bond between C3 and C4.

Forms A, B and C are present in native and recombinant HIV Env glycoproteins, and can be isolated from one another in accordance with the present invention. In particular, the present invention provides a method for isolating HIV Env antigens with Form A, or Form B, or Form C of the V1/V2 domain torn each other to provide HIV Env antigens with a homogeneous conformational form of the V1/V2 domain. In particular, the different forms in a native or recombinant composition of HIV Env proteins can be separated from one another by irmnunoprecipitation with antibodies that are specific for the different conformations. For example, human Mab 8.22.2 (He et al, 2002, J. Immunol. 169:595-605) is specific for Form C. One of skill in the art can identify antibodies thai are specific for the different conformations, and can perform inaminoprecipitation, including serial imimmoprecipitation, by methods known in the art to separate the different conformations. These forms can be fractionated by iimnunoafrmiry methods using monoclonal antibodies that, are specific for one of the, distinct forms. The homogeneous forms may be obtained either in the depleted fiaction or by elation of bound molecules.

Accordingly, in another embodiment, the present invention provides a composition of structurally and immunologically distinct HTV-^'l antigens in which VI /V2 domains possess alternate disulfide bond patterns, i.e. Form A, Form B, or Forai (^". or combinations thereof, as disclosed herein. The antigens include at least the V1/V2 domain, and optionally other domains of the Env protein or other fusion partners. These antigens may consist of fusion glycoproteins that express the isolated VT/V2 domains as well as native gpl20 and gpl4G proteins that have been fractionated so that they contain homogeneously folded V1/V2 domains.

The antigens of the present invention are useful as antigens in immunoassays to characterize the specificity of antibodies directed against the V1/V2 domain i infected or immunized people. Accordingly, the invention further provides compositions comprising the antigen of he invention for use in such assays.

The antigen of the present invention are also useful a protein therapeutic agents for inhibiting functions mediated by the V1/V2 domain, and as inmiimogens in HIV vaccines. Accordingly, the present invention further provides pharmaceutical compositions comprising the antigens of the mvention.

Formulations of the compositions useful in certain embodiments such as polypeptides, polynucleotides, or antibodies may be prepared for storage by mixing the selected composition having the desired degree of purity with optional physiologically phannaceutically-acceptable earners, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, ed.. Mack Publishing Company (1990)) in the form of a lyophilized cake or an aqueous solution. Acceptable carriers, excipients or stabilizers are nontoxic to recipients and are preferably inert at the dosages and concentrations employed, and include buffers such as phosphate, citrate, or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; ammo acids such as glycine, gJutanirne, asparagiiie, arginine or lysine; monosaccharides,, disaccharides, and oilier carbohydrates including glucose, mamiose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions suc as sodium; and/or nonionic surfactants such as Tween, Pmronics or polyethylene glycol (PEG).

Compositions to be used for in vivo adininistiatioii should be sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyopbilization and reeonstitution. The composition for parenteral adniimstration ordinarily will be stored in lyophilized form or in solution.

Therapeutic compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. The route of administration of the compositio is in accord with known methods, e.g. oral, injection or infusion by intravenous, intraperitoneal, intracerebral, intramascular, intraocular, intraarterial, or infralesional routes, or by sustained release systems or implantation device. Where desired, the compositions may be administered continuously by infusion, bolus injection or by implantation device.

An effective amount of the compositions to be employed therapeutically will depend, for example, upo the therapeutic objectives, the route of administration, and the condition of the patient. Accordingly, it may be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect. A typical daily dosage may range from about 1 ug/kg to up to 100 mg/kg or more, depending on the factors mentioned above. Typically, a clinician will administer the composition until a dosage is reached that achieves the desired effect. The progress of this therapy is easily monitored by conventional assays designed to evaluate blood glucose levels or other particular conditions of interest in a particular subject.

Pharmaceutical compositions may be produced by admixing a phamiaceiriicall effective amount of protein with one or more suitable carriers or adjuvants such as water, mineral oil, polyethylene glycol, starch, talcum, lactose, thickeners, stabilizers, suspending agents, etc. Such compositions may be in the form of solutions, suspensions, tablets, capsules, creams, salves, ointments, or other conventional forms. In certai embodiments, compounds are formulated with pharniaceutically acceptable diluents, adjuvants, exeipients, or carriers. The phrase "pharmaceutically or pharmacologically acceptable'^* refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reaction when administered to an animal or a human, e.g., orally, topically, transdemially, parenterally, by inhalation spray, vaginally, reetally, or by inhacranial injection. The term parenteral as used herein includes subcutaneous injections, intravenous, intiamuscular, intracistemal injection, or infusion techniques. Administration by intravenous, intradermal, intramusclar, intramammary, intraperitoneal, intrathecal, retrobulbar, mtrapulnionary injection and/or surgical implantation at a particular site is contemplated as well.) Generally, this will also entail preparing compositions that are essentially free of pyrogens, as well as othe impurities that could be harmful to humans or animals. The term "pharmaceuticaDy acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, liposomes, capsids, nanocapsules, microcapsules and the like. The use of such media and agents for pharmaceutically active substances is well known in the art.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial an antifungal agents, for example, parabens, clilorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluniinum monostearate and gelatin.

In certain embodiments, the present invention provides a method of treating a subject comprising administration of a composition. As used herein, the term "subject" is used to mean an animal, preferably a mammal, including a human. The terms "patient" and "subject" may be used interchangeably. The therapeutic compositions may be administered by any route that delivers an effective dosage to the desired site of action, with acceptable (preferably minimal) side-effects. Numerous routes of administration of agents are known, for example, oral rectal iransmucosal, or intestinal admimstration; parenteral delivery, meluding intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, intraperitoneal, intranasal, cutaneous or intradermal injections; inhalation, and topical application.

Therapeutic dosing is achieved by monitoring therapeutic benefit and monitoring to avoid side-effects. Preferred dosage provides a maximum localized tlierapeutic benefit with minimum local or systemic side-effects. Suitable human dosage ranges for the polynucleotides or polypeptides can be extrapolated from these dosages or from similar studies in appropriate animal models. Dosages can then be adjusted as necessary by the clinician to provide maximal therapeutic benefit for human subjects.

When a therapeutically effective amount of a composition of the present invention is administered by e.g., intradermal cutaneous or subcutaneous injection, the composition is preferably in the form of a pyrogen-free. parenterally acceptable aqueous solution. The preparation of such parenterally acceptable protein or polynucleotide solutions, having due regard to pH, isotonieity, stability, and the like, is within the skill in the art. A preferred composition should contain, in addition to protein or other active ingredient of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art. The composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art. The agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution. Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the banier to be permeated are used in the formulation. Such penetrants are generally known in the ait.

The compositions of the invention may be in the form of a complex of the protein(s) or other active ingredient of present invention along with protein or peptide antigens.

The composition may further contain other agents which either enhance the activity of the protein or other active ingredient or complement its activity or use in treatment. Such additional factors and/or agents may ^'be included in the pharmaceutical composition to produce a synergistic effect with protein or other active ingredient, or to minimize side effects.

Techniques for formulation and administration of the therapeutic compositions of the instant application may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., lates edition. Whe applied to an individual active ingredient, administered alone, a therapeutically effective dose refers to that ingredient alone. When applied to a combination, a therapeutically effective dose refers to combined amounts of die active ingredients that result in the therapeutic effect, whethe administered in combination, serially or simultaneously. The antigens of the present invention are useful for identifying, for example by screening, and for generating antibodies or antigen-binding fragments thereof that are specific for antigens with homogeneous conformational forms of the V1/V2 domain.

Antibodies may take the form of any type of relevant antibody fragment, antibody binding portion, specific binding member, a non-protein synthetic mimic, or an other relevant terminology known i the art which refers to an entity which at least substantially retains the binding specificity/neutralization activity. Thus, the term "antibody" as used in any context within this specification is meant to include, but not be limited to, any specific binding member, immunoglobulin class and/or isotype (e.g., IgGi, IgG₂, IgG₃, IgG₄, IgM, IgA, IgD. IgE and IgM); and biologically relevant fragment or specific binding member thereof, including but not limited to Fab, F(ab')2, Fv, and scFv (single chain or related entity). Therefore, it is well known in the art, and is included as review only, that an "antibody" refers to a glycoprotein comprising at least two hea (H chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof. A heavy chain is comprised of a heavy chain variable region (VH) and a heaw chain constant region (CHI , CH2 and CH3). A light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The variable regions of both the heavy and light chains comprise framework regions (FWR) and complementarity determining regions (CDR). The four FWR regions are relatively conversed while CDR regions (CDRl , CDR2 and CDR3) represent hypervariable regions and are arranged from NH.sub.2 femiinus to the COOH terminus as follows: FWR1, CDRl, FWR2, CDR2, FWR3, CDR3, FWR4. The variable regions of the heaw arid light chains contain a binding domain that interacts with an antigen while, depending of the isotype, the constat region(s) may mediate the binding of the innmmoglobulin to host tissues or factors. That said, also included in the working definition of "antibody" are chimeric antibodies, humanized antibodies, a recombinant antibody, as human antibodies generated from a transgenic non-human animal, as well as antibodies selected from libraries using emichmefit technologies available to the artisan. Antibody fragments^' are obtained using techniques readily known and available to those of ordinary skill in the art, as reviewed below. Therefore, an "antibody" is any such entity or specific binding member, which specifically binds the conformational epitope of the of the VI V2 domain, as described herein. Therefore, the term "antibody" describes an immunoglobulin, whether natural or partly or wholly synthetically produced; any polypeptide or protein having a binding domain which is, or is substantially homologous to, an antibody binding domain. These can be derived from natural sources, or they may be partly or wholly synthetically produced. Examples of antibodies are the immunoglobulin isotypes and their isotypic subclasses; fragments which comprise an antigen binding domain such as Fab, scFv, Fv, dAb. Fd and diabodies, as discussed without limitation, infra. It is known in the ait that it is possible to manipulate monoclonal and other antibodies and use techniques of recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody Such techniques may evolve introducing DNA encoding the immunoglobulin variable region, or the complementarity deteriimiiiig regions (CDRs), of an antibody to the constant regions, or constant regions plus framework regions, of a different nmimoglobuhn. A hyhridama or other cell producing an antibody may be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced. Antibodies can be modified in a number of ways, and the term "antibody" should be construed as covering any specific binding member or substance having a binding domain with the required specificity. Thus, this term covers antibody fragments, derivatives, functional equivalents and homologues of "antibody" including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Such an entity may be a binding fragment encompassed within the term "antigen-binding portion'^* or "specific binding member" of an antibody including but not limited to (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody (y) a dAb fragment, which comprises a VH domain; (vi) an isolated complementarity determining region (CDR); (vii) a ^"scAb^", an antibody fragment containing VH and VL as well as either CL or CH; and (viii) artificial antibodies based upon protein scaffolds, including but not limited to fibronectin type III polypeptide antibodies (e.g., see U.S. Pat. No. 6,703,199, issued to oide on Mar. 9, 2004 and PCX international Application Publication No. WO 02/32925). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chai in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv)). In one embodiment, the invention provides a method for generating HIV-1 Env proteins with structurally homogeneous V1/V2 domains, and the use of such uniform straetiires as antigens, immunogens and therapeutic reagents.

Polyclonal or monoclonal antibodies for use in accordance with the present invention may be raised by known techniques. Monospecific murine (mouse) antibodies showing specificity to a conformational epitope of a target of choice may be purified from mammalian antisera containing antibodies reactive against this region, or may be prepared as monoclonal antibodies using the technique of Kohler and Milstem (1975, Nature 256: 495-497). Monospecific antibody as used herein is defined as a single antibody species or multiple antibody species with homogenous binding characteristics, such as the mouse monoclonal antibodies exemplified herein with the 13C3 series of monoclonal antibodies. Hybiidoma cells are produced by mixing the splenic lymphocytes with an appropriate fusion partner, preferably myeloma cells, under conditions which will allow the formation of stable hybridomas. The splenic antibody producing cells and myeloma cells are fused, selected, and screened for antibody production. Hybridoma cells from antibody positive wells are cloned by a technique such as the soft agar technique of MacPherson (1973, Soft Agar Techniques, in Tissue Culture Methods and Applications, Krase and Paterson, Eds, Academic Press). Monoclonal antibodies are produced in vivo by injecting respective hydridoma cells into pristine primed mice, collecting ascite fluid after an interval of time, and prepared by techniques well known in the art.

Beyond species specific monoclonal antibodies described above, the antibodies of the present invention may also be in the form of a "chimeric antibody", a monoclonal antibody constructed from the variable regions derived from say, the murine source, and constant regions derived from me intended host source (e.g.. human; for a review, see Morrison and Oi. 1989, Advances in Immunology, 44: 65-92). For example, the variable light and heavy DNA sequences from the rodent (e.g.. mouse) antibody may b cloned into a. mammalian expression vector. These light and heavy "chimeric" expression vectors are cotransfected into a recipient cell Sine and selected and expanded by known techniques. This cell line may then be subjected to known cell culture techniques, resulting in production of both tiie light chain and heav chain of a chimeric antibody. Such chimeric antibodies have historically been shown to have the antigen- binding capacity of the original rodent monoclonal while significantly reducing iimnunogenicity problems upon host administration.

A logical improvement to the chimeric antibody is the "humanized antibody," which arguably reduces the chance of the patient mounting an immune response against a therapeutic antibody when compared to use of a chimeric or full murine monoclonal antibody The strategy of "humanizing" a murine Mab is based on replacing amino acid residues which differ from those in the human sequences by site directed mutagenesis of individual residues or by grafting of entire complementarity detennining regions (Jones et al., 1986, Nature 321: 522-526). This technology is again now well known in the art and is represented by numerous strategies to improve on this technology; namely by implementing strategies including, but not limited to, "reshaping" (see Verhoeyen, et al, 1988. Science 239: 1534-1536), "hyperchimerization" (see Queen, et al., 1991, Proc, Natl. Acad. Sci. 88:2869-2873) or "veneering" (Mark, et al., 1994, Derivation of Therapeutically Active Humanized and Veneered anti-CD 18 Antibodies Metcalf end Dalton, eds. Cellular Adhesion Molecular Definition to Therapeutic Potential. New York: Plenum Press, 291-312). These strategies all involve to some degree sequence comparison between rodent and human sequences to determine whether specific amino acid substitutions from a rodent to human consensus is appropriate. Whatever the variations, the central theme involved in generating a humanized antibody relies on CDR grafting, where these three antigen binding sites from both the light and heavy chain are effectively removed from the rodent expressing antibody clone and subcloned (or "grafted") into an expression vector coding for the framework region of the human antibody. For example, utilizing the above techniques a humanized antibody may be expressed wherein the CDRI, CDR2, and CDR3 regions of the variable light chain are prepared, and the CDRI , CDR2, and CDR3 regions of the variable heavy chain are prepared. Therefore, a "humanized antibody" is effectively an antibody constructed with only murine CDRs (minus any additional improvements generated by incorporating one or more of the above mentioned strategies), wife the remainder of the variable region and ail of fee constant region being derived from. a. human source.

The invention also encompasses nucleic acid molecules encoding the proteins of fee invention. Nucleic acid molecules within fee invention can be cDNA, genomic DNA, synthetic DNA, or ENA, and can be double-stranded or single-stranded (i.e., either a sense or an antisense strand). Fragments of these molecules, which are also considered within fee scope of the invention, can be produced, for example, by the polymerase chain reaction (PCR) or generated by treatment with one or more restriction endonucleases. A ribonucleic acid (RNA) molecule can be produced by in vitro transcription.

As used herein, both "protein" and "polypeptide" mean any chain of amino acid residues, regardless of length or post-traiislational modification (e.g., glycosylation or phosphorylation). The polypeptide can be a naturally occurring, synthetic, or a recombinant molecule consisting of a hybrid with one portion, for example, encoding all or a portion of a V1/V2 domain, and a second portion being encoded by all or part of a second gene.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in thei entirety.

EXAMPLES

EXAMPLE 1

Isolated VI/V2 domain of HIV- 1 Em exists in at least two structurally and immunologically distinct fawns

The native VLV2 domain of a clade B sequence (CaseA2) was expressed by fusion to the C-terminus of a 273 amnio acid sequence fragment of fee MuLV gp70 domain. This fusion glycoprotein was characterized by SDS-PAGE under various conditions, by radioimmunoprecipitation experiments with a panel of mAbs directed against Vl/V2-specific epitopes, and by MALDI-TOF analysis of immunologically fractionated forms.

The CaseA2 gp70-Vl V2 sequence is set forth in Fig. 14.

The sequence of P565 plasmid is set forth in Fig. 15. Plasmid has 1 Hiiidlll site at the KL site in the V2 region

CAG AAA GAA TAT GCA CTT TTT TAT AAG CTT GAT ATA GTA CCA ATA GAT AAT

(SEQ ID NO. 49}

Q K E Y A L F Y K L D I V P I D N

(SEQ ID NO. 50}

This can be mutated and a unique Hindlll site can be inserted by silent mutations into the highly conserved KL site at the beginning of the VI sequence

CCC CCC OCT AGC GTA AAA TTA ACC CCA CTC (SEQ ID NO. 51)

P P A S V K L T P L (SEQ ID NO. 52)

CCC CCC GCT AGC GTA A/Ag cTt ACC CCA CTC (SEQ ID NO. 6)

Hindlil

The gp7G-Vl V2 sequence of P565 is set forth in Fig. 16.

The VI /Y2 domai of a number of HIV- 1 Envs was expressed in isolated form as a disulfide-bonded glycoprotein joined to the C-tenninus to a 273 amino acid fragment of the Friend MuLV gp70 protein. The CaseA2 V1/V2 fusion protein was radiolabeled and immunoprecipitated with monoclonal antibodies (niAbs) directed against a target in the gp70 carrier domain by capture of the antibody Fc regions with PANSORBIN®., a commercial preparation of crosslinked Staph A. The immunoprecipitated proteins were removed from PANSORBIN® by boiling in the presence of 1% SDS, and analyzed by SDS-PAGE before and after removal of all of the attached N-linked carbohydrate siibstituents both in non-reduced form and after reduction of disulfide bonds with dithiothreotol (DTT). Under non-reducing conditions a closely-spaced doublet was resolved o 10% gels at the position expected for the deglycosylated protein. These two bands coalesced into a single band with slightly lower mobility than the doublet after reduction, indicating that the doublei represented different disulfide-bonded confor neric forms. Analysis of samples immunoprecipitated with a variety of niAbs directed against sites in the V1/V2 domain contained either the upper or lower band, indicating that these represented two antigenically distinct conformeric forms. Analysis of the structure of the mmiiinofraetionated forms by MALDI-TOF mass spectiOmetry confirmed mat these possessed distinct disulfide-bonded structures and allowed the assignment of the structures represented by the upper and lower band form.

These results demonstrate that the isolated Y1/V2 domain of HIV- 1 Env exists in at least two structurally and mnnuno logic ally distinct forms .

EXAMPLE 2

Heterogeneous forms of V1/V2 domains are not limited to the C se-A2 sequences.

The biochemical method described above (SDS-PAGE of deglycosylated gp70-Vl V2 proteins under non-reducing conditons) allowed good resolution of structurally disti ct V1 V2 forms of the Case-A2 sequence. However, applying the similar method to the BaL sequence resulted in a smaller separation between the two bands, and a modified SF162 sequence gave two bands that were barely distinguishable. Furthermore, for several other gp70-Vl/V2 fusion proteins (e.g.. the Co C sequence, or the Th023 clade A E sequence) only a single band was observed. However, sequential immunoprecipitation assays revealed the presence of distinct conformational forms, indicating that the diverse forms were present, but could not be separated by gel electrophoresis.

EXAMPLE 3

Direct binding studies with rgpl 20/rgpl40 proteins andfimctional studies

Evidence was also obtained for immunological heterogeneity in recombinant and native g l20 and gpl40 immunogens. Sequential immtinoprecipitations with monoclonal antibodies specific for distinct conformation epitopes in V1/V2 showed that these antibodies recognized only a fraction of the molecules (Fig. I). Native and recombinant gpI20 and gpl40 proteins were recognized by MAbs specific for either the upper band-specific or lower band-specific epitopes, indicating the presence of both structures in these antigens. Finally , evidence lias been presented that MAbs reactive with either upper band-specific or lower band-specific epitopes neutralized viral iiifectivity, indicating that both forms were present in functional Env proteins and suggesting that both contribute to viral binding and or entry.

Analyses by sequential immunoprecipitations revealed that a substantial fraction (--25%) of three rgpl 20s tested were non-reactive with 697D, directed against a conformational V2 epitope. (Fig. 1A). ClOSg is a niAb isolated .from an HIV-infected chimp that recognizes a gly can-dependent VI V2 epitope overlapping with the PG PG16 site and possesses potent neutralizing activity against a limited set. of viruses. ClOS also recognized only a fraction of BaL rgpl20 containing the correctly-folded V2 domain. (Fig. IB).

To facilitate the structural analysis of the native V1/V2 domain, this region of a primary clade B Env (CaseA2) sequence was expressed as a fusion protein, joined to an N-terminal fragment of the MnLV Env protein, gp70 (Fig. 2). This fusion protein system allowed the expression of the V1/Y2 domain as a native glycosylated and disulfide-bonded protein. Separation of the VI V2 domain from the gp70 domain by cleavage at a proteolytic site engineered between the two domains showed that these regions form distinct and independent folded domains (Fig. 3). The deglycosylated gp70-Vl V2ce_*A2 protein was resolved as a doublet by SDS-PAGE under non-reducing conditions, indicating distinct conformational forms (Fig. 4; see also Figure 6 for three distinct and possible confirmations of the VL/V2 domain). The two VT V2 conformational forms possess distinct immunoreactivities with Vl V2-speeific mAbs. Mabs isolated from infected humans and mice immunized with rg l20 all specifically recognize the V1/V2 lower band form. Mabs from rats inimiinized with V1/V2 fusion protein recognize upper band or both bands (Fig. 5).

Homogeneous conformational forms of the V1 V2 fusion protein were isolated by inmiunoaffmity chromatography and analyzed by mass spectrometry. The two forms were fractionated by inimunoaffinity on columns containing lower band- specific mab 238. The flow- through contained upper band forms, while the column eluate contained the lower band form. The upper band form contained peptides at mass of 1573 and 2634 predicted for the C form, while the lower band form contained the complex peptide with mass of 4206 cliaracteiistic of forms A and B (Fig. 7).

Alternate VI /V2 conformeric forms of the CaseA2 and BaL V1/V2 fusion proteins were analyzed (Fig. 8). For the CaseA2 protein, rat Mab K10A11 (directed against a site in the gp70 carrier domain) pulled down both forms; mouse Mab SC-258 recognized only lower form; and human Mab 8.22.2 (from immunized Xenomouse, which contained only human Ig genes) recognized predominantly upper band form. The two forms were also seen for BaL VI /V2 fusion protein, although they were not resolved as well. Again, K10A11 recognized both bands, rat Mab IG/76b, directed against a linear V2 epitope, recognized both upper and lower band equally well, while chimp Mab ClQSg recognized only the lower band form. The ClOSg epitope was not well- expressed in this protein, perhaps because of its glycan-dependence.

Figure 9 shows an analysis of reactivity of human vaccine sera with coiifonners of the gp70-VI/V2c∞eA2. protein. Titrations of EflV-1 -positive human immune sera show different patterns against specific VI/V2 confomiers (Fig, 10).

The wide variety of various pg 120 V1/V2 sequence sequences exemplified herein are aligned in Fig. 11. The respective SEQ ID NO., plasrnid number and corresponding names of Envs from which these sequences represent are provided within Fig. Ϊ. The respective sequences are divided to indicate different regions of the V1/V2 domain; the conserved flanking regions, the VI hypervariable region, the semi-conserved V2 region, the V2 h pervariable region and the V2 flank. It is also noted that these sequences are arranged by decreasing size of the VI hypervariable region, and this order is reflected in the gels shown in both Fig. 12 ((+PngaseF, Non-Reduced)) and Fig, 13 ((+PngaseF, Reduced - DTT). Figures 12 and 13 show an analysis of the conformational heterogeneity in 1/V2 domain, as demonstrated by SDS-PAGE analysis of deglycosylated g 70- fusion proteins, in both non-reduced (Fig. 12) and reduced (Fig.13) form. The plasrnid number and sequence name are indicated above each sample in both Fig. 12 and Fig. 13. Samples are arranged according to the listing of Fig. 11, described above, as according to size of the VI hypervariable region. The CaseA2 sequence (p3020) is repeated in each gel to serve as a standard. Protein markers are also included as size standards. All samples represented in both Fig. 12 and 13 were deglycosylated by treatment with PNGaseF before analysis; again, with the gels of Fig. 12 being run in unreduced form and the gels of Fig. 13 representing gels showing samples analyzed after reduction of disulfide bonds by treatment with DTT. Many of the samples demonstrate multiple bands, similar in complexity to those seen for the CaseA2 protein. These bands coalesce to a single form, after reduction (bottom gels). In general, samples with longer VI hypervariable regions (left-hand gels) tend to display greated conformational diversity than samples with short VI regions (right-hand gels).

INDUSTRIAL APPLICABILITY

The invention has applications in the treatment and diagnosis of HIV- 1 vims infection disease. All publications cited in the specification, both patent publications and non-patent piiblicatioiis, are indicative of tlie level of skill of those skilled in the art to which this invention pertains. All these piiblicatioiis are herein fully incorporated by reference to the same extent as if each mdividiiai publication were specifically and individually indicated as being incorporated by reference.

Although the invention, herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is therefore to be imderstood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as defined by the following claims.

Claims

CLAIMS What is claimed is :

1. An isolated and purified polypeptide comprising a V1 V2 domain of a HIV envelope protein having a structural configuration of a " ^* form, as shown in Figure 6 and Figure 7,

2. The polypeptide of cl aim 1 wherein the ΗΓν envelope protein is native g l 20.

3. The polypeptide of claim 1 wherein the ΗΓν envelope protein is native gpl 40.

4. The polypeptide of claim 1 wherein the HIV envelope protein is recombinant gpl 20.

5. The polypeptide of claim 1 wherein the HIV envelope protein is recombinant g l 40.

6. The polypeptide of claim 4 wherein recombinant g l 20 comprises a portion of the g l20 coding region, such that the antigenicity of the VI V2 structural configuration of the "C" form is maintained.

7. Hie polypeptide of claim 1 which is a fusion protein comprising an ainiiio- terminal sequence directly linked to a VI V2 domain of an HIV envelope protein .having a structural configuration of a "C" form, as shown in Figure 6 and Figure 7,

8. The polypeptide of claim 7 wherein the ainino-terniinal sequence represents a portion ofMuLV gp7Q.

9. The polypeptide of claim 8 having the amino acid sequence as set forth in Figure 14 (SEQ ID NO;2).

10. An isolated nucleic acid molecule encoding a polypeptide with at least .90% amino acid sequence identity to the polypeptide of claim 9, wherein said polypeptide maintains substantial antigenicity of the V1/V2 structural configuration of the "C" form, as shown in

Figure 6 and Figure 7.

11. A recombinant DNA molecule comprising the nucleic acid sequence of claim 10.

12. A cell line transfected with a recombinant DNA molecule of claim 11.

1J

13. A method of treating a HTV positive subject comprising administering to the subject: a pharmaceutical, composition comprising a polypeptide of claim. 1, said pharmaceutical composition optionally containing a. pliamiaceufically active carrier, wherein adniinistration of the pharmaceutical composition results in a reduction in -HIV^' iral load.

14 A method of vaccinating a human subject against infection or progression of .HIV which comprises administration of a pharmaceutical composition which comprises a polypeptide of claim 1, said pharmaceutical composition optionally containing a pharmaceutically active carrier, wherein administration of the pharmaceutical composition results in generation of an immune response against HW infection.

15. A pharmaceutical composition, comprising: (a) an effective amount of a polypeptide of claim Ϊ ; and, (b) optionally, a pharmaceutically acceptable carrier.

16. A method for inducing an enhanced immunological response against a HIV antigen in a mammalian host, said method comprising inoculating the mammalian host with a polypeptide of claim 1.

17. A method of identifying a HIV antiviral compound, comprising: (a) combining component (i)— a test compound, component (ii)— a "C" form-specific antibody, and component (iii)— a polypeptide of claim 1; (b) measuring the effect the test compound has on the affinity of component (ii) for component (iii) of step (a); and, (c) comparing the effect the test compound has on the affinity of component (ii) for component (iii) versus the affinity of component (ii) for component (iii) in the absence of the test compound.

18. The method of claim 17 wherein component (i) is selected from the group consisting of a peptide, a protein, a non-proteinaceous organic or inorganic molecule, DNA and R A.

19. An isolated and purified polypeptide comprising a V1/V2 domain of an HIV envelope protein having a structural configuration of a "B" form, as shown in Figure 6 and Figure 7.

20. The polypeptide of claim 1 wherein the FHV envelope protein is native gpl20.

21. The polypeptide of claim 19 wherein the HIV envelope protein is native gp 140.

22. The polypeptide of claim 19 wherein the HIV envelope protein is recombinant gpl 20.

23. The polypeptide of claim 19 wherein the HIV envelope protein is recombinant g l 40.

24. The polypeptide of claim 22 wherein recombinant g l 20 comprises a portion of the gpl 20 coding region, such that the antigenicity of the VI/V2 ^'structural configuration of the "B" forai is maintained.

25. The polypeptide of claim 1 which is a fusion protein comprising an ainino- terminal sequence directly linked to a VI V2 domain of an HIV envelope protein having a structural configuration of a "B" form, as shown in Figure 6 and Figure 7.

26 The polypeptide of claim 25 wherein die aiiiino-terininal sequence represents a portion of MuLV gp70.

27. The polypeptide of claim 26 having the amino acid sequence as set forth in Figure 14 (SEQ ID NO: 2).

28. An isolated nucleic acid molecule encoding a polypeptide with at least 90% amino acid sequence identity to the polypeptide of claim 27, wheiein said polypeptide maintains substantial antigenicity of the V1/V2 structural configuration of the "B" form, as shown in

Figure 6 and Figure 7.

29. A recombinant DNA molecule comprising the nucleic acid sequence of claim 28.

30. A cell line transfected with a recombinant DNA molecule of c laim 29.

31. A method of tr eating a HIV positive subject comprising administering to the subject a pharmaceutical composition comprising a polypeptide of claim 19, said pharmaceutical composition optionally containing a pharmaceutically active carrier, wherein administration of the pharmaceutical composition results in a reduction in HIV viral load.

32. A method of vaccinating a human subject against infection or progression of HIV which comprises administration of a pharmaceutical composition which comprises a polypeptide of claim 19, said pharmaceutical composition optionally containing a pharmaceutically active carrier, wherein administration of iiie pharmaceutical composition results in generation of an immune response against HIV infection.

33. A pharmaceutical composition, comprising: (a) an effective amount of a polypeptide of claim 19; and, (b) optionally, a pharmaceutically acceptable carrier.

34. A method for inducing an enhanced immunological response agains an HIV antigen in a mammalian host , said method comprising inoculating the mammalian host with a polypeptide of c laim Ϊ 9.

35. A method of identifying a HIV antiviral compound, comprising: (a) combining component (i)— a test compound, component (ii)— a "B" form-specific antibody, and component (iii)— a polypeptide of claim 19; (b) measuring the effect the test compound has on the affinity of component (ii) for component (iii) of step (a); and, (c) comparing the effect the test compound has on the affinity of component (ii) for component (iii) versus the affinity of component (ii) for component (iii) in the absence of the test compound.

36. The method of claim 35 wherein component (i) is selected from the group consisting of a peptide, a protein, a non-proteinaceous organic or inorganic molecule, DNA or RNA.

37. An isolated and purified polypeptide comprising a V1/V2 domain of a HIV envelope protein having a structural configuration of a "B^W "A" combined form, as shown in Figure 6 and Figure 7.

38. The polypeptide of claim 37 wherein the HIV envelope protein is native gp 120.

39. The polypeptide of claim 37 wherein the HIV envelope protein is na tive gpl40.

40. The polypeptide of claim 37 wherein the HIV envelope protein is recombinant gpl20.

41. The polypeptide of claim 37 wherein the HTV envelope protein is recombinant

42. The polypeptide of claim 40 wherein recombinant gpl2Q comprises a portion of the gp!20 coding region, such that the anfigemciiy of the I/V2 structural configuration of the "B'V'A" _comb_me{j form is maintained_.

75

43. The polypeptide of claim 37 which is a fusion protein comprising an aniino- termmal sequence directly linked to a VI V2 domain of an HIV envelope protein having a structural configuration of a "B"f'A" combined form, as shown in Figure 6 and Figure 7.

44. The polypeptide of claim 43 wherein the ammo-terminal sequence represents a. portion of uLV gp70.

45. The polypeptide of claim 44 having the amino acid sequence as set forth in Figure 14 (SEQ ID NO;2).

46. An isolated nucleic acid molecule encoding a polypeptide with at least 90% amino acid sequence identity to the polypeptide of claim 45, wherein said polypeptide maintains substantial antigenicity of the V1 V2 structural configuration of the "B'7"A" combined form, as shown in Figure 6 and Figure 7.

47. A recombinant DNA molecule comprising the nucleic acid sequence of claim 46.

48. A cell line transfected with a recombinant DNA molecule of claim 47.

49. A method of heating a HIV positive subject comprising administering to the subject a pharmaceutical composition comprising a polypeptide of claim 37, said pharmaceutical composition optionally containing a pharmaceutically active carrier, wherein administration of the pharmaceutical composition results in a reduction in HIV viral load.

50. A method of vaccinating a human subject against infection or progression of HIV which comprises administration of a pharmaceutical composition which comprises a polypeptide of claim 37, said pharmaceutical composition optionally containing a pharmaceutically active carrier, wherein adiniiiistration of the pharmaceutical composition results in generation of an immune response against HTV infection.

51. A phamiaceutical composition, comprising: (a) an effective amount of a polypeptide of claim 37: and, (b) optionally, a pharmaceutically acceptable earner.

52. A method for inducing an enhanced immunological response against a HTV antigen in a mammalian host, said method comprising inoculating the mammalian host with a poiypeptide of claim 37.

53. A method of identifying a HIV antiviral compound, comprising: (a) combhimg component (i)— a test compound, component (ii)— a "B" "A" combined form-specific antibody, and component (iii)— a polypeptide of claim 37; (b) measuring the effect the test compound has on the affinity of component (ii) for component (iii) of step (a); and, (c) comparing the effect the test compound has on the affinity of component (ii) for component (iii) versus the affinity of componen (ii) for component (iii) in the absence of the test compound.

54. The method of claim 53 wherein component (i) is selected from the group consisting of a peptide, a protein, a non-proteinaceous organic or inorganic molecule, DNA and RNA.