STABLE IMMUNOGEN BASED ON INNER DOMAIN OF HIV-1 GP120 FOR

INDUCING IMMUNITY AGAINST HIV

BACKGROUND OF INVENTION

[0001] Fc receptor-dependent effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) constitute an important bridge between innate and adaptive immunities to HIV-1 [1]. They trigger clearing of virus particles or virus-infected cells through mechanisms involving interactions of antibody constant (Fc) region and Fey receptors (FcyRs) on the surface of cells involved or potentially involved in HIV infection (natural killer cells (NKs), monocytes, macrophages, dendritic cells, and neutrophils) [2-4].

[0002] In contrast to broadly neutralizing antibody responses, which become detectable 2-4 years post-infection [5, 6], ADCC responses appear relatively early during acute infection [7, 8] and are detectable as early as 21 and 48 days after SIVmac251 [9-11] and acute HIV-1 [12] infection, respectively. These early ADCC responses, which in general are broadly reactive, precede the appearance of neutralizing antibody responses with similar breadth [13, 14]. ADCC ultimately leads to the target cell being killed by an effector cell, decreasing the probability of cell-to-cell transmission.

[0003] Considerable evidence supports a role of ADCC in preventing or modulating HIV-1 infection. ADCC responses in chronically HIV-1 infected individuals were shown to correlate with slower progression of HIV [15-18] or decreased virus replication [19]. HIV-specific antibodies capable of inducing ADCC and therefore important and serve to protect, for example, against infection in mother-to-infant transmission [20].

[0004] Another Fc-mediated effector function, antibody-dependent cell-mediated viral inhibition (ADCVI) coupled with the low-affinity IgG receptor CD 16 (FcyRIIIa) genotype, correlated with protection against HIV-1 transmission in a subset of volunteers in the Vax004 vaccine efficacy trial [21]. The picture is even stronger for non-human primate (NHP) infections where sustained ADCC responses predict delayed disease in SIV/SHIV infection [22, 23] and ADCC activity correlates with vaccine-induced protection, often in the absence of neutralizing antibodies [6, 10, 24-28]. Passive immunization studies using a variant of the neutralizing monoclonal antibody, bl2, that lacks full Fc receptor binding function but fully retains neutralization potency, resulted in reduced protection against SHIV challenge acquisition [29, 30]. To this end, the latest report on the protective effect of vaginal application of neutralizing and non-neutralizing antibodies against vaginal SHIVi6 _2p 3 challenge acquisition [31] clearly indicated that mAbs capable of Fc-dependent function provide post-infection control of the SHIV challenge virus, leading to in vivo anti-HIV effects that may strengthen those elicited by neutralizing mAbs.

[0005] With regard to vaccine-induced protection in humans, the recent vaccination strategy tested in the ALVACHIV/AIDSVAXB/E RV144 vaccine trial, which showed an estimated vaccine efficacy of 31.2% in preventing HIV-1 acquisition, is of particular importance [32]. It proved, for the first time, that vaccination can protect humans from HIV-1 infection, and that protection could be due to the generation of a blend of neutralizing and non-neutralizing antibody responses with the presence of very modest CD4 T cell responses [32, 33].

[0006] The RV144 immunization regimen selectively induced non-neutralizing IgG3 responses which showed highly coordinated Fc-mediated effector responses [34, 35]. These responses correlated with decreased risk of HIV- 1 infection in a blinded follow-up case control study [34]. The array of mAbs elicited in RV144 trial was narrow with specificities for the second variable (V2) loop region [33] as well as CD4-inducible epitopes within the first and second constant (C1/C2) domain of gpl20 [36]. Whereas V2-specific mAbs were suggested to act through both direct neutralization and Fc-mediated effector function [37, 38], the C1/C2- region specific mAbs were non-neutralizing and capable of potent ADCC [39]. Most importantly, ADCC emerged as a potential correlate of protection in secondary analyses in the subset vaccinated subjects with low plasma anti-HIV-1 Env IgA titers [40]. These studies clearly indicated that RV144 vaccine induced broadly reactive ADCC responses to the C1/C2 domain and that these responses might be partially responsible for the protection observed.

[0007] Together, this data suggests an intriguing link between vaccine-mediated protection and ADCC, and provides the first evidence that ADCC might contribute to HIV-1 vaccine efficacy in humans. A role for Fc-mediated effector function in protective immunity against HIV-1 is also indicated. Studies also point toward non-neutralizing, conformational epitopes in the C1/C2 region as important players in these responses (reviewed in [47, 50, 51]). It is well established that this transitional epitope region becomes exposed on the virion spike only upon binding to the CD4 receptor post-infection [48, 49, 52, 53]. In addition, binding to CD4 significantly enhances its exposure in context of monomeric full length gpl20 [44]. The fact that the ADCC responses were directed to these transitional CD4-induced epitopes in the RV144 trial consisting of immunization regiment of ALVACHIV vCP1521 prime and recombinant clade B/E monomeric gpl20 boost confirms high immunogenicity of this region.

[0008] Protein immunogens based on the C1/C2 region of gpl20 that have the ability to induce non-neutralizing antibody responses along with Fc-mediated effector responses could serve as effective components of HIV- 1 vaccines. The present invention is directed to the elucidation of such peptides as well as other important goals.

BRIEF SUMMARY OF INVENTION

[0009] Provided herein are stable protein immunogens that comprise portions of the inner domain of the Human Immunodeficiency Virus (HIV-1) gpl20 protein. These inner domain immunogens can be used to vaccinate subjects against infection with HIV, the causative agent of Acquired Immunodeficiency Syndrome (AIDS). The ID immunogens can also be used as targets in screening to identify small molecule or peptide inhibitors that block HIV- 1 viral entry and attachment steps (e.g., for therapeutic or research use). The ID immunogens can further be used as probes for identifying gpl20 first and second constant (C1/C2) region (or C1/C2 and V1V2 combined)- specific antibodies (such as monoclonal antibodies; mAbs), e.g., for use in ELISA, C1/C2 binding titer assays, and the like, and for clinical diagnostic or research applications.

[0010] The ID immunogens contain C1/C2- specific (or C1/C2 and V1V2 combined) gpl20 epitopes that are occluded in the native Env trimer of most HIV-1 isolates and only become exposed on the target cell surface during viral entry after Env trimers engage CD4 of the target cell. Importantly, the C1/C2 epitopes also persist on freshly infected cell surfaces for extended periods of time post-infection where they become excellent targets for Fc-mediated effector activity. These epitopes, marked by mAb A32 (A32-like epitopes), show poor immunogenicity in conventional gpl20-based vaccines. The ID immunogens selectively present these CD4- inducible epitopes (CD4i epitopes) within a minimal structural unit of gpl20. As compared to unliganded gpl20, the ID immunogens stably present CD4i epitopes within the C1/C2 gpl20 region and they are ideal vaccine candidates for stimulating a non-neutralizing Fc-mediated effector function antibody response to the C1/C2 region.

[0011] C1/C2 epitopes of gpl20 have been implicated as targets for antibodies mediating antibody-dependent cellular cytotoxicity (ADCC) that correlate with reduced HIV infection risk in a subset of vaccinated subjects. Utilization of the novel immunogens in vivo in a vaccination regimen allows selective elicitation of non-neutralizing Fc-mediated response to the C1/C2 region (or both C1/C2 and V1V2 regions) without the complication of neutralization epitopes. In addition, vaccination approaches with the ID immunogens will help overcome the major obstacle of passive immunization studies with antibodies acting through Fc-mediated effector function and relating to overdosing and an apparent prozone effect. It will allow evaluation of the protective spectrum of elicited non-neutralizing antibodies that mediate the Fc-mediated effector function in vaccine settings.

[0012] Each of the ID immunogens of the invention is based on a mature HIV-1 gpl20 polypeptide sequence, but lacks selected domains and includes particular amino acid changes. The mature gpl20-based ID immunogens are thus further characterized by two or more of the following features:

(i) deletion of a portion of the N-terminus of the protein (generally corresponding to residues 1-11 of gpl20 from the HIV-1 isolate HXBc2 set forth in SEQ ID NO: 1);

(ii) engineered cysteines at positions 65 and 115 corresponding to the sequence of the HXBc2 isolate, which leads to stabilization of the immunogen in a CD4-bound conformation upon disulfide bond formation between the cysteines (Ces-Cns);

(iii) deletion of the outer domain region of the protein (generally corresponding to amino acids 258-472 of the HXBc2 isolate) and replacement by a GlyGly dipeptide linker; and

(iv) deletion of the V1V2 loop of the protein (generally corresponding to amino acids 119-205 of the HXBc2 isolate) and replacement by a GlyGlyAla tripeptide linker.

[0013] As used herein, mature gpl20 refers to gpl20 polypeptide lacking the signal peptide (e.g., residues 1-30 of the sequence of full-length gpl20 from the HIV-1 isolate HXBc2 shown in SEQ ID NO:2; the sequence of mature gpl20 from HXBc2 is provided in SEQ ID NO: l).

[0014] Thus, and in a first embodiment, the invention is directed to a HIV-1 gpl20 inner domain (ID) immunogen, where the ID immunogen comprises:

(a) a mature HIV-1 gpl20 polypeptide having two or more of the features (i)-(iv):

(i) deletion of amino acids 1-11 of the N-terminus,

(ii) cysteine residues at amino acid positions 65 and 115,

(iii) deletion of amino acids 258-472 of the outer domain region and replacement by a GlyGly dipeptide linker, and (iv) deletion of amino acids 119-205 of the V1V2 loop and replacement by a

GlyGlyAla tripeptide linker,

wherein the amino acid numbering corresponds to the amino acid sequence set forth in SEQ ID NO: 1; or

(b) a variant having at least about 95% sequence identity with an immunogen of (a) over the entire length of the immunogen sequence

[0015] SEQ ID NO: l is the amino acid sequence of mature gpl20 from the HIV-1 isolate HXBc2.

[0016] In certain aspects, the ID immunogen of (a) has three or more of the features (i)-(iv). In other certain aspects, the ID immunogen of (a) has features (i)-(iii). In still further aspects, the ID immunogen of (a) has features (i)-(iv).

[0017] In non-limiting examples, the ID immunogen comprises an amino acid sequence set forth in any one of SEQ ID NOs:3-54 or a variant thereof having at least about 95% sequence identity with the immunogen over the entire length of the immunogen sequence

[0018] In certain aspects, the ID immunogens have a C65-C ₁₁₅ disulfide bond.

[0019] In certain aspects, the variants have the property of inducing Fc-mediated humoral response to C1/C2 or combined C1/C2 and V1/V2 regions of HIV-1 gpl20.

[0020] In a second embodiment, the invention is directed to an immunogenic composition comprising one or more ID immunogens as defined herein, and a pharmaceutically-acceptable carrier or diluent.

[0021] The immunogenic compositions of the invention can be comprised of only one specific ID immunogen, or two or more different ID immunogens (i.e., ID immunogens having different amino acid sequences). In a non-limiting example, the immunogenic composition comprises one or more of the ID immunogens set forth in SEQ ID NOs:3-54 or variants thereof, or both.

[0022] In a third embodiment, the invention is directed to a method of inducing an immune response to an ID immunogen in a subject, comprising administering to a subject an

immunogenic amount of an immunogenic composition as defined herein, thereby inducing an immune response in the subject.

[0023] As indicated above, the immunogenic compositions of the invention can be comprised of only one specific ID immunogen, or two or more different ID immunogens (i.e., ID immunogens having different amino acid sequences). In a non-limiting example, the immunogenic composition comprises one or more of the ID immunogens set forth in SEQ ID NOs:3-54 or variants thereof, or both.

[0024] In a fourth embodiment, the invention is directed to a method of vaccinating a subject with a protective antigen, comprising administering to a subject in need thereof an effective amount of an immunogenic composition as defined herein, thereby vaccinating a subject with a protective antigen.

[0025] As indicated above, the immunogenic compositions of the invention can be comprised of only one specific ID immunogen, or two or more different ID immunogens (i.e., ID immunogens having different amino acid sequences). In a non-limiting example, the

immunogenic composition comprises one or more of the ID immunogens set forth in SEQ ID NOs:3-54 or variants thereof, or both.

[0026] In a fifth embodiment, the invention is directed to a method of screening for antibodies with binding affinity for the C1/C2 region HIV-1 gpl20, comprising:

(a) contacting an ID immunogen as defined herein with a test antibody, and

(b) detecting binding of the ID immunogen by the test antibody.

[0027] Because antibodies can recognize and bind different antigens having similar sequences, the method of this embodiment can be conducted using one specific ID immunogen, or two or more different ID immunogens (i.e., ID immunogens having different amino acid sequences). In a non-limiting example, the ID immunogen is one or more of the ID immunogens set forth in SEQ ID NOs:3-54 or variants thereof, or both.

[0028] In some aspects of this embodiment, the antibody is one or more selected from a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, a single chain antibody (scFv), an Fab fragment, an F(ab')2 fragment, a multi- specific antibody, and a disulfide-linked antibody.

[0029] In some aspects of this embodiment, binding of the ID immunogen by the test antibody is detecting using an immunoassay, such as ELISA.

[0030] In a sixth embodiment, the invention is directed to a method of screening for inhibitors of HIV-1 viral attachment comprising:

(a) contacting an ID immunogen as defined herein with a test molecule, and

(b) detecting binding of the ID immunogen by the test molecule. [0031] The method of this embodiment can be conducted using one specific ID immunogen, or two or more different ID immunogens (i.e., ID immunogens having different amino acid sequences). In a non-limiting example, the ID immunogen is one or more of the ID immunogens set forth in SEQ ID NOs:3-54 or variants thereof, or both.

[0032] The test molecule may be any molecule that binds to an ID immunogen, such as an antibody or some other protein, peptide, nucleic acid, carbohydrate, lipid, fatty acid, small drug molecule, etc. After being identified as having binding affinity for an ID immunogen, the test molecule can be further screened to determine with the test molecule can interferes with the association of Env proteins displayed by HIV-1 and cell surface receptors. If this association can be inhibited or blocked, HIV-1 viral attachment and/or entry can be inhibited or blocked.

[0033] In some aspects of this embodiment, binding of the ID immunogen by the test molecule is detecting using an immunoassay such as ELISA.

[0034] The foregoing has outlined rather broadly the features and technical advantages of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described herein, which form the subject of the claims of the invention. It should be appreciated by those skilled in the art that any conception and specific embodiment disclosed herein may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present invention. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims. The novel features which are believed to be characteristic of the invention, both as to its organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that any description, figure, example, etc. is provided for the purpose of illustration and description only and is by no means intended to define the limits the invention.

BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1. Amino acid sequence alignment of ID immunogens based the consensus sequences of HIV-1 gpl20 proteins from HIV-1 clades Al (SEQ ID NO:3), A2 (SEQ ID NO:4), B (SEQ ID NO:5) , C (SEQ ID NO:6), D (SEQ ID NO:7), Fl (SEQ ID NO:8), F2 (SEQ ID NO:9), G (SEQ ID NO: 10), H (SEQ ID NO: 11), 01_AE (SEQ ID NO: 12), 02_AG (SEQ ID NO: 13), 03_AB (SEQ ID NO: 14), 04_CPX (SEQ ID NO: 15), 06_CPX (SEQ ID NO: 16),

08_BC (SEQ ID NO: 17), 10_CD (SEQ ID NO: 18), 11_CPX (SEQ ID NO: 19), 12_BF (SEQ ID NO:20), 14_BG (SEQ ID NO:21). The entry termed "CON_OF_CONS_VlV2" (SEQ ID NO:22) is an ID immunogen based on a consensus sequence of the other HIV-1 clade consensus sequences. Each of the immunogens comprises the V1V2 region of the protein.

[0036] Figure 2. Amino acid sequence alignment of ID immunogens based on the sequences of HIV-1 gpl20 proteins from particular HIV-1 strains AB097872 (SEQ ID NO:23), AB052867 (SEQ ID NO:24), YU2 (SEQ ID NO:25), HXBc2 (SEQ ID NO:26), HM623558 (SEQ ID NO:27) and DQ404010 (SEQ ID NO:28). Each of the immunogens comprises the V1V2 region of the protein.

[0037] Figure 3. Amino acid sequence alignment of ID immunogens based on HIV-1 gpl20 proteins from HIV-1 clades AI (SEQ ID NO:3), A2 (SEQ ID NO:4), B (SEQ ID NO:5) , C

(SEQ ID NO:6), D (SEQ ID NO:7), Fl (SEQ ID NO:8), F2 (SEQ ID NO:9), G (SEQ ID NO: 10), H (SEQ ID NO: 11), 01_AE (SEQ ID NO: 12), 02_AG (SEQ ID NO: 13), 03_AB (SEQ ID NO: 14), 04_CPX (SEQ ID NO: 15), 06_CPX (SEQ ID NO: 16), 08_BC (SEQ ID NO: 17), 10_CD (SEQ ID NO: 18), 11_CPX (SEQ ID NO: 19), 12_BF (SEQ ID NO:20), 14_BG (SEQ ID NO:21), and CON_OF_CONS_VlV2 (SEQ ID NO:22), and strains AB097872 (SEQ ID NO:23), AB052867 (SEQ ID NO:24), YU2 (SEQ ID NO:25), HXBc2 (SEQ ID NO:26),

HM623558 (SEQ ID NO:27) and DQ404010 (SEQ ID NO:28). Each of the immunogens comprises the V1V2 region of the protein.

[0038] Figure 4. Amino acid sequence alignment of ID immunogens based on the consensus sequences of HIV-1 gpl20 proteins from HIV-1 clades AI (SEQ ID NO:29), A2 (SEQ ID NO:30), B (SEQ ID NO:31), C (SEQ ID NO:32), D (SEQ ID NO:33), Fl (SEQ ID NO:34), F2 (SEQ ID NO:35), G (SEQ ID NO:36), H (SEQ ID NO:37), 01_AE (SEQ ID NO:38), 02_AG (SEQ ID NO:39), 03_AB (SEQ ID NO:40), 04_CPX (SEQ ID NO:41), 06_CPX (SEQ ID NO:42), 08_BC (SEQ ID NO:43), 10_CD (SEQ ID NO:44), 11_CPX (SEQ ID NO:45), 12_BF (SEQ ID NO:46), and 14_BG (SEQ ID NO:47). The entry termed "CON_OF_CONS" (SEQ ID NO:48) is an ID immunogen based on a consensus sequence of the other HIV-1 clade consensus sequences. The V1V2 region in each of the immunogens is replaced by the GGA trimer. [0039] Figure 5. Amino acid sequence alignment of ID immunogens based on the sequences of HIV- 1 gpl20 proteins from particular HIV-1 strains AB097872 (SEQ ID NO:49), AB052867 (SEQ ID NO:50), YU2 (SEQ ID NO:51), HXBc2 (SEQ ID NO:52), HM623558 (SEQ ID NO:53) and DQ404010 (SEQ ID NO:54). The V1V2 region in each of the immunogens is replaced by the GGA trimer.

[0040] Figure 6. Amino acid sequence alignment of ID immunogens based on HIV-1 gpl20 proteins from HIV-1 clades clades Al (SEQ ID NO:29), A2 (SEQ ID NO:30), B (SEQ ID

NO:31), C (SEQ ID NO:32), D (SEQ ID NO:33), Fl (SEQ ID NO:34), F2 (SEQ ID NO:35), G (SEQ ID NO:36), H (SEQ ID NO:37), 01_AE (SEQ ID NO:38), 02_AG (SEQ ID NO:39), 03_AB (SEQ ID NO:40), 04_CPX (SEQ ID NO:41), 06_CPX (SEQ ID NO:42), 08_BC (SEQ ID NO:43), 10_CD (SEQ ID NO:44), 11_CPX (SEQ ID NO:45), 12_BF (SEQ ID NO:46), 14_BG (SEQ ID NO:47) and CON_OF_CONS (SEQ ID NO:48), and strains AB097872 (SEQ ID NO:49), AB052867 (SEQ ID NO:50), YU2 (SEQ ID NO:51), HXBc2 (SEQ ID NO:52), HM623558 (SEQ ID NO:53) and DQ404010 (SEQ ID NO:54). The V1V2 region in each of the immunogens is replaced by the GGA trimer.

[0041] Figures 7A-7C. Co-localization of Cluster A epitopes within Env trimer and monomeric gpl20. Figures 7A and 7B show Cluster A epitopes are associated with the so-called non-neutralizing face of Env [76] which is partially occluded by gp41 in the oligomer and buried within the unliganded Env trimer. gpl20 is shown as oval shapes, with the outer neutralizing face in light shading and the inner noneutralizing face in darker shading. Carbohydrate moieties are depicted as tree-like structures. gp41 is comprised of N-terminal and C-terminal

transmembrane domains, depicted as tubes. The membrane-proximal gp41 region exposed on the trimer is shown as the darker shaped portion of the tubes. The figure is adapted from [76]. Figure 7C shows colocalization of Cluster A epitopes (highlighted with darker shading) within the full- length gpl20 monomer displayed with nonneutralizing face up. Cluster A epitopes are associated with the region proximal to the N, C termini of gpl20. The figure is adapted from [44].

[0042] Figure 8. Relative potencies of mAb groups in the bound virion ADCC assay.

[0043] Figures 9A-9D. Low-dose heterologous SHIV(162P3) challenge studies with rhFLSC. Six male rhesus macaques per group were immunized with 300 μg of rhFLSC formulated with RC529SE or Iscomatrix adjuvant at 0, 4, 24 and 59 weeks. Four weeks after last boost animals were challenged weekly with 50 TCID ₅₀ SHIV^s- Unimmunized naive animals served as controls. Figure 9A shows acquisition plots of all experimental groups. Figure 9B shows the correlation between the numbers of intrarectal challenges required to infect and ADCC EC ₅₀ titer. All animals immunized with rhFLSC are included. Figure 9C shows acquisition plots for N12i2 competitive titers. Figure 9D shows acquisition plots for competitive titers to A32 epitope. The correlations are shown for animals with IL2 ELISPOT counts above the mean. Figure from adopted from [81].

[0044] Figure 10. Crystal structures of mAb N5i5, N60i3, JR4 and 2.2c bound to sCD4- triggered gpl20 cores. The electrostatic potential displayed at the molecular surfaces of gpl20 (right).

[0045] Figures 11A-11C. Definition of Cluster A epitopes within the Env antigen. Fig. 11 A shows epitope footprints. The Ca atoms of the gpl20 residues involved in mAb binding are represented balls and displayed over the ribbon diagram of gpl20. Fig. 1 IB shows mapping of contact residues on the inner domain sequence. Residues of gpl20 (from strains 93TH057(for N5-i5, Ν60-Ϊ3 and JR4 footprints, these three sequences are identical and found in SEQ ID NO:58) and 89.6P (for 2.2c footprints; SEQ ID NO:59)) involved in binding to N5i5, N60i3, JR4 and 2.2c are highlighted. Fig. 11C shows sequence conservation of Cluster A epitopes. The height of the residue at each position is proportional to its frequency of distribution among the HIV-1 isolates as deposited Los Alamos data base (all clades are included).

[0046] Figures 12A-12B. Design of inner domain constructs. Fig. 12A shows the subsequent steps of design of the inner domain molecule; (left) structure of gpl20 _93TH057 core _e in CD4-bound conformation (from the N5-i5 Fab-gpl20 _93TH057 -dld2CD4 complex, Protein Data Bank (PDB) code: 4H8W); (middle and right) the putative IDl and ID2 immunogen structures. Asn-proximal NAG residues of glycosyl group and disulfide bonds of inner domain are shown as balls-and- sticks. Fig. 12B shows sequence of ID constructs IDl (SEQ ID NO:56) and ID2 (SEQ ID

NO:57) which are based on the gpl20 sequence (SEQ ID NO:55) of the HIV-1 isolate 93TH057. The -GG- and -GGA- linkers are indicated, as is the C ₆ 5-Cn ₅ disulfide introduced to IDl. Inner domain layers are shaded as in (A).

[0047] Figures 13A-13B. Physico-chemical characterization of IDl. Fig. 13A shows analytical RP-HPLC where IDl (left panel) and PNGF digestion product of IDl (right panel) were analyzed on Symmetry 300™ C4 column (2.1 x 150 mm, 3.5μιη) using a linear gradient of 5-65% of acetonitrile at a flow rate of 0.25 mL/min over 30 min. Fig. 13B shows SDS PAGE analysis of purified ID1 (line 1), ID1 subjected to cleavage with PNG F (line 2), protein standards (line 3).

[0048] Figures 14A-14B. Binding of Cluster A antibodies to ID2. Fig. 14A provides bar graph for binding affinity (K _D ) of Cluster A mAbs to FLSC (column 1), gp l20BaL (column 2), ID1 (column 3), and ID2 (column 4). The insert represents a magnified view of the higher binding affinity section of the graph. Units are in nM. Fig. 14B shows isothermal titration calorimetry (ITC) curves for A32 IgG against FLSC and ID2.

[0049] Figures 15A-15B. Functional properties of ID2. Fig. 15A shows results from anti- Cluster A ADCC competition. ADCC activities of Cluster A mAbs (at concentration 0.67nM) in the presence of increasing concentrations of ID2 protein were measured using gpl20-coated CEM GFP CCR5 snap cells as targets and PBMCs as effector cells. Fig. 15B shows results from ADCC competition of antibodies in sera from HIV-infected individuals. CEM.NKr cells infected with Nef-Vpu- VSV-G pseudotyped NL4.3 GFP ADA were used at 48h post-infection for surface staining (left) and FACS -based ADCC assay (right) using sera from HIV-1 infected individuals pre-incubated in absence or presence of 10 μg AV1V2V3V5 D368R or 2.66 μg inner domain protein/μΐ sera for 30 min at room temperature. Data shown are representative of at least three different experiments. Statistical significance was tested using paired one-way ANOVAs (** p<0.01, *** p<0.001, **** p<0.0001).

[0050] Figures 16A-16D. Humoral Immune Response Induced by ID2 in Rabbits. Fig. 16A shows the results of evaluation of IgG titers elicited in sera of immunized rabbits against ID2 protein. 10-fold dilutions of heat inactivated rabbits sera, were tested in a direct ELISA (see Material and Methods) Fig. 16B shows recognition and Fig. 16C shows ADCC response of antibodies elicited in sera of immunized rabbits against HIV-1 gpl20-coated cells. CEM.NKr cells were coated with 100 ng/ml of recombinant HIV-1 gpl20 and analysed to be

recognized/ ADCC killed (as measured by FACS-based ADCC assay [80] by antibodies elicited in rabibit sera of week 0, 12 and 15 (1: 100 and 1: 1000 dilution). Fig. 16D shows the ADCC response of antibodies elicited in rabibit sera against HIV-1 infected cells. CEM.NKr cells were infected with HIV-1 lacking Nef and Vpu (N- U-) for 48 h and the FACS-based ADCC assay with rabbit sera of weeks 0 and 15 (diluted at 1: 100) was performed. Alternatively, target cells were pre-incubated 30 min with 5 μg/ml of Fab A32 or Fab 17b prior incubation with effector cells and rabbit sera. Data shown represents the percentage of ADCC-mediated killing obtained from three independent experiments.

[0051] Figure 17. Specificity of antibodies elicited in immunized rabbits. Recognition of HIV-1 gpl20-coated cells by antibodies of sera of immunized rabbits (week 0, 12 and 15; 1: 100 dilution) was analyzed using CEM.NKr cells coated with 100 ng/ml of recombinant HIV-1 gpl20. The target cells were pre-incubated 30 min with Fabs of Cluster A mAbs A32 and N5-i5, co-receptor binding side mAb ΝΊ2-Ϊ2 and AV1V2V3V5 D368R gpl20 (a gpl20 variant bearing a D368R mutation abrogating the ability of gpl20 to bind to cell surface CD4 and lacking variable regions VI, V2, V3, and V5 as described in [41] before mixing with sera and analyzing for surface staining. Data shown are representative of at least two different experiments.

DETAILED DESCRIPTION OF THE INVENTION

/. Definitions

[0052] Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found, for example, in Benjamin Lewin, Genes VII, published by Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al. (eds.); The Encyclopedia of Molecular Biology, published by Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by Wiley, John & Sons, Inc., 1995 (ISBN

0471186341); and other similar technical references.

[0053] As used herein, "a" or "an" may mean one or more. As used herein when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more than one. As used herein "another" may mean at least a second or more. Furthermore, unless otherwise required by context, singular terms include pluralities and plural terms include the singular.

[0054] As used herein, "about" refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term "about" generally refers to a range of numerical values (e.g., +/- 5-10% of the recited value) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). In some instances, the term "about" may include numerical values that are rounded to the nearest significant figure. //. The Present Invention

[0055] As indicated above, provided herein are protein immunogens that selectively present the HIV-1 gpl20 polypeptide C1/C2 region (or both the C1/C2 and V1/V2 regions) in its CD4- bound state within a minimal structure. These immunogens can be used in vivo for inducing humoral Fc-mediated effector responses to C1/C2 region epitopes (or both C1/C2 and V1/V2 regions) without the complication of neutralization epitopes. The invention focuses on A32-like epitope targets because they persist on freshly infected cell surfaces for extended periods of time post-infection [52, 53]. The persistence of C1/C2 region epitopes on target cell surfaces in a model of cell-to-cell HIV-1 spread is unique as the exposure of other non-neutralizing conserved domains such as the Cluster I and II epitopes of gp41 is evanescent [54, 55]. The C1/C2 region epitopes are potentially better targets for ADCC antibodies capable of the blocking of HIV- 1 infection as opposed to epitopes exposed during viral budding. Passive immunization studies suggest that protective neutralizing antibodies have less than a twenty-four hour window to block transmission [56, 57]. This time frame is around the length of a single replication cycle of HIV-1 [58, 59] making it unlikely that transmission-blocking antibodies involve the recognition of budding virions from infected cells. The C1/C2 region- specific epitopes can also be important, along with those on budding virions, as ADCC targets in the post-infection control of viremia by Fc-mediated effector function is incorporated in vaccine strategy.

ID immunogens

[0056] The invention is directed to HIV-1 gpl20 polypeptide inner domain (ID)

immunogens which include immunogens designed to exclusively present epitopes within the C1/C2 region as well as immunogens designed to present epitopes within both the C1/C2 region and the VI V2 loop.

[0057] The ID immunogens of the invention are derived from HIV-1 gpl20 polypeptides, where selected regions or domains are deleted in order to limit the immunogens to amino acids of the gpl20 inner domain. In addition, it has been found that by altering the sequence of the immunogens to exhibit cysteine residues at specific positions, disulfide bond formation occurs that results in a stable immunogen which selectively presents CD4-inducible epitopes. Therefore, the ID immunogens of the invention are also engineered to have cysteine residues in selected positions. [0058] Each of the ID immunogens of the invention is based on a mature HIV-1 gpl20 polypeptide sequence, but lacks selected domains and includes particular amino acid changes. The mature gpl20-based ID immunogens are thus further characterized by two or more of the following features:

(i) deletion of a portion of the N-terminus of the protein (generally corresponding to residues 1-11 of gpl20 from the HIV-1 isolate HXBc2 set forth in SEQ ID NO: 1);

(ii) engineered cysteines at positions 65 and 115 corresponding to the sequence of the HXBc2 isolate, which leads to stabilization of the immunogen in a CD4-bound conformation upon disulfide bond formation between the cysteines (Ces-Cns);

(iii) deletion of the outer domain region of the protein (generally corresponding to amino acids 258-472 of the HXBc2 isolate) and replacement by a GlyGly dipeptide linker; and

(iv) deletion of the V1V2 loop of the protein (generally corresponding to amino acids 119-205 of the HXBc2 isolate) and replacement by a GlyGlyAla tripeptide linker.

[0059] More succinctly, the HIV-1 gpl20 ID immunogens of the invention are those that comprise a mature HIV-1 gpl20 polypeptide having two or more of the features (i)-(iv):

(i) deletion of amino acids 1-11 of the N-terminus,

(ii) cysteine residues at amino acid positions 65 and 115,

(iii) deletion of amino acids 258-472 of the outer domain region and replacement by a GlyGly dipeptide linker, and

(iv) deletion of amino acids 119-205 of the V1V2 loop and replacement by a

GlyGlyAla tripeptide linker,

wherein the amino acid numbering corresponds to the amino acid sequence set forth in SEQ ID NO: 1.

As discussed in detail below, the ID immunogens of the present includes also include variants having at least about 95% sequence identity with one of these immunogen over the entire length of the immunogen sequence.

[0060] In certain aspects of the invention, the ID immunogen has three of the features (i)- (iv). In particular aspects, the ID immunogen has features (i) and (ii), or features (i) and (iii), or features (i) and (iv), or features (ii) and (iii), or features (ii) and (iv), or features (iii) and (iv), or features (i), (ii) and (iii), or features (ii), (iii) and (iv), or features (i), (ii) and (iv), or features (i), (iii) and (iv), or features (i)-(iv). In exemplary aspects, the ID immunogen has features (i)-(iii) or features (i)-(iv).

[0061] Reference herein to "a mature HIV-1 gpl20 polypeptide" means a gpl20 polypeptide without the signal peptide. Thus, for example, reference to "amino acids 1-11 of the N- terminus...wherein the amino acid numbering corresponds to the amino acid sequence set forth in SEQ ID NO: l" indicates that SEQ ID NO: l is the amino acid sequence of a mature gpl20 protein lacking the signal peptide. The mature gpl20 protein provided in SEQ ID NO: l is mature gpl20 from the HIV-1 isolate HXBc2. The sequence of the full-length protein (i.e., containing the signal peptide at positions 1-30) is provided in SEQ ID NO:2.

[0062] In certain aspects, the ID immunogens have a C ₆₅ -Cn ₅ disulfide bond. In other aspects, the ID immunogens exist without a disulfide bond linking C ₆₅ -Cu ₅ - [0063] As will be clear from the present disclosure, the identity of the gpl20 polypeptide upon which the ID immunogens of the invention may be based is not a limiting factor in the invention. Therefore, any gpl20 polypeptide sequence, now known or later characterized, may serve as the basis for an ID immunogen of the invention. Thus, HIV-1 gpl20 sequences can be used without regard to the clade, isolate or strain from which they are obtained. However, exemplary clades from which gpl20 polypeptides may be selected include, but are not limited to, HIV-1 clades Al, A2, B, C, D, Fl, F2, G, H, 01_AE, 02_AG, 03_AB, 04_CPX, 06_CPX, 08_BC, 10_CD, 11_CPX, 12_BF, and 14_BG. Exemplary strains from which gpl20

polypeptides may be selected include, but are not limited to, strains AB097872 and AB052867 (clade A), strains YU2 and HXBc2 (clade B), strains HM623558 and DQ404010 (clade C), and strains 93TH057 and 92TH023 (clade AE).

[0064] Specific examples of ID immunogens of the invention based on the consensus sequence of different HIV- 1 clades are provided in Table 1 where the ID immunogens have each of features (i)-(iii) (i.e., retaining the V1V2 region) and where the clades are Al, A2, B, C, D, Fl, F2, G, H, 01_AE, 02_AG, 03_AB, 04_CPX, 06_CPX, 08_BC, 10_CD, 11_CPX, 12_BF, and 14_BG. The entry termed "CON_OF_CONS_VlV2" is an ID immunogen based on the consensus of consensus sequences of HIV-1 clades shown. An amino acid sequence alignment of these immunogens is provided in Figure 1. Table 1

[0065] Specific examples of ID immunogens of the invention based on different HIV-1 strains are provided in Table 2 where the ID immunogens have each of features (i)-(iii) (i.e., retaining the V1V2 region) and where the strains are strains AB097872 and AB052867 (clade A), strains YU2 and HXBc2 (clade B), and strains HM623558 and DQ404010 (clade C). An amino acid sequence alignment of these immunogens is provided in Figure 2.

Table 2

[0066] Specific examples of ID immunogens of the invention based on the consensus sequence of different HIV- 1 clades are provided in Table 3 where the ID immunogens have each of features (i)-(iv) (i.e., the V1V2 region is replaced by the GGA trimer) and where the clades are Al, A2, B, C, D, Fl, F2, G, H, 01_AE, 02_AG, 03_AB, 04_CPX, 06_CPX, 08_BC, 10_CD, 11_CPX, 12_BF, and 14_BG. The entry termed "CON_OF_CONS" is an ID immunogen based on the consensus of consensus sequences of HIV-1 clades shown. An amino acid sequence alignment of these immunogens is provided in Figure 4. Table 3

[0067] Specific examples of ID immunogens of the invention based on different HIV-1 strains are provided in Table 4 where the ID immunogens have each of features (i)-(iv) (i.e., the V1V2 region is replaced by the GGA trimer) and where the strains are strains AB097872 and AB052867 (clade A), strains YU2 and HXBc2 (clade B), and strains HM623558 and DQ404010 (clade C). An amino acid sequence alignment of these immunogens is provided in Figure 5.

Table 4

Variants

[0068] Due to the high degree of conservation between gpl20 polypeptide sequences from different HIV-1 clades and strains, minor variations can be made to the ID immunogens without altering the ability of the resulting variants to induce non-neutralizing Fc-mediated effector responses. Thus, ID immunogens of the invention include variants of selected gpl20 sequences. The variants can have any combination of amino acid additions, deletions or substitutions, but the variants of the invention will have at least about 80% sequence identity with an ID immunogen as defined herein over the entire length of the amino acid sequence of the selected ID immunogen. In certain aspects, the variants will have at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with an ID immunogen, e.g., the ID immunogens set forth in SEQ ID NOs:3- 54. Each of the variants of the invention has the property of inducing Fc-mediated humoral response to C1/C2 or combined C1/C2 and V1/V2 regions of HIV-1 gpl20.

[0069] Figure 1 provides an amino acid sequence alignment between the ID immunogens of Table 1. Figure 2 provides an amino acid sequence alignment between the ID immunogens of Table 2. Figure 3 provides an amino acid sequence alignment between the ID immunogens of Tables 1 and 2. Figure 4 provides an amino acid sequence alignment between the ID

immunogens of Table 3. Figure 5 provides an amino acid sequence alignment between the ID immunogens of Table 4. Figure 6 provides an amino acid sequence alignment between the ID immunogens of Tables 3 and 4. See also Figure 11C for a graphical representation of sequence conservation. These sequence alignments demonstrate the regions of identity (marked as "*" below a column of letters), conservative changes (marked as ":") and semi-conservative changes (marked as ".") between the ID immunogens. This knowledge with enable the skilled artisan to easily select residues and regions of the immunogens that may be varied via additions, deletions or substitutions.

[0070] In certain aspects of the invention, the amino acid sequence of a variant differs from the ID immunogen upon which it is based only in those residues or regions where there is no identity (marked as " "). In other aspects, the amino acid sequence of a variant differs from the ID immunogen upon which it is based in those residues or regions where there is no identity and/or there are semi-conservative changes. In still other aspects, the amino acid sequence of a variant differs from the ID immunogen upon which it is based in those residues or regions where there is no identity and/or there are semi-conservative changes and/or there are conservative changes. In still further aspects, the amino acid sequence of a variant differs from the ID immunogen upon which it is based in any residue or regions of the polypeptide. The cysteines present at positions corresponding to residues 65 and 115 of SEQ ID NO: l are not altered in the variants. Immunogenic Compositions

[0071] The present invention is also directed to immunogenic compositions comprising one or more of the ID immunogens as defined herein and pharmaceutically-acceptable carriers or diluents.

[0072] It will be evident that the immunogenic compositions can vary based on the number of different ID immunogens and their relative amounts present in each the composition. For example, immunogenic compositions may comprise an ID immunogen of a single selected sequence or they may comprise two or more different ID immunogens (i.e., ID immunogens having different amino acid sequences). In non-limiting examples, an immunogenic composition comprises one or more of the ID immunogens set forth in SEQ ID NOs:3-54, or variants thereof, or both.

[0073] It is contemplated that the immunogenic compositions will be administered as pharmaceutical formulations for use in inducing an immune response or vaccination of individuals, preferably humans. In addition to the ID immunogens, the immunogenic

compositions may thus include pharmaceutically-acceptable carriers or diluents. The

immunogenic compositions may also include other ingredients, such as an adjuvant.

[0074] The carriers and diluents must be pharmaceutically acceptable in the sense that they are compatible with the immunogens and other ingredients, and they are not unduly deleterious to the recipient. The other ingredients are provided in an amount and frequency necessary to achieve the desired immunological effect.

[0001] The pharmaceutically-acceptable carriers and diluents included in the immunogenic compositions will vary based on the amount of ID immunogens in the composition, the means used to administer the composition, the site of administration and the dosing schedule used, among other factors. Suitable examples of carriers and diluents are well known to those skilled in the art and include water-for-injection, saline, buffered saline, dextrose, water, glycerol, ethanol, propylene glycol, polysorbate 80 (Tween-80™), poly(ethylene)glycol 300 and 400 (PEG 300 and 400), PEGylated castor oil (e.g. Cremophor EL), poloxamer 407 and 188, hydrophilic and hydrophobic carriers, and combinations thereof. Hydrophobic carriers include, for example, fat emulsions, lipids, PEGylated phospholipids, polymer matrices, biocompatible polymers, lipospheres, vesicles, particles, and liposomes. The terms specifically exclude cell culture medium. Additional carriers include cornstarch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride, alginic acid, croscarmellose sodium, and sodium starch glycolate.

[0075] As a specific example, intramuscular preparations can be prepared and administered in a pharmaceutically acceptable diluent such as Water-for-Injection, 0.9% saline, or 5% glucose solution.

[0076] The immunogenic compositions of the present invention may also include an adjuvant. Suitable adjuvants include Freund' s Complete and Incomplete Adjuvant, Titermax, oil in water adjuvants, as well as aluminum compounds where antigens, normally proteins, are physically precipitated with hydrated insoluble salts of aluminum hydroxide or aluminum phosphate. Other adjuvants include liposome-type adjuvants comprising spheres having phospholipid bilayers that form an aqueous compartment containing the ID immunogen and protecting it from rapid degradation, and that provide a depot effect for sustained release. Surface active agents may also be used as adjuvants and include lipoteichoic acid of gram-positive organisms, lipid A, and TDM. Quil A and QS-21 (saponin-type adjuvants), monophosphoryl lipid A, and lipophilic MDP derivatives are suitable adjuvants that have hydrophilic and hydrophobic domains from which their surface-active properties arise. Compounds normally found in the body such as vitamin A and E, and lysolecithin may also be used as surface-active agents. Other classes of adjuvants include glycan analog, coenzyme Q, amphotericin B, dimethyldioctadecylammonium bromide (DDA), levamisole, and benzimidazole compounds. The immuno stimulation provided by a surface active agent may also be accomplished by either developing a fusion protein with non-active portions of the cholera toxin, exotoxin A, or the heat labile toxin from E. coli. Immunomodulation through the use of anti-IL-17, anti IFN-γ, anti-IL- 12, IL-2, IL-10, or IL-4 may also be used to promote a strong Th2 or antibody mediated response to the immunogenic compositions.

Methods of Inducing an Immune Response

[0077] The invention is also directed to methods of inducing an immune response to an ID immunogen in a subject comprising administering to a subject an immunogenic amount of an immunogenic composition as defined herein, thereby inducing an immune response in the subject. The immune response is preferably a protective immune response. [0078] As used herein, an "immunogenic amount" of an immunogenic composition is one that is sufficient to induce an immune response to an ID immunogen in the subject to which the immunogenic composition is administered. A "protective immune response" is one that confers on the subject to which the immunogenic composition is administered protective immunity against HIV- 1. The protective immunity may be partial or complete immunity.

[0079] As indicated above, the immunogenic compositions may comprise an ID immunogen of a single selected sequence, or two or more different ID immunogens (i.e., ID immunogens having different amino acid sequences). In non-limiting examples, an immunogenic composition comprises one or more of the ID immunogens set forth in SEQ ID NOs:3-54, or variants thereof, or both.

Methods of Vaccinating a Subject

[0080] In a related aspect, the invention is further directed to methods of vaccinating a subject with a protective antigen, comprising administering to a subject in need thereof an effective amount of an immunogenic composition as defined herein, thereby vaccinating a subject with a protective antigen.

[0081] As used herein, an "effective amount" of an immunogenic composition is one that is sufficient to induce a protective immune response to an ID immunogen in the subject to which the immunogenic composition is administered. A "protective immune response" is one that confers on the subject to which the immunogenic composition is administered protective immunity against HIV- 1. The protective immunity may be partial or complete immunity.

[0082] As indicated above, the immunogenic compositions may comprise an ID immunogen of a single selected sequence, or two or more different ID immunogens (i.e., ID immunogens having different amino acid sequences). In non-limiting examples, an immunogenic composition comprises one or more of the ID immunogens set forth in SEQ ID NOs:3-54, or variants thereof, or both.

[0083] In each of the relevant methods of the present invention, the immunogenic compositions are administered in a pharmaceutically acceptable form and in substantially nontoxic quantities. The immunogenic compositions may be administered to a subject using different dosing schedules, depending on the particular use to which the compositions are put (e.g., inducing an immune response or vaccinating a subject), the age and size of the subject, and the general health of the subject, to name only a few factors to be considered. In general, the immunogenic compositions may be administered once, or may be administered more than once as boosters over a dosing schedule. The timing between each dose in a dosing schedule may range between a six, 12, or 18 hours, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months. The same quantity of protein in the formulation may be administered in each dose of the dosing schedule, or the amounts in each dose may vary. The identity of the particular ID immunogens in the composition may also vary or remain the same in each dose in a dosing schedule.

[0084] The amount of protein (i.e., ID immunogen) administered to a subject in a dose when the methods of the present invention are practiced will vary based on the particular methods being practiced (e.g., inducing an immune response or vaccinating a subject), the means and formulation of administration, the age and size of the subject, and the general health of the subject, to name only a few factors to be considered. In general, however, the amount of protein administered to a subject in a dose will be sufficient to induce or boost an immune response in a subject to the components of the composition. For example, the immunogenic compositions may contain between about 1 to about 1000 ug of total ID immunogen protein per kg of body weight of the subject to which the dose of the vaccine formulation will be administered, more preferably between about 10 to about 200 ug, even more preferably between about 15 to about 100 ug.

[0085] Appropriate doses and dosing schedules can readily be determined by techniques well known to those of ordinary skill in the art without undue experimentation. Such a determination will be based, in part, on the tolerability and efficacy of a particular dose, by use of conventional antibody titer determination techniques, and conventional bioefficacy/biocompatibility protocols.

[0086] The immunogenic compositions may be administered to a subject by any suitable means and/or methods for delivering the ID immunogens to a corporeal locus of a subject where the immunogenic compositions are intended to be effective in triggering an immune response, for example, for oral, sublingual, intranasal, intraocular, rectal, transdermal, mucosal, pulmonary, topical or parenteral administration. Parenteral modes of administration include without limitation, intradermal, subcutaneous (s.c, s.q., sub-Q, Hypo), and intramuscular (i.m.). Any known device useful for parenteral injection or infusion of immunogenic compositions can be used to effect such administration. In preferred aspects of each of the embodiments on the invention, the immunogenic compositions are administered to a subject by sub-cutaneous or intraperitoneal injections.

[0087] The term "subject" is intended to mean an animal, such mammals, including humans and animals of veterinary or agricultural importance, such as dogs, cats, horses, sheep, goats, and cattle.

[0088] Kits comprising the necessary components of an immunogenic composition that can be used to elicit an immune response to an ID immunogen or vaccinate a subject, and

instructions for its use, are also within the purview of the present invention.

In vitro Uses for the ID Immunogens

[0089] The ID immunogens of the invention may also be used in a wide variety of research applications. For example, the immunogens can be used as targets in screening to identify small molecule or peptide inhibitors that block HIV-1 viral entry and/or attachment steps. The immunogens can also be used as probes for identifying gpl20 C1/C2 region (or C1/C2 and V1V2 combined)-specific antibodies (such as monoclonal antibodies; mAbs), e.g., for use in ELISA, C1/C2 binding titer assays, and the like, and for clinical diagnostic or research applications.

[0090] Thus, and in one aspect of the in vitro uses to which the ID immunogens may be put, the invention is directed to methods of screening for antibodies with binding affinity for the C1/C2 region HIV-1 gpl20, comprising:

(a) contacting one or more ID immunogens as defined herein with a test antibody, and

(b) detecting binding of the one or more ID immunogens by the test antibody.

[0091] Because antibodies can recognize and bind different antigens having similar sequences, the method of this embodiment can be conducted using one specific ID immunogen, or two or more different ID immunogens (i.e., ID immunogens having different amino acid sequences). In non-limiting examples, the ID immunogen is one or more of the ID immunogens set forth in SEQ ID NOs:3-54, or variants thereof, or both.

[0092] The antibody being screened in this aspect of the invention can be any type or form of antibody that might have binding affinity for an ID immunogen. Particular, non-limiting, examples include a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, a single chain antibody (scFv), an Fab fragment, an F(ab')2 fragment, a multi- specific antibody, and a disulfide-linked antibody.

[0093] Antibodies can be screened for binding to an ID immunogen, for example, by immunoassays and other techniques known to those of skill in the art including, but not limited to, western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay),

"sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays. Such assays are routine and well known in the art (see [85]).

[0094] Such assays may include the conjugation of a detectable substance to the ID immunogen or an antibody to be screened. Examples of detectable substances include, but are not limited to, various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals, and nonradioactive paramagnetic metal ions. The detectable substance may be coupled or conjugated either directly to the ID immunogen or the antibody or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of

bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include ¹²⁵ I, ¹³¹ I, ^lu In, or ⁹⁹ Tc.

[0095] In another aspect of the in vitro uses to which the ID immunogens may be put, the invention is directed to methods of screening for inhibitors of HIV- 1 viral attachment comprising:

(a) contacting one or more ID immunogens as defined herein with a test molecule, and

(b) detecting binding of the one or more ID immunogens by the test molecule.

[0096] The method can be conducted using one specific ID immunogen, or two or more different ID immunogens (i.e., ID immunogens having different amino acid sequences). In non- limiting examples, the ID immunogen is one or more of the ID immunogens set forth in SEQ ID NOs:3-54, or variants thereof, or both.

[0097] The test molecule may be any molecule that binds to an ID immunogen, such as an antibody or some other protein, peptide, nucleic acid, carbohydrate, lipid, fatty acid, small drug molecule, etc. After being identified as having binding affinity for an ID immunogen, the test molecule can be further screened to determine with the test molecule can interferes with the association of Env proteins displayed by HIV-1 and cell surface receptors. If this association can be inhibited or blocked, HIV-1 viral attachment and/or entry can be inhibited or blocked.

[0098] In some aspects of this embodiment, binding of the ID immunogen by the test molecule is detecting using an immunoassay such as ELISA.

[0099] Test molecules can be screened for binding to an ID immunogen, for example, by immunoassays and other techniques known to those of skill in the art including, but not limited to, western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay),

"sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays. Such assays are routine and well known in the art (see [85]).

[00100] Such assays may include the conjugation of a detectable substance to the ID immunogen or test molecule to be screened. Examples of detectable substances include, but are not limited to, various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals, and nonradioactive paramagnetic metal ions. The detectable substance may be coupled or conjugated either directly to the ID immunogen or test molecule or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of

bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include ¹²⁵ I, ¹³¹ I, ^lu In, or ⁹⁹ Tc. IV. Examples

1. The A32-subregion (Cluster A epitopes) serves as an immunodominant region for potent ADCC response during natural infection.

[00101] The present studies were built on available [44] and unpublished data on antibody responses specific for epitopes that are exposed on gpl20 consequent to CD4 binding (CD4 inducible, CD4i epitopes). Recently, a panel of human anti-envelope antibodies was isolated from plasma of both elite controllers and non-controllers [60-62] based on an assay that detects epitopes exposed on gpl20 consequent to CD4 binding during viral entry and attachment steps [44]. The ADCC assay used in the study was a fluorometric method [63], with target cells (CEMNKrCCR5+) sensitized with a saturating dose of gpl20 of the HIVI _BOL isolate to simulate the first steps of viral entry [44]. Studies on epitope specificities among mAbs in the panel [44] clearly indicated that the Fc-mediated effector function component focused on transitional non- neutralizing epitopes associated with the gpl20 face that are occluded by gp41 in the HIV spike (Cluster A epitopes in [44]; see Fig. 7C). It was shown that these Env targets are capable of mediating potent ADCC against envelope coated cells and/or bound virons ([44] and Fig. 8). When assayed in the bound virion ADCC assay, Cluster A mAbs showed average IC ₅₀ values of 0.15 nM and 100% killing [44]. Similar high ADCC potencies were reported for antibodies specific for A32-blockable epitopes and isolated from RV144 patients and induced by vaccination [36]. By contrast, none was neutralizing in the TZMbl assay done by the CAVD Neutralization Core. Taken together, these findings underscore that non-neutralizing epitopes within A32 sub-region are extremely good targets for mAbs capable of potent ADCC. As such they will have capacities to afford protection via ADCC, depending on the dynamics and timing of antigenic exposure within the transitional structures that arise during virus-cell

attachment/entry.

2. ADCC activity directed to A32 (Cluster A) subregion and induced by the single chain gpl20-CD4 complex subunit vaccine is associated with a reduced rate of acquisition.

[00102] A single chain gpl20-CD4 complex (rhFLSC) subunit vaccine was developed that comprises gpl20 of the HIVlsa _L isolate fused to the dld2 domains of rhesus CD4. The vaccine was found to elicit protective immunity against a rectal challenge with SHIV^s- rhFLSC induced antibody responses to highly conserved domains of the envelope that become exposed only upon binding to CD4 [66, 67]. These include epitopes within/around the co-receptor binding site but also transitional epitopes associated with the N and C termini of gpl20, including the A32 sub-region [66, 67]. These epitopes show poor immunogenicity in

conventional gpl20 based vaccines [60, 66, 67]. Parenteral immunization of rhesus macaques with rhFLSC allows the elite control of a single high-dose rectal SHIV^s infection to undetectable levels in two studies, ([60, 81]. Apparent non-sterilizing protection of rhesus macaques against repeated low-dose challenges with SHIV^s was observed in two additional rhFLSC immunization studies [81]. An example of one of the low-dose challenge studies is shown in Fig. 9. By the end of four challenges, all of the naive animals were infected whereas two groups immunized with rhLFLSC showed a clear trend towards reduced acquisition (Fig. 9A).

[00103] In this study, as in all other cases of rhFLSC challenge studies, protection correlated with the titers of antibodies specific for epitopes selectively exposed on gpl20 consequent to CD4 binding (CD4i epitopes, [60]) but not with conventional neutralizing antibodies. In all cases the control of SHIV replication afforded by rhFLSC immunizations correlated with ADCC [81]. As show in Fig. 9B, a strong correlation was apparent between the number of intrarectal challenges required to infect and ADCC EC ₅₀ titer. Specificity analysis confirmed that protection was mediated by antibody responses directed to two CD4i epitope sub-regions: the co-receptor binding site (CoRBS) (Fig. 9C) and A32 sub-region (Fig. 9D). As shown in Fig. 9C, slower acquisition rates were observed in animals where the cumulative competitive epitope titers to mAb N12i2, a recently described CoRBS antibody [44], were above the median. A correlation was also apparent between the number of intrarectal challenges and competitive binding titers to A32 epitope (Fig. 9D). Whereas mAb N12i2 was shown to act through both direct neutralization and ADCC [44], mAb A32 is non-neutralizing and capable only of potent ADCC [39]. Taken together, these data show that ADCC is associated with a reduced rate of acquisition with challenge virus and that A32 sub-region (Cluster A region) is one of two specificities conferring protection in low-dose heterologous SHIV^rs challenge studies with rhFLSC. Interestingly, in all these studies a higher rate of infection was observed for animals that exhibited IL2 ELISPOTs counts above the mean, indicating that tightly balanced T cell help is needed to establish effective protective immunity [81]. 3. Cluster A antibodies recognize highly conserved epitope surfaces within the inner domain of C1/C2 regions of gpl20.

[00104] Next, x-ray crystallography was employed to map Cluster A epitopes at the atomic level [82,83]. Structures of complexes of antigen binding fragments (Fabs) with CD4-triggered gpl20 cores of four A32-like antibodies were produced. These include: mAb N5i5 and N60i3, both isolated from a selected cohort [60-62]; mAb JR4, a recombinant hybridoma cell line clone of the rhesus macaque mAb 3.5B; mAb 2.2c, an antibody derived by Epstein-Barr virus (EBV) transformation of peripheral blood B cells from an HIVl-infected subject (RW/92/13) [44].

[00105] N5i5, N60i3 and JR4 Fabs were co-crystallized with Clade A/E gpl20 _93T H057 core _e triggered with dld2 domain CD4 (N5i5) or the CD4 peptide mimetic M48U1 or M48 (N60i3 and JR4, respectively). These mAbs cross-compete with each other and with mAb A32 for binding to monomeric gpl20 as tested by ELISA ([44] and unpublished data). Structures of complexes were solved by molecular replacement approach at a resolution in a range of 1.8 - 3.5 A [82,83]. 2.2c Fab was co-crystallized with Clade B gpl20yu ₂ core _e and the CD4 mimetic peptide M48U1 and the structure was solved at 3.5 A.

[00106] As shown in Fig. 10, mAbs N5i5, N60i3, JR4 and 2.2c bind to largely overlapping areas of gpl20, proximal to the N- and C-terminal extensions. These mAbs approach gpl20 from slightly different angles and target their epitopes by two binding modes as shown by a heavy and light chain variable region switch. The structures reveal precise complementarity between positively-charged Fab paratopes, contributed largely by CDR H2 and electronegative and glycan-free surfaces of the inner domain (Fig. 10). The epitope terrains contributed by short CDRFBs (10, 12, 14 and 11 residue long for N5i5, N60i3, JR4 and 2.2c, respectively) are relatively flat in all cases. The short CDRFBs in these mAbs are distinct from the extended CDRFBs that many broadly neutralizing mAbs often have [64, 65].

[00107] Analysis of CDR sequences of the entire Cluster A pool confirmed that mAbs in this group share the same characteristics of the antigen binding site with a moderate length of CDR H3 loops. In addition, a statistically significant inverse correlation was observed between CDRH3 length and ADCC activity (data not shown), confirming that effective ADCC potency of these antibodies is not associated with unique or unusual structural features such as a long CDR H3. It is also important to note that broadly neutralizing antibodies (bnAbs) typically exhibit high levels of somatic hypermutation (SHM) [66, 67], whereas Cluster A mAbs are only mutated modestly at averages of 5% to 10% at the protein level [82,83]. There is no correlation between the degree of V _R affinity maturation and ADCC potency (data not shown). This underscores the view that if these epitopes are targets of protective antibodies in vivo, these relatively low levels of SHM can be readily attained with standard vaccination regimens.

[00108] Detailed analysis of epitope footprints of N5i5, N60i3, JR4 and 2.2c within Env antigen (Fig. 11) indicates that they map exclusively to the inner domain of gpl20 within the CI and C2 regions (Fig. 11A and 11B). N5i5, N60i3 and JR4 bind to layer 1 (β2, βΐ-strands, β2α0- and βΐβθ-connecting coils) and layer 2 (al-helix, β4 strand and β4-β5- connecting coil) of C1/C2 region with an average of 70% of the epitope surface mapped to layer 1 (Fig. 11A and 1 IB). mAbs N60i3 and JR4 make a few additional contacts with the 7-stranded β-sandwich and layer 3. mAb 2.2c targets mainly residues of layer 1 (94 % of contact surface area) with few contacts within the β4-strand of layer 2 and no binding contacts to the al-helix (Fig. 11A and 1 IB). These epitope surfaces map to the highly conserved gpl20 region with many single residue/residue motifs being strictly conserved as shown in Fig. 11C.

[00109] High conservation of Cluster A region suggests that it is functionally relevant. Indeed analysis of the Cluster A epitope footprints in context of the native untriggered virion-associated HIV-1 trimers [68] and structures of a cleaved, soluble SOSIP gpl40 trimer [69, 70] indicate that they map to a region of gpl20 that is buried within the trimer interface and engaged directly in interactions with the gp41 central helix (HR1) of the trimer (data not shown, [82,83]). In addition, mutagenesis studies confirm involvement of residues forming Cluster A epitopes (such as Leu 52 , Phe 53 , Trp 69 , His 72 , Ala 73 and Asp 107 ) in stabilization of the unliganded Env trimer or contribution to layer 1 and 2 functions in conformational transitions upon CD4 receptor binding [71-73]. Taken together, these findings indicate that potent A32-like entry targets localize to highly conserved epitope surface within the C1/C2 gpl20 region, at the junction of the functional layer 1 and 2 of inner domain. These regions are known to interact with gp41 in untriggered trimeric Env thus are inaccessible for antibody recognition until interactions of Env with the host receptor CD4. 4. Stable inner domain construct IDl as Cluster A mAb target.

[00110] Isolation of the Cluster A epitope into the smallest, stable structural unit of gpl20 possible, and in one embodiment without epitopes recognized by neutralizing antibodies, was sought. Crystallographic analysis indicated that Cluster A mAbs bind to a conformationally- conserved surface, which is centered around β2, βΐ-strands and αθ- and al-helixes of layer 1 and 2 in the CD4-bound state and without involvement of the gpl20 outer domain and variable loops (Figs. 10 and 11). Based on this information, it was hypothesized that an inner domain (ID) immunogen with V1V2 loop deleted, preferentially stabilized in the CD4-bound conformation, would be a suitable minimal structure that could be evaluated without the complication of neutralization epitopes being present. The initial design was based on an observation that unliganded HIV-1 gpl20 extended core (core _e ) tended to spontaneously adopt the CD4-bound conformation. It was shown for several structures of unliganded gpl20 core _e s of different HIV-1 clades that gpl20 truncated to preserve its extended core resembles the CD4-bound conformation which represents a "ground state" for gpl20 when variable loops are removed [77,78].

[00111] A stable inner domain construct of gpl20 from HIV-1 clade A/E isolate 93TH057 (SEQ ID NO:55; "gpl20" of Fig. 12B), which is composed of the gpl20 core _e sequence with the outer domain sequence (residues 258-472, HxBc2 numbering) replaced within inner domain Layer 3 by a simple -GG- dipeptide linker (Fig. 12) was prepared. The first design of the inner domain immunogen is referred to "IDl" (SEQ ID NO:56). A synthetic IDl gene was fused to the signal peptide sequence, placed into pCMV6-A-puro vector, expressed by transient transfection of 293T cells (FreeStyle™ mammalian 293T and GnTi ^" 293T Expression System (Invitrogen, Carlsbad, CA)) and purified from cell supernatants via N5-i5 IgG affinity and gel filtration chromatography. The typical production and purification procedure yielded 1-2 mg of IDl per liter of culture. Shown in Fig. 13 is the final purified sample of IDl analyzed by RP-HPLC, SDS-PAGE and gel filtration chromatography.

[00112] The IDl construct contains eight cysteine residues and all eight are involved in disulfide bond formation (Fig. 12A and 12B). A single RP-HPLC protein peak (Fig. 13A) indicates that the IDl molecule, folded, fully oxidized and purified, consists of a homogeneous species in solution and not a mixture of different disulfide-bonded isomers. Purified IDl protein has a molecular weight of -31-32 kDa as determined by SDS-PAGE (Fig. 13B, line 1). This corresponds to the molecular weight of the protein backbone (MW of 21,886 Da as calculated on the basis of the average isotopic compositions of the protein) and 3 N-glycosyl groups attached to N88, N234 and N241. Removal of these groups with PNGF yielded a protein backbone of molecular weight in good agreement with the expected values calculated on the basis of the average isotopic compositions of the protein (Fig. 13B, line 2). Analysis of the IDl sequence identified no new potential sites of N- or O-linked glycosylation in the newly exposed (after the outer domain removal) solvent accessible surface. Size-exclusion chromatography analyses indicate that un-liganded IDl forms a monomer in solution (data not shown).

[00113] mAb A32 is capable of binding unliganded gpl20 but CD4-triggering significantly enhances its exposure and stability in the context of full length protein [44, 79]. Binding of Cluster A mAbs, including mAb A32 and anti-Cluster A mAbs N5-i5, Ν60-Ϊ3, 2.2c and JR4 [44] to monomeric full length gp l20 of BaL isolate (gp l20BaL) and to the single-chain gp l20BaL- sCD4 complex (FLSC) [46] was tested by surface plasmon resonance (SPR) on a Biacore T-100 (GE Healthcare) at 25°C. The kinetic constants were determined using a 1: 1 Langmuir model of binding with the BIAevaluation software (GE Healthcare) and the results are shown in Table 5. All mAbs tested showed increased affinities for the gp l20-CD4 complex as shown by a 8-32 fold lower affinity constant (K _D ) for FLSC as compared to unliganded gp l20 (Table 5). In all cases the increased affinity for the gp l20BaL-sCD4 complex resulted from the faster association rates and reduced dissociation rates confirming that the epitopes in the C1-C2 region is indeed inducible and stabilized in the context of monomeric gp l20 by CD4 triggering. The same antibodies were used set to test how the C1-C2 region epitopes are preserved within the IDl molecule. As shown in Table 5, IDl binds effectively to each of five antibodies tested, with a K _D value in a range of 1.1-9.3 nM thus with affinities comparable or slightly improved as compared to the full-length gp l20 (K _D value in a range of 2.0-12.0 nM). This indicates that the inner domain molecule IDl preserves the antigenic properties of monomeric gp l20 and is folded to effectively display A32-like epitopes within a minimal structural unit of gp l20. IDl represents a first stable construct of inner domain of gp l20 expressed independently of the outer domain. Table 5. Binding kinetics of mAb A32, N5-i5, N60-i3, JR4 and 2.2c to the FLSC, gpl20 and IDl as measured by SPR. The assay was run by passing the FLSC [45] monomeric full length gp l20BaL or IDl over the immobilized antibody at 0-200 nM concentrations. The binding kinetics (association rates (k _a ), dissociation rates (k _d ), and affinity constants (K _D ) were calculated with the BIAevaluation software. Standard deviations of k _a , k _d and K _D for two experiments are shown.

¹ fold change relative to gpl20, ² fold change relative to FLSC

5. Structure Based Design of ID2 and Evaluation of its Antigenic Properties

[00114] Structural analysis of IDl complexed with the Fab of JR4, a C1-C2 specific mAb recognizing the A32-C11 mixed epitope within Cluster A region [83] revealed that

conformational CD4i epitopes of the A32-like region are not fully formed within IDl design with regions around V1V2 stem and a0-helix flexible and not contributing to anti-Cluster A mAb binding. In gpl20 core _e structures, the shortened V1V2 loop forms half of the bridging sheet, but in IDl it forms a flexible loop that probably contributes to the disorder seen in layers 1 and 2 (Fig. 12). Therefore, a second inner domain immunogen was designed to remove this disorder with 12 additional residues of the V1V2 loop removed as shown in Fig. 12. This initial revision of the inner domain which had CI 19-C205 disulfide bond at the base of the V1V2 loop removed expressed well but showed a reduced affinity for A32-like antibodies as measured by SPR (data not shown). It was hypothesized that adding a disulfide bond at the bases of αθ- and αΐ-helix could restore and preserve CD4i epitopes in this region (Fig. 12). Therefore, positions V ₆ 5 and S ₁₁₅ were mutated to cysteines to form a single disulfide bond at the αθ- and al-helix junction. Similar mutations have been shown to increase CD4i epitope exposure in context of full length gpl20 [75]. This revised second independent inner domain molecule design with V1/V2 stem removed and stabilized by C65-C115 disulfide bond was designated"ID2". ID2 was expressed in mammalian cells and purified. ID2 protein (SEQ ID NO:57) similar to ID1 contains 8 cysteines forming four disulfide bonds and folds into one disulfide-bonded form with no additional misfolded/different disulfide isomers. Expression yields of ID2 in 293T cells were typically comparable with ID1 expression with 1-2 mg of protein per liter of culture. The affinity of ID2 for the panel of anti-Cluster A mAbs was tested using SPR (Fig. 14, Table 6).

Table 6. Binding kinetics of mAb A32, N5-i5, N60-i3, JR4 and 2.2c to the FLSC, gpl20 and ID2 as measured by SPR. The assay was run by passing the FLSC [45] monomeric full length gp l20BaL or ID2 over the immobilized antibody at 0-200 nM concentrations. The binding kinetics (association rates (k _a ), dissociation rates (k _d ), and affinity constants (K _D ) were calculated with the BIAevaluation software. Standard deviations of k _a , k _d and K _D for two experiments are shown.

¹ fold change relative to gpl20, ² fold change relative to FLSC [00115] As can be seen, ID2 bound the panel of mAbs tested with an average of 17-fold tighter affinities that ID1, and similar or higher than those of FLSC (Fig. 14A). The increase in affinity was largely attributable to an increase in k _a for the complex and to a lesser extent a decrease in k _< j suggesting that ID2 more closely resembles the CD4-triggered conformation of gpl20 recognized by these antibodies. To additionally test if indeed ID2 is folded to stably present A32-like epitopes in solution, the binding of mAb A32 to ID2 was tested by Isothermal Titration Calorimetry (ITC) and compared it to mAb A32 interaction with FLSC (Fig. 14B). These data clearly indicated that ID2 adopts the CD4-bound conformation in solution which closely resembles the presentation of A32-epitope as in context of CD4-trigerred gpl20. The binding kinetics of mAb A32 to ID2 and FLSC are very similar with K _D value of 9.0 and 11.5 nM and ΔΗ of -2.553(+0.18) and -3.15(+0.69) kcal/mol for mAb A32-ID2 and mAb A32-FLSC interaction, respectively. All together these studies indicate that ID2 stably present the desired epitopes within the C1-C2 gpl20 region and shows the antigenicity profile of the CD4-triggered gpl20. ID2 represents the first stable design of inner domain expressed independently of outer domain and stabilized in CD4 bound confirmation to exclusively express the non-neutralizing ADCC epitopes of the C1-C2 region.

[00116] To additionally test if ID2 engrafts epitopes involved in ADCC function to the C1-C2 region, an ADCC competition assay was performed in which ADCC activities of anti-Cluster A mAbs against gpl20 coated cells at the effective given concentration of 0.67 nM were tested in presence of increasing concentrations of ID2. ADCC measurements were performed using an RFADCC assay which is based on the rapid fluorimetric ADCC assay [84] optimized for high throughput efficiency. EGFP-CEM-NKr-CCR5-SNAP target cells (Orlandi C. et al., in preparation) were stained with SNAP-Surface Alexa Fluor 647 (BioLabs Cat. S9136S) for 20 min at 37° C with or without HIV- l _Ba i pl20 (50ug/ml). gpl20-sensitized targets were then washed once with R10 medium and added to a 96-well V-bottom plate (5,000 cells/well).

[00117] The results show that ID2 completely inhibited ADCC of all of the A32-like mAbs as well as the mixed A32-C11 mAb JR4 at a concentration range of 0.04-3 μg/ml (Fig. 15A). The ADCC activity of mAb CI 1 which recognizes an epitope involving residues of N- and C- termini and 7-stranded β-sandwich of gpl20 not present within the ID design, is not affected. This indicates that ID2 is folded to stably display functional A32-like and mixed A32-C11-like epitopes recognized by anti-Cluster A mAbs capable of potent ADCC function. [00118] Next, whether ID2 could compete the ADCC activities of antibodies present in the sera of HIV- 1 infected individuals was tested. It was shown previously that infected individuals frequently elicit antibodies specific for A32-like epitopes [41, 42, 43, 44, 48] and the predominant fraction of ADCC response in sera from HIV- 1 -infected individuals is directed at epitopes exposed on the CD4-triggered gpl20 core including A32 sub-region [41,43]. Whether ID2 could bind to and block ADCC activity of antibodies present in sera of HIV- 1 infected individuals was tested (Fig. 15B). Sera dilutions were pre-incubated with either purified soluble D368R gpl20 dVlV2V3V5 as described in [41] and ID2 protein before being assayed for their ability to bind to CEM.NKr cells infected with Nef-Vpu- NL4.3 virus (Fig. 15B, left panel) and to mediate ADCC (Fig. 15B, right panel). ID2 specifically binds antibodies present in HIV-1 individuals and adsorbs on average approximately 30.5% of ADCC activity mediated by sera. This indicates that ID2 stably engrafts the CD4i epitopes recognized by antibodies involved in ADCC function present in sera of infected individuals.

6. Evaluation of Humoral Immune Response Induced by ID2 in Rabbits.

[00119] A group of three rabbits was immunized on weeks 0, 4 and 12 with 300 ug/dose of ID2 protein in IDRI-EM098 Emulsion with 0.04 mg/ml TLR4 agonist as adjuvant. Serum samples collected before (preimmune) and week 4, 8 and 15 were evaluated in ELISA to determine the elicitation of antibody against ID2 protein. Briefly, plates were coated with ID2 (1 ^ig/mL,) in TBS t 4°C overnight. After blocking for Ihr with blocking buffer (5% dry milk, and 0.1% Nonidet P-40 in TBS), 10-fold dilutions of rabbit sera starting at 1 : 10 were added to the plates. After washing with TBS-T buffer (TBS with 1% Tween-20), alkaline phosphatase (AP)- goat anti-rabbit IgG (H+L) (Southern Biotech; catalog no.) was added to the plates at a 1 :1000 dilution and then detected with Blue Phos Microwell Phosphatase Substrate System (KPL 50- 88-00), After ISrnin, the reaction was stopped and plates were read at 620nrn. Reactions were considered positive when the optical density (OD) measured by the ELISA was higher than the O.D. + 3SD of the same dilution of pre-immunization sera.

[00120] All three rabbits showed anti-ID2 specific antibodies already after the first immunization with titers ranging from 1: 100 (Rabbit 2 and 3) to 1: 10.000 (Rabbit 1). At week 15, after 3 immunizations, animals showed two log increase in anti-ID2 specific antibodies titers (titers of 1: 100.000 and 1 : 10.000 for Rabbit 1/Rabbit 2 and 3 respectively). The increase in antibodies titer was statistically significant for all 3 animals as determined by unpaired t test (p < 0.005 for Rabbit 1 and p<0.05 for Rabbit 2 and 3). Although all thee rabbits mounted anti- ID2 response, only sera of two (rabbit 2 and 3) after three immunizations had antibodies directed at the desired CD4i Cluster A gpl20 epitopes. As shown in Fig. 16B, sera from immunized rabbit 2 and 3 at week 15 were able to effectively recognize and bind to the HIV-1 gpl20yu ₂ - coated CEM.NKr cells, cells developed to express the target cell-bound gpl20 Env epitopes and used as suitable targets to evaluate ADCC response specific for CD4-bound conformation of HIV-1 [80].

[00121] In addition, antibodies elicited in these two rabbits were directed specifically against the epitopes in the Cluster A region. As shown in Fig. 17, the sera binding to HIV-1 gpl20yu ₂ - coated CEM.NKr cells was abrogated entirely by AV1V2V3V5 D368R gpl20, a gpl20 core protein stabilized in CD4 bound conformation [41] and Fabs of either of two anti-Cluster A mAbs tested, mAb A32 or N5-i5 [44]. In contrast, the binding was not affected by pre-treating of gpl20-coated cells with Fab of mAb ΝΊ2-Ϊ2, CD4i antibody recognizing epitope in the co- receptor binding side region [44]. Finally, the overall ADCC activity potential in the 15 ^th - week sera of immunized rabbits was tested using the previously described fluorescence-activated cell sorting (FACS)-based ADCC assay [48,80] against CEM.NKr cells coated with HIV-1 gpl20 _Y u ₂ or infected with HIV-1 lacking Nef and Vpu (N- U-) (Fig. 16C and D). The cells infected with a virus lacking both accessory proteins Nef and Vpu were shown previously to be unable to down- regulate CD4 on the target cell surface thus more susceptible to ADCC killing by CD4i antibodies [41]. Sera of both animal 2 and 3 was capable of ADCC against gpl20yu ₂ -coated coated cells (Fig. 16C) with animal 2 sera showing ADCC potencies comparable with ADCC mediated by sera from HIV-infected individuals [41]. Furthermore, sera of rabbit 2 showed effective ADCC killing of cells infected with HIV-1 lacking Nef and Vpu. Moreover, although by design ID2 is not bearing known neutralizing epitopes, neutralizing activity of sera from pre- and post-3th immunization was tested against easy-to-neutralize tier 1 virus, SF162.LS MN.3 As expected (results not shown), no neutralizing activity was detected in any of sample sera. These results indicate that ID2 is immunogenic in an animal host, capable of inducing specific FcR- mediated humoral response directed at the desired A32 epitope sub-region without neutralizing activities. 7. Design of Additional ID Immunogens.

[00122] Based on the results from the earlier studies, additional ID immunogens were prepared that comprise the gpl20 core sequence with the following additional features:

1) 11 -amino acid deletion (residues 31-42 according to gpl20 sequence numbering of

HXBc2 isolate; SEQ ID NO:2) at the N-terminus;

2) Cys point mutations at position 65 and 115 (numbering according to gpl20 sequence of HXBc2 isolate; SEQ ID NO:2) introducing extra Ces-Cns disulfide;

3) -GlyGly- dipeptide linker replacing residues 258-472 (numbering according to gpl20 sequence of HXBc2 isolate; SEQ ID NO:2) of outer domain; and, in some cases,

4) -GlyGlyAla- linker encoding for residues 119-205 (numbering according to gpl20

sequence of HXBc2 isolate; SEQ ID NO:2) in VI V2 loop.

[00123] The design was applied to gpl20 from a number of different HIV-1 clades and strains. In Table 5, the first column provides the clade, the second column provides the strain, the third column provides those features defined in the paragraphs above found in the immunogen, and the fourth column provides the sequence identifier for the immunogen in the sequence listing.

Table 5

03_AB_VIV2 N/A D-3) SEQIDNO:14

04_CPX_VIV2 N/A D-3) SEQIDNO:15

06_CPX_VIV2 N/A D-3) SEQIDNO:16

08_BC_VIV2 N/A D-3) SEQIDNO:17

10_CD_VIV2 N/A D-3) SEQIDNO:18

11_CPX_VIV2 N/A D-3) SEQIDNO:19

12_BF_VIV2 N/A D-3) SEQ ID NO:20

14_BG_VIV2 N/A D-3) SEQ ID NO:21

CON_OF_CONS_ N/A D-3) SEQ ID NO:22 VIV2

A AB097872_VIV2 D-3) SEQ ID NO:23

A AB052867_VIV2 D-3) SEQ ID NO:24

B yu2gpl20_VIV2 D-3) SEQ ID NO:25

B HXB2_VIV2 D-3) SEQ ID NO:26

C HM623558_VIV2 D-3) SEQ ID NO:27

C DQ404010_VIV2 D-3) SEQ ID NO:28

Al N/A D-4) SEQ ID NO:29

A2 N/A D-4) SEQ ID NO:30

B N/A D-4) SEQIDNO:31

C N/A D-4) SEQ ID NO:32

D N/A D-4) SEQ ID NO:33

Fl N/A D-4) SEQ ID NO:34

F2 N/A D-4) SEQ ID NO:35

G N/A D-4) SEQ ID NO:36

H N/A D-4) SEQ ID NO:37

01_AE N/A D-4) SEQ ID NO:38

02_AG N/A D-4) SEQ ID NO:39

03_AB N/A D-4) SEQ ID NO:40

04_CPX N/A D-4) SEQ ID NO:41

06_CPX N/A D-4) SEQ ID NO:42 08_BC N/A l) - 4) SEQ ID NO:43

10_CD N/A l) - 4) SEQ ID NO:44

11_CPX N/A l) - 4) SEQ ID NO:45

12_BF N/A l) - 4) SEQ ID NO:46

14_BG N/A l) - 4) SEQ ID NO:47

CON_OF_CONS N/A l) - 4) SEQ ID NO:48

A AB097872 l) - 4) SEQ ID NO:49

A AB052867 l) - 4) SEQ ID NO:50

B yu2gpl20 l) - 4) SEQ ID NO:51

B HXB2 l) - 4) SEQ ID NO:52

C HM623558 l) - 4) SEQ ID NO:53

C DQ404010 l) - 4) SEQ ID NO:54

[00124] The Δ11 deletion at the inner domain N-terminus was introduced initially to follow up with the previously reported data on overall enhanced antigenicity and immunogenicity (also to the gpl20 CI region) of the RV144 A244 gpl20 immunogen which was modified by the same N-terminal deletion [74] . In addition, a group of the immunogens was designed to contain deletion of V1V2 loop and 'locked' in a CD4 bound state by a single C ₆₅ -Cn ₅ disulfide bond, which brings together layer 1 and 2 within the C1/C2 epitope surface. It was shown previously that C ₆₅ -C ₁₁₅ disulfide locks monomeric gpl20 in CD4 bound state [75]. Rabbits immunized with C ₆₅ -Cn ₅ disulfide- stabilized monomeric gpl20 elicit a higher proportion of antibodies directed against both CD4i and CD4 binding site epitopes than the wild-type protein [75].

* * * *

[00125] While the invention has been described with reference to certain particular embodiments thereof, those skilled in the art will appreciate that various modifications may be made without departing from the spirit and scope of the invention. The scope of the appended claims is not to be limited to the specific embodiments described. REFERENCES

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