WO2004053100A2 - Immunogenic mutant human immunodeficiency virus gp120 polypeptides, and methods of using same - Google Patents

Abstract

Immunogenic mutant HIV gp120 polypeptides that contain at least one mutation in at least one epitope that specifically binds non-neutralizing antibodies are provided. Also provided are methods of making such HIV neutralizing antibodies using such immunogenic mutant gp120 polypeptides. In addition, methods of inducing an HIV neutralizing antibody response in a subject are provided.

Description

IMMUNOGENIC MUTANT HUMAN IMMUNODEFICIENCY VIRUS gpl20 POLYPEPTIDES, AND METHODS OF USING SAME

[0001] This invention was made in part with government support under Grants

No. Al 33292 awarded by the National Institutes of Health. The government has certain rights in this invention.

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION [0002] The present invention relates generally to the field of immunology and specifically to human monoclonal antibodies which bind and neutralize human immunodeficiency virus (HIV), e.g., HIV type I (HIV-1), and immunogens useful for inducing neutralizing antibodies against an HIV such as HIV-1.

BACKGROUND INFORMATION [0003] HIV is the focus of intense studies as it is the causative agent for acquired immunodeficiency syndrome (AIDS). Immunotherapeutic methods are one of several approaches to prevention, cure or remediation of HIV infection and HIV-induced diseases. Specifically, the use of neutralizing antibodies in passive immunotherapies is of central importance to the present invention. Passive immunization of HIV-1 infected humans using human sera containing polyclonal antibodies immunoreactive with HIV has been reported (see, for example, Jackson et al., Lancet, Sep. 17:647-652, (1988); Karpas et al., Proc. Natl. Acad. Sci., USA, 87:7613-7616 (1990)).

[0004] Numerous groups have reported the preparation of human monoclonal antibodies that neutralize HIV isolates in vitro. The described antibodies typically have immunospecificities for epitopes on the HIV glycoprotein gpl60 or the related glycoproteins gpl20 or gp41 (see, for example Karwowska et al., AIDS Research and Human Retroviruses, 8:1099-1106 (1992); Takeda et al, J. Clin. Invest., 89:1952-1957 (1992); Tilley et al., AIDS Research and Human Retroviruses, 8:461-467 (1992); Laman et al, J. Virol., 66:1823-1831 (1992); Thali et al, J. Virol., 65:6188-6193 (1991); Ho et al., Proc. Natl. Acad. Sci., USA, 88:8949-8952 (1991); D'Souza et al, AIDS, 5:1061-1070 (1991); Tilley et al., Res. Virol., 142:247-259 (1991); Broliden et al, Immunol., 73:371-376 (1991); Posner et al., J. Immunol., 146:4325-4332 (1991); and Gorny et al, Proc. Natl. Acad. Sci., USA, 88:3238-3242 (1991)). Levy provides a review of pathogenesis of HIV infection and therapeutic modalities including the use of passive immunity with anti-HIV antibodies (Microbiol. Rev., 57:183-289 (1993)).

[0005] The majority of human monoclonal antibodies reported to date are not effective in passive immunization therapies. Further, as monoclonal antibodies, they all each react with an individual epitope on the HIV envelope surface glycoproteins, gρl20 or gpl60, or against the V3 loop of gpl20 or against the envelope transmembrane glycoprotein, gp41. The epitope against which an effective neutralizing antibody immunoreacts has not been identified.

[0006] There continues to be a need to develop human monoclonal antibody preparations with significant HIV neutralization activity. In addition, there is a need for monoclonal antibodies immunoreactive with additional and diverse neutralizing epitopes on HIV gpl20. Additional (new) epitope specificities are required because, upon passive immunization, the administered patient can produce an immune response against the administered antibody, thereby inactivating the particular therapeutic antibody. Furthermore, the well documented ability of HIV to mutate its envelope glycoprotein structure and thereby alter its reactivity with the immune system of an infected host produces variant "field isolates" which compromise the utility of individual antibody preparations immunoreactive with an individual laboratory strain of HIV. Existing antibody preparations tend to be less potent against primary field isolates of HIV than against laboratory strains (Moore et al., Perspectives in Drug Discovery and Design, 1 :235-250 (1993)).

[0007] Broadly neutralizing antibodies can protect against intravenous and mucosal challenge with immunodeficiency viruses in animal models (4, 38, 50, 65, 68, 88, 96, 98, 125). It has, therefore, become increasingly clear that eliciting such antibodies should be a major goal in efforts to develop a human immunodeficiency virus type 1 (HIV-1) vaccine (18, 21, 66, 85, 117, 145). The animal model studies provide a number of guidelines as to the types of antibody that should be elicited. First, protection is generally provided by antibodies that effectively neutralize virus in vitro (88, 95). Second, serum neutralizing antibody levels at the time of virus challenge need to be relatively high (around 1:100) to achieve sterile protection, although lower levels can provide benefit in terms of delayed and/or decreased viremia (88, 98, 125). Third, protection by broadly neutralizing human monoclonal antibodies (mAbs) against a number of viruses suggests that protection against many different strains of HIV-1 may be achievable (4, 97, 98).

[0008] The major problem to date, from a vaccine standpoint, is that no immunogen has been generated that can elicit reasonable levels of such broadly neutralizing antibodies. These antibodies should be targeted to relatively conserved and exposed regions of the HIV-1 envelope, but the paucity of broadly neutralizing antibodies in natural infection suggests that the virus presents these regions to the immune system in such a way as to minimize an effective antibody response (21, 100, 143, 145). A molecular understanding of regions on the HIV-1 envelope that are exposed and conserved and how they can be recognized by antibodies would be invaluable in the design of immunogens that can elicit broadly neutralizing antibodies.

[0009] The CD4 binding site (CD4bs) on the HIV-1 surface glycoprotein gpl20 is a highly conserved region that is known to be exposed for ligand binding (30, 54). In theory, this would seem to form an excellent target for neutralizing antibodies. Many mAbs that bind with high affinity to the CD4bs of monomeric gpl20 from various primary and T-cell line-adapted (TCLA) HIV-1 isolates have been isolated (see, hypertext transfer protocol (http), at URL "resdb.lanl.gov/ABDB/antibody_id.htm", which is incorporated herein by reference). These mAbs are characterized by their ability to compete with soluble CD4 and with one another (80).

[0010] Anti-CD4bs mAbs typically neutralize TCLA viruses with moderate efficacy, but neutralize primary isolates of HIV-1 very weakly if at all (102). However, one mAb, bl2, which interacts with the CD4bs, does neutralize many primary and TCLA viruses very efficiently (22, 29, 53, 69). MAb bl2 and non-neutralizing anti-CD4bs mAbs typically have very similar binding affinities for monomeric gpl20 from a number of isolates (78, 80). The differences between bl2 and the other mAbs in neutralizing activity against TCLA viruses, therefore, have been associated with different affinities for the mature envelope trimer expressed on virions (35, 110, 116). Typically, mAb bl2 is able to bind with comparable affinity to monomeric gpl20 and mature trimer on the surface of infected cells (99), which is believed to be identical to the functional envelope molecule on the surface of virions (116). Non-neutralizing anti-CD4bs mAbs, on the other hand, bind with lowered affinity to the mature trimer. The implication, therefore, is that bl2 is able to bind similarly to monomer gpl20 and to the native TCLA trimer and neutralize the virus effectively, whereas the other anti-CD4bs mAbs suffer some impediment in their access to the CD4bs on the mature TCLA trimer and, therefore, neutralize virus less effectively (103). Lower levels of envelope expression have made the investigation of the correlation between binding to mature trimer and neutralization more troublesome for primary HIV-1. Such a correlation has been reported (121) and, as disclosed herein, it was considered that the explanation for the efficacy of bl2 against primary viruses can be similar to that for TCLA viruses.

SUMMARY OF THE INVENTION

[0011] The present invention is based on the seminal discovery that although monomeric gpl20 binds bl2 and non-neutralizing anti-CD4bs antibodies equivalently, monomeric gpl20 can be mutated such that it can bind neutralizing antibodies such as bl2 to a greater extent that it can bind non-neutralizing mAbs. As disclosed herein, such mutated gpl20 polypeptides are useful as immunogens for stimulating an antibody response primarily comprising HIV neutralizing antibodies, including neutralizing antibodies specific for HIV type I (HIV-1), HIV type 2 (HIV-2), or both, particularly HIV-1. Accordingly, immunogenic mutant gpl20 polypeptides that can stimulate a neutralizing antibody response are provided, as are methods of using such immunogenic mutant gpl20 polypeptides, and antibodies raised using such immunogenic gρl20 polypeptides.

[0012] The invention provides immunogenic modified (mutant) HIV-1 gpl20 polypeptides, which can elicit an antibody response in a subject, particularly an antibody response characterized by the generation of neutralizing HIV-1 antibodies, including a much greater level of neutralizing antibody (as compared to non-neutralizing antibody) than that elicited by a wild-type gpl20 polypeptide. Preferably, the neutralizing antibody response is elicited in the absence of a non-neutralizing antibody response. In one aspect, wild-type gpl20 polypeptide was modified by the substitution of at least one alanine for the wild-type amino acid residue(s). In another aspect, variable loop deletions were made in the gp 120 molecule.

[0013] The present invention relates to an immunogenic mutant gρl20 polypeptide that can stimulate a neutralizing antibody response against a human immunodeficiency virus (HIV), wherein the mutant gpl20 polypeptide comprises an HIV (e.g., HIV-1) gpl20 polypeptide having at least one amino acid mutation in at least one epitope of the HIV gpl20 polypeptide specifically bound by a non-neutralizing antibody. As such, an immunogenic mutant gpl20 polypeptide of the invention can contain 2, 3, 4, 5, 6, 7, or more two amino acid mutations in 1, 2, 3, 4, 5, 6, or more epitopes of the HIV g l20 polypeptide specifically bound by a non-neutralizing antibody. Thus, an immunogenic mutant gpl20 polypeptide can have, for example, at least a first amino acid mutation in a first epitope of the HIV gρl20 polypeptide specifically bound by a non-neutralizing antibody, and at least a second amino acid mutation in a second epitope of the HIV gpl20 polypeptide specifically bound by a non-neutralizing antibody, wherein the first epitope and the second epitope can be the same or different. The epitope of the HIV gρl20 polypeptide specifically bound by a non-neutralizing antibody can include any epitope, including, for example, an epitope comprising Phe43 cavity perimeter amino acid residues, VI loop amino acid residues, V2 loop amino acid residues, V3 loop amino acid residues, non-neutralizing face amino acid residues, Cl domain amino acid residues, C5 domain amino acid residues, co-receptor binding site amino acid residues, or a combination thereof.

[0014] The amino acid mutation (or mutations) introduced into in epitopes bound by a non-neutralizing antibody generally is a substitution of a first amino acid residue for a second different amino acid residue, which can be a conservative substitution or a non-conservative substitution. In one embodiment, amino acid mutations in epitopes bound by a non-neutralizing antibody are exemplified by the substitution of an amino acid residue of HIV-1 gpl20 polypeptide with an alanine residue, for example, a substitution such as G473A, D474A, M475A, R476A, or a combination thereof, e.g., D474A and R476A; G473A and M475A; D474A, M475A and R476A; or G473 A, D474A, M475A and R476A; or a substitution such as DI 13A, V127A, D180A, N197A, or S256A, or any combination of the above-described mutations.

[0015] In another embodiment, the amino acid mutations in epitopes bound by a non- neutralizing antibody are exemplified by substitutions, including one or two substitutions, that generate a glycosylation motif (e.g., an N-glycosylation motif) at a position in the HIV-1 gpl20 polypeptide that otherwise is not subject to glycosylation. Such substitutions are exemplified by H92Ν and N94T; Ql 14N and Ll 15T; S143T; E150N and G152T; K171N and Y173T; Q246N; P313N and R315T; or I423N andN425T, and combinations thereof. For example, an immunogenic mutant HIV-1 gpl20 polypeptide can have Ql 14N, Ll 15T, S143T, E150N, and G152T substitutions and, if desired, can have one or more substitutions, e.g., H92N; K171N and Y173T; and/or I423N.

[0016] In still another embodiment, the amino acid mutations in epitopes bound by a non-neutralizing antibody are exemplified by substitutions that generate a glycosylation motif and, further, substitutions that do not generate a glycosylation motif, particularly substitutions that decrease the ability of a non-neutralizing antibody to bind an HIV-1 gρl20 epitope without substantially affecting the ability of a neutralizing antibody to bind an HIV-1 gpl20 epitope. An immunogenic mutant gpl20 polypeptide containing such combinations of mutations is exemplified by a gpl20 polypeptide containing one or more of G473A, D474A, M475A, and R476A substitutions and one or more of H92N, Ql 14N, Ll 15T, S143T, E150N, G152T, K171N, Y173T, and I423N substitutions, including an immunogenic mutant gpl20 polypeptide containing all of these substitutions. As disclosed herein, the mutations of an epitope bound by a non-neutralizing antibody also can be a deletion, for example, a deletion of Cl and/or C5 amino acid residues.

[0017] The present invention also provides neutralizing antibodies stimulated by a mutant HIV gpl20 immunogen. Such antibodies, which can be in an antiserum or an immunoglobulin fraction of such a serum or substantially purified antibodies, are useful for diagnostic and/or immunotherapeutic purposes. In one aspect, the antibodies have a specificity that is substantially similar to the bl2 monoclonal antibody (U.S. Pat. Nos. 5,652,138 and 5,804,440, each of which is incorporated herein by reference), particularly HIV neutralizing activity. As disclosed herein, the bl2 monoclonal antibody provides an exemplary neutralizing antibody that is useful for identifying an immunogenic mutant HIV-1 gpl20 polypeptide of the invention.

[0018] The present invention further provides a method of generating neutralizing antibodies for HIV in a subject. Such a method can utilize an immunogenic mutant HIV-1 gpl20 polypeptide of the invention as an immunogen in a subject, thereby generating an antibody response comprising primarily or preferentially HIV-1 neutralizing antibodies. The subject can be any subject capable of raising an immune response against such an immunogen, including a mammalian subject, particularly a human subject. For example, the invention provides a method of inducing antibodies that can neutralize HIV by immunizing a subject with an immunogenic mutant HIV gpl20 polypeptide that can stimulate a neutralizing antibody response against an HIV as disclosed herein, e.g., an HIV-1 gpl20 polypeptide having at least one amino acid mutation in at least one epitope of the gρl20 polypeptide specifically bound by a non-neutralizing antibody. The epitope of the HIV gpl20 polypeptide specifically bound by a non-neutralizing antibody can include, for example, Phe43 cavity perimeter amino acid residues, VI loop amino acid residues, V2 loop amino acid residues, V3 loop amino acid residues, non-neutralizing face amino acid residues, Cl domain amino acid residues, C5 domain amino acid residues, co-receptor binding site amino acid residues, or a combination of such epitopes.

[0019] The mutation (or mutation) in the epitope (or epitopes) can be a substitution or deletion, as disclosed herein, including, for example, one, two or three amino acid mutations that generate a glycosylation motif in the epitope. The immunogenic mutant gpl20 polypeptide used in a method of the invention can be any immunogenic mutant gpl20 polypeptide that preferentially induces an HIV neutralizing antibody response, for example, an immunogenic mutant HIV-1 gpl20 polypeptide containing DI 13A; V127A; D180A; N197A; S256A; G473A; D474A; M475A; R476A; H92N and N94T; Ql 14N and Ll 15T; S143T; E150N and G152T; K171N and Y173T; Q246N; P313N and R315T; I423N and N425T; or a combination thereof (e.g., an immunogenic mutant gpl20 polypeptide containing G473A, D474A, M475A, R476A, and, optionally, H92N and N94T; Ql 14N and Ll 15T; S143T; E150N and G152T; K171N and Y173T; Q246N; P313N and R315T; or I423N and N425T; or a combination thereof).

[0020] A method of inducing an HIV neutralizing antibody response in a subject can further include obtaining antiserum from the subject, wherein the antiserum comprises HIV neutralizing antibodies. In one embodiment, the antiserum contains HIV-1 neutralizing antibodies. In another embodiment, the subject is a human subject. Such a method can further include an HIV neutralizing antibody fraction from said antiserum and/or substantially purifying HIV neutralizing antibodies from the subject or from antiserum obtained from the subject. Accordingly, antiserum containing HIV neutralizing antibodies, an isolated HIV neutralizing antibody fraction, and a substantially purified HIV neutralizing antibody, which can be a polyclonal or monoclonal antibody (e.g., an anti-HIV- 1 neutralizing monoclonal antibody) obtained by a method of the invention is provided.

[0021] The present invention also provides methods of producing HIV neutralizing antibodies by contacting antibody producing cells with an immunogenic mutant HIV gpl20 polypeptide of the invention. In one embodiment, the method is performed, for example, by contacting antibody producing cells (e.g., spleen cells) in culture under conditions such that antibody producing cells in the culture are induced to express HIV neutralizing antibodies (e.g., HIV-1 neutralizing antibodies). Such a method can further include isolating the HIV neutralizing antibodies from the culture. Accordingly, the invention provides isolated HIV neutralizing antibodies produced by such a method, for example, isolated HIV-1 neutralizing antibodies.

[0022] In another embodiment, a method of producing HIV neutralizing antibodies is performed, for example, by administering the immunogenic mutant gpl20 polypeptide to a subject under conditions sufficient for the immunogenic mutant gpl20 polypeptide to stimulate an immune response in the subject. Such a method can further include obtaining blood, plasma, and/or serum from the subject, and/or obtaining antiserum containing the HIV neutralizing antibodies. In addition, the method can further include isolating an HIV neutralizing antibody fraction and/or substantially purifying HIV neutralizing antibodies from the antiserum, or from blood, plasma or serum of the subject. The method can also include isolating antibody producing cells from the subject, wherein the cells can be used as a source of the HIV neutralizing antibodies, for example, by immortalizing the antibody producing cells or generating hybridomas from the antibody producing cells, thus further providing a means to obtain monoclonal HIV neutralizing antibodies. Accordingly, the invention provides antiserum, an isolated HIV neutralizing antibody fraction; substantially purified HIV neutralizing antibodies produced by such methods, isolated antibody producing cells produced by such methods, and antibodies produced by such isolated antibody producing cells or cells derived therefrom.

[0023] The present invention also provides a method of treating a human subject having or at risk of having an HIV infection or HIV-induced disease. Such a method can be performed, for example, by administering to the subject a therapeutically effective amount of an immunogenic mutant gpl20 polypeptide of the invention, thereby actively inducing a neutralizing antibody response in the subject, and treating the infection or disease. Such a method can be useful as a prophylactic method, thus reducing the likelihood that a subject can become infected with HIV, or as a therapeutic for a subject infected with HIV, thus providing a means to ameliorate HIV infection.

[0024] The present invention also relates to a method of ameliorating an HIV infection in a subject. In one embodiment, the method is performed, for example, by administering an immunogenic mutant gpl20 polypeptide of the invention to the subject. The immunogenic mutant gpl20 polypeptide can be any immunogenic mutant gpl20 polypeptide that preferentially induces an HIV neutralizing antibody response, including, for example, an immunogenic mutant HIV-1 gpl20 polypeptide comprises D474A and R476A; G473 A and M475A; D474A, M475A and R476A; or G473 A, D474A, M475A and R476A, which, optionally, can further comprise H92N and N94T; Ql 14N and Ll 15T; S143T; E150N and G152T; K171N and Y173T; Q246N; P313N and R315T; or I423N and N425T; or a combination thereof, and/or a deletion of Cl domain amino acid residues, C5 domain amino acid residues, or a combination thereof, which can be useful for ameliorating an HIV-1 infection in a subject.

[0025] In another embodiment, a method of ameliorating an HIV infection in a subject is performed, for example, by administering HIV neutralizing antibodies to the subject, wherein the HIV neutralizing antibodies comprise antibodies stimulated in response to mutant gpl20 polypeptide of the invention. The HIV neutralizing antibodies can be obtained using any method as disclosed herein, including, for example, by immunizing an individual with the immunogenic mutant gpl20 polypeptide, and isolating the HIV neutralizing antibodies, or antiserum containing the antibodies, for the individual; or by immunizing an individual with the immunogenic mutant gpl20 polypeptide, thereafter generating hybridomas from antibody producing cells from the individual, and isolating the HIV neutralizing antibodies from hybridomas expressing the HIV neutralizing antibodies.

[0026] The present invention further relates to a method of reducing or preventing HIV infection in a subject. Such a method, which can be a prophylactic method, can be performed, for example, by administering an immunogenic mutant g l20 polypeptide of the invention to the subject under conditions that stimulate an HIV neutralizing antibody response in the subject.

[0027] The present invention further provides a method for identifying a neutralizing antibody effective for neutralizing HIV or related viruses. The method includes a competition assay whereby an immunogenic mutant gpl20 polypeptide is contacted with a bl2 or bl2-like antibody, followed by contacting with a test neutralizing antibody, particularly a monoclonal antibody. Such an assay also can be performed in reverse, wherein the test antibody is first contacted with the gpl20 molecule, followed by contacting with the bl2 antibody. A desired neutralizing antibody of the invention can be identified by detecting competition of a test antibody with a bl2 antibody for gpl20 binding, thereby indicating that the test antibody is a neutralizing antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figures 1 A to ID show the location of amino acid substitutions on gpl20, the spatial relationship of epitopes on g l20, and variability of gpl20 residues among primate immunodeficiency viruses and HIV-1. HIV gpl20 is exemplified by the gpl20 of HIV-1 isolates JR-CSF (GenBank Ace. No. AAB03749; for encoding nucleic acid, see GenBank Ace. No. M38429, each of which is incorporated herein by reference) and JR-FL (AAB05604; for encoding nucleic acid, see GenBank Ace. No. U63632, each of which is incorporated herein by reference). See also Appendix 5, Figures 1-15 in black and white.

[0029] Figure 1 A provides a ribbon amino acid sequence diagram of gpl20j_R-cs_F indicating the locations of the alanine substitutions in this study. Arrows indicate amino acids that were mutated to alanine. Variable loop deletions are indicated by white letters encircled by blue spheres. Light blue dots indicate the positions of amino acid mutations in bl2 neutralization escape mutants (74, 104). Red and yellow dots indicate primary and secondary bl2 contact residues, respectively, based on a computational docking model of bl2 and core gpl20_HXB2 (90). Primary bl2 contacts are gpl20 residues that, based on the docking model, contact mAb bl2; secondary contacts are gpl20 residues within 3-5 A of mAb bl2.

[0030] Figure IB shows the location of mutations (colored blue) mapped onto the gpl20 core of HxB2. The view is from the perspective of CD4.

[0031] Figure IC shows the approximate location of the faces of the gpl20 core, defined by the interaction of gpl20 and antibodies (61, 143, 145). The region in the CD4bs that is accessible to neutralizing ligands on primary HIV-1 isolates (i.e. mAbs CD4-IgG2 and bl2), termed neutralizing face, is shown in yellow. The region of gpl20 that is believed to be poorly accessible on oligomeric gρl20 and elicits non-neutralizing antibodies is shown in cyan. The location of the immunologically 'silent' face, which encompasses the epitope recognized by the broadly neutralizing mAb 2G12 (115, 119, 137), is shown in magenta. The co-receptor binding site is shown in light grey. Modeled carbohydrate chains are shown in dark grey and black. The approximate areas that are believed to be covered by the V2 and V3 loops (primarily the coreceptor binding site) are indicated. The locations of the Phe43 cavity (61), involved in CD4 binding, and the VI /N2 loop are also indicated.

[0032] Figure ID shows the molecular surface of gpl20 depicting the sequence variability of the amino acid residues among primate immunodeficiency viruses and HIN-1 : green, residues conserved among all primate immunodeficiency viruses; yellow, residues conserved among all HIN-1 isolates but not among all primate immunodeficiency viruses; grey, residues that are variable among HIV-1 isolates. Amino acid conservation defined as in (61).

[0033] Figures 2 A to 2C show the apparent affinity of mAbs for alanine mutants of gpl20j_R-csF relative to wild-type gpl20. Numbering is based on the sequence of HIV-1_HXB2 (HxB2) (57). The amino acid sequences for gpl20 of HIV-1 isolates JR-CSF (GenBanlc Ace. No. AAB03749; for encoding nucleic acid, see GenBank Ace. No. M38429, each of which is incorporated herein by reference; see Appendices 1 and 2, respectively) and JR-FL (GenBank Ace. No. AAB05604; for encoding nucleic acid, see GenBanlc Ace. No. U63632, each of which is incorporated herein by reference; see Appendices 3 and 4, respectively) are provided for comparison, wherem, for example, P313-X-R315 corresponds to positions 309-311 in GenBanlc Ace. No. AAB03749 and to positions 308-310 in GenBanlc Ace. No. AAB05604. On the x-axis, only every second amino acid residue listed in Table 1 (see below) is numbered. Red bars represent CD4 binding. A schematic of the conserved and variable regions of HIV-1 gpl20 is also shown below. Numbers indicate amino acid residues (HxB2 numbering). C, conserved domain; V, variable region.

[0034] Figure 2 A shows antibody b3 binding (green bars).

[0035] Figure 2B shows antibody b6 binding (grey bars).

[0036] Figure 2C shows antibody bl2 binding (blue bars).

[0037] Figure 3 shows the effects of alanine substitutions on antibody binding mapped onto the HxB2 gρl20 core structure. The view is from the perspective of CD4. Only substitutions that affect antibody binding are colored and labeled. Alanine substitution of residues that are colored yellow significantly enhanced mAb binding (>200% affinity relative to wild type), whereas those colored blue significantly reduced mAb or CD4 binding (<50% affinity relative to wild type). Amino acid substitutions in the V2 loop that affected antibody or CD4 binding are indicated by colored circles on the left of each structure.

[0038] Figure 4 provides epitope maps of the unique effects of alanine substitutions on mAb binding affinity. Color scheme and labeling as in the legend to Figure 3, except that, in this figure, amino acid mutations which did not significantly affect antibody binding are also indicated (colored black). Top panel: differential map of alanine point mutations for which the effect on mAb binding was unique as compared to the other two mAbs. Bottom panel: differential map of the unique effects of alanine substitutions on binding by mAbs b3 and b6, respectively, in comparison to bl2.

[0039] Figure 5 depicts alanine mutants that were selected for neutralization assays. Labeled amino acids that are colored grey indicate mutant pseudovirions for which there was a reasonable correlation between bl2 binding to monomeric gpl20 and neutralization efficiency. Residues labeled and colored yellow indicate pseudovirions, which were neutralized equally well or better than wild-type virus despite a decrease in bl2 affinity for monomeric gpl20 of the respective mutant. Residues labeled and colored red indicate pseudovirions which were neutralized less well than wild-type virus despite an increase in bl2 binding affinity for monomeric gpl20 for the respective mutant.

[0040] Figures 6 A to 6D show antibody binding in the context of the functional envelope trimer.

[0041] Figure 6A depicts the trimeric gpl20 model as proposed by Kwong et al. (62); gpl20 is depicted as viewed from the virus.

[0042] Figure 6B shows a docking model of mAb bl2 (yellow) to gpl20.

[0043] Figures 6C and 6D depict how two hypothetical non-neutralizing anti-CD4bs mAbs (pink, Figure 6C; and green, Figure 6d) can interact with gpl20. Note how bl2 and the non-neutralizing anti-CD4bs mAbs are able to interact with monomeric gpl20, but that only bl2 binds at an orientation that also allows the interaction with gpl20 in the context of a functional envelope trimer^{" •} For clarity, only the ■antibody Fab fragments are shown.

[0044] Figures 7A to 7D show binding of CD4 binding site (CD4bs) mAbs (left) and non-CD4bs mAbs (right) to gpl20jR-FL of mutant GDMR in ELISA. Supernatants containing monomeric gpl20 were captured onto ELISA plate wells and probed with varying concentrations of mAb, starting at 10 μg/ml. Bound antibody was detected with alkaline phosphatase-conjugated secondary antibody. Absorbance was measured at 405 nm.

[0045] Figures 7A and 7B show antibody binding to wild-type gpl20.

[0046] Figures 7C and 7D show antibody binding to gpl20 of mutant GDMR.

[0047] Figure 8 A to 8D show binding of V3 loop mAbs to wild-type gp 120_JR._PL and glycoprotein of mutant P313N by western blot analysis and in an ELISA.

[0048] Figures 8 A and 8B show western blot analyses of wild-type gpl20 (lane 1) and mutant glycoprotein (lane 2) reacted with mAb bl2 (1 μg/ml; Figure 8 A) or with mAbs 19b, 447-52D and loop 2 (pooled mixture; 1 μg/ml each; Figure 8B). Molecular weight indicators (bars) and the average molecular weight of wild type and mutant glycoproteins (arrows) are shown on the left; lcDa, kilodaltons.

[0049] Figures 8C and 8D show MAb binding to wild-type gpl20 (Figure 8C) and mutant P313N (Figure 8D) as determined by ELISA.

[0050] Figure 9 shows binding of mAb bl2 to gpl20 with added N-glycosylation sequons. Wild-type gpl20 and mutant glycoproteins (labeled I-NII) were captured onto ELISA wells and probed with mAb bl2 (10 ng/ml). HINIG (1 μg/ml) was used to ensure that equivalent amounts of protein were captured. Bound antibody was detected with peroxidase-conjugated secondary antibody and absorbance measured at 450 nm. [0051] Figures 10A to 10C show the location of introduced N-glycan attachment sites and alanine substitutions, mapped onto the gpl20 core. N-linked oligosaccharides (yellow) were modeled onto the core structure (61) according to the most likely glycoforms, as inferred from a previous study (151). The putative locations of the VI and V2 loops are also indicated in each panel. The 20 A-bar represents the average width of a typical antibody-combining site. Figures were made with the RasMol™ program (118) and modified using Adobe PHOTOSHOP.

[0052] Figure 10A depicts the gpl20 core shown from the perspective of CD4. The attachment sites of the extra glycans are labeled and colored dark blue; dark blue-colored spheres indicate those in the VI and V2 loops. The alanine substitutions at positions 473-476 (GDMR) are labeled and colored red. The perspectives shown in Figure 10B and 10C are also indicated by arrows.

[0053] Figure 10B shows a view of the gpl20 outer domain (61, 143). The spatial location of the V3 and V4 loops, which are proposed to extend from the protein surface, are also indicated.

[0054] Figure 10C shows a view of the proposed gp41-gpl20 interface (61, 143).

[0055] Figure 11 shows a determination of the molecular weight of mutant mCHO*-GDMR by western blot. Wild-type gpl20jR-FL and mutant glycoprotein were separated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane and probed with a polyclonal antibody preparation generated against the C5 region of gpl20 (5 μg/ml). Lane 1, wild type; lane 2, mutant mCHO*-GDMR. Molecular weight indicators are shown on the left and the average molecular weight of each glycoprotein is denoted on the right; lcDa, kilodaltons.

[0056] Figures 12A to 12H show binding of anti-gpl20 mAbs to wild type (Figures 12A-D, left) and mutant mCHO*-GDMR (Figures 12E-H, right) glycoproteins. Captured glycoproteins were probed with mAb concentrations indicated on the x-axis. Absorbance was measured at 405 nm. [0057] Figures 12A and 12E show binding of mAbs to the Cl, C5, C1-C4, and C1/C5 domains.

[0058] Figures 12B and 12F show binding mAbs to the CD4bs.

[0059] Figures 12C and 12G show binding of mAbs to the coreceptor binding site.

[0060] Figures 12D and 12H show binding of mAbs to the V2 and V3 loops.

[0061] Figure 13 shows glycoproteins of mutants mCHO*-GDMR and mCHO*-GDMR N/C (N and C termini deleted), captured onto ELISA plate wells with indicated antibodies (10 μg/ml) and detected with biotinylated mAb 2G12 (1 μg/ml). Grey bars represent mutant mCHO*-GDMR; dark bars represent mutant mCHO*-GDMR ,N/C. Absorbance (optical density, OD) was measured at 450 nm.

[0062] Figure 14 shows the reactivity of HIVIG (open symbols) and mAb bl2 (filled symbols) in ELISA with wild-type gpl20 and mutant glycoproteins. wt, wild-type gpl20; GDMR, mutant GDMR in which residues at positions 473-476 in gpl20 have been substituted by alanine (see Example 2); GDMR-P313N, similar to mutant GDMR with the addition of an N-glycosylation site in the N3 loop; mCHO*-GDMR, similar to mutant GDMR-P313Ν, with the addition of six glycosylation motifs (2 in the V 1 loop, 1 in the V2 loop, and 3 on the gpl20 core).

[0063] Figure 15 shows binding of CD4bs mAbs and mAb A32 to glycoprotein of mutant mCHO*. This mutant contains all the glycosylation sequons that are also present in mutant mCHO*-GDMR, but lacks the alanine substitutions on the edge of the Phe43 cavity.

DETAILED DESCRIPTION OF THE INVENTION

[0064] Irrespective of the underlying mechanisms responsible for neutralization differences, bl2 and non-neutralizing anti-CD4bs mAbs constitute probes to distinguish presentations of gpl20 that are desirable for vaccine purposes from those that are less desirable. As a first step, alanine scanning mutagenesis was used to identify amino acid residues on HIV-1 gpl20 that modulate binding by bl2 and to compare these to residues that affect binding of two representative non-neutralizing anti-CD4bs mAbs that previously were characterized in detail, namely b3 and b6. Amino acid changes that had an effect on antibody binding were compared to those that affected CD4 binding. The results show that the mAbs and CD4 bind gpl20 with a similar footprint. The footprint for CD4 was very close to that which would be expected from the crystal structure of a complex of CD4 and the core of gp 120 (61). The footprint for antibody bl2 also corresponded well with that expected from a docking model of the bl2 structure and the gpl20 core structure (90). However, a number of differences observed in the epitope maps between bl2 and the non-neutralizing anti-CD4bs mAbs suggest that HIV-1 gpl20 can be engineered to generate a molecule that is more disposed to eliciting bl2-like antibodies.

[0065] Amino Acid Residue: An amino acid formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide linkages. The amino acid residues described herein are preferably in the "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property is retained by the polypeptide. NH₂ refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide. In keeping with standard polypeptide nomenclature (described in J. Biol. Chem., 243:3552-59 (1969) and adopted at 37 C.F.R 1.822(b)(2)), standard one letter and three letter abbreviations for amino acid residues are used, as shown in the "Table of Correspondence" (below).

[0066] It should be noted that all amino acid residue sequences represented herein by formulae have a left-to-right orientation in the conventional direction of amino terminus to carboxy terminus. In addition, the phrase "amino acid residue" is broadly defined to include the amino acids listed in the Table of Correspondence and modified and unusual amino acids, such as those listed in 37 C.F.R. 1.822(b)(4), and incorporated herein by reference. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino acid residues or a covalent bond to an amino-terminal group such as NH₂ or acetyl or to a carboxy- terminal group such as COOH. Table of Correspondence:

SYMBOL l-Letter/3-Letter/AMINO ACID

Y Tyr tyrosine

G Gly glycine

F Phe phenylalanine

M Met methionine

A Ala alanine

S Ser serine

I He isoleucine

L Leu leucine

T Thr threonine

V Val valine

P Pro proline

K Lys lysine

H His histidine

Q Gin glutamine

E Glu glutamic acid

Z Glx Glu and/or Gin w Trp tryptophan

R Arg arginine

D Asp aspartic acid

N Asn asparagine

B Asx Asn and/or Asp

C Cys cysteine

X Xaa Unknown or other [0067] Recombinant DNA (rDNA) molecule: A DNA molecule produced by operatively linking two DNA segments. Thus, a recombinant DNA molecule is a hybrid DNA molecule comprising at least two nucleotide sequences not normally found together in nature. rDNA's not having a common biological origin, i.e., evolutionarily different, are said to be "heterologous".

[0068] Vector: A rDNA molecule capable of autonomous replication in a cell and to which a DNA segment, e.g., gene or polynucleotide, can be operatively linked so as to bring about replication of the attached segment. Vectors capable of directing the expression of genes encoding for one or more polypeptides are referred to herein as "expression vectors". Particularly important vectors allow cloning of cDNA (complementary DNA) from mRNAs produced using reverse transcriptase.

[0069] Antibody: The term antibody in its various grammatical forms is used herein to refer to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antibody combining site or paratope. Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and portions of an immunoglobulin molecule, including those portions known in the art as Fab, Fab', F(ab')₂ and F(v).

[0070] Antibody Combining Site: An antibody combining site is that structural portion of an antibody molecule comprised of a heavy and light chain variable and hypervariable regions that specifically binds (immunoreacts with) an antigen. The term "immunoreact" in its various forms means specific binding between an antigenic determinant-containing molecule and a molecule containing an antibody combining site such as a whole antibody molecule or a portion thereof.

[0071] Monoclonal Antibody: A monoclonal antibody in its various grammatical forms refers to a population of antibody molecules that contain only one species of antibody combining site capable of immunoreacting with a particular epitope. A monoclonal antibody thus typically displays a single binding affinity for any epitope with which it immunoreacts. A monoclonal antibody may therefore contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different epitope, e.g., a bispecific monoclonal antibody. Although historically a monoclonal antibody was produced by immortalization of a clonally pure immunoglobulin secreting cell line, a monoclonally pure population of antibody molecules can also be prepared by the methods of the present invention.

[0072] Global efforts to obtain an effective vaccine against HIV-1 have thus far failed. The induction of antibodies with broad anti-viral activity, considered a highly beneficial feature of a future vaccine (18, 19, 66, 85, 117, 143, 145), has proven particularly problematic. The use of soluble monomeric HIV-1 gpl20, the major component of the viral envelope spike, has yielded antibodies that bind solely to monomeric gpl20 or only to a narrow range of HIV-1 isolates (9, 27, 63). The crystal structures of the gpl20 core in complex with CD4 and an antibody Fab fragment (60, 61, 143) have shed light on why it may be difficult to elicit antibodies that are capable of recognizing gpl20 as presented on the virion surface. Conserved sequences, such as found in the CD4 binding domain, lie recessed within the core and are partially occluded by (hyper)variable loops, which then reduces antibody recognition (61, 143, 145). Furthermore, although other conserved regions, such as the interface between gρl20 and the transmembrane unit glycoprotein gp41 (61), can be readily exposed on monomeric gρl20, these epitopes are most likely occluded on the envelope spike (143, 145).

[0073] Because of the disappointing results with monomeric gpl20, new approaches are being explored for eliciting broadly neutralizing antibodies. Two main approaches are currently being investigated using HIV envelope glycoproteins. One strategy focuses on the preservation or reconstruction of the trimeric envelope spike. For example, virions have been chemically inactivated by modification of the zinc finger domains of the nucleocapsid region, while maintaining the native envelope structure (3, 113). In another approach, soluble gpl40 oligomers containing the ectodomain of gp41 covalently linked to gpl20 have been generated by fusing GCN4 trimerization domains or T4 bacteriophage fibritin trimeric motifs to the C-terminus of soluble, uncleaved gpl40 glycoproteins (147-149). In other studies cysteine residues have been incorporated into gpl20 and gp41 (11, 114) to prevent dissociation of the two subunits through the formation of an intersubunit disulfide bridge upon expression of cleaved gpl40. More recently, proteoliposomes have been generated containing native, trimeric uncleaved gpl60,CT (cytoplasmic tail-deleted) glycoproteins (44). Although all of these approaches appear promising, such attempts to mimic native HIV envelope trimers have the limitation that key cross-neutralizing epitopes may be of relatively low immunogenicity on the trimer (143, 145).

[0074] A second strategy for obtaining broadly neutralizing antibodies with recombinant envelope glycoproteins focuses on the use of monomeric, but slightly modified, gpl60 or gpl40 glycoproteins. For example, various envelope glycoproteins have been generated in which the V2 loop has been deleted, with the aim of increasing the exposure of neutralizing epitopes (128). In other studies, partially deglycosylated recombinant gpl60 (13), or recombinant viruses expressing gpl20 glycosylation mutants, have been generated (107). Unfortunately, all of these approaches have so far failed to provide immunogens that elicit the desired level of neutralizing antibodies (23, 107), most likely because the elicited antibodies are unable to recognize their cognate epitopes on wild-type virus particles.

[0075] Neutralizing antibodies should target conserved regions on the HIV-1 envelope because such antibodies are most likely to be cross-reactive and useful in protection against HIV. The CD4-binding site (CD4bs) on gpl20 of HIV-1 is a particularly attractive target for vaccine design because it displays a high degree of conservation (61), and is accessible to neutralizing monoclonal antibodies (mAbs) on the surface of primary HIV-1 isolates prior to CD4 binding (110). One mAb, bl2, can be particularly useful as a model for the design of a vaccine capable of inducing potently neutralizing antibodies targeted to the CD4bs. MAb bl2 was isolated from a combinatorial phage display library developed from bone marrow donated by an individual who had been HIV positive for more than 6 years, but had not yet developed clinical symptoms (20). MAb bl2 effectively neutralizes a broad range of primary isolates from various HIV-1 clades as well as T-cell-line-adapted viruses (22, 29, 53). It has been inferred that this capability stems from bl2 being able to bind functional oligomeric gpl20 with comparable affinity to monomeric gpl20 (99, 116, 121); non-neutralizing mAbs bind with substantially lower affinity, or not at all, to functional oligomeric gρl20, presumably due to epitope occlusion or steric hindrance by vicinal gpl20 protomers on the viral surface (103, 143, 145).

[0076] Monomeric gpl20 thus contains all of the antigenic determinants to elicit a broadly neutralizing antibody such as bl2. However, the use of monomeric gρl20 is jeopardized by the exposure of non-neutralizing epitopes that are normally occluded on oligomeric gpl20 or that reside in the variable regions, in particular the V2 and V3 loops (100, 103, 145). These antigenic determinants are immunologically dominant over more conserved neutralizing epitopes (70, 86). Furthermore, they may induce antibodies that interact with gpl20 in a manner that is not permitted on native envelope spikes. As disclosed herein, monomeric HIV-1 gpl20 was engineered to restrict antibody binding to broadly neutralizing antibodies, e.g., bl2. As a first step, alanine scanning mutagenesis was used to identify residues on gpl20 that are important for binding of bl2, CD4 and the non-neutralizing CD4bs mAbs b3 and b6 (Pantophlet et al., J. Virol. 77:642-658 (2003), which is incorporated herein by reference; see Example 1). Although these tliree antibodies and CD4 bind monomeric gpl20 with a similar footprint, four residues were identified on the perimeter of the Phe43 cavity (61) that, when substituted by alanine, abrogated binding of the two non-neutralizing antibodies and CD4 (94). The binding of another non-neutralizing mAb, F105, was also abolished, whereas that of two further non- neutralizing CD4bs mAbs, F91 and 15e, was reduced. In contrast, the binding of mAb bl2 relative to wild type monomeric gpl20 was slightly enhanced (94).

[0077] The modified gpl20 polypeptides containing alanine substitutions provide promising prospective immunogens. However, considering that the variable loops on monomeric gpl20 also can serve as immune decoys (34, 70, 86, 122) gpl20 was further altered so as to focus the antibody response on the CD4bs; the concept of refocusing B-cell responses has been postulated by Delves et al. (32), who suggested that epitope-specific molecules could be elicited by selectively mutating 'undesired' epitopes, while preserving the overall fold of the protein and, hence, the desired B-cell (or T-cell) epitope(s). In a recent report (24), the e-chain of human chorionic gonadotrophin was used as a model system to show the feasibility of this strategy. This study showed that a single amino acid substitution that does not disrupt the overall conformation of the protein is sufficient to shift the immune response away from an unwanted epitope and towards a weakly immunogenic determinant (24).

[0078] Rather than inserting single mutations, a previously described approach (37) was adopted, wherein undesired epitopes are masked through the incorporation of extra N-linked glycans. As disclosed herein, this strategy resulted in blocking of the binding of non-neutralizing and weakly neutralizing antibodies, whereas binding of the broadly neutralizing antibody bl2 was retained (Example 2). These reengineered HIV-1 gpl20 polypeptides provide potential immunogens useful, for example, as a component of an HIV-1 vaccine, and can serve as a basis for alternative approaches to the development of candidate vaccines against other viral pathogens.

[0079] The present invention relates to mutant gpl20 molecules, referred to generally herein as immunogenic mutant gpl20 polypeptides, which stimulate neutralizing antibodies, including human antibodies that are specific for and neutralize an human immunodeficiency virus (HIV), particularly HIV-1. In an embodiment of the invention, epitopic polypeptide . sequences in glycoprotein gpl20 of HIV-1 are provided that are capable of stimulating neutralizing antibodies for HIV. In another embodiment, the gρl20 immunogen binds only neutralizing antibodies, and not non-neutralizing antibodies. Stimulation of such human monoclonal antibodies with a claimed specificity, and like human monoclonal antibodies with like specificity, are useful in the diagnosis and therapy of HIV-induced disease.

[0080] The term "HIV-induced disease" means any disease caused, directly or indirectly, by HIV. An example of a HIV-induced disease is acquired immunodeficiency syndrome (AIDS) caused by HIV-1 infection, and any of the numerous conditions associated generally with AIDS and caused by HIV infection. Thus, in one aspect, the present invention is directed to modified or mutant gρl20 molecules that provide an HIV neutralization site and to monoclonal antibodies stimulated by the invention immunogenic mutant HIV-1 gρl20 molecules. The monoclonal antibody referred to as "bl2", as disclosed in U.S. Patent Νos. 5,652,138 and 5,804,440, herein incorporated by reference in their entirety, is an exemplary neutralizing antibody to utilize to compare other antibodies against for purposes of the present invention. Thus, if a modified or mutant gpl20 molecule elicits a human monoclonal antibody response and the antibody generated binds and neutralizes HIV, particularly HIV-1, in a manner similar to a bl2 human monoclonal antibody then the mutant gpl20 molecule is a candidate immunogen for eliciting a neutralizing antibody response.

[0081] A "modified" or "mutant" gρl20 molecule is a molecule that has at least one deletion, substitution or the like such that the molecule is different at the amino acid residue level from a wild-type gρl20 molecule from which it was derived. It should be understood that gpl20 molecules may also naturally differ within a population of molecules. A molecule prior to mutagenesis will be considered "wild-type" for purposes of this invention.

[0082] It is also possible to determine, without undue experimentation, if a mutant gpl20 molecule of the invention elicits a neutralizing human monoclonal antibody response similar to has the same (i.e., equivalent) ability to elicit a response including antibodies having specificity as a human monoclonal antibody such as bl2 by ascertaining whether the former or test antibodies prevent the bl2 antibody from binding to HIV. If the neutralizing human monoclonal antibody being tested competes with a bl2 or bl2-like human monoclonal antibody, as shown by a decrease in binding by the non-bl2 human monoclonal antibody in standard competition assays for binding to a solid phase antigen, for example, to HIV-1 gρl20, then it is likely that the two monoclonal antibodies bind to the same, or a closely related, epitope. In that case, the mutant gpl20 molecule utilized to elicit the test monoclonal antibody is a candidate immunogen for eliciting a neutralizing monoclonal antibody response.

[0083] The ability to neutralize HIV at one or more stages of virus infection is a desirable quality of an elicited human monoclonal antibody described herein. Virus neutralization can be measured by a variety of in vitro and in vivo methodologies. Exemplary methods described herein for determining the capacity for neutralization are the in vitro assays that measure inhibition of HIV-induced syncytia formation, plaque assays and assays that measure the inhibition of output of core p24 antigen from a cell infected with HIV (see also the Examples section below).

[0084] A number of previous mutagenesis studies have reported on the effects of amino acid substitutions on binding by anti-CD4bs antibodies (49, 71, 130, 133, 135). However, only the study by Roben et al. (110) describes such an analysis applied to mAb bl2. In that study, wild-type and mutant gpl20 were tested for their ability to bind a saturating concentration of each antibody, the results being expressed as the ratio of antibody bound to mutant gpl20 and wild-type gpl20. This approach is most satisfactory for substitutions that produce large effects in binding, but may be less reliable for detecting smaller effects, since a change in saturation does not necessarily indicate a change in antibody affinity.

[0085] The invention provides methods and compositions for stimulating high levels of broadly neutralizing antibodies. As disclosed herein, the immunospecificity of a human monoclonal antibody of this invention can be directed to epitopes that are shared across serotypes and/or strains of HIV, or can be specific for a single strain of HIV, depending upon the epitope. Thus, a preferred human monoclonal antibody can immunoreact with HIV-1, HIV-2, or both, and can immunoreact with one or more of the HIV-1 strains IIIB, MN, RF, SF-2, Z2, Z6, CDC4, ELI and the like strains. In addition, a preferred human monoclonal antibody can immunoreact and neutralize a majority of field isolates of HIV, as described further herein.

[0086] The immunospecificity of an antibody, its HIV-neutralizing capacity, and the attendant affinity the antibody exhibits for the epitope, are defined by the epitope with which the antibody immunoreacts. The epitope specificity is defined at least in part by the amino acid residue sequence of the variable region of the heavy chain of the immunoglobulin the antibody, and in part by the light chain variable region amino acid residue sequence. Preferred human monoclonal antibodies immunoreact with the CD4 binding site of HIN-1 gpl20.

[0087] Also disclosed are immunogenic mutant HIV-1 gpl20 molecules having a specified amino acid sequence, which sequence confers the ability to elicit broadly neutralizing human monoclonal antibodies that bind a specific unique neutralizing epitope and to neutralize HIV when the virus is bound by these antibodies. Such mutant gpl20 molecules include, for example, HIV-1 gpl20 polypeptides having a substitution of an amino acid corresponding to Gly-473, Asp-474, Met-475, Arg-476, or a combination thereof, for example, a substitution such as G473 A, D474A, M475A, R476A, or a combination thereof, for example, at least two amino acid mutations (e.g., D474A and R476A; or G473A and M475A; or D474A, M475A and R476A; or G473 A, D474A, M475A and R476A), as well as mutant gpl20 polypeptides having DI 13A, V127A, D180A, N197A, or S256A substitution, or any combination of the above exemplified substitutions. In one embodiment, one or a combination of mutations modifies the gpl20 polypeptide so as to generate a glycosylation site (e.g., an N-glycosylation site). Such mutations are exemplified by HIN-1 gpl20 comprising H92Ν and N94T; Ql 14N and L115T; S143T; E150N and G152T; K171N and Y173T; Q246N; P313N and R315T; I423N and N425T; as well as combinations of such mutations that generate a glycosylation site (e.g., Ql 14N, Ll 15T, S143T, E150N, and G152T), any or all of which can further include a H92N, K171N, Y173T, and/or I423N substitution. In another embodiment, the mutant gpl20 polypeptide comprises combinations of mutations some of which can introduce a glycosylation site into the gpl20 polypeptide and others that do not, for example, an immunogenic mutant HIN-1 gpl20 polypeptide comprising two or more of G473A, D474A, M475A, R476A, H92Ν, Q114N, L115T, S143T, E150N, G152T, K171N, Y173T, and I423N.

[0088] In one aspect, neutralizing antibodies, including neutralizing monoclonal antibodies, elicited by mutant gpl20 molecules of the invention exhibit a potent capacity to neutralize HIV, particularly neutralizing monoclonal antibodies elicited by an immunogenic mutant HIV-1 gpl20 that are specific for HIV-1. The capacity to neutralize HIV is expressed as a concentration of antibody molecules required to reduce the infectivity titer of a suspension of HIV when assayed in an typical in vitro infectivity assay such as is disclosed herein. A monoclonal antibody of this invention has the capacity to reduce HIV infectivity titer in an in vitro virus infectivity assay by 50% at a concentration of less than about 700 nanograms (ng) of antibody per milliliter (ml) of culture medium in the assay, and preferably reduces infectivity titers 50% at a concentration of less than about 300 ng/ml, and more preferably at concentrations less than about 10 ng/ml.

[0089] Exemplary monoclonal antibodies described herein are effective at 3-700 ng/ml, and therefore are particularly well suited for inhibiting HIV in vitro and in vivo. Particularly preferred human monoclonal antibodies of this invention immunoreact with gpl20 in its "mature" form, which form is to be distinguished from antigenic determinants present on the HIV envelope precursor glycoprotein designated gpl60. gpl60 is processed during virus biogenesis by cleavage into two polypeptides, gp41 and gρl20. "Mature" gpl20 refers to the processed protein that is found in mature HIV virus particles, and can be detected on the surface of HIV-infected cells.

[0090] Thus, a preferred antibody of this invention binds mature gpl20 preferentially over HIV precursor glycoprotein gpl60. By "binds preferentially" is meant that the antibody immunoreacts with (binds) substantially more mature gpl20 than gpl60 in an immunoreaction admixture. Substantially more typically indicates that at least greater than 50% of the total mass of immunoprecipitated material is gpl20, and preferably indicates that at least greater than 75%, more preferably 90%, of the immunoprecipitated material is gpl20.

[0091] Methods for determining immunoreaction of a subject antibody with gpl20 are well known in the art, and the invention need not be so limited. However, preferred methods for determining the relative amounts of envelope glycoprotein antigens include radio-immunoprecipitation (RIP) of cell-surface labeled HIV-infected cells, followed by molecular weight analysis of the labeled products by polyacrylamide gel electrophoresis (PAGE).

[0092] A particularly useful mutant HIV-1 gpl20 molecule of the invention can elicit neutralizing human monoclonal antibodies that have the capacity to neutralize a majority of field isolates. As is well understood, the field (i.e., clinically isolated) strains of HIV-1 are typically different to some degree antigenically from laboratory strains. Therefore, it is well understood that useful neutralizing antibodies must immunoreact with, and be neutralizing against, field isolates of HIV. Preferably, the useful antibody neutralized a large percentage of field isolates, thereby increasing its effectiveness when new strains are encountered.

[0093] U.S. Patent No. 5,652,138 and 5,804,440 demonstrate that the human monoclonal antibody bl2 has the ability to neutralize a majority of the field isolates tested. By "majority" is meant that in a representative and diverse collection of field isolates, the antibody is capable of neutralizing at least more than 50% of the strains, and particularly at least about 75%) of the strains tested. In this context, "neutralizing" means an effect of reducing the HIV infectivity titre in an in vitro virus infectivity assay as described herein at the antibody concentrations described. A preferred antibody is an antibody having the binding specificity of the bl2 monoclonal antibody described herein. Preferred are human antibodies having the binding specificity of the immunoglobulin heavy and light chain polypeptides produced by ATCC 69079.

[0094] The term "conservative variation" as used herein denotes the replacement of an amino acid residue by another, biologically similar residue. Examples of conservative variations include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like. The term "conservative variation" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that antibodies having the substituted polypeptide also neutralize HIV. By analogy, another embodiment of the invention relates to polynucleotides that encode the above noted heavy and/or light chain polypeptides and to polynucleotide sequences that are complementary to these polynucleotide sequences. Complementary polynucleotide sequences include those sequences which hybridize to the polynucleotide sequences of the invention under stringent hybridization conditions.

[0095] By using a neutralizing human monoclonal antibody of the invention, it is now possible to produce anti-idiotypic antibodies that can be used to screen human monoclonal antibodies to identify whether the antibody has the same binding specificity as a human monoclonal antibody of the invention and also used for active immunization (Herlyn et al., Science, 232:100 (1986), which is incorporated herein by reference). Such anti-idiotypic antibodies can be produced using well-known hybridoma techniques (Kohler et al., Nature, 256:495 (1975), which is incorporated herein by reference). An anti-idiotypic antibody is an antibody which recognizes unique determinants present on the human monoclonal antibody produced by the cell line of interest. These determinants are located in the hypervariable region of the antibody. It is this region which binds to a given epitope and, thus, is responsible for the specificity of the antibody. An anti-idiotypic antibody can be prepared by immunizing an animal with the monoclonal antibody of interest. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody and produce an antibody to these idiotypic determinants. By using the anti-idiotypic antibodies of the immunized animal, which are specific for the human monoclonal antibody of the invention produced by a cell line which was used to immunize the second animal, other clones with the same idiotype as the antibody of the hybridoma used for immunization can be identified. Idiotypic identity between human monoclonal antibodies of two cell lines demonstrates that the two monoclonal antibodies are the same with respect to their recognition of the same epitopic determinant. Thus, by using anti-idiotypic antibodies, other hybridomas expressing monoclonal antibodies having the same epitopic specificity can be identified.

[0096] Anti-idiotype technology also can be used to produce monoclonal antibodies that mimic an epitope. For example, an anti-idiotypic monoclonal antibody made to a first monoclonal antibody will have a binding domain in the hypervariable region which is the "image" of the epitope bound by the first monoclonal antibody. Thus, the anti-idiotypic monoclonal antibody can be used for immunization, since the anti-idiotype monoclonal antibody binding domain effectively acts as an antigen.

[0097] In one embodiment, the invention contemplates a truncated immunoglobulin molecule comprising a Fab fragment derived from a human monoclonal antibody of this invention. The Fab fragment, lacking Fc receptor, is soluble, and affords therapeutic advantages in serum half life, and diagnostic advantages in modes of using the soluble Fab fragment. The preparation of a soluble Fab fragment is generally known in the immunological arts and can be accomplished by a variety of methods. A preferred method of producing a soluble Fab fragment is described herein.

[0098] In another embodiment, the invention contemplates an immunoglobulin molecule comprising a Fab fragment derived from a human monoclonal antibody of this invention and the fragment crystallizable (Fc) domain of a human immunoglobulin molecule. The entire (i.e., complete) immunoglobulin (Ig) molecule comprising a Fab fragment with the Fc domain may afford therapeutic and diagnostic advantages, and can be any of the several Ig species depending upon the ultimate use, including IgG, IgA, IgD, IgE, IgM, and isotypes thereof. The immunoglobulin molecule would be capable of effector functions associated with the Fc domain when used in passive immunotherapy. These effector functions include antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cellular cytotoxicity (CDCC) which promote the death of the cell to which the immunoglobulin molecule is specifically bound. The effector functions may therefore be desirable in therapeutic applications. Diagnostic assays include the ability to detect the presence of the immunoglobulin molecule. These assays rely on the cross-linking of red cells or beads in agglutinations, the activation of complement in plaque assays, or the antigenic properties of the Fc region of the heavy chain as detected by secondary antibodies in ELISA or RIA procedures to detect the presence of the immunoglobulin molecule. Such diagnostic assays can only be performed with the entire immunoglobulin molecule. The isolation of the immunoglobulin molecule is also facilitated by the presence of the Fc domain in that commonly used methods of immunoglobulin purification are based upon interaction of reagents with the Fc domain. The preparation of a Fab fragment with the Fc domain is generally known in the immunological arts and can be accomplished by a variety of methods. A preferred method of producing a Fab fragment with the Fc domain is described herein.

[0099] A mutant gpl20 polypeptide engineered to elicit neutralizing human monoclonal antibodies can also be used immunotherapeutically for HIV disease. The term "immunotherapeutically" or "immunotherapy" as used herein in conjunction with an immunogenic mutant gpl20 polypeptide such as. a mutant HIV-1 g l20 polypeptide of the invention denotes both prophylactic as well as therapeutic administration. Thus, the gpl20 mutants can be administered to high-risk patients in order to lessen the likelihood and/or severity of HIV-induced disease, administered to patients already evidencing active HIV infection, or administered to patients at risk of HIV infection.

[0100] The present invention therefore contemplates therapeutic compositions useful for practicing the therapeutic methods described herein. Therapeutic compositions of the present invention contain a physiologically tolerable carrier together with at least one species of gpl20 mutant or neutralizing antibodies as described herein, dissolved or dispersed therein as an active ingredient. In a preferred embodiment, the therapeutic composition is not immunogenic for non-neutralizing antibodies when administered to a human patient for therapeutic purposes.

[0101] As used herein, the terms "pharmaceutically acceptable", "physiologically tolerable" and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a human without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like. The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art. Typically such compositions are prepared as sterile injectables either as liquid solutions or suspensions, aqueous or non-aqueous, however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared. The preparation can also be emulsified.

[0102] The active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance tl e effectiveness of the active ingredient.

[0103] The therapeutic composition of the present invention can include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

[0104] Physiologically tolerable carriers are well known in the art. Exemplary of liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, propylene glycol, polyethylene glycol and other solutes. Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, organic esters such as ethyl oleate, and water-oil emulsions.

[0105] A therapeutic composition contains an HIV-neutralizing amount of a human monoclonal antibody of the present invention or a mutant gpl20 molecule of the invention, typically an amount of at least 0.1 weight percent of antibody per weight of total therapeutic composition. A weight percent is a ratio by weight of antibody to total composition. Thus, for example, 0.1 weight percent is 0.1 grams of antibody per 100 grams of total composition.

[0106] For in vivo modalities, the method comprises administering to a patient a therapeutically effective amount of a physiologically tolerable composition containing a human monoclonal antibody or mutant g l20 of the invention. Thus, the present invention describes in one embodiment a method for providing passive immunotherapy to HIV disease in a human comprising administering to the human an immunotherapeutically effective amount of the monoclonal antibody of this invention.

[0107] A representative patient for practicing the present passive immunotherapeutic methods is any human exhibiting symptoms of HIV-induced disease, including AIDS or related conditions believed to be caused by HIV infection, particularly HIV-1 infection, and humans at risk of HIV infection. Patients at risk of infection by HIV include babies of HIV- infected pregnant mothers, recipients of transfusions known to contain HIV, users of HIV contaminated needles, individuals who have participated in high risk sexual activities with known HIV-infected individuals, and the like risk situations. In one embodiment, the passive immunization method comprises administering a composition comprising more than one species of human monoclonal antibody of this invention, preferably directed to non-competing epitopes or directed to distinct serotypes or strains of HIV, particularly HIV-1, so as to afford increased effectiveness of the passive immunotherapy.

[0108] A therapeutically (immunotherapeutically) effective amount of a human monoclonal antibody is a predetermined amount calculated to achieve the desired effect, i.e., to neutralize the HIV present in the sample or in the patient, and thereby decrease the amount of detectable HIV in the sample or patient. In the case of in vivo therapies, an effective amount can be measured by improvements in one or more symptoms associated with HIV-induced disease occurring in the patient, or by serological decreases in HIV antigens.

[0109] Thus, the dosage ranges for the administration of the monoclonal antibodies of the invention are those large enough to produce the desired effect in which the symptoms of the HIV disease are ameliorated or the likelihood of infection decreased. The dosage should not be so large as to cause adverse side effects, such as hyperviscosity syndromes, pulmonary edema, congestive heart failure, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. [0110] The dosage can be adjusted by the individual physician in the event of any complication. A therapeutically effective amount of an antibody of this invention is typically an amount of antibody such that when administered in a physiologically tolerable composition is sufficient to achieve a plasma concentration of from about 0.1 microgram (ϊg) per milliliter (ml) to about 100 ϊg/ml, preferably from about 1 ϊg/ml to about 5 ϊg/ml, and usually about 5 ϊg/ml. Stated differently, the. dosage can vary from about 0.1 mg/kg to about 300 mg/kg, preferably from about 0.2 mg/kg to about 200 mg/kg, most preferably from about 0.5 mg/kg to about 20 mg/kg, in one or more dose administrations daily, for one or several days.

[0111] The human monoclonal antibodies of the invention, mutant gpl20 or combination thereof, can be administered parenterally by injection or by gradual infusion over time. Although the HIV infection is typically systemic and therefore most often treated by intravenous administration of therapeutic compositions, other tissues and delivery means are contemplated where there is a likelihood that the tissue targeted contains infectious HIV. Thus, human monoclonal antibodies of the invention can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, transdermally, and can be delivered by peristaltic means.

[0112] The therapeutic compositions containing a mutant gpl20 or human monoclonal antibody of this invention are conventionally administered intravenously, as by injection of a unit dose, for example. The term "unit dose" when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.

[0113] The compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered depends on the subject to be treated, capacity of the subject's system to utilize the active ingredient, and degree of therapeutic effect desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosage ranges for systemic application are disclosed herein and depend on the route of administration. Suitable regimes for administration are also variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusion sufficient to maintain concentrations in the blood in the ranges specified for in vivo therapies are contemplated.

[0114] The invention also relates to a method for preparing a medicament or pharmaceutical composition comprising the human monoclonal antibodies of the invention, the medicament being used for immunotherapy of HIV disease.

[0115] In a related diagnostic embodiment, the invention contemplates screening HIV- infected patients for the presence of circulating anti-HIV antibodies immunoreactive with gpl20 that have a similar epitope immunospecificity when compared to a neutralizing antibody of this invention. Such a screening method indicates that the HIV-infected patient is exhibiting a significant immune response to the virus, and provides useful information regarding disease status and prognosis. The presence of anti-HIV antibodies cross-reactive with a neutralizing antibody of this invention indicates that the patient has some degree of HIV neutralizing activity, as defined herein.

[0116] The diagnostic assay involves determining whether the patient contains human anti-HIV antibodies immunoreactive with the same, similar or overlapping epitopes as a neutralizing antibody of the invention, such that there is a likelihood that there is a useful neutralizing immune response in the patient. There are a variety of immunological assay formats that can be utilized to determine cross-reactivity of test and control antibodies, and the invention need not be so limiting. Particularly preferred are competition assays for a common antigen, preferably in the solid phase.

[0117] The present invention also describes a diagnostic system, preferably in kit form, for assaying for the presence of HIV or an anti-HIV antibody in a sample according to the diagnostic methods described herein. A diagnostic system includes, in an amount sufficient to perform at least one assay, a subject human monoclonal antibody, as a separately packaged reagent. In another embodiment, a diagnostic system is contemplated for assaying for the presence of an anti-HIV monoclonal antibody in a body fluid sample such as for monitoring the fate of therapeutically administered antibody. The system includes, in an amount sufficient for at least one assay, a subject antibody as a control reagent, and preferably a preselected amount of HIV antigen, each as separately packaged immunochemical reagents. "Instructions for use" typically include a tangible expression describing the reagent concentration or at least one assay method parameter such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions and the like.

[0118] The present invention provides methods for producing novel HIV-neutralizing human monoclonal antibodies. The methods are based generally on the use of combinatorial libraries of antibody molecules which can be produced from a variety of sources, and include naive libraries, modified libraries, and libraries produced directly from human donors exhibiting an HIV-specific immune response. The combinatorial library production and manipulation methods have been extensively described in the literature, and will not be reviewed in detail herein, except for those feature required to make and use unique embodiments of the present invention. However, the methods generally involve the use of a filamentous phage (phagemid) surface expression vector system for cloning and expressing antibody species of the library. Various phagemid cloning systems to produce combinatorial libraries have been described by others. See, for example the preparation of combinatorial antibody libraries on phagemids as described by Kang et al., Proc. Natl. Acad. Sci., USA, 88:4363-4366 (1991); Barbas et al., Proc. Natl. Acad. Sci., USA, 88:7978- 7982 (1991); Zebedee et al., Proc. Natl. Acad. Sci., USA, 89:3175-3179 (1992); Kang et al., Proc. Natl. Acad. Sci., USA, 88:11120-11123 (1991); Barbas et al., Proc. Natl. Acad. Sci., USA, 89:4457-4461 (1992); and Gram et al., Proc. Natl. Acad. Sci., USA, 89:3576-3580 (1992), which references are hereby incorporated by reference.

[0119] A method for producing a human monoclonal antibody generally involves (1) preparing separate H and L chain-encoding gene libraries in cloning vectors using human immunoglobulin genes as a source for the libraries, (2) combining the H and L chain encoding gene libraries into a single dicistronic expression vector capable of expressing and assembling a heterodimeric antibody molecule, (3) expressing the assembled heterodimeric antibody molecule on the surface of a filamentous phage particle, (4) isolating the surface- expressed phage particle using immunoaffinity techniques such as panning of phage particles against a preselected antigen, thereby isolating one or more species of phagemid containing particular H and L chain-encoding genes and antibody molecules that immunoreact with the preselected antigen. As disclosed herein, the resulting phagemid library can be manipulated to increase and/or alter the immunospecificities of the monoclonal antibodies of the library to produce and subsequently identify additional, desirable, human monoclonal antibodies of the present invention. For example, the H chain and L chain immunoglobulin molecule encoding genes can be randomly mixed (shuffled) to create new HL pairs in an assembled immunoglobulin molecule. Additionally, either or both the H and L chain encoding genes can be mutagenized in the complementarity determining region (CDR) of the variable region of the immunoglobulin polypeptide, and subsequently screened for desirable immunoreaction and neutralization capabilities.

[0120] In one embodiment, the H and L chain genes can be cloned into separate, monocistronic expression vectors, referred to as a "binary" system described is further herein. In this method, step (2) above differs in that the combining of H and L chain encoding genes occurs by the co-introduction of the two binary plasmids into a single host cell for expression and assembly of a phagemid having the surface accessible antibody heterodimer molecule.

[0121] In the present methods, the antibody molecules are monoclonal because the cloning methods allow for the preparation of clonally pure species of antibody producing cell lines. In addition, the monoclonal antibodies are human because the H and L chain encoding genes are derived from human immunoglobulin producing immune cells, such as spleen, thymus, bone marrow, and the like. [0122] The method of producing a HIV-neutralizing human monoclonal antibody also requires that the resulting antibody library, immuiioreactive with a preselected HIV antigen, is screened for the presence of antibody species which have the capacity to neutralize HIV in one or more of the assays disclosed herein for determining neutralization capacity. Thus, a preferred library of antibody molecules is first produced which binds to an HIV antigen, preferably gpl60, gpl20, gρ41, the V3 loop region of gpl60, or the CD4 binding site of gpl20 and gp41, and then is screened for the presence of HIV-neutralizing antibodies as described herein.

[0123] As a further characterization of the present invention the nucleotide and corresponding amino acid residue sequence of the antibody molecule's H or L chain encoding gene is determined by nucleic acid sequencing. The primary amino acid residue sequence information provides essential information regarding the antibody molecule's epitope reactivity.

[0124] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting between different genetic environments another nucleic acid to which it has been operatively linlced. Preferred vectors are those capable of autonomous replication and expression of structural gene products present in the DNA segments to which they are operatively linlced. Vectors, therefore, preferably contain the replicons and selectable markers described earlier.

[0125] As used herein with regard to DNA sequences or segments, the phrase "operatively linlced" means the sequences or segments have been covalently joined, preferably by conventional phosphodiester bonds, into one strand of DNA, whether in single or double stranded form. The choice of vector to which transcription unit or a cassette of this invention is operatively linlced depends directly, as is well known in the art, on the functional properties desired, e.g., vector replication and protein expression, and the host cell to be transformed, these being limitations inherent in the art of constructing recombinant DNA molecules.

[0126] The following examples are intended to illustrate but not limit the invention. EXAMPLE 1

FINE MAPPING OF NEUTRALIZING AND NON-NEUTRALIZING

MONOCLONAL ANTIBODY BINDING TO HIV-1 g_P120

[0127] This example demonstrates that mutations to gpl20 can be used to obtain an immunogenic mutant gpl20 polypeptide having increased binding specificity with respect to HIV neutralizing antibodies as compared to non-neutralizing antibodies (see, also, (94)).

[0128] Alanine scanning mutagenesis was performed on monomeric HIV-1 gpl20 to systematically identify residues important for gpl20 recognition by neutralizing and non-neutralizing monoclonal antibodies (mAbs) to the CD4 binding site (CD4bs). Substitutions that affected binding of broadly neutralizing antibody bl2 were compared to substitutions that affected the binding of CD4 and of two non-neutralizing anti-CD4bs antibodies (b3 and b6) with affinities for monomeric gpl20 comparable to that of bl2. Not surprisingly, the sensitivities to a number of amino acid changes were similar for the mAbs and for CD4. However, in contrast to what was seen for the mAbs, no enliancing mutations were observed for CD4, suggesting that the virus has evolved toward an optimal gpl20- CD4 interaction. Although the epitope maps of the mAbs overlapped, a number of key differences between bl2 and the other two antibodies were observed. These differences can explain why bl2, in contrast to non-neutralizing antibodies, can interact not only with monomeric gpl20, but also with functional oligomeric gpl20 at the virion surface. Neutralization assays performed with pseudovirions bearing envelopes from a selection of alanine mutants showed a reasonable correlation between the effects of the mutations on bl2 binding to monomeric gpl20 and neutralization efficacy. However, some mutations produced an effect on bl2 neutralization counter to that predicted from gpl20 binding data; these mutations appeared to have different effects on the bl2 epitope on monomeric gpl20 and functional oligomeric gpl20. To determine whether monomeric gpl20 can be engineered to preferentially bind mAb bl2, recombinant gpl20 polypeptides were generated containing combinations of alanine substitutions shown to uniquely enhance bl2 binding. Whereas bl2 binding was maintained or increased, binding by five non-neutralizing anti-CD4bs mAbs (b3, b6, F105, 15e, and F91) was reduced or completely abolished. As such, the mutant engineered gpl20 polypeptides can provide useful immunogens for eliciting broadly neutralizing antibodies.

Materials and Methods

Antibodies

[0129] Monoclonal antibodies (mAbs) b3, b6 and bl2 were isolated as Fab fragments from a phage display library derived from a single donor and have been characterized (6, 7,

20, 22, 110). All tliree antibodies recognize discontinuous epitopes overlapping the CD4bs on HIV-l gpl20.

[0130] CD4-IgG2 is a recombinant antibody-like fusion protein in which the heavy- chain and light-chain variable domains of human IgG2 have been replaced with the D1D2 domains of human CD4 (1). This molecule was used as a surrogate for CD4. MAbs 15e and F91 react with respective epitopes overlapping the CD4bs on g l20 (49, 76, 78, 111), and mAb 17b recognizes an epitope overlapping the coreceptor binding site on gp 120 (131, 134, 144).

[0131] Human IgG purified from pooled plasma obtained from healthy asymptomatic seropositive individuals (HIVIG) and mAb F105, another anti-CD4bs antibody (105, 106, 135), were obtained from the NIH AIDS Research and Reference Reagent Program (NIH ARRRP). MAb 2G12 recognizes an epitope involving El— »2 mannose residues on the carbohydrate-rich "silent" face (61, 143) of gpl20 (115, 119, 137).

Plasmid constructs and mutagenesis

[0132] Alanine mutations (Table 1, below; Fig. IA) were generated using the QUIKCHANGE mutagenesis kit (Stratagene). For antibody mapping experiments, plasmid pSVIIIexE7pA^"jR-csF was used as the template. This plasmid was derived from pSVIIIexE7pA^"HχB2 (47), which was modified as described previously (154) in order to subclone the env gene of HIV-1 _JR-_CSF, a molecularly cloned primary HIV-1 virus (59). Two variable loop-deleted mutants were also generated, using pS VIIIexE7pA^"jR-csF as the PCR template. A Vl-deleted (,V1) mutant (deletion of residues 134-154) was constructed by PCR using the primer pair csfl20-f (5'-GTCTGAGTCGGAGCTAGCGTAGAAAAGTTGTGGGTCA-3'; SEQ ID NO.T), and csfdVl-r (5'-GTCTGAGTCGGAACCGGACCCATCTTTGCAATTTAAAGTA-3'; SEQ

ID NO:2); and the primer pair csfdVl-f (5'-GTCTGAGTCGGATCCGGTTCTGGGAAAAACTGCTCTTT-3'; SEQ ID

NO:3), and csfl20-r (5'- GTCTGAGTCGGACTCGAGTTTTCTCTTTGCACCACTCTTC-3'; SEQ ID

NO:4). Primers csfdVl-f and csfdVl-r both contain a BsaW I restriction site (bold).

[0133] The PCR products were cloned into pSVIIIexE7pA^"jR.csF using Kpnl, BsaW I and Mfel in a 2-step ligation reaction. A V3 -mutant (deletion of residues 303-324) was generated in a similar manner using the primers csfl20-f (SEQ ID NO:l) and csfdN3-r (5'-GTCTGAGTCGGAACCGGACCCATTGTTGCTGGGCCTTGT-3'; SEQ ID

NO: 5), and the primers csfdV3-f (5'-GTCTGAGTCGGATCCGGTTCTGGGGATATAAGACAAGCCC -3'; SEQ

ID NO:6) and csfl20-r (SEQ ID NO:4). Primers csfdV3-f and csfdV3-r also contain unique

BsaW I restriction sites.

[0134] To generate a Nl/V2-mutant (V2 loop deleted from residues 160-193), the pSVIIIexE7pA^"j_R-csF-,Vl mutant was used as a template. First, the BsaW I site introduced into the ^l-mutant was changed by site-directed mutagenesis, whereby the amino acid sequence was retained. Deletion of the V2 sequences was performed in an analogous manner as for the ,V1- and ,V3-mutants, using the primers csfl20-f (SEQ ID ΝO:l) and csfdV2-r2 (5'-GTCTGAGTCGGAACCGGACCCGAAAGAGCAGTTTTT-3'; SEQ ID NO: 7) and the primers csfdN2-f (5'-GTCTGAGTCGGATCCGGTTCTGGGATAAGTTGTAACACC-3'; SEQ ID ΝO:8) and csfl20-r (SEQ ID NO:4). The unique BsaW I restriction sites in primers csfdV2-r2 and csfdV2-f are shown in bold. PCR fragments were cloned into pSVIIIexE7pA^" _JR-_CSF using Kpnl, BsaW I and Mfel. In all variable loop-deleted mutants, the deleted sequences were replaced by a Gly-Ser-Gly-Ser-Gly linker. [0135] Mutants containing multiple alanine substitutions were also generated using the QUIKCHANGE mutagenesis kit, except that the plasmid pCMV-Tag-tpajR-FLg_Pi20 was used as the template. This plasmid was derived from plasmid pCMV-Tag4A (Stratagene). First, a DNA fragment encoding the tissue plasminogen activator protein (Tpa) was generated by PCR using the primers tpal

(5'-CGTTGAATTCGCCGCCACCATGGATGCAATGAAGAGAGGGCTCTGCTGTGT GCTGCTGCTGTGTGGAGCAGTCTTCGTTTCG-3'; SEQ ID NO:9), which contains an EcoR I restriction site (bold) and tpa2

(5'-GCACCTCGAGGCGCGCTCCTCTTCTGAATCGGGCATGGATTTCCTGGCTGGG CGAAACGAAGACTGCTCCACACAG-3'; SΕQ ID NO: 10), which contains Mol and BssH II restriction sites (bold and bold/italics, respectively). This fragment was cloned into pCMV-Tag4A using EcoR I and_ϊ7.oI, to generate pCMV-Tag-tpa. The env gene from HIV-1 R-FL (59) was amplified by PCR using as template the plasmid pSyngp 140J_R.PL (obtained from the NIH ARRRP; 2, 43(2)), in which most wild-type gpl40 codons were replaced with codons from highly expressed human genes. For PCR, the primers jr-fi5' (5'-CGTTGCGCGCGTGGAGAAGCTGTGGGTG-3'; SΕQ ID NO: 11), which contains a BssH II restriction site (bold), and flXho-r (5'-GCAGAGGGAGAAGCGCCTCGAGGCTGTGGGCATTGG-3'; SΕQ ID NO:12), which contains aXhol restriction site (bold), were used. The PCR fragment was cloned into pCMVtag-tpa using BssH II and Xhol, to generate pCMVtag-tpajR.p_Lgpno- All plasmids and mutations generated in this study were verified by DNA sequencing.

Generation of recombinant HIV-1 virions

[0136] To produce recombinant virions, 293T cells grown in Dulbecco's modified Eagle's media (Gibco) supplemented with penicillin, streptomycin, L-glutamine and fetal bovine serum (10%) were transiently transfected with wild-type or mutant pSVIIIexE7pA^" _JR-_CSF plasmids (2 ϊg) along with the luciferase reporter plasmid pNL4-3.Luc.R^~E^" (4 ϊg, obtained from the NIH ARRRP (26, 46)) using FuGENE6 transfection reagent (Roche). At 24 hr post-transfection, the culture supernatant was replaced with serum-free media and incubation was continued for another 24 hr. Cell culture supernatants, containing pseudovirions, were subsequently harvested and stored at -80°C for neutralization assays (see below). Alternatively, recombinant virions were lysed by the addition of detergent to the harvested culture supernatant, which was then stored at -20°C until further use.

Expression of recombinant gpl20

[0137] pCMVtag-tpajR.pL_gpi20 plasmids expressing wild-type or mutant gpl20 were used to transiently transfect subconfluent 293T cells grown in serum-containing media as described above, except that no pNL4.3Luc was used. Two days post-transfection, culture supernatants containing recombinant gpl20 proteins were collected and stored at -20°C.

ELISA assays

[0138] For ELISA, microtiter plate wells (flat bottom, Costar type 3690, Corning Inc.) were coated overnight at 4°C with anti-gpl20 antibody D7324 (International Enzymes, Inc.) at a concentration of 5 ϊg/ml (250 ng/well, diluted in PBS). Subsequent incubation steps were performed at room temperature. Coated plates were washed twice with PBS supplemented with 0.05% Tween (PBS-T), blocked for 1 hr with PBS supplemented with 3% BSA and subsequently incubated for 2-4 hr with cell culture supernatant that was diluted 1 :3 in PBS containing 1% BSA and 0.02% TWEEN-20 detergent (PBS-B-T). Plates were washed with PBS-T (10 times), then incubated with mAb serially diluted in PBS-B-T (starting at a concentration of 10 ϊg/ml). HIVIG (1 ϊg/ml, diluted in PBS-B-T) was used as a control to ensure that similar amounts of envelope protein were captured. After washing as before, peroxidase-conjugated or alkaline phosphatase-conjugated goat anti-human IgG (F(ab') -specific, Pierce) was added (diluted 1:1000 in PBS-B-T) and incubation continued for another hour. Plates were washed again, followed by incubation with TMB-substrate (Pierce) when peroxidase-conjugated secondary antibody was used or j^-nitrophenyl phosphate (Sigma) when alkaline phosphatase-conjugated secondary antibody was used. The color reaction was stopped by adding 2 M sulfuric acid (in the case of TMB) and absorbances were measured at 450 nm. Assays developed with j nitrophenyl phosphate were measured at 405 nm without stopping the reaction. Apparent affinities were calculated as the antibody concentration at half-maximal binding; percent changes in affinity relative to wild type were expressed as (apparent affinity of the wild type/apparent affinity of the mutant) x 100.

HIV-1 neutralization assays

[0139] Recombinant virions competent for a single round of infection were generated as described above. Neutralization assays were performed essentially as described previously (154), using an initial seeding density of between 1-3 x 10⁴ target cells (U87.CD4.CCR5, obtained from the NIH ARRRP). The degree of virus neutralization by antibody was determined by measuring the luciferase activity.

[0140] The percent neutralization at a given antibody concentration was expressed as {(luciferase activity in the absence of antibody — . luciferase activity in the presence of a given antibody concentration)/luciferase activity in the absence of antibody} x 100. To determine the degree of correlation between neutralization efficiency and the change in antibody binding affinity for each mutant relative to wild type, a neutralization index was defined. This index was expressed as {(antibody concentration required to achieve 90% neutralization of the wild type x apparent antibody affinity for wild-type-gpl20)/(antibody concentration required to achieve 90% neutralization of the mutant x apparent antibody affinity for mutant gpl20)}. Neutralization indexes of between 0.2 and 5 were considered indicative of a reasonable correlation between the change in antibody affinity for monomeric gpl20 and neutralization efficiency.

RESULTS

[0141] Alanine mutagenesis was performed on monomeric gpl20 to define in more detail which residues, on gpl20 influence or modulate bl2 reactivity. In parallel, CD4-IgG2 (used as a surrogate for CD4) and two non-neutralizing anti-CD4bs mAbs (b3 and b6 (6)) were assayed to compare the effects of alanine mutations on binding by neutralizing versus non-neutralizing mAbs, as well as to distinguish between amino acid substitutions that uniquely affect mAb binding and mutations that affect the binding of anti-CD4bs ligands in general. Mutagenesis was performed using gpl20 from HIV-I_JR-_CSF as the parent. A total of 81 mutants containing single alanine substitutions and 3 variable loop-deleted mutants were generated (Fig. 1 A). The residues that were mutated to alanine were selected primarily from a list of likely contact residues based on the docking model of the crystal structure of mAb bl2 and the CD4-complexed gpl20 core structure of HxB2 (90). Most mutations were in or adjacent to the CD4bs (Fig. IB), encompassing the neutralizing as well as the non-neutralizing faces of gpl20 (Fig. IC). However, a number of amino acids that were selected for mutagenesis were also located on the silent face. As shown in Fig. ID, amino acids that were selected for mutagenesis ranged from those that are highly conserved among primate immunodeficiency viruses to residues that vary significantly among HIV-1 isolates.

[0142] To determine apparent antibody affinities, mutant monomeric gpl20 from pseudovirions was captured onto ELISA plate wells and probed with varying concentrations of antibody to generate a binding curve for each mutant. Apparent binding affinities were determined from the antibody concentration at half-maximal binding. The apparent antibody affinity for each mutant gpl20 was then related to that for wild-type gpl20 (Table 1, below). Changes in relative affinity greater than 200% were designated as increases, whereas those below 50% were designated as decreases. Intermediate values were recorded as having no or limited effect on antibody binding.

[0143] Tliree variable loop-deleted gpl20 mutants (Nl, ,V1/V2 and ,V3) were investigated. Deletion of the VI loop alone or together with the V2 loop had an adverse effect on the binding of CD4 and all three mAbs to gρl20, whereas deletion of the V3 loop decreased the binding affinity of CD4 and mAbs b6 and bl2, but not b3 (Table 1, below). Nineteen alanine substitutions in gpl20 reduced the affinity for CD4 and all three mAbs. Tliree substitutions (at D180, 1184, F176) are located in the V2 loop, one (at K207) is located at the base of the VI /V2 loop-stem, one (at 1213) is located on the non-neutralizing face at the putative gpl20-gp41 interface (61, 143), six (at T257, E370, 1371, Y384, M426, G472) line, i.e., the Phe43 cavity (61) of gpl20, four (at N386, P470, R476, W479) are in close proximity to the CD4 binding pocket (61), three (at R350, W395, T450) are on the carbohydrate-rich silent face of gpl20 and one (at 1439) is close to the junction of silent and neutralizing faces (61, 143), adjacent to the base of the stem of the VI /V2 loop. Except for R350 and W395, these residues are all conserved among primate immunodeficiency viruses or HIV-1 isolates in terms of identity or similarity of the amino acid side chain (Table 1, below). Four of the nineteen residues listed above (E370, 1371, M426 and G472) are CD4 contact residues (Table 1, below; (61)), and their conservation is probably required for the optimal interaction of gpl20 with CD4. For the remaining residues, conservation may be associated with maintenance of the structural integrity of gpl20 (61, 143) and it is possible that substitution by alanine leads to local misfolding of monomeric gpl20.

[0144] To determine whether monomeric gpl20 from these mutants was globally perturbed, the binding of mAb 2G12, which recognizes a carbohydrate-dependent conformational epitope on the silent face of gl20 (115, 119, 137), also was investigated. For most mutant glycoproteins, 2G12 binding was unchanged (Table 1, below). It would thus appear that these proteins are not globally misfolded. Interestingly, mutating residue F176 (V2 loop) to alanine caused a fourfold increase in 2G12 binding relative to that seen with wild-type gpl20, indicating that mutations in the V2 loop of gpl20 can have some effect on 2G12 binding to its carbohydrate epitope. Alanine substitutions of tliree residues (N386, W395, and W479) caused moderate to significant decreases in 2G12 relative affinity (Table 1, below). None of these residues is believed to be a contact residue for mAb 2G12 (115, 119, 137), suggesting that these alanine replacements may cause significant perturbations of the gpl20 structure.

[0145] The decrease in CD4 binding affinity observed with for the W395A mutant was somewhat striking, considering that this residue shows significant variability among HIV-1 isolates (61). However, alignment of the sequences from HIV-1 clade B isolates showed that this amino acid is identical in 106 of the 107 isolates listed in the HIV sequence database (see hypertext transfer protocol (http), at URL hiv-web.lanl.gov/content/hiv- dp/mainpage.html). In the single clade B isolate in which this amino acid is not Trp, it is replaced by a His residue. Position 395 may thus play a role in preserving a structural conformation that is required for optimal CD4 binding in clade B isolates and that is achieved by the incorporation of aromatic amino acids. The same may hold true for R350, which is 60% conserved as arginine in clade B isolates but 80% conserved as a positively charged side chain.

[0146] The remaining 62 alanine substitutions had varying effects on CD4- and antibody-reactivity (Fig. 2). A noticeable difference between the effects of alanine substitution on ligand binding was that, whereas some substitutions enhanced mAb binding, CD4 binding was always either decreased or unchanged. Many substitutions produced similar effects on mAb and CD4 binding (Table 1, below; Fig. 2). For 31 mutations, the effects on b3 binding were similar to the effects on CD4 binding. In comparison, the effects on b6 and bl2 binding were similar to the effect on CD4 binding for 24 and 26 mutations, respectively. However, if a focus was directed on substitutions that decreased ligand binding, then the greatest correspondence was between mAb bl2 and CD4, closely followed by mAb b6 and CD4, and with the greatest discrepancy being between mAb b3 and CD4. Thus, excluding the previous 19 alanine substitutions which uniformly reduced binding by CD4 and the three mAbs, 18 substitutions decreased binding of both bl2 and CD4, 17 decreased binding for both b6 and CD4, and only 11 decreased binding for CD4 and for b3.

[0147] Amino acid substitutions that affected mAb binding were mapped onto the crystal structure of the gρl20 core of HIV-1 _{H B} , to obtain a better understanding of the spatial arrangement among the amino acid residues that were mutated (Fig. 3). Although the mutagenesis studies used gpl20jR.csF, this approach was thought to be valid since the structure of the core seems to be highly conserved among HIV-1 isolates (60). One caveat to note is that the structure of gpl20 is that of a core molecule complexed to CD4 and Fab 17b; some differences in the conformation of this molecule and the corresponding unliganded molecule have been proposed (61, 143, 145), but no structural data to address this issue yet exists. The epitope maps for the antibodies, particularly mAbs b6 and bl2, were highly similar (Fig. 3). This is not surprising, given that anti-CD4bs antibodies compete with each other for binding to gp 120 (80). The map observed for CD4 was in good agreement with the CD4 footprint derived from the crystal structure of the CD4-gpl20 complex (61, 143). Notably, the map for bl2 was also in agreement with the footprint obtained from a docking model of the bl2 structure and the gpl20 core structure (90). [0148] To gain a better understanding of the differences between each mAb, only those alanine substitutions for which the effect (or lack of effect) on antibody reactivity was unique compared to the other two antibodies were mapped onto the gpl20_HXB2 core structure (Fig. 4, top panel). The differential map obtained for mAb b3 shows a cluster of several residues (G366A, G367A, T388A, K421A, D457A, G458A and R469A) on the neutralizing face (61, 143) and close to the V1/V2 loop stem (61, 143) of gpl20 (K121A, V430A) that, when mutated to alanine, uniquely do not affect b3 binding. This result implied that the epitope recognized by b3 may not involve these particular regions. The differential map for mAb b6 shows that, in contrast to b3 and bl2, it is not affected by certain substitutions (D279A and E462A) in the upper region of the neutralizing face of g l20 or by some alanine mutations which cluster around the Phe43 cavity (D368A, W427A, G473 A). The differential map for mAb bl2, in contrast to the maps of the other two antibodies, shows little clustering of amino acid mutations that uniquely do not affect bl2 binding. Rather, alanine substitutions to which bl2 is uniquely insensitive are located at the tip of the Vl/N2-stem (N127A), at the junction of the neutralizing and non-neutralizing faces (D474A and M475A) and in the upper half of the neutralizing face (I467A). Interestingly, most mutations that uniquely reduce bl2 binding were located in close proximity to the Phe43 cavity (Fig. 4, top panel).

[0149] In addition, those alanine substitutions that had a unique effect on b3 or b6 (non-neutralizing antibody) binding compared to bl2 (neutralizing antibody) binding were mapped (Fig. 4, bottom panel). The differential map for b3 showed that it was not affected by many alanine substitutions, particularly those which are on the perimeter of the CD4bs, as compared to bl2. The strikingly high number of colored residues in the map for b3 suggests that b3 and bl2 differ significantly in gρl20 contact residues. Indeed, many of the residues that, when substituted by alanine, uniquely caused a decrease in b3 binding as compared to bl2 are located on the non-neutralizing face and inside face of the bridging sheet which faces the non-neutralizing face. The differential map for b6 showed fewer colored residues as compared to the map obtained for b3, which suggests that there are few differences between b6 and bl2 in their interaction with gpl20. [0150] Mutants which are able to escape neutralization by mAb bl2 have been generated in vitro and in vivo (74, 104) and are characterized by amino acid mutations in the N2 loop (D167Ν, DI 85N), as well as in the C3 region (P369L, P369Q), adjacent to the Phe43 cavity. To determine the extent to which alanine substitutions in the current study resulted in neutralization escape, 19 alanine mutants with varying effects on bl2 binding affinity for monomeric gpl20 were selected to encompass the entire gpl20 envelope and used in an assay in which an ew-defective HIV-1 provirus encoding the firefly luciferase gene (pNL4.3Luc) is complemented for a single round of infection by a plasmid encoding wild- type or mutant envelope glycoproteins (Table 2, below). A neutralization index was defined to determine the degree of correlation between antibody affinity for monomeric gpl20 and neutralization efficiency.

[0151] For 12 mutants (K97A, T123A, L125A, R166A, F210A, R252A, T283A, Q337A, P369A, G459A, G471 A and D474A), there was a reasonable correlation between neutralization sensitivity of pseudovirions expressing mutant gpl20 and antibody affinity for monomeric gpl20 of the corresponding mutant. For the remaining seven alanine mutants, discrepancies were observed between the change in bl2 binding affinity for monomeric gpl20 and neutralization efficiency (Table 2, below; Fig. 5). Strikingly, for five mutants (DI 13A, V127A, D180A, N197A and S256A), neutralization efficiency was maintained or increased despite a decrease in antibody affinity for monomeric gρl20. For instance, whereas the binding affinity of bl2 for monomeric gpl20 from the DI 13A virus was reduced 100-fold compared to wild-type gpl20, DI 13 A virus was neutralized as efficiently as wild-type virus by bl2. In contrast, the neutralization indices for the remaining two mutants, N276A and S365A, were low (0.14 and 0.04, respectively), because of decreased neutralization efficiency despite an increase in binding affinity for monomeric gpl20. These results suggest that antibody affinity changes observed with amino acid substitutions on monomeric gpl20 are not always maintained in the context of functional oligomeric gpl20 as present on the surface of the virus.

[0152] To determine whether the observed discrepancies between binding to monomeric gpl20 and neutralization were only applicable to bl2, mAbs CD4-IgG2, 2G12, 15e and 17b also were tested with six of the seven mutants described above in neutralization assays. Mutant V127A was excluded because the observed neutralization index was only slightly higher than the cut-off value and thus considered only marginally discrepant. MAb 15e recognizes an epitope overlapping the CD4bs on gp 120 (111), whereas mAb 17b recognizes an epitope which overlaps the coreceptor binding site on gpl20 and is better exposed upon CD4 binding (134). Both mAbs, which neutralize HIV-1 primary isolates poorly (35, 72, 136), were not able to achieve 90% neutralization of any of the mutant viruses or wild-type virus at concentrations up to 100 ϊg/ml, although both mAbs bound monomeric gpl20 from all mutants with affinities comparable to wild-type gρl20. This result suggested that the introduced mutations did not dramatically increase the neutralization sensitivity of JR-CSF to these mAbs. Further, all six mutants remained similarly sensitive to neutralization by mAb 2G12 as the wild-type virus (Table 3, below). Therefore, it is likely that the changes in neutralization efficiency observed with mutant pseudovirions are restricted to the b 12 epitope. Interestingly, for mutant S365A, a neutralization index <0.2 for mAb 2G12 was observed; 2G12 had a higher affinity for monomeric gpl20 from this mutant, but the corresponding pseudovirions were not neutralized better than wild-type virus. In view of the location of the 2G12 epitope on gpl20 (115, 119, 137) and considering that residue S365 is located in the CD4bs of gpl20, mutating this residue to alanine appears to cause a conformational change in monomeric gρl20 that leads to better presentation of one or more glycans on the silent face of gpl20 to which 2G12 binds. However, this conformational change did not appear to take effect on the viral spike, since mutant viruses were not neutralized more efficiently than wild-type viruses.

[0153] For mAb CD4-IgG2, neutralization efficiency was decreased with respect to wild-type virus with two (S256A and N276A) of the six mutant pseudovirions that were tested (Table 3, below). This decrease correlated well with the observed decrease in CD4- IgG2 affinity for monomeric gpl20 (Table 3, below). For the remaining 4 mutants, there was a discrepancy between neutralization efficiency and antibody affinity for gpl20. For mutants DI 13A, D180A and N197A, the same discrepancy was observed as with bl2 - an increase in neutralization efficiency despite a decrease in binding affinity for monomer. Interestingly, for mutant S365A, the effects on CD4-IgG2 and bl2 binding and neutralization diverged. Monomeric gpl20 of mutant S365 A showed enhanced affinity for bl2 and reduced affinity for CD4-IgG2. However, whereas the corresponding virus was neutralized by CD4-IgG2 equally well as wild type, a decrease in neutralization efficacy was observed for bl2 with pseudovirions from mutant S365A. Thus, although mutations in gpl20 can affect the binding of bl2 and CD4-IgG2 to monomeric gρl20 differently, these differences are apparently not necessarily maintained when gρl20 is oligomerized.

[0154] Although mAb bl2 and non-neutralizing anti-CD4bs antibodies bind monomeric gpl20 with similar affinity, it was hypothesized monomeric gpl20 could be mutated so that it preferentially binds bl2, but not non-neutralizing mAbs. Based on structural analysis (Fig. 4), it was apparent that a region encompassing amino acids G473-R476, which partially line the Phe43 cavity, uniquely affected bl2 binding as compared to mAbs b3 and b6 (Fig. 4, right panel). Accordingly, a small panel of recombinant gpl20 proteins with multiple alanine substitutions at these amino acid positions was generated to determine whether the unique differences in the effects observed with the single alanine mutations could be retained. For this, a plasmid was constructed encoding the gpl20 segment of a codon-optimized env gene of the primary isolate JR-FL, which is 94% identical in amino acid sequence to JR-CSF. A tissue plasminogen activator leader sequence was placed upstream of the env gene to ensure secretion of g l20 into the culture media. Four mutants (GDMR, DMR, DR and GM) were generated and tested on a panel of anti-CD4bs mAbs (Table 4, below). The results show that, with the double (DR, GM) and triple (DMR) mutants, bl2 binding affinity was similar to wild-type gpl20, whereas with the quadruple mutant (GDMR), bl2 binding was increased. In contrast, the binding affinities of mAbs b3, b6 and F105 were severely reduced with all four mutants. CD4 binding was also severely diminished with the GDMR, DMR and GM mutants, but not with the DR double mutant (twofold reduction in affinity relative to wild type). Two other anti-CD4bs antibodies, mAbs F91 and 15e, were not as susceptible to alanine substitutions in this region as compared to the other mAbs; none of the mutations produced a decrease of more than 50% with either of the two antibodies. The binding affinity of mAb F91 was affected most by mutant DMR, whereas the binding of mAb 15e was affected most by mutants GDMR and GM. This result suggested that these mAbs might bind to gpl20 differently than other anti-CD4bs antibodies. Indeed, the binding of mAb F91 is uniquely enhanced in the presence of anti-V2 and -N3 loop antibodies (80), whereas both mAbs enhance the binding of several anti-V2 and anti-V3 loop mAbs (80). The other anti-CD4bs antibodies tested herein do not generally display similar effects. Binding of mAb bl2, for example, is decreased in the presence of a number of anti-V2 loop antibodies (80).

[0155] The CD4bs on gpl20 is a particularly attractive target for HIV-1 vaccine design because CD4 is the primary receptor on target cells for virtually all naturally occurring viruses studied to date (30, 54), thus suggesting a common structural framework for the binding site among all HIV-1 isolates. This notion is supported by the limited sequence variability among amino acids which make up the CD4 binding pocket (58, 61, 91), as well as by the high degree of similarity between the crystallized gpl20 core structures of a TCLA strain and a primary isolate (60). Of the three broadly neutralizing anti-gpl20 mAbs that have been described to date, including bl2, 2G12 and Fab X5, only the former binds to an epitope directly overlapping the CD4bs (6, 20, 22, 110).

[0156] As disclosed herein, the amino acid residues of gpl20 that affect binding were systematically defined using the broadly neutralizing mAb bl2, CD4 and two non-neutralizing anti-CD4bs antibodies. Selected residues were changed to alanine because alanine generally does not significantly alter the main-chain conformation or impose extreme electrostatic or steric effects and so permits identification of amino acid side chains, which may be important for ligand binding. To determine antibody affinity changes, antibody binding curves from ELISA data were generated, and the apparent antibody affinity for each gpl20 mutant was determined and related to that for wild-type gpl20.

[0157] The effects of alanine substitutions on CD4 binding were determined using CD4- IgG2 because it allowed the same detection system to be used as for the antibodies. No increase in affinity was observed for CD4 with any of the alanine mutants tested (see Table 1, below; Figures 2 and 3), consistent with a drive towards selection of a particular ensemble of residues during viral evolution for optimal CD4 binding. In fact, of the 28 amino acid substitutions that diminished CD4 binding to less than 20%> of wild type (Table 1, below), all but one (1467) were conserved (as defined by amino acid identity or conservation of the amino acid type) among primate immunodeficiency viruses or HIV-1 isolates, and/or are CD4 contact residues. The decrease in CD4 reactivity can reflect the loss of certain functional or structural features required to maintain the integrity of the CD4bs. Twenty-nine mutations caused a moderate decrease (20-50%)) in CD4 binding affinity. Of these 29 residues, 21 are conserved among primate immunodeficiency viruses or HIV-1, and/or are CD4 contact residues. The other 8 residues (K171, F210, R252, R273, N276, R350, W395, E462) showed moderate to significant variability among HIV-1 isolates; the affinity changes observed with CD4 when these amino acids were mutated to alanine can relate specifically to the HIV isolate, i.e. JR-CSF.

[0158] Although the results disclosed herein are largely in agreement with those of Olshevslcy et al. (91), wherein a panel of gpl20 mutants was tested for binding to CD4⁺- target cells, a direct comparison between that study and the present results is difficult due to the different assay formats (Olshevslcy et al. used densitometric quantitation of autoradiograms of immunoprecipitated gρl20 mutants after incubation of the mutants with CD4⁺-target cells). Minor discrepancies also can be due to differences in amino acid substitutions, wherein Olshevslcy et al. substituted by many different amino acids, whereas the present study utilized only alanine.

[0159] As disclosed herein, many amino acid changes affected b3, b6 and bl2 binding similarly (Figure 2 and 3), indicating a high degree of overlap in gpl20 determinants recognized by these anti-CD4bs antibodies. This result was not surprising, considering that anti-CD4bs antibodies compete with each other for binding (80). Increases in gpl20 binding affinity were observed with a number of alanine substitutions, in contrast to CD4. An increase in affinity may indicate that the amino acid in the wild type sterically hinders antibody binding. Typically, alanine substitutions result in a smaller side chain at a given position that may decrease steric interference and increase antibody affinity. However, most mutations had an adverse effect on their binding ability (Figures 2 and 3).

[0160] Deletion of the VI and/or V2 loops diminished binding by all three mAbs as well as CD4. Deletion of the V3 loop also diminished binding by CD4 and by mAbs b6 and bl2. These results indicate that the variable loops can affect binding of anti-CD4bs antibodies (146). Alanine substitutions in the V2 loop and the C-terminal strand of the Vl/N2-stem generally decreased the binding affinity of bl2 more severely than of b3 and b6. Previous studies showed that bl2 is sensitive to changes in the Vl/V2-stem loop structure (12), to deletion of V1/V2 (12) and to mutations in the V2 loop (74, 110). The results obtained here are consistent with the previous observations, although it is noted that mAb bl2 does not appear to be uniquely sensitive to N1/N2 deletion. These results are further supportive of the assumption (143) that the V2 loop in particular is in close proximity to the Phe43 cavity on the gpl20 core. The mutagenesis results are also consistent with those from previous studies, since many of the substitutions that adversely affect antibody binding (e.g. DI 13, S256, T257, Ν262, E370, D368, K421, D477) diminish binding by other anti-CD4bs mAbs (49, 71, 130, 133, 135).

[0161] Unique differences between each antibody were mapped onto the gpl20 core structure to gain better insight into how the neutralizing antibody bl2 differs from the other non-neutralizing anti-CD4bs antibodies (Figure 4). The large cluster of residues on the neutralizing face of gpl20, which uniquely do not affect b3 binding, suggests that the non-neutralizing face and inner domain (143) of gpl20, and to a lesser extent the neutralizing face, form a major contact region for the b3 antibody. This is also supported by the insensitivity of mAb b3 to removal of the V3 loop.

[0162] For mAb b6, some substitutions that uniquely affected antibody binding were located close to or facing the non-neutralizing face, suggesting that b6 may interact with an epitope extending across the non-neutralizing and neutralizing faces, but at an angle inclined toward the non-neutralizing face. Based on its unique differences with mAbs b3 and bl2, b6 appeared to contact very few residues that line the Phe43 cavity, suggesting that this region is less involved in the b6 epitope.

[0163] For bl2, no spatial clustering was observed for residues that uniquely do not affect binding (Fig. 4). Most mutations that uniquely affected bl2 binding were located in the neutralizing face. This observation and consideration of the epitope map obtained for bl2 indicate that the epitope recognized by bl2 is located primarily in this region. This results supports the computational docking model of the gρl20 core structure and mAb bl2 (90), in which bl2 binds to an epitope extending from the stem of the VI /V2 loops across the neutralizing face of gpl20, with little contact to the non-neutralizing face. Figure 4 (top panel) also shows that a number of mutations around the Phe43 cavity on gpl20 uniquely diminished bl2 binding, supporting recent results that residues comprising the antigen binding region, particularly those in the extended finger-like loop of the third complementarity determining region (CDR) of the heavy chain of Fab bl2, make crucial contacts with the residues close to the CD4 binding pocket on the gpl20 surface (90, 153).

[0164] A model was sought to explain the differences between bl2 and non-neutralizing antibodies, based on the data and the epitope maps obtained here. Figure 6 depicts how two hypothetical non-neutralizing antibodies can be excluded from interacting with trimeric gpl20, in contrast to bl2 which binds effectively. As shown in Figures 6C and 6D, non-neutralizing antibodies binding to gpl20 molecules in a functional trimer can be hindered by the close proximity of a neighboring gpl20 molecule. In contrast, mAb bl2, in this view, is not hindered by adjacent gpl20 protomers because the angle of interaction permits binding to both monomeric as well as oligomeric gpl20 (Figure 6B).

[0165] The neutralization sensitivity of pseudovirions from a select number of alanine mutants was determined, to investigate how well changes in bl2 binding affinity for monomeric gpl20 correlated with neutralizing ability. For this, a neutralization index was defined: mutants with high indices are neutralized better or equally well by mAb bl2 as compared to wild type virus, despite a decrease in bl2 binding affinity for monomeric gpl20, whereas mutants with very low indices are neutralized worse than wild type pseudovirions, despite an increase in bl2 binding affinity for monomer. Although there was a reasonable correlation for most mutants, e.g. an increase in bl2 binding affinity and an increase in neutralization efficacy, discrepancies were observed for 7 of the 19 mutants that were selected (Table 2). This result indicates that the effects of certain substitutions on antibody binding to monomeric gpl20 are nullified or reversed when presented in the context of a functional oligomeric gpl20 complex on the viral surface, because the ability of an antibody to bind functional oligomeric gpl20 is generally believed to correlate with neutralization efficacy (35, 95, 100, 116). Interestingly, mutants for which there was a discrepancy between binding to monomer and neutralization were located on opposite sides of the core (Figure 5).

[0166] Three of the five mutants (DI 13A, N197A and V127A) for which bl2 binding affinity for monomeric gpl20 was reduced, but which were still neutralized efficiently by the antibody (neutralization indices of 233, 56 and 5.65, respectively; Table 2), were located in close proximity to or on the VI /V2 stem on the inside face of the bridging sheet which faces the non-neutralizing face. Considering that the location of the V1/V2 loop may affect bl2 binding, the substitutions in the stem region can lead to a repositioning of the VI /V2 loops, the effect of which is markedly different for monomeric and functional oligomeric gpl20; i.e., the substitutions lead to increased obstruction of the CD4bs for monomeric g l20, but have little effect for oligomeric gpl20. These observations are reminiscent of results obtained recently by Kolchinsky et al. (55, 56), in which mutant pseudovirions of the primary isolate ADA with an N→-K or Q substitution at position 197 become highly sensitive to neutralization by various anti-gpl20 mAbs (56). These mutations, which eliminate an N-linked glycan at position 197, may cause movement of the N2 loop, as inferred from the ability of these viruses to infect target cells independent of CD4 (55, 56). Surprisingly, the Ν197A mutant was not sensitive to neutralization by mAbs 15e and 17b, which neutralized ADA viruses containing the N197K mutation well (56). The exceptional sensitivity of the ADA mutant pseudovirions to antibody neutralization may relate specifically to ADA, and not apply to other HIV isolates to the same extent. In this respect, preliminary results indicated that the N197A mutant of JR-CSF was not able to infect target cells that do not express CD4.

[0167] The other two mutants, DI 80A and S256A, for which a similar discrepancy was observed between binding to monomer and virus neutralization, are located in the V2 loop and line the Phe43 cavity, respectively. It is likely that changing residue D 180 to alanine also influences the position of the V2 loop. The reason for the observed discrepancy with mutant S256A is not readily apparent. Residue S256 lies recessed in the Phe43 cavity of the CD4-liganded conformation of gpl20, at the interface between the inner and outer domains (61), and, therefore, is unlikely to contact bl2 directly. Replacing this residue with alanine may affect the spatial orientation of the inner and outer domains on monomeric gpl20, whereas on functional envelope spikes this is counteracted by oligomerization.

[0168] The opposite effect between binding to monomeric gpl20 and neutralization was observed with mutants N276A and S365A than for the previous five mutants; i.e., although binding to monomeric gpl20 was increased, neutralization efficiency was lost (neutralization indices were 0.44 and 0.04, respectively). Residue N276 is an N-linked glycosylation site that is part of the D-loop on gpl20 (61), which is believed to provide important residues for bl2 binding (152, 153). Removal of the glycan may facilitate the interaction of bl2 with monomeric gpl20, whereas on the native trimer, the absence of the glycan may adversely affect the conformation of the D-loop (61) and lead to the observed decrease in neutralization efficiency. Residue S365 is part of a ridge formed by residues 364-368 that make direct contact with CD4 (61) and, based on the bl2-gpl20 docking model, may fit into a cleft formed by the heavy chain CDR2 and CDR3 of bl2 (90). The S365A mutation, like the Ν276A mutant, may cause a conformational change on monomeric gpl20 that favors bl2 binding to monomer, but that has a negative effect on bl2 binding to functional oligomeric gpl20.

[0169] A feature of bl2 neutralization escape mutants selected in vivo is a mutation at position 369 (Pro) to Gin or Leu (74, 104). When mutated to alanine (P369A), no change in binding affinity for monomeric gpl20 and no significant change in neutralization sensitivity was observed (Table 2, below), indicating that this residue does not normally form part of the bl2 epitope. Rather, it is more likely that mutating this amino acid to a larger residue caused the steric impairment of the interaction between bl2 and gpl20, either directly or by altering peptide backbone conformation.

[0170] Based on the mutagenesis results, four recombinant gpl20 molecules containing multiple alanine mutations between amino acid residues G473-R476 were generated and tested on a panel of five non-neutralizing anti-CD4bs mAbs, bl2, and CD4 (Table 4, below). The quadruple mutant GDMR increased bl2 binding, but completely abolish binding by three (b3, b6, F105) of the five non-neutralizing anti-CD4bs mAbs tested. Of the two remaining mAbs, the binding affinity of one mAb, 15e, was reduced for two of the four mutants, whereas with the other mAb, F91, a slight decrease in binding affinity was observed. These results demonstrate that gpl20 can be engineered so as to make it less prone to recognition by non-neutralizing antibodies.

EXAMPLE 2 HYPERGLYCOSYLATED MUTANT gpl20 POLYPEPTIDES

[0171] This example demonstrates that mutation of gpl20 polypeptide sequences to generate glycosylation sites, and glycosylation of such mutant polypeptides, reduces or inhibits non-neutralizing antibody binding.

[0172] The ability to induce broadly neutralizing antibodies should be a key component of any forthcoming vaccine against human immunodeficiency virus type 1. One potential vaccine candidate, monomeric gpl20, has generally failed to elicit such antibodies. As disclosed in Example 1, above, a better HIN immunogen was prepared by engineering gpl20 to preferentially bind known broadly neutralizing antibodies. For example, four alanine substitutions on the perimeter of the Phe43 cavity of gpl20 reduced binding of weakly neutralizing CD4-binding site antibodies, while slightly enhancing binding of the potent, broadly neutralizing antibody bl2.

[0173] The present example extends the results described in Example 1 by introducing N-glycosylation motifs at select positions into the hypervariable loops and the gpl20 core, and examining the mutant glycosylated gpl20 polypeptides for the ability to further reduce or abrogate the binding of a wider range of non-neutralizing antibodies. As disclosed herein, a hyperglycosylated mutant gpl20 polypeptide containing seven extra glycosylation sequons and four alanine substitutions as described in Example 1 did not bind an extensive panel of non-neutralizing and weakly neutralizing antibodies, including a polyclonal immunoglobulin preparation (HINIG) of low neutralizing potency. Binding of bl2, at lowered affinity, and of four antibodies to the Cl and C5 regions was maintained. Removal of Ν-terminal and C-terminal residues in the Cl and C5 regions, respectively, reduced or abolished binding of the four antibodies, but also adversely affected bl2 binding. Thus, the hyperglycosylated mutant gpl20 polypeptides and analogues thereof provide previously undescribed antigens that can provide a new approach to eliciting antibodies with bl2-like neutralizing properties.

MATERIALS AND METHODS Plasmids and mutagenesis

[0174] The generation of plasmid pCMV-Tag4A-tpa _R._FLgpi2_0wthas been described (94). This plasmid, which is derived from plasmid pCMV-Tag4A (Stratagene), contains a tissue plasminogen activator leader sequence immediately upstream of the env gene to ensure secretion of gpl20 envelope glycoprotein into the culture supernatant. The env gene of HIV-1 JR-FL was obtained by PCR amplification using as template a plasmid (pSyngp 140_JR._PL) encoding the codon-optimized gpl40 gene (2, 45) of this HIV isolate. Site-directed mutagenesis to substitute wild-type residues for alanine and to incorporate N-glycosylation sequence motifs was performed using the QUIKCHANGE mutagenesis kit (Stratagene).

[0175] The generation of gpl20 containing deletions of residues in the N and C termini (52 and 19 residues, respectively) was performed by PCR. The primers flCORE-5 (5'-GGAGGTCAACAGCACCGCGCGCGAGGTGGTGCTGGAGAATGTGAC-3'; SEQ ID NO: 13), which contains a BssU II restriction site (bold), and flCORE-3 (5'-GGAGGTCAACAGCACCCTCGAGTTAATTAATTAACTCAATCTTCACCACCTT GTA-3'; SEQ ID NO: 14), which contains an Xho I site (bold) were used. Plasmids encoding wild-type or mutant gρl20 were used as templates. The PCR products were cloned into pCMN-Tag4A-tpa using restriction enzymes BssH II and Xlio I according to standard protocols. DΝA sequencing prior to use verified that the plasmids and mutations generated in this study were correct.

Antibodies

[0176] A total of 22 mAbs and one polyclonal immunoglobulin preparation were used. MAbs b3, b6, bl2, X5 and loop 2 were as described (6, 7, 20, 22, 83, 110). The remaining antibodies were obtained from their respective source or from the IH AIDS Research and Reference Reagent Program (ΝIH ARRRP). Primary citations for these mAbs are as follows: Cll, 212A, 19b, 17b, 48d, F91, 15e, A32 (49, 76-78, 81, 82, 111, 112, 123, 133, 134, 144); 2G12 (16, 137); G3-4, G3-136 (36, 48, 132); 447-52D (17, 41, 42); F105 (105, 106, 135); M90 (33); 133/290 and 133/237 (71, 82, 87); 522-149 (80). The epitopes recognized by the antibodies are summarized in the HIV sequence database (see hypertext transfer protocol (http), at URL "resdb.lanl.gov/ABDB/antibody_id.htm", which is incorporated herein by reference). Human IgG purified from pooled plasma obtained from healthy asymptomatic seropositive individuals (HIVIG) was obtained from the ΝIH AlrU RP. This manufactured product (ΝABI, Boca Raton, Florida) is a 50 mg/ml solution that contains 98% monomeric IgG (28). An affinity-purified polyclonal antibody preparation against the C5 region of gpl20, APTKAKRRVVQREKR (SEQ ID NO: 15), was purchased from Cliniqa (Fallbrook, CA).

Transient expression of recombinant HIV glycoproteins

[0177] Monomeric wild-type gpl20 and mutant glycoproteins were obtained by transiently transfecting 293T cells (94). Culture supernatants containing recombinant glycoproteins were stored at -20°C.

MAb biotinylation

[0178] MAb 2G12 was conjugated to biotin using EZ-Linlc biotinylation reagents (Pierce) according to the manufacturer's instructions. Biotin-labeled mAb was tested in ELISA to assure that the conjugation did not adversely affect binding to gpl20.

Enzyme-linked immunosorbent assays (ELISA)

[0179] Assays were performed essentially as described (94). Glycoproteins were captured onto ELISA plate wells using the anti-C5 polyclonal antibody preparation, unless otherwise indicated. Antibodies against CD4-induced epitopes were tested in the absence of soluble CD4. MAb 2G12, or in some cases HIVIG, was used to ensure that similar amounts of envelope proteins were captured in each experiment. In general, plates were developed with p-nitrophenyl phosphate (Sigma) and absorbance measured at 405 nm. When peroxidase-conjugated secondary antibody (Pierce) was used, plates were developed with 3, 3', 5, 5' tetramethyl benzidine/hydrogen peroxide substrate (TMB, Pierce) and absorbance measured at 450 nm. In this case, the color reaction was stopped with sulfuric acid (2 M) prior to measurement. For detection of biotinylated 2G12, peroxidase-conjugated streptavidin (Jackson ImmunoResearch) was used in combination with the TMB system. ELISA assays were performed in duplicate and repeated on at least two different occasions.

SDS-PAGE and Western blot analysis

[0180] SDS-PAGE and western blot analyses were performed essentially as described (93, 140). For electrophoresis, supernatants from 293T cells transiently expressing wild- type or mutant glycoproteins (typically one 6-well plate for each glycoprotein) were pooled and concentrated ~10 times using YM-50 concentrators (Amicon). The supernatants were diluted 1:2 with concentrated sample buffer (125 mM Tris/HCl, 4% SDS, 20% glycerol, 0.02% bromphenol blue; pH 6.8) and vortexed briefly. Of this mixture, 10 ϊl were loaded onto a 5% separating gel. After electrophoresis, the proteins were transferred to a polyvinylidene difluoride membrane by tank blotting (BioRad). Blots were incubated overnight with primary antibody (1 or 5 ϊg/ml, as indicated), then with alkaline phosphatase- conjugated secondary antibody (Pierce) and developed using 5-bromo-4-chloro-3-indolyl- phosphate and nitro blue tetrazolium (BCIP, NBT) as substrates (Sigma).

RESULTS

Effect of multiple alanine substitutions on the perimeter of the Phe43 cavity of gpl20 on mAb binding

[0181] As disclosed in Example 1, the effects of multiple alanine substitutions on the perimeter of the Phe43 cavity (positions 473 -476) of gp 120_JR._FL on the binding of a panel of seven CD4bs antibodies was examined. For one mutant, GDMR, in which four gpl20 amino acid residues were substituted by alanine, binding of mAbs b3, b6, CD4-IgG2 and F105 was essentially abolished, whereas the apparent binding affinity of mAbs F91 and 15e was reduced relative to wild type. In contrast, the binding affinity of mAb bl2 was unaffected or slightly enhanced.

[0182] In the present example, a larger panel of antibodies was tested with the GDMR mutant. A total of 14 anti-gpl20 antibodies were selected, including the seven antibodies described above. One of the antibodies, mAb A32, binds to a discontinuous epitope involving the C1-C4 domains on gpl20 and competes with CD4bs antibodies for binding (80, 144). The affinity of this antibody is increased in the presence of soluble CD4, thus qualifying it nominally as a "CD4-induced" antibody. However, mAb A32 does not compete with other CD4-induced mAbs, e.g. 17b and 48d, for binding to gpl20 (144). The other antibodies bind to continuous and discontinuous epitopes in the Cl and C5 domains of gpl20 (mAb Cl 1), the V2 loop (mAbs G3-4 and G3-136), and the V3 loop (mAbs loop 2, 19b and 447-52D). To determine apparent binding constants, cell culture supernatants containing recombinant glycoproteins were captured onto ELISA plate wells using a polyclonal antibody preparation to the C5 region of gpl20. The specificity of this polyclonal antibody is similar to that of mAb D7324, and it does not compete for binding with any of the antibodies tested here (80). Captured glycoproteins were then probed with varying concentrations of antibody to generate binding curves. Apparent affinities were determined from the antibody concentration at half-maximal binding, and related to that determined for wild-type gpl20.

[0183] Most antibodies bound with high affinity to wild-type gpl20j_R-F_L (Figures 7A and 7B). However, mAbs G3-4 and G3-136, which recognize similar conformational epitopes in the V2 loop, and mAb Cl 1 bound with only moderate affinity to monomeric gpl20j_R-p_L. The moderate binding affinity of mAbs G3-4 and G3-136 to gpl20j_R-FL, in contrast to binding to gpl20m_B, was likely due to sequence differences between the two gpl20s. T he sequence -STSIRGKVKEYAFFYKLDI- (SEQ ID NO: 16), which is believed to be involved in antibody binding to gpl20ui_B (101, 150), is replaced by the sequence -TTSIRDEVQKEYALFYKLDV- (SEQ ID NO:17) in gpl20j_R-_FL (residues in JR-FL that differ from those in IIIB are in bold). The indicated residues can cause slight changes in the epitope recognized by these two antibodies, thus diminishing antibody binding. The same may hold true for mAb Cl 1, although the epitope of mAb Cl 1 has not been clearly defined.

[0184] As expected, mAbs b3, b6 and F105 did not bind to gpl20 of mutant GDMR, whereas the binding of mAbs F91 and 15e was reduced relative to wild type (55% and 45%, respectively; Figure 7C). The binding of mAb A32 was slightly reduced (70%> relative to wild-type gpl20; Figure 7D). In contrast, binding of the other antibodies, including bl2, was largely unaffected by the alanine substitutions (Figure 7C and 7D). Based on these results, it was apparent that, although introduction of the four alanine mutations was sufficient to abrogate or reduce binding to gpl20 of non-neutralizing or weakly neutralizing mAbs against epitopes close to or overlapping the CD4bs, additional mutations can further eliminate the reactivity of undesired antibodies against other gpl20 epitopes.

Effect of the introduction of an N-linked glycan in the V3 loop on reactivity with V3 loop antibodies

[0185] First, it was determined whether introduction of an N-glycan in the N3 loop could inhibit reactivity by N3 loop mAbs. For this purpose, a mutant, termed P313Ν, was generated. In the P313N mutant , which also included an R315T substitution (P313N/R315T), Arg and Thr were substituted for Pro and Arg at positions 313 and 315, respectively, in the "tip" of the V3 loop (-Gly-Pro-Gly-Arg-Ala-Phe-; SEQ ID NO: 17) segment. This N-glycosylation sequon (ΝXT) permits the addition of a potential N-linked glycan at position 313. SDS-PAGE and western blotting were used to investigate the presence of the extra glycan (Figure 8A); a slight increase in average molecular weight was observed for the mutant glycoprotein in comparison to wild-type gpl20, suggesting the presence of an additional glycan in the mutant.

[0186] The effect of the mutation on antibody reactivity was determined for tliree N3 loop mAbs, namely loop 2, 19b and 447-52D. These three antibodies pooled together were unable to react with glycoprotein of mutant P313Ν as determined by western blot (Figure 8B). ELISA, using individual mAbs, confirmed these results (Figure 8C and 8D). In contrast, the apparent affinity of mAb bl2, relative to wild type, was unaffected by the introduced mutation. The glycoprotein of mutant P313N was stained much more strongly by bl2 in comparison to wild-type gpl20 (Figure 8 A), perhaps due to a difference in the amount of glycoprotein present in the respective preparations, which were not standardized prior to SDS-PAGE. To determine whether the inability of the three N3 loop antibodies to bind mutant P313Ν was due solely to incorporation of an N-linked glycan, the Asn at position 313 was replaced by Gin (the Arg-to-Thr substitution at position 315 was left unchanged); the three antibodies were also unable to react with this mutant, indicating that, even though the N3 loop mAbs may be blocked from binding to their respective epitopes by the introduced glycan, reactivity also is abrogated due to substitution of the proline residue alone or in combination with the arginine residue. The mutations most likely disrupt the e-turn at the apex of the V3 loop comprising the -Gly-Pro-Gly-Arg- segment (40, 129).

Effect of introducing N-linked glycans in gpl20 on reactivity with anti-gpl20 mAbs

[0187] Next, other epitopes that could be recognized by non-neutralizing antibodies were blocked using the same glycosylation strategy. First, N-glycosylation sequons were introduced individually at various positions in gρl20, primarily on the non-neutralizing face (143) and in the Nl and V2 loops, to determine the effect on bl2 binding. The ΝXT glycosylation sequon (X is any amino acid except proline) was used because the asparagine in this motif is 2-fold more likely to be glycosylated than the asparagine in the NXS motif (124). In some cases two substitutions, at the 'N and T positions, were required to introduce the glycosylation motif, whereas in other instances only one of the two positions needed to be substituted by the appropriate residue. The mutations incorporated were as follows: Ql 14N/L116T and Q246N (masking of the non-neutralizing face and the putative gp41-gpl20 interface), S143T and E150N/G152T (masking of the VI loop), K171N/Y173T (masking of the V2 loop), S365T (masking of potential epitopes on the outer perimeter of the CD4bs) and R440N/Q442T (masking of potential epitopes close to the base of the V3 loop, adjacent to the silent face). The S143T and S365T mutations were introduced to enhance the likelihood of glycan attachment at residues N141 and 363, respectively.

[0188] Binding of mAb bl2 was not significantly affected by mutations Ql 14N/L116T and S143T, whereas binding was somewhat decreased by the E150N/G152T mutation (Figure 9). Binding was more strongly affected by the mutation in the V2 loop (K171N/Y173T) and by the Q246N mutation. With the two remaining mutants, S365T and R440N, no reactivity with bl2 was observed. Expression of the mutant with the R440N/Q442T substitution was very poor, suggesting that introduction of a glycan at position 440 interferes with the proper folding or intracellular processing of gpl20.

[0189] Glycosylation motifs that did not affect bl2 binding, or affected it only slightly, i.e., mutations Q114N/L116T, S143T and E150N/G152T, were then combined. The P313N and R315T substitutions (mutant P313N), respectively, and the alanine substitutions at positions 473-476 (mutant GDMR; see Example 1) were also incorporated to generate a hyperglycosylated mutant (Figure 10). In addition, the K171N/Y173T mutation was inserted, despite its negative effect on bl2 binding, because this was the only mutation in the V2 loop that could potentially hinder the binding of V2 loop antibodies. Additional N-glycosylation sites were also introduced at positions 92 and 423, without prior testing with bl2. Residue H92 lies on the non-neutralizing face of gpl20 and residue 1423 lies in the coreceptor-binding site. [0190] Western blots were performed to confirm that the glycoprotein of this mutant, termed mCHO*-GDMR, was hyperglycosylated. The molecular weight of the modified glycoprotein was ~137 kDa (Figure 11), suggesting that most, if not all, extra glycosylation sites are occupied in this mutant. The largest oligosaccharide that can be incorporated at each site is a fully sialylated, fucosylated and galactosylated tetraantennary complex glycan (~3.5 lcDa), which would increase the average molecular weight of wild-type gpl20 by -25 kDa. However, this complex glycan type is observed in less than 1% of the total glycans on gpl20jR-FL (119). Rather, recombinant gpl20 R-FL expressed in mammalian cells appears to contain predominantly galactosylated, fucosylated biantennary complex glycans (sialylated and non-sialylated) and high oligomannose glycans (Man-8) (119). Such glycans would add an average of ~2 kDa per glycosylation site, thus increasing the molecular weight of wild-type gρl20 to ~134 lcDa, which approximates the average mass determined from the SDS gel for the mutant mCHO*-GDMR. However, the possibility that some sites are not glycosylated cannot be excluded from this determination; mass spectrometric analyses of the mutagenized protein (151) can address this issue in detail.

[0191] To investigate the antigenic properties of this glycoprotein mutant, binding affinities were determined for bl2 and for a large panel of non-neutralizing anti-gpl20 mAbs. Most mAbs bound wild-type gpl20j_R-_FL with high affinity (Figure 12A to 12D). Saturation levels were not obtained with certain mAbs against the Cl domain (133/237, 133/290 and 522-149) and, as already observed, by mAbs Cl l, G3-4 and G3-136. MAbs 133/237 and 133/290 bind better to denatured gpl20 than to native glycoprotein (79) and here display moderate affinity for gpl20 in ELISA. Whether this is also the case for mAb 522-149 is not Icnown. It is also possible the moderate binding affinity stems from sequence differences between the homologous antigen and gpl20jR-_FL- Antibodies 17b and 48d also bound with lower affinity to wild-type gpl20, due to the absence of sCD4 in these experiments. When tested with mutant mCHO*-GDMR, binding of virtually all mAbs was abolished, whereas binding of mAb bl2 was reduced in comparison to wild-type gpl20. Unfortunately, but not unexpectedly, the binding of only two of the five Cl antibodies was inhibited. In this study, no glycan attachment motifs were introduced in the Cl region because glycosylation of the N-terminus is reported to be rare (39). Binding of the three other Cl antibodies to mutant mCHO*-GDMR was nearly twofold higher than to wild-type gpl20 (Figure 12E). Removal of N-terminal residues in the Cl region reduced or abolished the ability of these antibodies to bind gpl20, and deletion of residues in the C-terminus of the C5 region reduced the binding of the anti-C5 polyclonal antibody preparation used to capture gpl20 (Figure 13). Surprisingly, bl2 binding was also severely diminished (Figure 13), indicating that the combined removal of these N-terminal and C-terminal residues dramatically influences the conformation of the bl2 epitope in mutant mCHO*-GDMR.

Epitope masking by introduction of N-glycans abolishes reactivity with a polyclonal antibody preparation

[0192] To test the degree to which this approach can mask non-neutralizing epitopes on gpl20, the effects of the introduced glycans on binding of a preparation of polyclonal antibodies was examined. This preparation, HIVIG, which is derived from pooled plasma of asymptomatic individuals that was selected based on high antibody titers to the HIV structural protein p24, neutralizes primary HIV-1 isolates poorly (28). When tested with mutant GDMR, a 2-fold reduction in relative binding affinity was observed, in comparison to wild-type gpl20j_R-F_L (Figure 14). However, binding was much more reduced to the mutant with an extra glycosylation site in the V3 loop, thus suggesting the presence of a large percentage of V3 loop antibodies in this preparation. No binding of HIVIG to mutant mCHO*-GDMR was observed, indicating that a number of epitopes on gpl20 that have the potential to induce non-neutralizing antibodies successfully were masked.

Influence of the N-glycosylation motif in the V2 loop on bl2 binding [0193] To examine whether the lowered affinity of mAb bl2 for mutant mCHO*-GDMR was caused by the K171Ν/Y173T mutation in the V2 loop, the mutation was reverted to wild-type sequence. Antibody bl2 binding affinity for this mutant, termed K171Nx, was similar to that for mutant mCHO*-GDMR. Therefore, the lower affinity of b 12 for mutant mCHO*-GDMR in comparison to wild-type glycoprotein likely stems from the introduction of a particular combination of glycans in mutant mCHO*-GDMR, rather than from steric hindrance by a specific glycan. Interestingly, the V2 loop antibody G3-4 did not react with mutant K171Nx, indicating that the extra V2 loop-glycan may not be required for masking antigenic determinants in this loop. Rather, the result with mAb G3-4 suggests that neighboring glycans that were inserted are sufficient to block the binding of V2 loop antibodies.

Influence of alanine substitutions at positions 473-476 in mutant mCHO*-GDMR on binding of mAbs to epitopes overlapping the CD4bs

[0194] Although epitopes of various non-neutralizing and weakly neutralizing antibodies were successfully masked, it was unclear whether blockage of CD4bs antibodies and mAb A32 could be accomplished by the glycans alone, i.e., without the need for the introduced alanine substitutions at positions 473-476. Therefore, another variant of mutant mCHO*-GDMR was generated. In this variant, termed mCHO*, the alanine substitutions at positions 473-476 were reverted to wild type. As shown in Figure 15, binding of mAbs 15e, F91, F105 and A32 was significantly reduced or completely abolished, indicating that the added glycans, alone, were sufficient to block binding of many non-neutralizing or weakly neutralizing CD4bs antibodies; the weakly neutralizing antibodies b3 and b6 were able to bind the mCHO* mutant glycoprotein, albeit with lower affinity than mAb bl2. Thus, the extra glycans, while not completely blocking access to the CD4bs, appear to have confined the space available to antibodies for interaction with this site.

[0195] A major concern for HIV-1 vaccine design is that at present no immunogen, or combination of immunogens, is capable of eliciting the levels of broadly neutralizing antibodies that are likely needed to contribute to significant protection against infection. Nevertheless, the anti-gpl20 and anti-gp41 mAbs that have been isolated from natural infection and from immunization studies serve as valuable tools to screen prospective antigenic formulations for their suitability as candidate immunogens (19, 145). However, antigens that preferentially bind neutralizing antibodies, but not non-neutralizing ones, would be most desirable. As disclosed in Example 1, binding of the broadly and potently neutralizing human antibody bl2 was unaffected or slightly enhanced by the introduction of four alanine mutations on the perimeter of the Phe43 cavity on gpl20 (mutant GDMR), whereas binding of CD4 and five weakly neutralizing CD4bs antibodies was abolished or reduced (see, also, (94)).

[0196] As disclosed herein, the GDMR gpl20 mutant was further examined. As a first step, mutant GDMR was tested in ELISA with a selection of mAbs against various linear and discontinuous epitopes on gpl20. Only non-neutralizing or weakly neutralizing CD4bs mAbs were significantly affected by the alanine substitutions (Figure 7). It is unclear whether this result correlates with how these antibodies interact with gpl20 and their lack of neutralizing potency. However, considering that antibodies often form salt-bridges or hydrogen bonds with available polar groups on the antigen, most probably because this compensates for the loss in entropy upon antigen interaction (10, 14, 15, 126, 138, 139), it is noteworthy that mAbs b3, b6 and F 105 are particularly sensitive to substitution of residues at positions 474 (Asp) and 476 (Arg) by alanine (94). The results obtained using the GDMR mutant suggest that these aspartate and arginine residues are ideal contact residues for many non-neutralizing antibodies. These residues may be more accessible on monomeric gpl20 than on the trimeric envelope spikes of primary HIV isolates, thus explaining why these antibodies are unable to neutralize virions potently.

[0197] The observation that non-CD4bs antibodies were not inhibited from binding to mutant GDMR prompted the pursuit of further means for blocking epitopes recognized by non-neutralizing antibodies. The concept of diverting B-cell immune responses away from undesired epitopes has been discussed (32). A recent study (24) showed that, by introducing a single amino acid mutation in the e-chain of human chorionic gonadotrophin, cross-reactive antibodies to lutenizing hormone, which are normally elicited upon immunization with wild-type human chorionic gonadotrophin e-chain, could be eliminated. As such, the immune response was refocused to epitopes that are normally only weakly immunogenic.

[0198] In the case of HIV, such a strategy is likely to be tedious and time-consuming, particularly considering the vast genetic diversity that is manifested among HIV isolates (8). Therefore, a decision was made to insert N-glycosylation sequons into the gpl20 sequence with the aim of selectively incorporating additional N-glycans onto the glycoprotein to mask undesired epitopes. Masking epitopes in the (hyper)variable loops of HIN was of particular importance, since antibodies to such sites are often induced upon immunization with gpl20 or during natural infection (25, 34, 43, 64, 67, 70, 75, 122, 127, 141). During infection, these antibodies are detected relatively early, suggesting the presence of immunodominant epitopes within the loops (34, 70, 86, 122). The masking of epitopes by glycans is not, in itself, novel and, in fact, is a strategy 'employed by HIN to avoid facile recognition by the host immune system (5, 31, 92, 107, 108, 120, 143). For example, a large portion of gpl20 is covered by N-glycans, thus rendering those regions of the antigen immunologically 'silent' (143). Furthermore, changes in the number and placement of N-linked glycans in g l20 can modulate the exposure of antigenic determinants. For example, elimination of an N-glycan in the N3 loop by site-directed mutagenesis increases viral sensitivity to neutralizing antibodies (5). Also, when virus lacking an N-linked glycan in the N3 loop is grown in the presence of a N3 loop antibody, it rapidly reverts to a variant in which a glycan is reincorporated at that position (120). However, in terms of vaccine design, the approach of epitope masking by the incorporation of additional N-linked glycans is not widespread and, in fact, has been applied in only one previous study, wherein incorporation of an N-glycosylation site in the N3 loop resulted in a shift in the immune response towards epitopes in the Nl loop (37).

[0199] In the present study, N-linked glycans were introduced based primarily, but not solely, on the following criteria: (i) the side chain of the residue to be mutated should be solvent-exposed, (ii) the introduced glycan should block a region on gpl20 that might elicit non-neutralizing antibodies, (iii) the site selected for introduction of the N-glycan should contain residues that might tolerate mutation, yet still retain a similar conformation, and (iv) the potential glycosylation site should not be hindered by neighboring sequence elements. For incorporation of the N-glycans, an ΝXT glycosylation sequence was selected rather than an ΝXS motif because the asparagine in the ΝXT sequon is more likely to be glycosylated (39, 52, 73, 124). Furthermore, in an ΝXT glycosylation motif, more residues are tolerated at position X in terms of glycosylation efficiency compared to an NXS sequence (52).

[0200] First, a determination was made whether introduction of an N-glycan in the V3 loop could abolish binding of V3 loop antibodies. Considering that a glycan at the apex of V3 may potentially mask a substantial portion of the loop on both sides, a glycosylation site was incorporated at position 313 (Pro). Indeed, none of the three V3 loop mAbs tested were able to bind to this mutant. However, antibody binding to a second mutant, in which the Asn in the glycosylation sequon was replaced by Gin, was also abrogated. These results indicate that incorporation of the extra glycosylation sequon serves two functions. First, incorporation of the glycosylation motif allows potential antibody epitopes to be masked due to the presence of the glycan. Whether the glycan indeed masks the entire loop is uncertain, since there are currently no antibodies available that are reactive with residues down- or upstream from the introduced glycosylation site in the V3 loop of JR-FL. The second effect of replacing the proline and arginine residues is that this most likely eliminates or modifies the e-turn and e-type hairpin, which are characteristic of the apex of the V3 loop and confer immunodominance to the loop (40, 109). Lowering this predominant characteristic of the V3 loop may further increase the potential to obtain antibodies against epitopes on gpl20 that are normally only weakly immunogenic.

[0201] Next, the glycosylation strategy was extended to other epitopes that could elicit non-neutralizing antibodies. Seven sites were selected for the incorporation of N-glycosylation motifs (Figure 9). The mutations were generated in mdividual mutants first and tested with bl2, to ensure that none severely compromised gpl20 folding as detected by antibody binding. Of the seven mutations, three were eliminated from subsequent studies because they abolished bl2 binding. The added glycosylation site in the N2 loop also reduced bl2 binding, but was still selected for further studies since no other glycosylation motifs had been incorporated in the V2 loop for epitope masking. This mutation and the others that did not significantly affect bl2 binding were combined together with the glycosylation sequon in the V3 loop and the alanine substitutions in the Phe43 cavity to generate a hyperglycosylated mutant glycoprotein. Two additional glycosylation motifs were introduced at positions 92 and 423, to further mask the non-neutralizing face on gpl20 and the coreceptor-binding site, respectively. This hyperglycosylated gpl20 mutant, mCHO*-GDMR, blocked the binding of virtually all non-neutralizing and weakly neutralizing antibodies examined, including a polyclonal antibody preparation (HIVIG) of low neutralizing potency. Importantly, bl2 binding was maintained, albeit at lowered affinity. However, this reduced binding was not caused by the N-glycan that was incorporated in the V2 loop, since reversion to the wild-type sequence did not alter bl2 affinity. Binding to mutant mCHO*-GDMR was also still observed with tliree mAbs against the Cl region and a polyclonal antibody against the C5 region. Although the binding of these antibodies could be reduced or abolished by removal of residues in Cl and C5, respectively, bl2 binding was also severely reduced (Figure 13). Thus, in the mutant mCHO*-GDMR, bl2 binding can not be maintained at the expense of the remaining Cl -reactive and C5-reactive antibodies by truncating the Ν and C termini because of the apparent negative effect on the conformation of the bl2 epitope.

[0202] In summary, a series of mutant gpl20 polypeptides have been generated that diminish or abolish the binding of non-neutralizing or weakly neutralizing antibodies to varying degrees, but retain, at least to some extent, bl2 binding. The mutant gpl20 polypeptides have the potential to induce broadly neutralizing antibodies, particularly those targeted to the CD4bs. Although it may be considered that, because of the extra glycans, the mutant gpl20 is conformationally different from the gpl20 conformation on the viral surface, the ability of bl2 to bind to mutant mCHO*-GDMR indicates that it is unlikely that the extra oligosaccharide moieties significantly alter the overall gpl20 core structure, including the CD4bs. Since the majority of broadly neutralizing antibodies appear to bind equally well to monomeric and oligomeric gpl20 (35, 99, 116), antibodies that recognize the CD4bs on gpl20 in a bl2-like manner should also be reactive with oligomeric gpl20. In fact, the introduction of the extra N-glycans may have created spatial constraints around the CD4 binding domain that antibodies are likely to encounter with native, virion- associated oligomeric gpl20. [0203] Besides epitope masking, the extra glycans also can serve an additional function. Recent studies suggest that monomeric gpl20 contains unusually high intrinsic entropy, which might undermine efforts to obtain broadly neutralizing antibodies (84). Considering that asparagine-linked glycosylation influences protein structure and folding (51, 52, 89, 142) and that the addition of carbohydrate moieties may minimize the conformational flexibility of proteins (89, 142), the extra glycans incorporated into the gρl20 mutants can reduce the overall flexibility of monomeric gpl20 and, therefore, further promote the induction of antibodies with neutralizing properties superior to those obtained with unmodified gpl20.

[0204] Accordingly, a panel of mutant gpl20 polypeptides is provided that, based on their antigenic properties, can be useful as immunogens for stimulating primarily a neutralizing antibody response.

TABLE 1. Alanine and variab e loop-del< -ted mutants generated in this study and the ir binding to mAbs and CD4.

Domain⁰ Mutant" Apparent affinity relative to wild-type gpl20_JR._CSF (%)^c for. b3 b6 bl2 CD4^d 2G12

gρl20 domain. C, constant domain; V, variable loop.

^AAmino acid numbering is relative to HIV-l_HxB2, where 1 is the initial methionine (57). Δ denotes amino acid deletions: ΔV1 (amino acids 134-154); ΔV1/V2 (amino acids 134-154 and 160-193); ΔV3 (amino acids 303-

324). White circles (O) indicate that an amino acid is identical among 51-98% of all HIV-1 isolates. Black circles (•) indicate that an amino acid is identical among 99-100%) of all HIV-1 isolates (amino acid identity determined from sequence alignment of HIV-1 isolates listed in the HIV sequence database at http://hiv- web.lanl.gov/content/hiv-db/mainpage.html).

'Apparent affinities were calculated as the antibody concentration at half-maximal binding. Apparent affinities relative to those for wild-type gpl20 were calculated with the formula: (apparent affinity for the wild type/apparent affinity for tlie mutant) x 100. The color scheme is the same as in Fig. 3; substitutions which resulted in apparent affinity <50% relative to wild type are colored blue, those which resulted apparent affinity between 50-200%) relative to wild type are colored grey, and those that resulted in apparent affinity >200%> relative to wild type are colored yellow. ^dCD4-IgG2 was used as a surrogate for CD4 in this study. ^eND, not determined. Amino acid residues conserved among all HIV-1 isolates. Amino acid conservation is defined as in reference

(61): single amino acid changes are allowed, as are larger substitutions, as long as the character of the side chain is maintained (e.g., Lys to Arg or Phe to Leu). ^fiAmino acid residues conserved among all primate immunodeficiency viruses. ^ACD4 contact residues [as determined from the crystal structure of the gpl20-CD4-17b complex by Kwong et al. (61)].

Data derived from reference (119)

Table 2. Neutralization of wildtype and mutant pseudovirions of HIV-1_JR._CSF by mAb bl2.

Pseudovirus IC, Apparent affinity Neutralization index relative to wildtype gpl20. JR-CSF \ wildtype 14 100 1

K97A 12 134 0.87

D113A 6 1 233

T123A 4 233 1.50

L125A 5 383 0.73

V127A 4 62 5.65

R166A 8 52 3.37

D180A 10 1 140

N197A 1 25 56

F210A 4 77 4.55

R252A 43 17 1.92

S256A 16 13 6.73

N276A 46 225 0.14

T283A 13 55 1.96

Q337A 9 83 1.87

S365A 64 534 0.04

P369A 8 129 1.36

G459A 9 54 2.88

G471A 11 37 3.44

D474A 11 91 1.40

TC₉₀, antibody concentration yielding 90%> neutralization efficiency.

''See the text for details.

TABLE 3. Neutralization of wildtype and select mutant pseudovirions of JR-CSF by mAbs CD4-IgG2 and

2G12. Pseudovirus ICgo (μg/ml) for mAb": Apparent affinity relative Neutralization index for mAb: to wildtype gpl20_JR._CsF

(%) for mAb:

CD4 -IgG2 2G12 CD4-IgG2 2G12 CD4-IgG2 2G12 wildtype 13 6 100 100 1 1

D113A 5 3 9 169 28.9 1.18

D180A 1 14 31 108 41.9 0.40

N197A 1 6 3 318 433 0.31

S256A 20 6 47 108 1.38 0.93

S365 A 6 9 23 1800 9.42 0.04

N276A 30 3 27 169 1.60 1.18

^σICgo, antibody concentration yielding 90%) neutralization efficiency.

TABLE 4. Binding affinities of a panel of anti-CD4bs mAbs to recombinant gpl20 _R._FL containing multiple alanine substitutions.

Mutant" Apparent antibody affinity (%>) relative to wildtype gpl20_JR-F for: bl2 b6 b3 CD4 F91 15e F105

GDMR 250 0.1 0.1 0.1 82 63 0.1

DMR 167 0.1 0.1 0.1 64 112 0.1

DR 167 0.1 0.1 43 90 112 0.1

GM 120 9 0.1 0.1 75 68 0.1

"Mutants are denoted by the amino acids that were changed to alanine. G, G473; D, D474; M, M475; R, R476.

[0205] Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the claims, which follow the List of References.

REFERENCES

Each of references 1 to 154 as follows is incorporated herein by reference. 1. Allaway, G. P., K. L. Davis-Bruno, G. A. Beaudry, E. B. Garcia, E. L. Wong, A. M. Ryder, K. W. Hasel, M.-C. Gauduin, R. A. Koup, J. S. McDougal, and P. J. Maddon. 1995. Expression and characterization of CD4-IgG2, a novel heterotetramer that neutralizes primary HIN type 1 isolates. AIDS Res.Hum.Retroviruses 11:533-539.

2. Andre, S., B. Seed, J. Eberle, W. Schraut, A. Bultmann, and J. Haas. 1998. Increased immune response elicited by DΝA vaccination with a synthetic gpl20 sequence with optimized codon usage. J.Nirol. 72:1497-1503.

3. Arthur, L. O., J. W. Bess, Jr., E. Ν. Chertova, J. L. Rossio, M. T. Esser, R. E. Benveniste, L. E. Henderson, and J. D. Lifson. 1998. Chemical inactivation of retroviral infectivity by targeting nucleocapsid protein zinc fingers: a candidate SIN vaccine. AIDS Res. Hum. Retroviruses 14 (Suppl 3):S311-S319.

4. Baba, T. W., V. Liska, R. Hofmann-Lehmann, J. Vlasak, W. Xu, S. Ayehunie, L. A. Cavacini, M. R. Posner, H. Katinger, G. Stiegler, B. J. Bernacky, T. A. Rizvi, R. Schmidt, L. R. Hill, M. E. Keeling, Y. Lu, J. E. Wright, T. C. Chou, and R. M. Ruprecht. 2000. Human neutralizing monoclonal antibodies of the IgGl subtype protect against mucosal simian-human immunodeficiency virus infection. at. Med. 6:200-206.

5. Back, N. K. T., L. Smit, J.-J. de Jong, W. Keulen, M. Schutten, J. Goudsmit, and M. Tersmette. 1994. An N-glycan within the human immunodeficiency virus type 1 gρl20 V3 loop affects virus neutralization. Virology 199:431-438.

6. Barbas, C. F. Ill, E. Bjorling, F. Chiodi, Ν. Dunlop, D. Cababa, T. M. Jones, S. L. Zebedee, M. A. Persson, P. L. Νara, E. Νorrby, and D. R. Burton. 1992. Recombinant human Fab fragments neutralize human type 1 immunodeficiency virus in vitro. Proc. Νatl. Acad. Sci. USA 89:9339-9343. 7. Barbas, C. F. Ill, T. A. Collet, W. Amberg, P. Roben, J. M. Binley, D. Hoekstra, D. Cababa, T. M. Jones, R. A. Williamson, G. R. Pilkington, N. L. Haigwood, E. Cabezas, A. C. Satterthwait, I. Sanz, and D. R. Burton. 1993. Molecular profile of an antibody response to HIV-1 as probed by combinatorial libraries. J. Mol. Biol. 230:812-823.

8. Beer, B. E., E. Bailes, P. M. Sharp, and V. M. Hirsch. 1999. Diversity and evolution of primate lentiviruses, p. 460-474. In C. L. Kuiken, B. Foley, B. Hahn, B. Korber, F. McCutchan, P. A. Marx, J. W. Mellors, J. I. Mullins, J. Sodroski, and S. Wolinsky (ed.), Human retroviruses and AIDS 1999. Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, N. Mex.

9. Belshe, R. B., G. J. Gorse, M. J. Mulligan, T. G. Evans, M. C. Keefer, J. L. Excler, A. M. Duliege, J. Tartaglia, W. I. Cox, J. McNamara, K. L. Hwang, A. Bradney, D. Montefiori, and K. J. Weinhold. 1998. Induction of immune responses to HIV-1 by canarypox virus (ALVAC) HIV-1 and gpl20 SF-2 recombinant vaccines in uninfected volunteers. AIDS 12:2407-2415.

10. Bhat, T. N., G. A. Bentley, G. Boulot, M. I. Greene, D. Tello, W. Dall'Acqua, H. Souchon, F. P. Schwarz, R. A. Mariuzza, and R. J. Poljak. 1994. Bound water molecules and conformational stabilization help mediate an antigen-antibody association. Proc. Natl. Acad. Sci. USA 91:1089-1093.

11. Binley, J. M., R. W. Sanders, B. Clas, N. Schuelke, A. Master, Y. Guo, F. Kajumo, D. J. Anselma, P. J. Maddon, W. C. Olson, and J. P. Moore. 2000. A recombinant human immunodeficiency virus type 1 envelope glycoprotein complex stabilized by an intermolecular disulfide bond between the gρl20 and gp41 subunits is an antigenic mimic of the trimeric virion-associated structure. J. Virol. 74:627- 643.

12. Binley, J. M., R. Wyatt, E. Desjardins, P. D. Kwong, W. Hendrickson, J. P. Moore, and J. Sodroski. 1998. Analysis of the interaction of antibodies with a conserved, enzymatically deglycosylated core of the HIV type 1 envelope glycoprotein 120. AIDS Res. Hum. Retroviruses 14:191-198.

13. Bolmstedt, A., S. Sjolander, J.-E. S. Hansen, L. Akerblom, A. Hemming, S.-L. Hu, B. Morein, and S. Olofsson. 1996. Influence of N-linked glycans in V4-5 region of human immunodeficiency virus type 1 glycoprotein g l60 on induction of a virus-neutralizing humoral response. J. Acquir. Immune Def. Syndr. 12:213-220.

14. Braden, B. C, B. A. Fields, and R. J. Poljak. 1995. Conservation of water molecules in an antibody-antigen interaction. J. Mol. Recognit. 8:317-325.

15. Braden, B. C. and R. J. Poljak . 1995. Structural features of the reactions between antibodies and protein antigens. FASEB J. 9:9-16.

16. Buchacher, A., R. Predl, C. Tauer, M. Purtscher, G. Gruber, R. Heider, F. Steindl, A. Trkola, A. Jungbauer, and H. Katinger. 1992. Human monoclonal antibodies against gp41 and gpl20 as potential agents for passive immunization, p. 191-194. In F. Brown, R. Chanock, H. S. Ginsberg, and R. Lerner (eds.), Vaccines '92: modern approaches to new vaccines including prevention of AIDS. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

17. Buchbinder, A., S. Karwowska, M. K. Gorny, S. T. Burda, and S. Zolla-Pazner.

1992. Synergy between human monoclonal antibodies to HIV extends their effective biological activity against homologous and divergent strains. AIDS Res.Hum.Retroviruses 8:425-427.

18. Burton, D. R. 1997. A vaccine for HIV type 1 : the antibody perspective. Proc. Natl. Acad. Sci. USA 94:10018-10023.

19. Burton, D. R. 2002. Antibodies, viruses and vaccines. Nat. Rev. Immunol. 2:706-713. 20. Burton, D. R., C. F. Barbas III, M. A. A. Persson, S. Koenig, R. M. Chanock, and R. A. Lerner. 1991. A large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals. Proc. Natl. Acad. Sci. USA 88:10134-10137.

21. Burton, D. R. and D. C. Montefiori. 1997. The antibody response in HIV-1 infection. AIDS 11 (suppl A):S87-S98.

22. Burton, D. R., J. Pyati, R. Koduri, S. J. Sharp, G. B. Thornton, P. W. H. I. Parren, L. S. W. Sawyer, R. M. Hendry, N. Dunlop, P. L. Nara, M. Lamacchia,

E. Garratty, E. R. Stiehm, Y. J. Bryson, Y. Cao, J. P. Moore, D. D. Ho, and C.

F. Barbas. 1994. Efficient neutralization of primary isolates of HIV-1 by a recombinant human monoclonal antibody. Science 266:1024-1027.

23. Cherpelis, S., I. Shrivastava, A. Gettie, X. Jin, D. D. Ho, S. W. Barnett, and L. Stamatatos. 2001. DNA vaccination with the human immunodeficiency virus type 1 SF162ΔV2 envelope elicits immune responses that offer partial protection from simian/human immunodeficiency virus infection to CD8+ T-cell-depelted rhesus macaques. J. Virology 75:1547-1550.

24. Chiesa, M. D., P. M. Martensen, C. Simmons, N. Porakishvili, J. Justesen, G. Dougan, I. M. Roitt, P. J. Delves, and T. Lund. 2001. Refocusing of B-cell responses following a single amino acid substitution in an antigen. Immunology 103:172-178.

25. Clements-Mann, M. L., K. Weinhod, T. J. Matthews, B. S. Graham, G. J. Gorse, M. C. Keefer, M. J. McElrath, R. H. Hsieh, J. Mestechy, S. Zolla- Pazner, R. Belshe, R. Dolin, S. Jackson, S. Xu, P. Fast, M. C. Walker, D. Stablein, J. L. Excler, and J. Tartaglia. 1998. Immune responses to human immunodeficiency virus (HIV) type 1 induced by canarypox expressing HIV-1MN gpl20, HIV-1 SF2 recombinant gpl20, or both vaccines in seronegative adults. JJnfectDis. 177:1230-1246.

26. Connor, R. I., B. K. Chen, S. Choe, and N. R. Landau. 1995. Vpr is required for efficient replication of human immunodeficiency virus type-1 in mononuclear phagocytes. Virology 206:935-944.

27. Connor, R. I., B. T. M. Korber, B. S. Graham, B. H. Hahn, D. D. Ho, B. D. Walker, A. U. Neumann, S. H. Vermund, J. Mestecky, S. Jackson, E. Fenamore, Y. Cao, F. Gao, S. Kalams, K. J. Kunstman, D. McDonald, N. McWilliams, A. Trkola, J. P. Moore, and S. M. Wolinsky. 1998. Immunological and virological analyses of persons infected by human immunodeficiency virus type 1 while participating in trials of recombinant gpl20 subunit vaccines. JNirol. 72:1552-1576.

28. Cummins, L. M., K. J. Weinhold, T. J. Matthews, A. J. Langlois, C. F. Perno, R. M. Condie, and J. P. Allain. 1991. Preparation and characterization of an intravenous solution of IgG from human immunodeficiency virus-seropositive donors. Blood 77:1111-1117.

29. D'Souza, M. P., D. Livnat, J. A. Bradac, S. Bridges, The AIDS Clinical Trials Group Antibody Selection Working Group, and Collaborating Investigators.

1997. Evaluation of monoclonal antibodies to HIV-1 primary isolates by neutralization assays: Performance criteria for selecting candidate antibodies for clinical trials. J. Infect. Dis. 175:1056-1062.

30. Dalgleish, A. G., P. C. L. Beverly, P. R. Clapham, D. H. Crawford, M. F. Greaves, and R. A. Weiss. 1984. The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus. Nature 312:763-767.

31. Davis, D., D. M. Stephens, C. Willers, and P. J. Lachmann. 1990. Glycosylation governs the binding of antipeptide antibodies to regions of hypervariable amino acid sequence within recombinant gpl20 of human immunodeficiency virus type 1. J. Gen. Virol. 71:2889-2898.

32. Delves, P. J., T. Lund, and I. M. Roitt. 1997. Can epitope-focused vaccines select advantageous immune responses? Mol. Med. Today 3:55-60.

33. di Marzo Veronese, F. R., R. Rahman, R. Pal, C. Boyer, J. Romano, V. S. Kalyanaraman, B. C. Nair, R. C. Gallo, and M. G. Sarngadharan. 1992. Delineation of immunoreactive, conserved regions in the external envelope glycoprotein of the human immunodeficiency virus type I. AIDS Res. Hum. Retroviruses 8:1125-1132.

34. Ebenbichler, C, T. McNearney, H. Stoiber, J. Most, R. Zangerle, W. Vogetseder, J. R. Patsch, L. Ratner, and M. P. Dierich. 1995. Sera from HIV-1 infected individuals in all stages of disease preferentially recognize the V3 loop of the prototypic macrophage-tropic glycoprotein gρl20 ADA. Mol. Immunol. 32:1039-1045.

35. Fouts, T. R., J. M. Binley, A. Trkola, J. E. Robinson, and J. P. Moore. 1997. Neutralization of the human immunodeficiency virus type 1 primary isolate JR-FL by human monoclonal antibodies correlates with antibody binding to the oligomeric form of the envelope glycoprotein complex. J. Virol. 71:2779-2785.

36. Fung, M. S. C, C. R. Y. Sun, W. L. Gordon, R.-S. Liou, T. W. Chang, W. N. C. Sun, E. S. Daar, and D. D. Ho. 1992. Identification and characterization of a neutralization site within the second variable region of human immunodeficiency virus type 1 gpl20. JNirol. 66:848-840.

37. Garrity, R. R., G. Rimmelzwaan, A. Minassian, W. P. Tsai, G. Lin, J. J. de Jong, J. Goudsmit, and P. L. Νara. 1997. Refocusing neutralizing antibody response by targeted dampening of an immunodominant epitope. J. Immunol. 159:279-289.

38. Gauduin, M. C, P. W. H. I. Parren, R. Weir, C. F. Barbas III, D. R. Burton, and R. A. Koup. 1997. Passive immunization with a human monoclonal antibody protects hu-PBL-SCID mice against challenge by primary isolates of HIN-1. Nat. Med. 3:1389-1393.

39. Gavel, Y. and G. von Heijne. 1990. Sequence differences between glycosylated and non-glycosylated Asn-X-Thr/Ser acceptor sites: implications for protein engineering. Protein Eng. 3:433-442.

40. Ghiara, J. B., E. A. Stura, R. L. Stanfield, A. T. Profy, and I. A. Wilson. 1994. Crystal structure of the principal neutralization site of HIN-1. Science 264:82-85.

41. Gorny, M. K., A. J. Conley, S. Karwowska, A. Buchbinder, J.-Y. Xu, E. A. Emini, S. Koenig, and S. Zolla-Pazner . 1992. Neutralization of diverse human immunodeficiency virus type 1 variants by an anti-V3 human monoclonal antibody. J. Virol. 66:7538-7542.

42. Gorny, M. K., J. Y. Xu, S. Karwowska, A. Buchbinder, and S. Zolla-Pazner.

1993. Repertoire of neutralizing human monoclonal antibodies specific for the V3 domain of HIV-1 gpl20. J.Immunol. 150:635-643.

43. Graham, B. S., M. C. Keefer, M. J. McElrath, G. J. Gorse, D. H. Schwartz, K. Weinhold, T. J. Matthews, J. R. Esterlitz, F. Sinangil, P. E. Fast, and NIAID AIDS Vaccine Evaluation Group. 1996. Safety and immunogenicity of a candidate HIV-1 vaccine in healthy adults: Recombinant glycoprotein (rgp) 120 - A randomized, double-bind trial. Ann.Int.Med. 125:270-279.

44. Grundner, C, T. Mirzabekov, J. Sodroski, and R. Wyatt. 2002. Solid-phase proteoliposomes containing human immunodeficiency virus envelope glycoproteins. J. Virol. 76:3511-3521.

45. Haas, J., E. C. Park, and B. Seed. 1996. Codon usage limitation in the expression of HIV-1 envelope glycoprotein. Curr Biol 6:315-324.

46. He, J., S. Choe, R. Walker, P. Di Marzio, D. O. Morgan, and N. R. Landau.

1995. Human immunodeficiency virus type 1 viral protein R (Vpr) arrests cells in the G2 phase of the cell cycle by inhibiting p34cdc2 activity. J. Virol. 69:6705-6711.

47. Helseth, E., M. Kowalski, D. Gabuzda, U. Olshevsky, W. Haseltine, and J. Sodroski. 1990. Rapid complementation assays measuring replicative potential of human immunodeficiency virus type 1 envelope glycoprotein mutants. J. Virol. 64:2416-2420.

48. Ho, D. D., M. S. Fung, Y. Z. Cao, X. L. Li, C. Sun, T. W. Chang, and N. C. Sun.

1991. Another discontinuous epitope on glycoprotein gpl20 that is important in human immunodeficiency virus type 1 neutralization is identified by a monoclonal antibody. Proc.Natl.Acad.Sci.USA 88:8949-8952.

49. Ho, D. D., J. A. McKeating, X. L. Li, T. Moudgil, E. S. Daar, N. C. Sun, and J. E. Robinson. 1991. Conformational epitope on gpl20 important in CD4 binding and human immunodeficiency virus type 1 neutralization identified by a human monoclonal antibody. J. Virol. 65:489-493.

50. Hofmann-Lehmann, R., J. Vlasak, R. A. Rasmussen, B. A. Smith, T. W. Baba, V. Liska, F. Ferrantelli, D. C. Montefiori, H. M. McClure, D. C. Anderson, B. J. Bernacky, T. A. Rizvi, R. Schmidt, L. R. Hill, M. E. Keeling, H. Katinger, G. Stiegler, L. A. Cavacini, M. R. Posner, T. C. Chou, J. Andersen, and R. M. Ruprecht. 2001. Postnatal passive immunization of neonatal macaques with a triple combination of human monoclonal antibodies against oral simian-human immunodeficiency virus challenge. J. Virology 75:7470-7480. 51. Imperiali, B. and K. W. Rickert. 1995. Conformational implications of asparagine- linlced glycosylation. Proc. Natl. Acad. Sci. USA 92:97-101.

52. Kasturi, L., H. Chen, and S. H. Shakin-Eshleman. 1997. Regulation of N-linked core glycosylation: use of a site-directed mutagenesis approach to identify Asn-Xaa- Ser/Thr sequons that are poor oligosaccharide acceptors. Biochem. J. 323:415-419.

53. Kessler, J. A., P. M. McKenna, E. A. Emini, C. P. Chan, M. D. Patel, S. K. Gupta, G. E. MarkllL, C. F. Barbas, D. R. Burton, and A. J. Conley. 1997. Recombinant human monoclonal antibody IgGl bl2 neutralizes diverse human immunodeficiency virus type 1 primary isolates. AIDS Res. Hum. Retroviruses 13:575-581.

54. Klatzmann, D., E. Champagne, S. Chamaret, J. Gruest, D. Guetard, T. Hercend, J. C. Glukman, and L. Montagnier. 1984. T-lymphocyte T4 molecule behaves as the receptor for human retrovirus LAV. Nature 312:767-768.

55. Kolchinsky, P., E. Kiprilov, P. Bartley, R. Rubinstein, and J. Sodroski. 2001. Loss of a single N-linked glycan allows CD4-independent human immunodeficiency virus type 1 infection by altering the position of the gpl20 V1/V2 variable loops. J. Virol. 75:3435-3443.

56. Kolchinsky, P., E. Kiprilov, and J. Sodroski. 2001. Increased neutralization sensitivity of CD4-independent human immunodeficiency virus variants. J. Virol. 75:2041-2050.

57. Korber, B., Foley, B. T., Kuiken, C, Pillai, S. K., and Sodroski, J. G. 1998. Numbering positions in HIV relative to HXB2CG, p. III-102-III-111. /?. B. Korber, CL. Kuiken, B. Foley, B. Hahn, F. McCutchan, J.W. Mellors, and J. Sodroski (ed.), Human Retroviruses and AIDS 1998. Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, N. Mex. 58. Kowalski, M., J. Potz, L. Basiripour, T. Dorfman, W. C. Goh, E. Terwilliger, A. Dayton, C. Rosen, W. Haseltine, and J. Sodroski. 1987. Functional regions of the envelope glycoprotein of human immunodeficiency virus type 1. Science 237:1351- 1355.

59. Koyanagi, Y., S. Miles, R. T. Mitsuyasu, J. E. Merrill, H. V. Vinters, and I. S. Chen. 1987. Dual infection of the central nervous system by AIDS viruses with distinct cellular tropisms. Science 236:819-822.

60. Kwong, P. D., R. Wyatt, S. Majeed, J. Robinson, R. W. Sweet, J. Sodroski, and W. A. Hendrickson. 2000. Structures of HIV-1 gpl20 envelope glycoproteins from laboratory-adapted and primary isolates. Structure Fold Des. 8:1329-1339.

61. Kwong, P. D., R. Wyatt, J. Robinson, R. W. Sweet, J. Sodroski, and W. A. Hendrickson. 1998. Structure of an HIV gρl20 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature 393:648-659.

62. Kwong, P. D., R. Wyatt, Q. J. Sattentau, J. Sodroski, and W. A. Hendrickson.

2000. Oligomeric modeling and electrostatic analysis of the gpl20 envelope glycoprotein of human immunodeficiency virus. J. Virol. 74:1961-1972.

63. Letvin, N. L., D. H. Barouch, and D. C. Montefiori. 2002. Prospects for vaccine protection against HIV-1 infection and AIDS. Annu. Rev. Immunol. 20:73-99.

64. Markham, R. B., J. Coberly, A. J. Ruff, D. Hoover, J. Gomez, E. Holt, J. Desormeaux, R. Boulos, T. C. Quinn, and N. A. Halsey. 1994. Maternal IgGl and IgA antibody to V3 loop consensus sequence and maternal-infant HIV-1 tranmission. Lancet 343:390-391.

65. Mascola, J. R., M. G. Lewis, G. Stiegler, D. Harris, T. C. VanCott, D. Hayes, M. K. Louder, C. Brown, C. V. Sapan, S. S. Frankel, Y. Lu, M. L. Robb, H. Katinger, and D. L. Birx. 1999. Protection of macaques against pathogenic SHIV- 89.6PD by passive transfer of neutralizing antibodies. J. Nirol. 73:4009-4018.

66. Mascola, J. R., S. Schlesinger Frankel, and K. Broliden. 2000. HIN-1 entry at the mucosal surface: role of antibodies in protection. AIDS 14 [Suppl 3]:S167-S174.

67. Mascola, J. R., S. W. Snyder, O. S. Weislow, S. M. Belay, R. B. Belshe, D. H. Schwartz, M. L. Clements, R. Dolin, B. S. Graham, G. J. Gorse, M. C. Keefer, M. J. McElrath, M. C. Walker, K. F. Wagner, J. G. McNeil, F. E. McCutchan, and D. S. Burke. 1996. Immunization with envelope subunit vaccine products elicits neutralizing antibodies against laboratory-adapted but not primary isolates of human immunodeficiency virus type 1. J nfect.Dis. 173:340-348.

68. Mascola, J. R., G. Stiegler, T. C. VanCott, H. Katinger, C. B. Carpenter, C. E. Hanson, H. Beary, D. Hayes, S. S. Frankel, D. L. Birx, and M. G. Lewis. 2000. Protection of macaques against vaginal transmission of a pathogenic HIV-1/SIV chimeric virus by passive infusion of neutralizing antibodies. Nat. Med. 6:207-210.

69. Mclnerney, T. L., L. McLain, S. J. Armstrong, and N. J. Dimmock. 1997. A human IgGl (bl2) specific for the CD4 binding site of HIV-1 neutralizes by inhibiting the virus fusion entry process, but bl2 Fab neutralizes by inl ibiting a postfusion event. Virology 233:313-326.

70. McKeating, J. A., C Shotton, S. Jeffs, C. Palmer, A. Hammond, J. Lewis, K. Oliver, J. May, and P. Balfe. 1996. Immunogenicity of full length and truncated forms of the human immunodeficiency virus type I envelope glycoprotein. Immunol. Lett. 51:101-105.

71. McKeating, J. A., M. Thali, C. Furman, S. Karwowska, M. K. Gorny, J. Cordell, S. Zolla-Pazner, J. Sodroski, and R. A. Weiss. 1992. Amino acid residues of the human immunodeficiency virus type 1 gpl20 critical for the binding of rat and human neutralizing antibodies that block the gpl20-sCD4 interaction. Virology 190:134-142.

72. McKeating, J. A., Y. J. Zhang, C. Arnold, R. Frederiksson, E. M. Fenyδ, and P. Balfe. 1996. Chimeric viruses expressing primary envelope glycoproteins of human immunodeficiency virus type 1 show increased sensitivity to neutralization by human sera. Virology 220:450-460.

73. Mellquist, J. L., L. Kasturi, S. L. Spitalnik, and S. H. Shakin-Eshleman. 1998. The amino acid following an asn-X-Ser/Thr sequon is an important determinant of N-linked core glycosylation efficiency. Biochemistry 37:6833-6837.

74. Mo, H., L. Stamatatos, J. E. Ip, C. F. Barbas III, P. W. H. I. Parren, D. R. Burton, J. P. Moore, and D. D. Ho . 1997. Human immunodeficiency virus type 1 mutants that escape neutralization by human monoclonal antibody IgGl bl2. JNirol. 71:6869-6874.

75. Montefiori, D. C, B. S. Graham, J. Zhou, D. H. Schwartz, L. A. Cavacini, and M. R. Posner. 1993. V3-specific neutralizing antibodies in sera from HIV-1 gpl60- immunized volunteers block virus fusion and act synergistically with human monoclonal antibody to the conformation-dependent CD4 binding site of gpl20. J.ClinJnvest. 92:840-847.

76. Moore, J. P. and D. D. Ho. 1993. Antibodies to discontinuous or conformationally sensitive epitopes on the gpl20 glycoprotein of human immunodeficiency virus type 1 are highly prevalent in sera of infected humans. JNirol. 67:863-875.

77. Moore, J. P. and D. D. Ho. 1995. HIN-1 neutralization: the consequences of viral adaptation to growth on transformed T cells. AIDS 9 (suppl A):S117-S136.

78. Moore, J. P., F. E. McCutchan, S. W. Poon, J. Mascola, J. Liu, Y. Cao, and D. D. Ho. 1994. Exploration of antigenic variation in gpl20 from clades A through F of human immunodeficiency virus type 1 by using monoclonal antibodies. JNirol. 68:8350-8364.

79. Moore, J. P., Q. J. Sattentau, R. Wyatt, and J. Sodroski. 1994. Probing the structure of the human immunodeficiency virus surface glycoprotein gpl20 with a panel of monoclonal antibodies. J. Nirol. 68:469-484.

80. Moore, J. P. and J. Sodroski. 1996. Antibody cross-competition analysis of the human immunodeficiency virus type 1 gpl20 exterior envelope glycoprotein. J. Nirol. 70:1863-1872.

81. Moore, J. P., A. Trkola, B. Korber, L. J. Boots, J. A. I. Kessler, F. E. McCutchan, J. Mascola, D. D. Ho, J. Robinson, and A. J. Conley. 1995. A human monoclonal antibody to a complex epitope in the N3 region of gpl20 of human immunodeficiency virus type 1 has broad reactivity within and outside clade B. J. Nirol. 69:122-130.

82. Moore, J. P., R. L. Willey, G. K. Lewis, J. Robinson, and J. Sodroski. 1994. Immunological evidence for interactions between the first, second, and fifth conserved domains of the gpl20 surface glycoprotein of human immunodeficiency virus type 1. J. Virol. 68:6836-6847.

83. Moulard, M., S. K. Phogat, Y. Shu, A. F. Labrijn, X. Xiao, J. M. Binley, M. Y. Zhang, I. A. Sidorov, C. C. Broder, J. Robinson, P. W. H. I. Parren, D. R. Burton, and D. S. Dimitrov. 2002. Broadly cross-reactive HIV-1 -neutralizing human monoclonal Fab selected for binding to gpl20-CD4-CCR5 complexes . Proc. Νatl. Acad. Sci. USA 99:6913-6918.

84. Myszka, D. G., R. W. Sweet, P. Hensley, M. Brigham-Burke, P. D. Kwong, W. A. Hendrickson, R. Wyatt, J. Sodroski, and M. L. Doyle. 2000. Energetics of the HIV gpl20-CD4 binding reaction. Proc. Natl. Acad. Sci. USA 97:9026-9031.

85. Nabel, G. J. and N. J. Sullivan. 2000. Antibodies and resistance to natural HIV infection. New Eng. J. Med 343:1263-1265.

86. Nara, P. L., R. R. Garrity, and J. Goudsmit. 1991. Neutralization of HIV-1 : a paradox of humoral proportions. FASEB J. 5:2437-2455.

87. Niedrig, M., H. P. Harthus, J. Hinkula, M. Broker, H. Bickhard, G. Pauli, H. R. Gelderblom, and B. Wahren. 1992. Inhibition of viral replication by monoclonal antibodies directed against human immunodeficiency virus gpl20. J. Gen. Virol. 73:2451-2455.

88. Nishimura, Y., T. Igarashi, N. Haigwood, R. Sadjadpour, R. J. Plishka, A. Buckler- White, R. Shibata, and M. A. Martin. 2002. Determination of a statistically valid neutralization titer in plasma that confers protection against simian-human immunodeficiency virus challenge following passive transfer of high- titered neutralizing antibodies. J. Virol. 76:2123-2130.

89. O'Connor, S. E. and B. Imperiali. 1996. Modulation of protein structure and function by asparagine-linked glycosylation. Chem. Biol. 3:803-812.

90. Ollmann Saphire, E., P. W. H. I. Parren, R. Pantophlet, M. B. Zwick, G. M. Morris, P. M. Rudd, R. A. Dwek, R. L. Stanfield, D. R. Burton, and I. A. Wilson. 2001. Crystal structure of a neutralizing human IgG against HIV-1 : A template for vaccine design. Science 293:1155-1159.

91. Olshevsky, U, E. Helseth, C. Furman, J. Li, W. Haseltine, and J. Sodroski.

1990. Identification of individual human immunodeficiency virus type 1 gpl20 amino acids important for CD4 receptor binding. JNirol. 64:5701-5707. 92. Overbaugh, J. and L. M. Rudensey. 1992. Alterations in potential sites for glycosylation predominate during evolution of the simian immunodeficiency virus envelope gene in macaques. J. Virology 66:5937-5948.

93. Pantophlet, R., L. Brade, L. Dijkshoorn, and H. Brade. 1998. Specificity of rabbit antisera against lipopolysaccharide of Acinetobacter. J. Clin. Microbiol. 36:1245-1250.

94. Pantophlet, R., E. O. Saphire, P. Poignard, P. W. H. I. Parren, I. A. Wilson, and D. R. Burton. 2003. Fine mapping of the interaction of neutralizing and non- neutralizing monoclonal antibodies with the CD4 binding site of human immunodeficiency virus type 1 gρl20. J. Virol. 77:642-658.

95. Parren, P. W. H. I. and D. R. Burton. 2001. The anti-viral activity of antibodies in vitro and in vivo. Adv. Immunol. 77:195-262.

96. Parren, P. W. H. I., H. J. Ditzel, R. J. Gulizia, J. M. Binley, C. F. Barbas III, D. R. Burton, and D. E. Mosier. 1995. Protection against HIV-1 infection in hu-PBL- SCID mice by passive immunization with a neutralizing human monoclonal antibody against the gpl20 CD4-binding site. AIDS 9:F1-F6.

97. Parren, P. W. H. I., M. C. Gauduin, R. A. Koup, P. Poignard, Q. J. Sattentau, P. Fisicaro, and D. R. Burton. 1997. Relevance of the antibody response against human immunodeficiency virus type 1 envelope to vaccine design. Immunol. Lett. 58:125-132.

98. Parren, P. W. H. I., P. A. Marx, A. J. Hessell, A. Luckay, J. Harouse, C. Cheng- Mayer, J. P. Moore, and D. R. Burton. 2001. Antibody protects macaques against vaginal challenge with a pathogenic R5 simian/human immunodeficiency virus at serum levels giving complete neutralization in vitro. J. Virol. 75:8340-8347. 99. Parren, P. W. H. L, I. Mondor, D. Nanϊche, H. J. Ditzel, P. J. Klasse, D. R. Burton, and Q. J. Sattentau. 1998. Neutralization of HIV-1 by antibody to gpl20 is determined primarily by occupancy of sites on the virion irrespective of epitope specificity. J. Virol. 72:3512-3519.

100. Parren, P. W. H. I., J. P. Moore, D. R. Burton, and Q. J. Sattentau. 1999. The neutralizing antibody response to HIV-1 : viral evasion and escape from humoral immunity. AIDS 13 (Suppl A):S137-S162.

101. Poignard, P., T. Fouts, D. Naniche, J. P. Moore, and Q. J. Sattentau. 1996. Neutralizing antibodies to human immunodeficiency virus type-1 gpl20 induce envelope glycoprotein subunit dissociation. J.Exp.Med. 183:473-484.

102. Poignard, P., P. J. Klasse, and Q. J. Sattentau. 1996. Antibody neutralization of HIV-1. Immunol.Today 17:239-246.

103. Poignard, P., E. OUmann Saphire, P. W. H. I. Parren, and D. R. Burton. 2001. gpl20: Biologic aspects of structural features. Annu. Rev. Immunol. 19:253-274.

104. Poignard, P., S. Sabbe, G. R. Picchio, M. Wang, R. J. Gulizia, H. Katinger, P. W. H. I. Parren, D. E. Mosier, and D. R. Burton. 1999. Neutralizing antibodies have limited effects on the control of established HIV-1 infection in vivo. Immunity 10:431-438.

105. Posner, M. R., L. A. Cavacini, C. L. Ernes, J. Power, and R. Byrn. 1993. Neutralization of HIV-1 by F105, a human monoclonal antibody to the CD4 binding site of gpl20. J.Acquir.Immune Def.Syndr. 6:7-14.

106. Posner, M. R., H. S. Elboim, T. Cannon, L. Cavacini, and T. Hideshima. 1992. Functional activity of an HIV-1 neutralizing IgG human monoclonal antibody: ADCC and complement-mediated lysis. AIDS Res.Hum.Retroviruses 8:553-558. 107. Quinones-Kochs, M. I., L. Buonocore, and J. K. Rose. 2002. Role of N-linlced glycans in a human immunodeficiency virus envelope glycoprotein: effects on protein function and the neutralizing antibody response. J. Virol. 76:4199-4211.

108. Reitter, J. N, R. E. Means, and R. C. Desrosiers. 1998. A role for carbohydrates in immune evasion in AIDS. Nat. Med. 4:679-684.

109. Rini, J. M., R. L. Stanfield, E. A. Stura, P. A. Salinas, A. T. Profy, and I. A. Wilson. 1993. Crystal structure of a human immunodeficiency virus type 1 neutralizing antibody, 50.1, in complex with its V3 loop peptide antigen. Proc. Natl. Acad. Sci. USA 90:6325-6329.

110. Roben, P., J. P. Moore, M. Thali, J. Sodroski, C. F. Barbas III, and D. R. Burton. 1994. Recognition properties of a panel of human recombinant Fab fragments to the CD4 binding site of gpl20 that show differing abilities to neutralize human immunodeficiency virus type 1. J. Virol. 68:4821-4828.

111. Robinson, J. E., D. Holton, S. Pacheco-Morell, J. Liu, and H. McMurdo. 1990. Identification of conserved and variant epitopes of human immunodeficiency virus type 1 (HIV-1) gpl20 by human monoclonal antibodies produced by EBV- transformed cell lines. AIDS Res.Hum.Retroviruses 6:567-2107.

112. Robinson, J. E., H. Yoshiyama, D. Holton, S. Elliott, and D. D. Ho. 1992. Distinct antigenic sites on HIV gpl20 identified by a panel of human monoclonal antibodies. J.Cell.Biochem. 16E(suppl.):71.

113. Rossio, J. L., M. T. Esser, K. Suryanarayana, D. K. Schneider, J. W. Bess, Jr., G. M. Vasquez, T. A. Wiltrout, E. Chertova, M. K. Grimes, Q. Sattentau, L. O. Arthur, L. E. Henderson, and J. D. Lifson. 1998. Inactivation of human immunodeficiency virus type 1 infectivity with preservation of conformational and functional integrity of viron surface proteins. JNirol. 72:7992-8001. 114. Sanders, R. W., L. Schiffner, A. Master, F. Kajumo, Y. Guo, T. Dragic, J. P. Moore, and J. M. Binley. 2000. Variable-loop-deleted variants of the human immunodeficiency virus type 1 envelope glycoprotein can be stabilized by an intermolecular disulfide bond between the gpl20 and gp41 subunits. J. Virol. 74:5091-5100.

115. Sanders, R. W., M. Venturi, L. Schiffner, R. Kalyanaraman, H. Katinger, K. O. Lloyd, P. D. Kwong, and J. P. Moore. 2002. The mannose-dependent epitope for neutralizing antibody 2G12 on human immunodeficiency virus type 1 glycoprotein gpl20. J. Virol. 76:7293-7305.

116. Sattentau, Q. J. and J. P. Moore. 1995. Human immunodeficiency virus type 1 neutralization is determined by epitope exposure on the gpl20 oligomer. J. Exp. Med. 182:185-196.

117. Sattentau, Q. J., M. Moulard, B. Brivet, F. Botto, J. C. Cuillemot, I. Mondor, P. Poignard, and S. Ugolini. 1999. Antibody neutralization of HIV-1 and the potential for vaccine design. Immunol. Lett. 66:143-149.

118. Sayle, R. A. and E. J. Milner-White. 1995. RASMOL: biomolecular graphics for all. Trends Biochem. Sci. 20:374-376.

119. Scanlan, C. N., R. Pantophlet, M. R. Wormwald, E. O. Saphire, R. Stanfield, I. A. Wilson, H. Katinger, R. A. Dwek, P. M. Rudd, and D. R. Burton. 2002. The broadly neutralizing anti-human immunodeficiency virus type 1 antibody 2G12 recognizes a cluster of alphal~>2 mannose residues on the outer face of gpl20. J. Virol. 76 :7306-7321.

120. Schønning, K., B. Jansson, S. Olofsson, and J. E. S. Hansen. 1996. Rapid selection for an N-linked oligosacharide by monoclonal antibodies directed against the V3 loop of human immunodeficiency virus type 1. J. Gen. Virol. 77:753-758. 121. Schønning, K., O. Lund, O. S. Lund, and J. E. S. Hanssen. 1999. Stochiometry of monoclonal antibody neutralization of T-cell line-adapted human immunodeficiency virus type 1. J. Virol. 73:8364-8370.

122. Schreiber, M., H. Petersen, C. Wachsmuth, H. Mϋller, F. T. Hufert, and H. Schmitz. 1994. Antibodies of symptomatic human immunodeficiency virus type 1- infected individuals are directed to the V3 domain of noninfectious and not of infectious virions present in autologous serum. J. Virol. 68:3908-3916.

123. Scott, C. F.,Jr., S. Silver, A. T. Profy, S. D. Putney, A. Langlois, K. Weinhold, and J. E. Robinson. 1990. Human monoclonal antibody that recognizes the V3 region of human immunodeficiency virus gpl20 and neutralizes the human T- lymphotropic virus type III _N strain. Proc.Natl.Acad.Sci.USA 87:8597-8601.

124. Shakin-Eshleman, S. H., S. L. Spitalnik, and L. Kasturi. 1996. The amino acid at the X position of an Asn-X-Ser sequon is an important determinant of N-linked core-glycosylation efficiency. J. Biol. Chem. 271:6363-6366.

125. Shibata, R., T. Igarashi, N. Haigwood, A. Buckler-White, R. Ogert, W. Ross, R. Willey, M. W. Cho, and M. A. Martin. 1999. Neutralizing antibody directed against the HIV-1 envelope glycoprotein can completely block HIV-1 /SI V chimeric virus infections of macaque monkeys. Nat. Med. 5:204-210.

126. Slagle, S. P., R. E. Kozack, and S. Subramaniam. 1994. Role of electrostatics in antibody-antigen association: anti-hen egg lysozyme/lysozyme complex (HyHEL- 5/HEL). J. Biomol. Struct. Dyn. 12:439-456.

127. Spear, G. T., D. M. Takefman, S. Sharpe, M. Ghassemi, and S. Zolla-Pazner.

1994. Antibodies to the HIV-1 V3 loop in serum from infected persons contribute a major proportion of immune effector functions including complement activation, antibody binding and neutralization. Virology 204:609-615. 128. Stamatatos, L. and C. Cheng-Mayer. 1998. An envelope modification that renders a primary neutralization resistant Clade B human immunodificiency virus type 1 isolate highly susceptible to neutralization by sera from other clades. JNirol. 72:7840-7845.

129. Stanfield, R. L., T. M. Fiester, R. A. Lerner, and I. A. Wilson. 1990. Crystal structures of an antibody to a peptide and its complex with peptide antigen at 2.8 A. Science 248:712-719.

130. Sugiura, W., C. C. Broder, B. Moss, and P. L. Earl. 1999. Characterization of conformation-dependent anti-gpl20 murine monoclonal antibodies produced by immunization with monomeric and oligomeric human immunodeficiency virus type 1 envelope proteins. Virology 254:257-267.

131. Sullivan, IS., Y. Sun, Q. Sattentau, M. Thali, D. Wu, G. Denisova, J. Gershoni, J. Robinson, J. Moore, and J. Sodroski. 1998. CD4-Induced conformational changes in the human immunodeficiency virus type 1 gpl20 glycoprotein: consequences for virus entry and neutralization. JNirol. 72:4694-4703.

132. Sullivan, Ν., M. Thali, C. Furman, D. D. Ho, and J. Sodroski. 1993. Effect of amino acid changes in the VI /N2 region of the human immunodeficiency vims type 1 gpl20 glycoprotein on subunit association, syncytium formation, and recognition by a neutralizing antibody. JNirol. 67:3674-3679.

133. Thali, M., C. Furman, D. D. Ho, J. Robinson, S. Tilley, A. Pinter, and J. Sodroski. 1992. Discontinuous, conserved neutralization epitopes overlapping the CD4 binding region of the HIV-1 gpl20 envelope glycoprotein. J. Virol. 66:5635- 5641.

134. Thali, M., J. P. Moore, C. Furman, M. Charles, D. D. Ho, J. Robinson, and J. Sodroski. 1993. Characterization of conserved human immunodeficiency virus type 1 gpl20 neutralization epitopes exposed upon gpl20-CD4 binding. J. Virol. 67:3978-3988.

135. Thali, M., U. Olshevshy, C. Furman, D. Gabuzda, M. Posner, and J. Sodroski.

1991. Characterization of a discontinuous human immunodeficiency virus type 1 gpl20-epitope recognised by a broadly reactive neutralizing human monoclonal antibody. J. Virol. 65:6188-6193.

136. Trkola, A., T. Dragic, J. Arthos, J. M. Binley, W. C. Olson, G. P. AUaway, C. Cheng-Mayer, J. Robinson, P. J. Maddon, and J. P. Moore. 1996. CD4- dependent, antibody-sensitive interactions between HIV-1 and its co-receptor CCR- 5. Nature 384:184-187.

137. Trkola, A., M. Purtscher, T. Muster, C. Ballaun, A. Buchacher, N. Sullivan, K. Srinivasan, J. Sodroski, J. P. Moore, and H. Katinger. 1996. Human monoclonal antibody 2G12 defines a distinctive neutralization epitope on the gρl20 glycoprotein of human immunodeficiency virus type I. J. Virol. 70:1100-1108.

138. Tsumoto, K., K. Ogasahara, Y. Ueda, K. Watanabe, K. Yutani, and I. Kumagai. 1996. Role of salt bridge formation in antigen-antibody interaction. Entropic contribution to the complex between hen egg white lysozyme and its monoclonal antibody HyHELlO. J. Biol. Chem. 271:32612-32616.

139. Tsumoto, K., Y. Ueda, K. Maenaka, K. Watanabe, K. Ogasahara, K. Yutani, and I. Kumagai. 1994. Contribution to antibody-antigen interaction of structurally perturbed antigenic residues upon antibody binding. J. Biol. Chem. 269:28777- 28782.

140. Vinogradov, E. V., R. Pantophlet, L. Dijkshoorn, L. Brade, O. Hoist, and H. Brade. 1996. Structural and serological characterisation of two O-specific polysaccharides of Acinetobacter. Eur. J. Biochem. 239:602-610. 141. Vogel, T., R. Kurth, and S. Norley. 1994. The majority of neutralizing abs in HIV- 1 infected patients recognize linear V3 loop sequences. Studies using HIV-I_MN multiple antigenic peptides. J.lmmunol. 153:1895-1904.

142. Wormald, M. R. and R. A. Dwek . 1999. Glycoproteins: glycan presentation and protein-fold stability. Structure Fold. Des. 7:R155-R160.

143. Wyatt, R., P. D. Kwong, E. Desjardins, R. W. Sweet, J. Robinson, W. A. Hendrickson, and J. G. Sodroski. 1998. The antigenic structure of the HIV gpl20 envelope glycoprotein. Nature 393:705-711.

144. Wyatt, R., J. Moore, M. Accola, E. Desjardin, J. Robinson, and J. Sodroski.

1995. Involvement of the V1/V2 variable loop structure in the exposure of human immunodeficiency virus type 1 gpl20 epitopes induced by receptor binding. J. Virol. 69:5723-5733.

145. Wyatt, R. and J. Sodroski. 1998. The HIV-1 envelope glycoproteins: fusogens, antigens, and immunogens. Science 280:1884-1888.

146. Wyatt, R., N. Sullivan, M. Thali, H. Repke, D. Ho, J. Robinson, M. Posner, and J. Sodroski. 1993. Functional and immunologic characterization of human immunodeficiency virus typ 1 envelope glycoproteins containing deletions of the major variable regions. JNirol. 67:4557-4565.

147. Yang, X., M. Farzan, R. Wyatt, and J. Sodroski. 2000. Characterization of stable, soluble trimers containing complete ectodomains of human immunodeficiency virus type 1 envelope glycoproteins. J. Nirol. 74:5716-5725.

148. Yang, X., L. Florin, M. Farzan, P. Kolchinsky, P. D. Kwong, J. Sodroski, and R. Wyatt. 2000. Modifications that stabilize human immunodeficiency virus envelope glycoprotein trimers in solution. JNirol. 74:4746-4754. 149. Yang, X., J. Lee, E. M. Mahony, P. D. Kwong, R. Wyatt, and J. Sodroski. 2002. Highly stable trimers formed by human immunodeficiency virus type 1 envelope glycoproteins fused with the trimeric motif of T4 bacteriophage fibritin. J. Virol. 76:4634-4642.

150. Yoshiyama, H., H. Mo, J. P. Moore, and D. D. Ho. 1994. Characterization of mutants of human immunodeficiency virus type 1 that have escaped neutralization by a monoclonal antibody the gpl20 V2 loop. JNirol. 68:974-978.

151. Zhu, X., C. Borchers, R. J. Bienstock, and K. B. Tomer. 2000. Mass spectrometric characterization of the glycosylation pattern of HIN-gpl20 expressed in CHO cells. Biochemistry 39:11194-11204.

152. Zwick, M. B., L. L. C. Bonnycastle, A. Menendez, M. B. Irving, C. F. Barbas, P. W. H. I. Parren, D. R. Burton, and J. K. Scott. 2001. Identification and characterization of a peptide that specifically binds the human, broadly neutralizing anti-human immunodeficiency virus type 1 antibody bl2. J. Virol. 75:6692-6699.

153. Zwick, M. B., Parren, P. H. W. X, Saphire, E. O., Wang, M., Scott, J. K., Dawson, P. E., Wilson, I. A., and Burton, D. R. 2003. Molecular features of the broadly neutralizing IgGl bl2 required for recognition of HIV-1 gpl20 by mutational analysis. J. Virol, in press.

154. Zwick, M. B., M. Wang, P. Poignard, G. Stiegler, H. Katinger, D. R. Burton, and P. W. H. I. Parren. 2001. Neutralization synergy of human immunodeficiency virus type 1 primary isolates by cocktails of broadly neutralizing antibodies. J. Virol. 75:12198-12208.

Claims

What is claimed is:

1. An immunogenic mutant HIV-1 gpl20 polypeptide that can stimulate a neutralizing antibody response against a panel of human immunodeficiency virus type 1 (HIV-1) comprising HIV-1 primary isolates of at least two different clades, which mutant HIV-1 gpl20 polypeptide has at least one amino acid mutation in at least one epitope of the HIV-1 gpl20 polypeptide specifically bound by a non-neutralizing antibody, which mutation reduces binding affinity of the non-neutralizing antibody.

2. The immunogenic mutant HIV-1 gpl20 polypeptide of claim 1, wherein said mutation increases the apparent binding affinity of a neutralizing antibody to at least 120% of the unmutated HIV-1 gpl20 polypeptide.

3. The immunogenic mutant HIV-1 gpl20 polypeptide of claim 1 or 2, wherein the panel of human immunodeficiency virus type 1 (HIV-1) comprises HIV-1 primary isolates of at least five different clades selected from clades A, B, C, D, E, F, and G.

4. The immunogenic mutant HIV- 1 gp 120 polypeptide of anyone of claims 1 -3 , wherein said mutation reduces the apparent binding affinity of the non-neutralizing antibody to 1% to 0.1% of the unmutated HIV-1 gpl20 polypeptide.

5. The immunogenic mutant HIV-1 g l20 polypeptide of anyone of claims 1-4, wherein the unmutated HIV-1 gpl20 polypeptide is derived from HIV-1 primary isolate JR-CSF or JR-FL.

6. An immunogenic mutant gp 120 polypeptide that can stimulate a neutralizing antibody response against a human immunodeficiency virus (HIV), said mutant gpl20 polypeptide comprising an HIV gpl20 polypeptide having at least one amino acid mutation in at least one epitope of the HIV gρl20 polypeptide specifically bound by a non-neutralizing antibody.

7. The immunogenic mutant gpl20 polypeptide of claim 6, comprising an HIV gpl20 polypeptide having at least two amino acid mutations in at least one epitope of the HIV gpl20 polypeptide specifically bound by a non-neutralizing antibody.

8. The immunogenic mutant gpl20 polypeptide of claim 7, wherein the at least two amino acid mutations comprise at least a first amino acid mutation in a first epitope of the HIV gpl20 polypeptide specifically bound by a non-neutralizing antibody and at least a second amino acid mutation in a second epitope of the HIV gpl20 polypeptide specifically bound by a non-neutralizing antibody.

9. The immunogenic mutant gpl20 polypeptide of claim 8, wherein the first epitope and the second epitope are different.

10. The immunogenic mutant gpl20 polypeptide of claim 9, wherein the epitope of the HIV gpl20 polypeptide specifically bound by a non-neutralizing antibody comprises F43 cavity perimeter amino acid residues, VI loop amino acid residues, V2 loop amino acid residues, V3 loop amino acids residues, non-neutralizing face amino acid residues, Cl domain amino acid residues, C5 domain amino acid residues, co- receptor binding site amino acid residues, or a combination thereof.

11. The immunogenic mutant gpl20 of claim 6, wherein the at least one amino acid mutation is a substitution of a first amino acid residue for a second amino acid residues, wherein the second amino acid residue is different from the first amino acid residue.

12. The immunogenic mutant gpl20 of claim 6, wherein the at least one amino acid mutation is a substitution of an amino acid residue of the gpl20 polypeptide with an alanine.

13. The immunogenic mutant gpl20 of claim 6, wherein the at least one amino acid mutation generates a glycosylation motif.

14. The immunogenic mutant gpl20 of claim 13, wherein the glycosylation motif comprises an N-linlced glycosylation motif.

15. The immunogenic mutant gpl20 of claim 6, wherein the HIV is HIV type 1 (HIV- 1).

16. The immunogenic mutant gpl20 of claim 15, wherein at least one mutation comprises a substitution of G473, D474, M475, R476, or a combination thereof.

17. The immunogenic mutant gpl20 of claim 15, wherein at least one mutation comprises a substitution of G473 A, D474A, M475A, R476A, or a combination thereof.

18. The immunogenic mutant gpl20 of claim 17, comprising at least two amino acid mutations.

19. The immunogenic mutant gpl20 of claim 18, which comprises D474A and R476A; G473 A and M475A; D474A, M475 A and R476A; or G473 A, D474A, M475A and R476A.

20. The immunogenic mutant gpl20 of claim 15, comprising G473A, D474A, M475A and G476A.

21. The immunogenic mutant gpl20 of claim 15, wherein the amino acid mutation comprises DI 13 A, V127A, DI 80A, N197A, S256A, or a combination thereof.

22. The immunogenic mutant gpl20 of claim 15, wherein at least one amino acid mutation generates a glycosylation motif.

23. The immunogenic mutant gpl20 of claim 22, wherein the glycosylation motif comprises an N-linlced glycosylation motif.

24. The immunogenic mutant gpl20 of claim 23, wherein the at least one amino acid mutation comprises H92N and N94T; Ql 14N and Ll 16T; S143T; E150N and G152T; K171N and Y173T; Q246N; P313N and R315T; or I423N andN425T; or a combination thereof.

25. The immunogenic mutant HIV-1 gpl20 of anyone of the claims 1-5, which comprises the amino acid mutations D474A and R476A; and furthermore comprises at least one amino acid mutation selected from H92N and N94T; Ql 14N and Ll 16T; S143T; E150N and G152T; K171N and Y173T; Q246N; P313N and R315T; I423N a d N425T; or any combination thereof.

26. The immunogenic mutant HIV-1 gρl20 of anyone of the claims 1-5, which comprises the amino acid mutations G473A and M475A; and furthermore comprises at least one amino acid mutation selected from H92N and N94T; Ql 14N and Ll 16T; S143T; E150N and G152T; K171N and Y173T; Q246N; P313N and R315T; I423N and N425T; or any combination thereof.

27. The immunogenic mutant HIV-1 gρl20 of anyone of the claims 1-5, which comprises the amino acid mutations D474A, M475A and R476A; and furthermore comprises at least one amino acid mutation selected from H92N and N94T; Ql 14N and L116T; S143T; E150N and G152T; K171N and Y173T; Q246N; P313N and R315T; I423N and N425T; or any combination thereof.

28. The immunogenic mutant HIV-1 gpl20 of anyone of the claims 1-5, which comprises the G473 A, D474A, M475A and R476A; and furthermore comprises at least one amino acid mutation selected from H92N and N94T; Ql 14N and Ll 16T; S143T; E150N and G152T; K171N and Y173T; Q246N; P313N and R315T; I423N and N425T; or any combination thereof.

29. The immunogenic mutant gpl20 polypeptide of claim 23, comprising Ql 14N, L116T, S143T, E150N, and G152T.

30. The immunogenic mutant gpl20 polypeptide of claim 23, comprising H92N, N94T, K171N, Y173T, I423N andN425T.

31. The immunogenic mutant gpl20 polypeptide of claim 29, further comprising G373A, D474A, M475A, and R476A.

32. The immunogenic mutant gρl20 polypeptide of claim 19, further comprising H92N, N94T, Q114N, L116T, S143T, E150N, G152T, K171N, Y173T, Q246N, P313N, R315T, I423N and N425T.

33. The immunogenic mutant gpl20 polypeptide of claim 10, which comprises a deletion of Cl domain amino acid residues, C5 domain residues, or a combination thereof.

34. A method of inducing antibodies that can neutralize human immunodeficiency virus type 1 (HIV-1), comprising immunizing a subject with an immunogenic mutant HIV-1 gρl20 polypeptide that can stimulate a neutralizing antibody response against a panel of human immunodeficiency virus type 1 (HIV-1) comprising HIV-1 primary isolates of at least two different clades, which mutant comprises an HIV-1 gpl20 polypeptide having at least one amino acid mutation in at least one epitope of the HIV-1 gpl20 polypeptide specifically bound by a non-neutralizing antibody, which mutation reduces binding affinity of the non-neutralizing antibody.

35. The method of claim 34, wherein said mutation in the immunogenic mutant HIV-1 gpl20 polypeptide increases the apparent binding affinity of a neutralizing antibody to at least 120% of the unmutated HIV-1 gpl20 polypeptide.

36. The method of claims 34 and 35, wherem the immunogenic mutant HIV-1 gpl20 polypeptide can stimulate a neutralizing antibody response against a panel of human immunodeficiency virus type 1 (HIV-1) comprising HIV-1 primary isolates of at least five different clades selected from clades A, B, C, D, E, F, and G.

37. The method of the claims 34-36, wherein said mutation in the immunogenic mutant HIV-1 gpl20 polypeptide reduces the apparent binding affinity of the non- neutralizing antibody to 1% to 0.1% of the unmutated HIV-1 gpl20 polypeptide.

38. The method of anyone of the claims 34-37, wherein the unmutated HIV-1 gpl20 polypeptide parent of the immunogenic mutated HIN-1 gpl20 polypeptide is derived from HIV-1 primary isolate JR-CSF or JR-FL.

39. A method of inducing antibodies that can neutralize human immunodeficiency virus (HIV), comprising immunizing a subject with an immunogenic mutant gpl20 polypeptide that can stimulate a neutralizing antibody response against a human immunodeficiency virus (HIV), said mutant gρl20 polypeptide comprising an HIV gpl20 polypeptide having at least one amino acid mutation in at least one epitope of the HIV gpl20 polypeptide specifically bound by a non-neutralizing antibody.

40. The method of claim 39, wherein the epitope of the HIV gpl20 polypeptide specifically bound by a non-neutralizing antibody comprises F43 cavity perimeter amino acid residues, VI loop amino acid residues, V2 loop amine acid residues, V3 loop amine acid residues, non-neutralizing face amine acid residues, Cl domain amino acid residues, C5 domain amino acid residues, co-receptor binding site amino acid residues, or a combination thereof.

41. The method of claim 39, comprising at least two amino acid mutations in at least one epitope of the HIV gρl20 polypeptide specifically bound by a non-neutralizing antibody.

42. The method of claim 39, comprising at least two amino acid mutations in at least two epitopes of the HIV gpl20 polypeptide specifically bound by a non-neutralizing antibody.

43. The method of claim 39, wherein the at least one amino acid mutation comprises at least one amino acid substitution.

44. The method of claim 43, wherein the at least one amino acid substitution generates a glycosylation motif.

45. The method of claim 44, wherein the glycosylation motif comprises an N-linlced glycosylation motif.

46. The method of claim 39, wherein the HIV is HIV type 1 (HIV-1).

47. The method of claim 46, wherein the immunogenic mutant gpl20 polypeptide comprises D113A; V127A; D180A; Ν197A; S256A; G473A; D474A; M475A; R476A; H92N andN94T; Q114N and L116T; S143T; E150N and G152T; K171N and Y173T; Q246N; P313N and R315T; I423N and N425T; or a combination thereof.

48. The method of claim 46, wherein the immunogenic mutant gpl20 polypeptide comprises D474A and R476A; G473 A and M475A; D474A, M475A and R476A; or G473 A, D474A, M475A and R476A.

49. The method of claim 46, wherein the immunogenic mutant gpl20 polypeptide comprises H92N andN94T; Q114N and L116T; S143T; E150N and G152T; K171N and Y173T; Q246N; P313N and R315T; or I423N and N425T; or a combination thereof.

50. The method of claim 46, wherein the immunogenic mutant gpl20 polypeptide further comprises G473 A, D474A, M475A and R476A.

51. The method of claim 50, wherein the immunogenic mutant gpl20 polypeptide further comprises H92N, N94T, Q114N, L116T, S143T, E150N, G152T, K171N, Y173T, Q246N, P313N, R315T, I423N and N425T.

52. The method of anyone of the claims 34-38, wherein the immunogenic mutant HIV-1 gpl20 comprises the amino acid mutations D474A and R476A; and furthermore comprises at least one amino acid mutation selected from H92N and N94T; Ql 14N and Ll 16T; S143T; E150N and G152T; K171N and Y173T; Q246N; P313N and R315T; I423N and N425T; or any combination thereof.

53. The method of anyone of the claims 34-37, wherein the immunogenic mutant HIV-1 gpl20 comprises the amino acid mutations G473A and M475M; and furthermore comprises at least one amino acid mutation selected from H92N and N94T; Ql 14N and L116T; S143T; E150N and G152T; K171N and Y173T; Q246N; P313N and R315T; I423N and N425T; or any combination thereof.

54. The method of anyone of the claims 34-37, wherein the immunogenic mutant HIV-1 gpl20 comprises the amino acid mutations D474A, M475A and R476A; and furthermore comprises at least one amino acid mutation selected from H92N and N94T; Q114N and L116T; S143T; E150N and G152T; K171N and Y173T; Q246N; P313N and R315T; I423N and N425T; or any combination thereof.

55. The method of anyone of the claims 34-37, wherein the immunogenic mutant HIV-1 gpl20 comprises the amino acid mutations G473A, D474A, M475A and R476A; and furthermore comprises at least one amino acid mutation selected from H92N andN94T; Q114N and L116T; S143T; E150N and G152T; K171N and Y173T; Q246N; P313N and R315T; I423N and N425T; or any combination thereof.

56. A vaccine comprising one or more mutant gpl20 polypeptides of anyone of the claims 1-5 or 25-28.

57. The method of claim 50, wherein the immunogenic mutant gpl20 polypeptide further comprises a deletion of Cl domain amino acid residues, C5 domain amino acid residues, or a combination thereof.

58. The method of claim 39, further comprising obtaining antiserum from the subject, said antiserum comprising HIV neutralizing antibodies.

59. The method of claim 58, wherein the antiserum comprises HIV-1 neutralizing antibodies.

60. The method of claim 60, wherein the subject is a human subject.

61. The method of claim 58, further comprising isolating an HIV neutralizing antibody fraction from said antiserum.

62. The method of claim 39, further comprising substantially purifying HIV neutralizing antibodies from the subject.

63. Antiserum obtained by the method of claim 58.

64. An isolated HIV neutralizing antibody fraction obtained by the method of claim 61.

65. A substantially purified HIV neutralizing antibody obtained by the method of claim 62.

66. The substantially purified HIV neutralizing antibody of claim 65, which comprises polyclonal HIV-1 neutralizing antibodies.

67. The substantially purified HIV neutralizing antibody of claim 65, which comprises a monoclonal HIV neutralizing antibody.

68. The substantially purified HIV neutralizing antibody of claim 65, which comprises a monoclonal HIV-1 neutralizing antibody.

69. A method of ameliorating a human immunodeficiency virus (HIV) infection in a subject, comprising administering an immunogenic mutant gpl20 polypeptide that can stimulate a neutralizing antibody response against HIV to the subject, said mutant gpl20 polypeptide comprising an HIV gpl20 polypeptide having at least one amino acid mutation in at least one epitope of the HIV gpl20 polypeptide specifically bound by a non-neutralizing antibody.

70. The method of claim 69, wherein the HIV is an HIV type 1 (HIV -1).

71. A method of preventing HIV-1 infection or ameliorating HIV-1 infection in a human subject, comprising administering one or more mutant gpl20 polypeptides of anyone of the claims 1-5 or 25-28.

72. The method of claim 69, wherein the epitope of the HIV gpl20 polypeptide specifically bound by a non-neutralizing antibody comprises F43 cavity perimeter amino acid residues, VI loop amino acid residues, V2 loop amino acid residues, V3 loop amino acid residues, non-neutralizing face amino acid residues, Cl domain amino acid residues, C5 domain amino acid residues, co-receptor binding site amino acid residues, or a combination thereof.

73. The method of claim 70, wherein the iinmunogenic mutant gpl20 polypeptide comprises D113A; V127A; D180A; N197A; S256A; G473A; D474A; M475A; R476A; H92N andN94T; Q114N and L116T; S143T; E150N and G152T; K171N and Y173T; Q246N; P313N and R315T; I423N and N425T; or a combination thereof.

74. The method of claim 70, wherein the immunogenic mutant gpl20 polypeptide comprises D474A and R476A; G473 A and M475A; D474A, M475A and R476A; or G473 A, D474A, M475A and R476A.

75. The method of claim 70, wherein the immunogenic mutant gρl20 polypeptide comprises H92N andN94T; Q114N and L116T; S143T; EI50N and G152T; K171N and Y173T; Q246N; P313N and R315T; or I423N and N425T; or a combination thereof.

76. The method of claim 70, wherein the immunogenic mutant gpl20 polypeptide comprises G473A, D474A, M475A and R476A.

77. The method of claim 76, wherein the immunogenic mutant gpl20 polypeptide further comprises H92N, N94T, Q114N, L116T, S143T, E150N, G152T, K171N, Y173T, Q246N, P313N, R315T, I423N, and N425T.

78. The method of claim 76, wherein the immunogenic mutant gpl20 polypeptide further comprises a deletion of Cl domain amino acid residues, C5 domain amino acid residues, or a combination thereof.

79. A method of ameliorating a human immunodeficiency virus (HIV) infection in a subject, comprising administering HIV neutralizing antibodies to the subject, wherein the HIV neutralizing antibodies comprise antibodies stimulated in response to mutant gpl20 polypeptide comprising an HIV gpl20 polypeptide having at least one amino acid mutation in at least one epitope of the HIV gρl20 polypeptide specifically bound by a non-neutralizing antibody.

80. The method of claim 79, wherein the HIV is HIV type 1 (HIV-1).

81. A method of preventing HIV-1 infection or ameliorating HIV-1 infection in a human subject, comprising administering HIV-1 neutralizing antibodies to the subject, wherein the HIV-1 neutralizing antibodies comprise antibodies stimulated in response to the vaccine of claim 56.

82. The method of claim 79, wherein said HIV neutralizing antibodies are obtained by immunizing an individual with the immunogenic mutant gpl20 polypeptide, and isolating the HIV neutralizing antibodies, or antiserum containing the antibodies, for the individual.

83. The method of claim 79, wherein said HIV neutralizing antibodies are obtained by immunizing an individual with the immunogenic mutant gpl20 polypeptide, generating hybridomas from antibody producing cells from the individual following said immunizing, and isolating the HIV neutralizing antibodies from hybridomas expressing the HIV neutralizing antibodies.

84. The method of claim 83, wherein the antibody producing cells comprise spleen cells.

85. A method of reducing or preventing human immunodeficiency virus (HIV) infection in a subject, comprising administering an immunogenic mutant gpl20 polypeptide that can stimulate a neutralizing antibody response against HIV to the subject, said mutant gpl20 polypeptide comprising an HIV gρl20 polypeptide having at least one amino acid mutation in at least one epitope of the HIV gpl20 polypeptide specifically bound by a non-neutralizing antibody.

86. The method of claim 85, wherein the HIV is HIV type 1 (HIV-1).

87. The method of claim 86, wherein the epitope of the HIV gpl20 polypeptide specifically bound by a non-neutralizing antibody comprises F43 cavity perimeter amino acid residues, VI loop amino acid residues, V2 loop amino acid residues, V3 loop amino acid residues, non-neutralizing face amino acid residues, Cl domain amino acid residues, C5 domain amino acid residues, co-receptor binding site amino acid residues, or a combination thereof.

88. The method of claim 86, wherein the immunogenic mutant gpl20 polypeptide comprises at least two amino acid mutations in at least one epitope of the HIV gpl20 polypeptide specifically bound by a non-neutralizing antibody.

89. The method of claim 86, wherein the immunogenic mutant gpl20 polypeptide comprises at least two amino acid mutations in at least two epitopes of the HIV gpl20 polypeptide specifically bound by a non-neutralizing antibody.

90. The method of claim 86, wherein the at least one amino acid mutation comprises an amino acid substitution that generates a glycosylation motif.

91. The method of claim 90, wherein the glycosylation motif comprises an N-linlced glycosylation motif.

92. The method of claim 86, wherein the immunogenic mutant gpl20 polypeptide comprises DI 13A, V127A,D180A, N197A; S256A; G473A; D474A; M475A; R476A; H92N and N94T; Q114N and L116T; S143T; E150N and G152T; K171N and Y173T; Q246N; P313N and R315T; I423N andN425T; or a combination thereof.

93. The method of claim 86, wherein the immunogenic mutant gpl20 polypeptide comprises D474A and R476A; G473A and M475A; D474A, M475A and R476A; or G473 A, D474A, M475A and R476A.

94. The method of claim 86, wherein the immunogenic mutant gpl20 polypeptide comprises H92N and N94T; Ql 14N and Ll 16T; S143T; E150N and G152T; K171N and Y173T; Q246N; P313N and R315T; or I423N and N425T; or a combination thereof.

95. The method of claim 86, wherein the immunogenic mutant gpl20 polypeptide comprises G473 A, D474A, M475A and R476A.

96. The method of claim 95, wherein the immunogenic mutant gpl20 polypeptide further comprises H92N, N94T, Q114N, L116T, S143T, E150N, G152T, K171N, Y173T, Q246N, P313N, R315T, I423N, and N425T.

97. The method of claim 95, wherein the immunogenic mutant gpl20 polypeptide further comprises a deletion of Cl domain amine acid residues, C5 domain amine acid residues, or a combination thereof.

98. A method of producing HIV neutralizing antibodies, comprising contacting antibody producing cells with an immunogenic mutant gpl20 polypeptide comprising an HIV gpl20 polypeptide having at least one amine acid mutation in at least one epitope of the HIV gpl20 polypeptide specifically bound by a non-neutralizing antibody, whereby the antibody producing cells are stimulated to pro duce HIV neutralizing antibodies.

99. The method of claim 98, wherein said contacting comprises contacting antibody producing cells in culture.

100. The method of claim 99, wherein the antibody producing cells comprise spleen cells.

101. The method of claim 99, further comprising isolating HIV neutralizing antibodies from the culture.

102. HIV neutralizing antibodies produced by the method of claim 98.

103. The HIV neutralizing antibodies of claim 102, which are HIV -1 neutralizing antibodies.

104. Isolated HIV neutralizing antibodies obtained by the method of claim 101.

105. The method of claim 98, wherein said contacting comprises administering the immunogenic mutant gpl20 polypeptide to a subject under conditions sufficient for the immunogenic gpl20 polypeptide to stimulate an immune response in the subject.

106. A method of eliciting HIV-1 neutralizing antibodies in a human antibody transgenic mouse by administering a vaccine comprising one or more mutant gpl20 polypeptides of anyone of the claims 1-5 or 25-28.

107. Isolated HIV-1 neutralizing antibodies obtained by harvesting spleen and lymph nodes from the mouse immunized by the method of claim 106.

108. The method of claim 105, further comprising obtaining antiserum comprising the HIV neutralizing antibodies from the subject.

109. The method of claim 108, further comprising isolating an HIV neutralizing antibody fraction from the antiserum.

110. The method of claim 108, further comprising substantially purifying HIV neutralizing antibodies from the antiserum.

111. The method of claim 105, further comprising isolating antibody producing cells from the subject.

112. Antiserum obtained by the method of claim 108.

113. The antiserum of claim 112, which comprises HIV-1 neutralizing antibodies.

114. An isolated HIV neutralizing antibody fraction obtained by the method of claim 109.

115. The isolated HIV neutralizing antibody fraction antiserum of claim 114, which comprises HIV -1 neutralizing antibodies.

116. A substantially purified HIV neutralizing antibody obtained by the method of claim 110.

117. The substantially purified HIV neutralizing antibody of claim 116, which comprises an HIV -1 neutralizing antibody.