Probing the structure of the human immunodeficiency virus surface glycoprotein gp120 with a panel of monoclonal antibodies.

We have probed the structures of monomeric and oligomeric gp120 glycoproteins from the LAI isolate of human immunodeficiency virus type 1 (HIV-1) with a panel of monoclonal antibodies (MAbs); most of these MAbs are directed against continuous epitopes. On native monomeric gp120, most of the first conserved (C1) domain is accessible to MAbs, although some regions of C1 are relatively inaccessible. All of the MAbs directed against the C2, C3, and C5 domains bind preferentially to denatured monomeric gp120, indicating that these regions of gp120 are poorly accessible on the native monomer, although the extreme C terminus in C5 is well exposed. Segments of the V1, V2, and V3 loops are exposed on the surface of monomeric gp120, although the base of the V3 loop is inaccessible. A portion of C4 is also available for MAb binding on monomeric gp120, as is the extreme C terminus in C5. However, on oligomeric gp120-gp41 complexes, only the V2 and V3 loops (and perhaps V1) are well exposed and a segment of the C4 region is partially exposed; continuous epitopes in C1 and C5 that are accessible to antibodies on monomeric gp120 are occluded on the oligomer. Although deletion of the V1, V2, and V3 loops resulted in increased exposure of several discontinuous epitopes overlapping the CD4-binding site, the exposure of most continuous epitopes on the monomeric gp120 glycoprotein was not affected. These results imply a HIV-1 gp120 structure in which the conserved continuous determinants are inaccessible; in some cases, this inaccessibility is due to intramolecular interactions between conserved regions, and in other cases, it is due to intermolecular interactions with other components of the glycoprotein spike. These findings have implications for the design of subunit vaccines based on gp120.     

Neutralizing monoclonal antibodies against human immunodeficiency virus type 2 gp120.

Monoclonal antibodies (MAbs) were obtained by immunizing mice with synthetic peptides corresponding to the third variable (V3) or the third conserved (C3) domain of the external envelope protein (gp120) of human immunodeficiency virus type 2 (HIV-2ROD). One MAb, designated B2C, which was raised against V3 peptide NKI26, bound to the surface of HIV-2-infected cells but not to their uninfected counterparts. B2C was capable of neutralizing cell-free and cell-associated virus infection in an isolate-specific fashion. The antibody-binding epitope was mapped to a 6-amino-acid peptide in the V3 variable domain which had the core sequence His-Tyr-Gln. Two MAbs, 2H1B and 2F19C, which were raised against the C3 peptide TND27 reacted with gp120 of HIV-2ROD in a Western immunoblot assay. The C3 epitopes recognized by these two MAbs appeared inaccessible because of their poor reactivity in a surface immunofluorescence assay. Although partial inhibition of syncytium formation was observed in the presence of the anti-C3 MAbs, their neutralizing activity appeared weak. Finally, the effects of these MAbs against CD4-gp120 binding were assessed. Partial inhibition of CD4-gp120 binding was observed in the presence of high concentrations of B2C. On the other hand, no inhibition of CD4-gp120 binding was observed in the presence of anti-C3 MAbs. Since complete neutralization could be achieved at a concentration corresponding to that of partial binding inhibition by B2C, some different mechanisms may be involved in the B2C-mediated neutralization. These results, taken together, indicated that analogous to the function of the V3 region of HIV-1, the V3 region of HIV-2ROD contained at least a type-specific fusion-inhibiting neutralizing epitope. In this respect, the V3 sequence of HIV-2 may be a useful target in an animal model for HIV vaccine development.     

Epitope mapping of the human immunodeficiency virus type 1 gp120 with monoclonal antibodies.

A soluble form of recombinant gp120 of human immunodeficiency virus type 1 was used as an immunogen for production of murine monoclonal antibodies. These monoclonal antibodies were characterized for their ability to block the interaction between gp120 and the acquired immunodeficiency syndrome virus receptor, CD4. Three of the monoclonal antibodies were found to inhibit this interaction, whereas the other antibodies were found to be ineffective at blocking binding. The gp120 epitopes which are recognized by these monoclonal antibodies were mapped by using a combination of Western blot (immunoblot) analysis of gp120 proteolytic fragments, immunoaffinity purification of fragments of gp120, and antibody screening of a random gp120 gene fragment expression library produced in the lambda gt11 expression system. Two monoclonal antibodies which blocked gp120-CD4 interaction were found to map to adjacent sites in the carboxy-terminal region of the glycoprotein, suggesting that this area is important in the interaction between gp120 and CD4. One nonblocking antibody was found to map to a position that was C terminal to this CD4 blocking region. Interestingly, the other nonblocking monoclonal antibodies were found to map either to a highly conserved region in the central part of the gp120 polypeptide or to a highly conserved region near the N terminus of the glycoprotein. N-terminal deletion mutants of the soluble envelope glycoprotein which lack these highly conserved domains but maintain the C-terminal CD4 interaction sites were unable to bind tightly to the CD4 receptor. These results suggest that although the N-terminal and central conserved domains of intact gp120 do not appear to be directly required for CD4 binding, they may contain information that allows other parts of the molecule to form the appropriate structure for CD4 interaction.     

The human immunodeficiency virus type 1 gp120 V2 domain mediates gp41-independent intersubunit contacts

The envelope protein of human immunodeficiency virus type 1 HIV-1 undergoes proteolytic cleavage in the Golgi complex to produce subunits designated gp120 and gp41, which remain noncovalently associated. While gp41 has a well-characterized oligomeric structure, the maintenance of gp41-independent gp120 intersubunit contacts remains a contentious issue. Using recombinant vaccinia virus to achieve high-level expression of gp120 in mammalian cells combined with gel filtration analysis, we were able to isolate a discrete oligomeric form of gp120. Oligomerization of gp120 occurred intracellularly between 30 and 120 min after synthesis. Analysis by sedimentation equilibrium unequivocally identified the oligomeric species as a dimer. In order to identify the domains involved in the intersubunit contact, we expressed a series of gp120 proteins lacking various domains and assessed the effects of mutation on oligomeric structure. Deletion of the V1 or V3 loops had little effect on the relative amounts of monomer and dimer in comparison to wild-type gp120. In contrast, deletion of either all or part of the V2 loop drastically reduced dimer formation, indicating that this domain is required for intersubunit contact formation. Consistent with this, the V2 loop of the dimer was less accessible than that of the monomer to a specific monoclonal antibody. Previous studies have shown that while the V2 loop is not an absolute requirement for viral entry, the absence of this domain reduces viral resistance to neutralization by monoclonal antibodies or sera. We propose that the quaternary structure of gp120 may contribute to resistance to neutralization by limiting the exposure of conserved epitopes.     

Characterization of the outer domain of the gp120 glycoprotein from human immunodeficiency virus type 1

The core of the gp120 glycoprotein from human immunodeficiency virus type 1 (HIV-1) is comprised of three major structural domains: the outer domain, the inner domain, and the bridging sheet. The outer domain is exposed on the HIV-1 envelope glycoprotein trimer and contains binding surfaces for neutralizing antibodies such as 2G12, immunoglobulin G1b12, and anti-V3 antibodies. We expressed the outer domain of HIV-1(YU2) gp120 as an independent protein, termed OD1. OD1 efficiently bound 2G12 and a large number of anti-V3 antibodies, indicating its structural integrity. Immunochemical studies with OD1 indicated that antibody responses against the outer domain of the HIV-1 gp120 envelope glycoprotein are rare in HIV-1-infected human sera that potently neutralize the virus. Surprisingly, such outer-domain-directed antibody responses are commonly elicited by immunization with recombinant monomeric gp120. Immunization with soluble, stabilized HIV-1 envelope glycoprotein trimers elicited antibody responses that more closely resembled those in the sera of HIV-1-infected individuals. These results underscore the qualitatively different humoral immune responses elicited during natural infection and after gp120 vaccination and help to explain the failure of gp120 as an effective vaccine.     

Generation and characterization of monoclonal antibodies to the putative CD4-binding domain of human immunodeficiency virus type 1 gp120.

A panel of seven monoclonal antibodies against the relatively conserved CD4-binding domain on human immunodeficiency virus type 1 (HIV-1) gp120 was generated by immunizing mice with purified gp120. These monoclonal antibodies reacted specifically with gp120 in an enzyme-linked immunosorbent assay and Western blots (immunoblots). By using synthetic peptides as antigens in the immunosorbent assay, the epitopes of these seven monoclonal antibodies were mapped to amino acid residues 423 to 437 of gp120. Further studies with radioimmunoprecipitation assays showed that they cross-reacted with both gp120 and gp160 of diverse HIV-1 isolates (HTLV-IIIB, HTLV-IIIRF, HTLV-IIIAL, and HTLV-IIIWMJ). They also bound specifically to H9 cells infected with HTLV-IIIB, HTLV-IIIRF, HTLV-IIIAL, HTLV-IIIZ84, and HTLV-IIIZ34 in indirect immunofluorescence studies. In addition, they blocked effectively the binding of HIV-1 to CD4+ C8166 cells. Despite the similarity of these properties, the monoclonal antibodies differed in neutralizing activity against HTLV-IIIB, HTLV-IIIRF, and HTLV-IIIAL, as demonstrated in both syncytium-forming assays and infectivity assays. Our findings suggest that these group-specific monoclonal antibodies to the putative CD4-binding domain on gp120 are potential candidates for development of therapeutic agents against acquired immunodeficiency disease syndrome.     

Evidence for a functional interaction between the V1/V2 and C4 domains of human immunodeficiency virus type 1 envelope glycoprotein gp120.

The domains of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein that are required for envelope function have been partially characterized. Little is known, however, about the nature of the interactions between these domains. To identify regions of the HIV-1 envelope glycoprotein that are involved in interactions necessary for proper envelope function, we constructed a series of 14 envelope recombinants between the env genes of two HIV-1 isolates. The envelope chimeras were examined for their ability to induce syncytia, to be proteolytically processed, and to function during a spreading viral infection. Our results demonstrate that the exchange between the two isolates of the first and second hypervariable regions (V1/V2) of gp120 results in defects in envelope glycoprotein processing, syncytium formation, and infectivity. Long-term passage of cultures infected with virus bearing a V1/V2 chimeric envelope glycoprotein leads to the emergence of a revertant virus with replication characteristics comparable to those of the wild type. Analysis of the revertant indicated that an Ile-->Met change in the C4 region of gp120 (between hypervariable regions V4 and V5) is responsible for the revertant phenotype. This single amino acid change restores infectivity without significantly affecting gp160 processing, CD4 binding, or the levels of virion-associated gp120. While the Ile-->Met change in C4 greatly enhances the fusogenic potential of the V1/V2 chimeric envelope glycoprotein, it has a detrimental effect on syncytium formation when analyzed in the context of the wild-type envelope. These results suggest that an interaction required for proper envelope glycoprotein function occurs between the V1/V2 and C4 regions of gp120.     

Presentation of native epitopes in the V1/V2 and V3 regions of human immunodeficiency virus type 1 gp120 by fusion glycoproteins containing isolated gp120 domains.

The immune response to viral glycoproteins is often directed against conformation- and/or glycosylation-dependent structures; synthetic peptides and bacterially expressed proteins are inadequate probes for the mapping of such epitopes. This report describes a retroviral vector system that presents such native epitopes on chimeric glycoproteins in which protein fragments of interest are fused to the C terminus of the N-terminal domain of the murine leukemia virus surface protein, gp70. The system was used to express two disulfide-bonded domains from gp120, the surface protein of human immunodeficiency virus type 1 (HIV-1), that include potent neutralization epitopes. The resulting fusion glycoproteins were synthesized at high levels and were efficiently transported and secreted. A fusion protein containing the HXB2 V1/V2 domain was recognized by an HIVIIIB-infected patient serum as well as by 17 of 36 HIV-1 seropositive hemophiliac, homosexual male and intravenous drug user patient sera. Many of these HIV+ human sera reacted with V1/V2 domains from several HIV-1 clones expressed in fusion glycoproteins, indicating the presence of cross-reactive antibodies against epitopes in the V1/V2 domain. Recognition of gp(1-263):V1/V2HXB2 by the HIVIIIB-infected human patient serum was largely blocked by synthetic peptides matching V1 but not V2 sequences, while recognition of this construct by a broadly cross-reactive hemophiliac patient serum was not blocked by individual V1 or V2 peptides or by mixtures of these peptides. A construct containing the V3 domain of the IIIB strain of HIV-1, gp(1-263):V3HXB2, was recognized by sera from a human and a chimpanzee that had been infected by HIVIIIB but not by sera from hemophiliac patients who had been infected with HIV-1 of MN-like V3 serotype. The reactive sera had significantly higher titers when assayed against gp(1-263):V3HXB2 than when assayed against matching V3 peptides. Immunoprecipitation of this fusion glycoprotein by the human serum was only partially blocked by V3 peptide, indicating that this infected individual produced antibodies against epitopes in V3 that were expressed on the fusion glycoprotein but not by synthetic peptides. These data demonstrated that the chimeric glycoproteins described here effectively present native epitopes present in the V1/V2 and V3 domains of gp120 and provide efficient methods for detection of antibodies directed against native epitopes in these regions and for characterization of such epitopes.     

Replication and neutralization of human immunodeficiency virus type 1 lacking the V1 and V2 variable loops of the gp120 envelope glycoprotein.

A human immunodeficiency virus type 1 (HIV-1) mutant lacking the V1 and V2 variable loops in the gp120 exterior envelope glycoprotein replicated in Jurkat lymphocytes with only modest delays compared with the wild-type virus. Revertants that replicated with wild-type efficiency rapidly emerged and contained only a few amino acid changes in the envelope glycoproteins compared with the parent virus. Both the parent and revertant viruses exhibited increased sensitivity to neutralization by antibodies directed against the V3 loop or a CD4-induced epitope on gp120 but not by soluble CD4 or an antibody against the CD4 binding site. This result demonstrates the role of the gp120 V1 and V2 loops in protecting HIV-1 from some subsets of neutralizing antibodies.     

Strain specificity and binding affinity requirements of neutralizing monoclonal antibodies to the C4 domain of gp120 from human immunodeficiency virus type 1.

The binding properties of seven CD4-blocking monoclonal antibodies raised against recombinant gp120 of human immunodeficiency virus type 1 strain MN (HIV-1MN) and two CD4-blocking monoclonal antibodies to recombinant envelope glycoproteins gp120 and gp160 of substrain IIIB of HIVLAI were analyzed. With a panel of recombinant gp120s from seven diverse HIV-1 isolates, eight of the nine antibodies were found to be strain specific and one was broadly cross-reactive. Epitope mapping revealed that all nine antibodies bound to epitopes located in the fourth conserved domain (C4) of gp120. Within this region, three distinct epitopes could be identified: two were polymorphic between HIV-1 strains, and one was highly conserved. Studies with synthetic peptides demonstrated that the conserved epitope, recognized by antibody 13H8, was located between residues 431 and 439. Site-directed mutagenesis of gp120 demonstrated that residue 429 and/or 432 was critical for the binding of the seven antibodies to gp120 from HIV-1MN. Similarly, residues 423 and 429 were essential for the binding of monoclonal antibody 5C2 raised against gp120 from HIV-1IIIB. The amino acids located at positions 423 and 429 were found to vary between strains of HIV-1 as well as between molecular clones derived from the MN and LAI isolates of HIV-1. Polymorphism at these positions prevented the binding of virus-neutralizing monoclonal antibodies and raised the possibility that HIV-1 neutralization serotypes may be defined on the basis of C4 domain sequences. Analysis of the binding characteristics of the CD4-blocking antibodies demonstrated that their virus-neutralizing activity was directly proportional to their gp120-binding affinity. These studies account for the strain specificity of antibodies to the C4 domain of gp120 and demonstrate for the first time that antibodies to this region can be as effective as those directed to the principal neutralizing determinant (V3 domain) in neutralizing HIV-1 infectivity.     

Mutations in human immunodeficiency virus type 1 gp41 affect sensitivity to neutralization by gp120 antibodies.

Three closely related molecular human immunodeficiency virus type 1 (HIV-1) clones, with differential neutralization phenotypes, were generated by cloning of an NcoI-BamHI envelope (env) gene fragment (HXB2R nucleotide positions 5221 to 8021) into the full-length HXB2 molecular clone of HIV-1 IIIB. These env gene fragments, containing the complete gp120 coding region and a major part of gp41, were obtained from three different biological clones derived from a chimpanzee-passaged HIV-1 IIIB isolate. Two of the viruses thus obtained (4.4 and 5.1) were strongly resistant to neutralization by infection-induced chimpanzee and human polyclonal antibodies and by HIV-1 IIIB V3-specific monoclonal antibodies and weakly resistant to soluble CD4 and a CD4-binding-site-specific monoclonal antibody. The third virus (6.8) was sensitive to neutralization by the same reagents. The V3 coding sequence and the gp120 amino acid residues important for the discontinuous neutralization epitope overlapping the CD4-binding site were completely conserved among the clones. However, the neutralization-resistant clones 4.4 and 5.1 differed from neutralization-sensitive clone 6.8 by two mutations in gp41. Exchange experiments confirmed that the 3' end of clone 6.8 (nucleotides 6806 to 8021; amino acids 346 to 752) conferred a neutralization-sensitive phenotype to both of the neutralization-resistant clones 4.4 and 5.1. From our study, we conclude that mutations in the extracellular portion of gp41 may affect neutralization sensitivity to gp120 antibodies.     

Antibody cross-competition analysis of the human immunodeficiency virus type 1 gp120 exterior envelope glycoprotein.

Forty-six monoclonal antibodies (MAbs) able to bind to the native, monomeric gp120 glycoprotein of the human immunodeficiency virus type 1 (HIV-1) LAI (HXBc2) strain were used to generate a competition matrix. The data suggest the existence of two faces of the gp120 glycoprotein. The binding sites for the viral receptor, CD4, and neutralizing MAbs appear to cluster on one face, which is presumably exposed on the assembled, oligomeric envelope glycoprotein complex. A second gp120 face, which is presumably inaccessible on the envelope glycoprotein complex, contains a number of epitopes for nonneutralizing antibodies. This analysis should be useful for understanding both the interaction of antibodies with the HIV-1 gp120 glycoprotein and neutralization of HIV-1.     

Characterization of neutralizing monoclonal antibodies to linear and conformation-dependent epitopes within the first and second variable domains of human immunodeficiency virus type 1 gp120.

A number of linear and conformation-dependent neutralizing monoclonal antibodies (MAbs) have been mapped to the first and second variable (V1 and V2) domains of human immunodeficiency virus type 1 (HIV-1) gp120. The majority of these MAbs are as effective at neutralizing HIV-1 infectivity as MAbs to the V3 domain and the CD4 binding site. The linear MAbs bind to amino acid residues 162 to 171, and changes at residues 183/184 (PI/SG) and 191/192/193 (YSL/GSS) within the V2 domain abrogate the binding of the two conformation-dependent MAbs, 11/68b and CRA-4, respectively. Surprisingly, a change at residue 435 (Y/H or Y/S), in a region of gp120 near the CD4 binding site (M. Kowalski, J. Potz, L. Basiripour, T. Dorfman, W. C. Goh, E. Terwilliger, A. Dayton, C. Rosen, W. Haseltine, and J. Sodroski, Science 237:1351-1355, 1987; L. A. Lasky, G. M. Nakamura, D. H. Smith, C. Fennie, C. Shimasaki, E. Patzer, P. Berman, T. Gregory, and D. Capon, Cell 50:975-985, 1987; and U. Olshevsky, E. Helseth, C. Furman, J. Li, W. Haseltine, and J. Sodroski, J. Virol. 64:5701-5707, 1990), abrogated gp120 recognition by both of the conformation-dependent MAbs. However, both MAbs 11/68b and CRA-4 were able to bind to HIV-1 V1V2 chimeric fusion proteins expressing the V1V2 domains in the absence of C4, suggesting that residues in C4 are not components of the epitopes but that amino acid changes in C4 may affect the structure of the V1V2 domains. This is consistent with the ability of soluble CD4 to block 11/68b and CRA-4 binding to both native cell surface-expressed gp120 and recombinant gp120 and suggests that the binding of the neutralizing MAbs to the virus occurs prior to receptor interaction. Since the reciprocal inhibition, i.e., antibody inhibition of CD4-gp120 binding, was not observed, the mechanism of neutralization is probably not a blockade of virus-receptor interaction. Finally, we demonstrate that linear sequences from the V2 region are immunogenic in HIV-1-infected individuals, suggesting that the primary neutralizing response may be directed to both V2 and V3 epitopes.     

Analysis of the interaction of the human immunodeficiency virus type 1 gp120 envelope glycoprotein with the gp41 transmembrane glycoprotein.

The human immunodeficiency virus type 1 (HIV-1) gp120 exterior envelope glycoprotein interacts with the viral receptor (CD4) and with the gp41 transmembrane envelope glycoprotein. To study the interaction of the gp120 and gp41 envelope glycoproteins, we compared the abilities of anti-gp120 monoclonal antibodies to bind soluble gp120 and a soluble glycoprotein, sgp140, that contains gp120 and gp41 exterior domains. The occlusion or alteration of a subset of gp120 epitopes on the latter molecule allowed the definition of a gp41 "footprint" on the gp120 antibody competition map. The occlusion of these epitopes on the sgp140 glycoprotein was decreased by the binding of soluble CD4. The gp120 epitopes implicated in the interaction with the gp41 ectodomain were disrupted by deletions of the first (C1) and fifth (C5) conserved gp120 regions. These deletions did not affect the integrity of the discontinuous binding sites for CD4 and neutralizing monoclonal antibodies. Thus, the gp41 interface on the HIV-1 gp120 glycoprotein, which elicits nonneutralizing antibodies, can be removed while retaining immunologically desirable gp120 structures.     

Immunochemical analysis of the gp120 surface glycoprotein of human immunodeficiency virus type 1: probing the structure of the C4 and V4 domains and the interaction of the C4 domain with the V3 loop.

We have probed the structure of the C4 and V3 domains of human immunodeficiency virus type 1 gp120 by immunochemical techniques. Monoclonal antibodies (MAbs) recognizing an exposed gp120 sequence, (E/K)VGKAMYAPP, in C4 were differentially sensitive to denaturation of gp120, implying a conformational component to some of the epitopes. The MAbs recognizing conformation-sensitive C4 structures failed to bind to a gp120 mutant with an alteration in the sequence of the V3 loop, and their binding to gp120 was inhibited by both V3 and C4 MAbs. This implies an interaction between the V3 and C4 regions of gp120, which is supported by the observation that the binding of some MAbs to the V3 loop was often enhanced by amino acid changes in an around the C4 region.     

Variant-specific monoclonal and group-specific polyclonal human immunodeficiency virus type 1 neutralizing antibodies raised with synthetic peptides from the gp120 third variable domain.

The third variable (V3) domain of the human immunodeficiency virus type 1 (HIV-1) external membrane glycoprotein gp120 is of crucial importance in eliciting neutralizing antibodies in infected persons. Polyclonal (PAb) and monoclonal (MAb) antibodies directed against selected epitopes in the V3 domain are valuable tools for analysis of the involvement of such sequences in neutralization and for definition of the relation between amino acid variability and immunological cross-reactions. The aim of this study was to obtain such site-specific antibodies. By using synthetic peptides derived from the V3 domain, a group-specific neutralizing PAb, two high-affinity HIV-1 IIIB neutralizing MAb, and two nonneutralizing MAb were raised. A 15-amino-acid peptide overlapping the tip of the V3 domain of HIV-1 MN was used to produce a rabbit PAb (W0/07). This PAb inhibited syncytium formation induced by HIV-1 IIIB and four field isolates. A similar IIIB-derived peptide was used to generate two murine immunoglobulin G1 (IgG1) MAb (IIIB-V3-13 and IIIB-V3-34). Pepscan analysis mapped the binding site of IIIB-V3-34 to the sequence IRIQRGPGR. The Kds of IIIB-V3-13 and IIIB-V3-34 for gp120 were 6.8 x 10(-11) and 1.6 x 10(-10) M, respectively. These MAb neutralized IIIB but not MN and inhibited syncytium formation induced by IIIB. They are applicable in enzyme-linked immunosorbent assays, immunocytochemistry, and flow cytometry. A peptide covering the left base of the V3 domain was used to generate two murine IgG1 MAb (IIIB-V3-21 and IIIB-V3-26). The binding site of IIIB-V3-21 was mapped to the sequence INCTRPN. These MAb did not neutralize HIV-1 and did not inhibit syncytium formation. This study supports the notion that HIV-1 neutralizing antibodies suitable for multiassay performance can be obtained with synthetic peptides and that high-affinity MAb can be generated. Such site-specific antibodies are useful reagents in the analysis of HIV-1 neutralization. In addition, the cross-neutralization of different viral strains by PAb generated through single-peptide immunization is directly relevant to vaccine development.     

Human monoclonal antibody 2G12 defines a distinctive neutralization epitope on the gp120 glycoprotein of human immunodeficiency virus type 1.

We have isolated and characterized human monoclonal antibody 2G12 to the gp120 surface glycoprotein of human immunodeficiency virus type 1 (HIV-1). This antibody potently and broadly neutralizes primary and T-cell line-adapted clade B strains of HIV-1 in a peripheral blood mononuclear cell-based assay and inhibits syncytium formation in the AA-2 cell line. Furthermore, 2G12 possesses neutralizing activity against strains from clade A but not from clade E. Complement- and antibody-dependent cellular cytotoxicity-activating functions of 2G12 were also defined. The gp120 epitope recognized by 2G12 was found to be distinctive; binding of 2G12 to LAI recombinant gp120 was abolished by amino acid substitutions removing N-linked carbohydrates in the C2, C3, V4, and C4 regions of gp120. This gp120 mutant recognition pattern has not previously been observed, indicating that the 2G12 epitope is unusual. consistent with this, antibodies able to block 2G12 binding to recombinant gp120 were not detected in significant quantities in 16 HIV-positive human serum samples.     

Functional interaction of constant and variable domains of human immunodeficiency virus type 1 gp120.

A previously reported amino acid substitution within the second conserved domain of the human immunodeficiency virus type 1 (HIV-1) gp120 envelope results in the production of noninfectious particles. Molecular characterization of spontaneous revertant viruses, which arose during long-term cocultures of this env mutant, revealed that an amino acid change within another region of gp120 could functionally compensate for the mutation and restore infectivity. In the current study, we have introduced a conservative amino acid substitution at this second-site revertant codon and observed a marked reduction in HIV-1 infectivity. During the passage of this defective virus in cocultures, yet another revertant appeared which contained an amino acid change within a variable region of gp120 which restored infectivity to near wild-type levels. These results, in combination with other point mutations that have been introduced into the HIV-1 envelope, suggest that at least three discrete regions of gp120 may interact during the establishment of a productive viral infection. This critical step occurs subsequent to the adsorption of virions to the cell surface and either prior to or concomitant with the fusion of viral and cellular membranes.     

Resistance to neutralization by broadly reactive antibodies to the human immunodeficiency virus type 1 gp120 glycoprotein conferred by a gp41 amino acid change.

A neutralization-resistant variant of human immunodeficiency virus type 1 (HIV-1) that emerged during in vitro propagation of the virus in the presence of neutralizing serum from an infected individual has been described. A threonine-for-alanine substitution at position 582 in the gp41 transmembrane envelope glycoprotein of the variant virus was responsible for the neutralization-resistant phenotype (M.S. Reitz, Jr., C. Wilson, C. Naugle, R. C. Gallo, and M. Robert-Guroff, Cell 54:57-63, 1988). The mutant virus also exhibited reduced sensitivity to neutralization by 30% of HIV-1-positive sera that neutralized the parental virus, suggesting that a significant fraction of the neutralizing activity within these sera can be affected by the amino acid change in gp41 (C. Wilson, M. S. Reitz, Jr., K. Aldrich, P. J. Klasse, J. Blomberg, R. C. Gallo, and M. Robert-Guroff, J. Virol. 64:3240-3248, 1990). It is shown here that the change of alanine 582 to threonine specifically confers resistance to neutralizing by antibodies directed against both groups of discontinuous, conserved epitopes related to the CD4 binding site on the gp120 exterior envelope glycoprotein. Only minor differences in binding of these antibodies to wild-type and mutant envelope glycoproteins were observed. Thus, the antigenic structure of gp120 can be subtly affected by an amino acid change in gp41, with important consequences for sensitivity to neutralization.     

Sequence diversity of V1 and V2 domains of gp120 from human immunodeficiency virus type 1: lack of correlation with viral phenotype.

We analyzed by PCR and direct sequencing 57 viral sequences from 47 individuals infected with human immunodeficiency virus type 1, focussing on the V1 and V2 regions of gp120. There was extensive length polymorphism in the V1 region, which rendered sequence alignment difficult. The V2 hypervariable locus also displayed considerable length variations, whereas flanking regions were relatively conserved. Two-thirds of the amino acid residues in these flanking regions were highly conserved (> 80%), presumably reflecting their critical contribution to V2 structure or function. We also characterized the syncytium-inducing properties of the isolates from which we derived sequence information. There was no correlation between V1 or V2 sequences and the viral phenotype, contrary to a previous report (M. Groenink, R. A. M. Fouchier, S. Broersen, C. H. Baker, M. Koot, A. B. van't Wout, H. G. Huisman, F. Miedema, M. Tersmette, and H. Schuitemaker, Science 260:1513-1516, 1993). The sequence heterogeneity described in this study provides information to suggest that it would be most difficult to exploit the V1 and V2 domains for vaccine development.