Characterization and epitope mapping of neutralizing monoclonal antibodies produced by immunization with oligomeric simian immunodeficiency virus envelope protein

In an attempt to generate broadly cross-reactive, neutralizing monoclonal antibodies (MAbs) to simian immunodeficiency virus (SIV), we compared two immunization protocols using different preparations of oligomeric SIV envelope (Env) glycoproteins. In the first protocol, mice were immunized with soluble gp140 (sgp140) from CP-MAC, a laboratory-adapted variant of SIVmacBK28. Hybridomas were screened by enzyme-linked immunosorbent assay, and a panel of 65 MAbs that recognized epitopes throughout the Env protein was generated. In general, these MAbs detected Env by Western blotting, were at least weakly positive in fluorescence-activated cell sorting (FACS) analysis of Env-expressing cells, and preferentially recognized monomeric Env protein. A subset of these antibodies directed toward the V1/V2 loop, the V3 loop, or nonlinear epitopes were capable of neutralizing CP-MAC, a closely related isolate (SIVmac1A11), and/or two more divergent strains (SIVsmDeltaB670 CL3 and SIVsm543-3E). In the second protocol, mice were immunized with unfixed CP-MAC-infected cells and MAbs were screened for the ability to inhibit cell-cell fusion. In contrast to MAbs generated against sgp140, the seven MAbs produced using this protocol did not react with Env by Western blotting and were strongly positive by FACS analysis, and several reacted preferentially with oligomeric Env. All seven MAbs potently neutralized SIVmac1A11, and several neutralized SIVsmDeltaB670 CL3 and/or SIVsm543-3E. MAbs that inhibited gp120 binding to CD4, CCR5, or both were identified in both groups. MAbs to the V3 loop and one MAb reactive with the V1/V2 loop interfered with CCR5 binding, indicating that these regions of Env play similar roles for SIV and human immunodeficiency virus. Remarkably, several of the MAbs generated against infected cells blocked CCR5 binding in a V3-independent manner, suggesting that they may recognize a region analogous to the conserved coreceptor binding site in gp120. Finally, all neutralizing MAbs blocked infection through the alternate coreceptor STRL33 much more efficiently than infection through CCR5, a finding that has important implications for SIV neutralization assays using CCR5-negative human T-cell lines.     

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.     

Epitope mapping of two immunodominant domains of gp41, the transmembrane protein of human immunodeficiency virus type 1, using ten human monoclonal antibodies.

Immunogenic regions of the gp41 transmembrane protein of human immunodeficiency virus type 1 (HIV-1) were previously mapped by examining polyclonal sera from HIV-infected patients and rodent polyclonal and monoclonal antibodies (MAbs) to peptides of gp41. To define the epitopes within these regions to which infected humans respond during the course of infection, the specificity of human MAbs to these regions had to be studied. Using 10 human MAbs identified initially by their reactivity to whole gp41 in HIV-1 lysates, the epitopes within the immunodominant region of gp41 and within a second immunogenic region of gp41 have been mapped. Thus, five MAbs (from five different patients) to the immunodominant domain of gp41 in the vicinity of the cysteines at positions 598 and 604 (hereinafter designated cluster I) reacted with a stretch of 11 amino acids from positions 590 to 600. Four of these five MAbs were reactive with linear epitopes, while one MAb required the conformation conferred by the disulfide bridge between the aforementioned cysteines. Three MAbs to cluster I revealed dissociation constants ranging from 10(-6) to 10(-8) M, depending on the MAb tested and the size of the synthetic or recombinant peptide used in the assay. Five additional MAbs reacted with a second immunogenic region between positions 644 and 663 (designated cluster II). Four of these five MAbs were specific for conformational determinants. Titration of sera from HIV-infected patients showed that there was about 100-fold more antibody to cluster I than to cluster II in patients' sera, confirming the immunodominance of cluster I.     

Humoral response to oligomeric human immunodeficiency virus type 1 envelope protein.

The humoral immune response to human immunodeficiency virus type 1 (HIV-1) is often studied by using monomeric or denatured envelope proteins (Env). However, native HIV-1 Env complexes that maintain quaternary structure elicit immune responses that are qualitatively distinct from those seen with monomeric or denatured Env. To more accurately assess the levels and types of antibodies elicited by HIV-1 infection, we developed an antigen capture enzyme-linked immunosorbent assay using a soluble, oligomeric form of HIV-1IIIB Env (gp140) that contains gp120 and the gp41 ectodomain. The gp140, captured by various monoclonal antibodies (MAbs), retained its native oligomeric structure: it bound CD4 and was recognized by MAbs to conformational epitopes in gp120 and gp41, including oligomer-specific epitopes in gp41. We compared the reactivities of clade B and clade E serum samples to captured Env preparations and found that while both reacted equally well with oligomeric gp140, clade B seras reacted more strongly with monomeric gp120 than did clade E samples. However, these differences were minimized when gp120 was captured by a V3 loop MAb, which may lead to increased exposure of the CD4 binding site. We also measured the ability of serum samples to block binding of MAbs to epitopes in gp120 and gp41. Clade B serum samples consistently blocked binding of oligomer-dependent MAbs to gp41 and, to a slightly lesser extent, MAbs to the CD4 binding site in gp120. Clade E serum samples showed equivalent or greater blocking of oligomer-dependent gp41 antibodies and considerably less blocking of CD4-binding-site MAbs. Finally, we found that < 5% of the antibodies in clade B sera bound to epitopes present only in monomeric gp120, 30% bound to epitopes present in both monomeric gp120 and oligomeric gp140, and 70% bound to epitopes present in oligomeric gp140, which includes gp41. Thus, captured oligomeric Env closely reflects the antigenic characteristics of Env protein on the surface of virions and infected cells, retains highly conserved epitopes that are recognized by antibodies raised against different clades, and makes it possible to detect a much greater fraction of total anti-HIV-1 Env activity in sera than does native monomeric gp120.     

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.     

Native oligomeric human immunodeficiency virus type 1 envelope glycoprotein elicits diverse monoclonal antibody reactivities.

We synthesized and purified a recombinant human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein, lacking the gp120/gp41 cleavage site as well as the transmembrane domain, that is secreted principally as a stable oligomer. Mice were immunized with separated monomeric and oligomeric HIV-1 Env glycoproteins to analyze the repertoire of antibody responses to the tertiary and quaternary structure of the protein. Hybridomas were generated and assayed for reactivity by immunoprecipitation of nondenatured Env protein. A total of 138 monoclonal antibodies (MAbs) were generated and cloned, 123 of which were derived from seven animals immunized with oligomeric Env. Within this group, a significant response was obtained against the gp41 ectodomain; 49 MAbs recognized epitopes in gp41, 82% of which were conformational. The influence of conformation on gp120 antigenicity was less pronounced, with 40% of the anti-gp120 MAbs binding to conformational epitopes, many of which blocked CD4 binding. Surprisingly, less than 7% of the MAbs derived from mice immunized with oligomeric Env recognized the V3 loop. In addition, MAbs to linear epitopes in the C-terminal domain of gp120 were not obtained, suggesting that this region of the protein may be partially masked in the oligomeric molecule. A total of 15 MAbs were obtained from two mice immunized with monomeric Env. Nearly half of these recognized the V3 loop, suggesting that this region may be a less predominant epitope in the context of oligomeric Env than in monomeric protein. Thus, immunization with oligomeric Env generates a large proportion of antibodies to conformational epitopes in both gp120 and gp41, many of which may be absent from monomeric Env.     

gp120-independent fusion mediated by the human immunodeficiency virus type 1 gp41 envelope glycoprotein: a reassessment.

In a natural context, membrane fusion mediated by the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins involves both the exterior envelope glycoprotein (gp120) and the transmembrane glycoprotein (gp41). Perez et al. (J. Virol. 66:4134-4143, 1992) reported that a mutant HIV-1 envelope glycoprotein containing only the signal peptide and carboxyl terminus of the gp120 exterior glycoprotein fused to the complete gp41 glycoprotein was properly cleaved and that the resultant gp41 glycoprotein was able to induce the fusion of even CD4-negative cells. In the studies reported herein, mutant proteins identical or similar to those studied by Perez et al. lacked detectable cell fusion activity. The proteolytic processing of these proteins was very inefficient, and one processed product identified by Perez et al. as the authentic gp41 glycoprotein was shown to contain carboxyl-terminal gp120 sequences. Furthermore, no fusion activity was observed for gp41 glycoproteins exposed after shedding of the gp120 glycoprotein by soluble CD4. Thus, evidence supporting a gp120-independent cell fusion activity for the HIV-1 gp41 glycoprotein is currently lacking.     

Determinants of human immunodeficiency virus type 1 envelope glycoprotein oligomeric structure.

Oligomerization of the human immunodeficiency virus type 1 envelope (env) glycoproteins is mediated by the ectodomain of the transmembrane glycoprotein gp41. We report that deletion of gp41 residues 550 to 561 resulted in gp41 sedimenting as a monomer in sucrose gradients, while the gp160 precursor sedimented as a mixture of monomers and oligomers. Deletion of the nearby residues 571 to 582 did not affect the oligomeric structure of gp41 or gp160, but deletion of both sequences resulted in monomeric gp41 and predominantly monomeric gp160. Deletion of residues 655 to 665, adjacent to the membrane-spanning sequence, partially dissociated the gp41 oligomer while not affecting the gp160 oligomeric structure. In contrast, deletion of residues 510 to 518 from the fusogenic hydrophobic N terminus of gp41 did not affect the env glycoprotein oligomeric structure. Even though the mutant gp160 and gp120 molecules were competent to bind CD4, the mutations impaired fusion function, gp41-gp120 association, and gp160 processing. Furthermore, deletion of residues 550 to 561 or 550 to 561 plus 571 to 582 modified the antigenic properties of the proximal residues 586 to 588 and the distal residues 634 to 664. Our results indicate that residues 550 to 561 are essential for maintaining the gp41 oligomeric structure but that this sequence and additional sequences contribute to the maintenance of gp160 oligomers. Residues 550 to 561 map to the N terminus of a putative amphipathic alpha-helix (residues 550 to 582), whereas residues 571 to 582 map to the C terminus of this sequence.     

Epitope mapping of monoclonal antibodies against human immunodeficiency virus type 1 structural proteins by using peptides.

Murine monoclonal antibodies directed against the structural proteins p17 and p24 of human immunodeficiency virus type 1 were investigated in an epitope mapping system. Overlapping peptides consisting of 15 amino acids of the p17 and p24 protein, respectively, were used as competitors in an enzyme-linked immunosorbent assay. Three different immunogenic regions (A, B, and C) could be defined, one on p17 and two on p24. Twenty monoclonal antibodies reacted with the human immunodeficiency virus type 1 peptides of region B, although differences in the reactivity of these antibodies with human immunodeficiency virus type 2 and simian immunodeficiency virus strain mac were detectable. Recognized epitopes were characterized by computer analysis as described by T.P. Hopp and K.R. Woods (Proc. Natl. Acad. Sci. USA 78:3824-3828, 1981) and P.Y. Chou and G.D. Fasman (Biochemistry 13:222-245, 1974).     

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.     

Differential recognition of epitopes present on monomeric and oligomeric forms of gp160 glycoprotein of human immunodeficiency virus type 1 by human monoclonal antibodies.

The mechanism of infectivity neutralization of human immunodeficiency virus type 1 (HIV-1) by Ig is poorly understood. Three human monoclonal antibodies (mAbs 1b12, 2G12 and 2F5) that are able to neutralize primary isolates of HIV-1 in vitro have been shown to act synergistically. In the present study this synergy was analyzed by measuring the epitope accessibility and binding kinetics for these three mAbs with respect to monomeric and oligomeric env protein gp160 IIIB using surface plasmon resonance. The results indicate that oligomerization of gp160 affects the accessibility of some of the epitopes recognized by the mAbs and provide some insight into the mechanism of synergy between different anti-(HIV-1) mAbs.     

The N-terminal 31 amino acids of human immunodeficiency virus type 1 envelope protein gp120 contain a potential gp41 contact site.

We have compared the expression of full-length gp160 envelope protein from human immunodeficiency virus type 1 with that of a deletion mutant lacking the N-terminal 31 amino acids of the mature protein (gp160 delta 32). The gp160 and gp160 delta 32 proteins are processed to yield gp41 and gp120 or gp120 delta 32, respectively. In contrast to full-length gp120, gp120 delta 32 failed to associate with gp41 at the cell surface, despite conformational integrity as judged by soluble CD4 binding. Thus, the N-terminal 31 amino acids of gp120, which contain hyperconserved sequences, are likely involved in forming a contact site for gp41.     

Binding of anti-membrane-proximal gp41 monoclonal antibodies to CD4-liganded and -unliganded human immunodeficiency virus type 1 and simian immunodeficiency virus virions

The broadly neutralizing monoclonal antibodies (MAbs) 4E10, 2F5, and Z13e1 target membrane-proximal external region (MPER) epitopes of HIV-1 gp41 in a manner that remains controversial. The requirements for initial lipid bilayer binding and/or CD4 ligation have been proposed. To further investigate these issues, we probed for binding of these MAbs to human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) virions with protein A-conjugated gold (PAG) nanoparticles using negative-stain electron microscopy. We found moderate levels of PAG associated with unliganded HIV-1 and SIV virions incubated with the three MAbs. Significantly higher levels of PAG were associated with CD4-liganded HIV-1 (epitope-positive) but not SIV (epitope-negative) virions. A chimeric SIV virion displaying the HIV-1 4E10 epitope also showed significantly higher PAG association after CD4 ligation and incubation with 4E10. MAbs accumulated rapidly on CD4-liganded virions and slowly on unliganded virions, although both reached similar levels in time. Anti-MPER epitope-specific binding was stable to washout. Virions incubated with an irrelevant MAb or CD4-only (no MAb) showed negligible PAG association, as did a vesicle-rich fraction devoid of virions. Preincubation with Fab 4E10 inhibited both specific and nonspecific 4E10 IgG binding. Our data provide evidence for moderate association of anti-MPER MAbs to viral surfaces but not lipid vesicles, even in the absence of cognate epitopes. Significantly greater MAb interaction occurs in epitope-positive virions following long incubation or CD4 ligation. These findings are consistent with a two-stage binding model where these anti-MPER MAbs bind first to the viral lipid bilayer and then to the MPER epitopes following spontaneous or induced exposure.     

Dissection of human immunodeficiency virus type 1 entry with neutralizing antibodies to gp41 fusion intermediates

Human immunodeficiency virus type 1 (HIV-1) entry requires conformational changes in the transmembrane subunit (gp41) of the envelope glycoprotein (Env) involving transient fusion intermediates that contain exposed coiled-coil (prehairpin) and six-helix bundle structures. We investigated the HIV-1 entry mechanism and the potential of antibodies targeting fusion intermediates to block Env-mediated membrane fusion. Suboptimal temperature (31.5 degrees C) was used to prolong fusion intermediates as monitored by confocal microscopy. After transfer to 37 degrees C, these fusion intermediates progressed to syncytium formation with enhanced kinetics compared with effector-target (E/T) cell mixtures that were incubated only at 37 degrees C. gp41 peptides DP-178, DP-107, and IQN17 blocked fusion more efficiently (5- to 10-fold-lower 50% inhibitory dose values) when added to E/T cells at the suboptimal temperature prior to transfer to 37 degrees C. Rabbit antibodies against peptides modeling the N-heptad repeat or the six-helix bundle of gp41 blocked fusion and viral infection at 37 degrees C only if preincubated with E/T cells at the suboptimal temperature. Similar fusion inhibition was observed with human six-helix bundle-specific monoclonal antibodies. Our data demonstrate that antibodies targeting gp41 fusion intermediates are able to bind to gp41 and arrest fusion. They also indicate that six-helix bundles can form prior to fusion and that the lag time before fusion occurs may include the time needed to accumulate preformed six-helix bundles at the fusion site.     

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.     

Cross-reactivity of anti-human immunodeficiency virus type 1 gp41 antibodies with human astrocytes and astrocytoma cell lines.

An antigen expressed by astrocytes in human brain tissue and by various human astrocytoma cell lines was shown to cross-react with a monoclonal antibody generated against amino acids (aa) 584 to 609 of the transmembrane protein gp41 of human immunodeficiency virus type 1 (HIV-1). This region is an immunodominant segment of gp41, and high levels of antibodies against this epitope have been detected in both serum and cerebrospinal fluid of HIV-infected individuals at all stages of HIV infection. Immunohistochemistry with this monoclonal antibody demonstrated the presence of a cross-reactive antigen in human brain tissue, with an increased frequency and intensity of staining in HIV-positive individuals when compared with HIV-negative controls. By using a panel of HIV-positive and -negative sera, we show that antibodies in HIV-positive serum specifically bound to the surfaces of human astrocytoma cells. HIV-positive sera depleted of antibodies recognizing gp41 aa 584 to 609 showed a significant diminution in cell surface binding. Conversely, the serum antibodies that bound to and were eluted from the aa 584 to 609 peptide also bound to the astrocyte cell surface. To identify the target antigen, the immunoreactivity of three astrocytoma cell lines was examined. By immunoprecipitation of metabolically labeled cell lysates and Western blot (immunoblot) analysis, we identified a protein of approximately 100 kDa as the target antigen. Cross-reactive antibodies between HIV proteins and astrocyte epitopes, such as this 100-kDa protein and others previously reported, suggests that an autoimmune response against these target antigens may disrupt the normal functions of astrocytes.     

A mutation in the human immunodeficiency virus type 1 Gag protein destabilizes the interaction of the envelope protein subunits gp120 and gp41

The Gag protein of human immunodeficiency virus type 1 (HIV-1) associates with the envelope protein complex during virus assembly. The available evidence indicates that this interaction involves recognition of the gp41 cytoplasmic tail (CT) by the matrix protein (MA) region of Pr55(Gag). Here we show that substitution of Asp for Leu at position 49 (L49D) in MA results in a specific reduction in particle-associated gp120 without affecting the levels of gp41. Mutant virions were markedly reduced in single-cycle infectivity despite a relatively modest defect in fusion with target cells. Studies with HIV-1 particles containing decreased levels of envelope proteins suggested that the L49D mutation also inhibits a postentry step in infection. Truncation of the gp41 tail, or pseudotyping by vesicular stomatitis virus glycoprotein, restored both the fusion and infectivity of L49D mutant virions to wild-type levels. Truncation of gp41 also resulted in equivalent levels of gp120 on particles with and without the MA mutation and enhanced the replication of the L49D mutant virus in T cells. The impaired fusion and infectivity of L49D mutant particles were also complemented by a single point mutation in the gp41 CT that disrupted the tyrosine-containing endocytic motif. Our results suggest that an altered interaction between the MA domain of Gag and the gp41 cytoplasmic tail leads to dissociation of gp120 from gp41 during HIV-1 particle assembly, thus resulting in impaired fusion and infectivity.     

Antibodies with specificity to native gp120 and neutralization activity against primary human immunodeficiency virus type 1 isolates elicited by immunization with oligomeric gp160.

Current human immunodeficiency virus type 1 (HIV-1) envelope vaccine candidates elicit high antibody binding titers with neutralizing activity against T-cell line-adapted but not primary HIV-1 isolates. Serum antibodies from these human vaccine recipients were also found to be preferentially directed to linear epitopes within gp120 that are poorly exposed on native gp120. Systemic immunization of rabbits with an affinity-purified oligomeric gp160 protein formulated with either Alhydrogel or monophosphoryl lipid A-containing adjuvants resulted in the induction of high-titered serum antibodies that preferentially bound epitopes exposed on native forms of gp120 and gp160, recognized a restricted number of linear epitopes, efficiently bound heterologous strains of monomeric gp120 and cell surface-expressed oligomeric gp120/gp41, and neutralized several strains of T-cell line-adapted HIV-1. Additionally, those immune sera with the highest oligomeric gp160 antibody binding titers had neutralizing activity against some primary HIV-1 isolates, using phytohemagglutinin-stimulated peripheral blood mononuclear cell targets. Induction of an antibody response preferentially reactive with natively folded gp120/gp160 was dependent on the tertiary structure of the HIV-1 envelope immunogen as well as its adjuvant formulation, route of administration, and number of immunizations administered. These studies demonstrate the capacity of a soluble HIV-1 envelope glycoprotein vaccine to elicit an antibody response capable of neutralizing primary HIV-1 isolates.     

Oligomeric structure of gp41, the transmembrane protein of human immunodeficiency virus type 1.

We characterized the structural forms of the human immunodeficiency virus env-encoded proteins with a panel of monoclonal and polyclonal antibodies. Western blot (immunoblot) assays with antibodies specific for gp41 invariably recognized a major component of 160 kilodaltons and a less intense component of 120 kilodaltons in viral lysates. We demonstrated that these species are noncovalently associated tetramers and trimers of gp41 which represent the native form of this protein in virions. These complexes were stable when boiled in the presence of low concentrations of sodium dodecyl sulfate but were dissociated to gp41 monomers at high sodium dodecyl sulfate concentrations. Moreover, two human monoclonal antibodies preferentially recognized the oligomeric complexes over monomeric gp41 in Western blots, indicating the presence of epitopes recognized by the human immune system on the gp41 multimers which are not efficiently expressed by the dissociated monomers. The demonstration of the existence of multimeric env complexes and the enhanced and altered antigenicity of such multimers may be relevant to the design of subunit and recombinant human immunodeficiency virus env vaccines.