Neutralization of HIV-1 primary isolates by polyclonal and monoclonal human antibodies

To examine antibody-mediated neutralization of HIV-1 primary isolates in vitro, we tested sera and plasma from infected individuals against four clade B primary isolates. These isolates were analyzed further for neutralization by a panel of several human anti-HIV-1 mAb in order to identify the neutralizing epitopes of these viruses. Each of the HIV-1+ serum and plasma specimens tested had neutralizing activities against one or more of the four primary isolates. Of the three individual sera, one (FDA-2) neutralized all of the four isolates, while the other two sera were effective against only one virus. The pooled plasma and serum samples reacted broadly with these isolates. Based on the neutralizing activities of the mAb panel, each virus isolate exhibited a distinct pattern of reactivity, suggesting antigenic diversity among clade B viruses. Neutralizing epitopes were found in the V3 loop and CD4- binding domain of gp120, as well as near the transmembrane region (cluster II epitope) of gp41. A mAb directed to the cluster I epitope of gp41 near the immunodominant disulfide loop weakly neutralized one primary isolate. None of the mAb in the panel affected one primary isolate, US4, although this virus was sensitive to neutralization by some of the polyclonal antibody specimens. This isolate was also resistant to neutralization by a cocktail of 10 mAb, most of which individually inhibited at least one of the other three viruses tested. These results suggest that neutralizing activity for this latter virus is present in certain HIV-1+ sera/plasma, but is not exhibited by the mAb in the panel. Thus, effective neutralizing antibodies against primary isolates can be generated by humans upon exposure to HIV-1, but not all of these antigenic specificities are represented in a large panel of human anti-HIV-1 mAb.      

Identification of an N-linked glycan in the V1-loop of HIV-1 gp120 influencing neutralization by anti-V3 antibodies and soluble CD4

Summary Glycosylation is necessary for HIV-1 gp120 to attain a functional conformation, and individual N-linked glycans of gp120 are important, but not essential, for replication of HIV-1 in cell culture. We have constructed a mutant HIV-1 infectious clone lacking a signal for N-linked glycosylation in the V1-loop of HIV-1 gp120. Lack of an N-linked glycan was verified by a mobility enhancement of mutant gp120 in SDS-gel electrophoresis. The mutated virus showed no differences in either gp120 content per infectious unit or infectivity, indicating that the N-linked glycan was neither essential nor affecting viral infectivity in cell culture. We found that the mutated virus lacking an N-linked glycan in the V1-loop of gp120 was more resistant to neutralization by monoclonal antibodies to the V3-loop and neutralization by soluble recombinant CD4 (sCD4). Both viruses were equally well neutralized by ConA and a conformation dependent human antibody IAM-2G12. This suggests that the N-linked glycan in the V1-loop modulates the three-dimensional conformation of gp120, without changing the overall functional integrity of the molecule.     

Epitope mapping and characterization of a novel CD4-induced human monoclonal antibody capable of neutralizing primary HIV-1 strains

Human immunodeficiency virus (HIV-1) enters target cells by binding its gp120 exterior envelope glycoprotein to CD4 and one of the chemokine receptors, CCR5 or CXCR4. CD4-induced (CD4i) antibodies bind gp120 more efficiently after CD4 binding and block the interaction with the chemokine receptor. Examples of CD4i antibodies are limited, and the prototypes of the CD4i antibodies exhibit only weak neutralizing activity against primary, clinical HIV-1 isolates. Here we report the identification of a novel antibody, E51, that exhibits CD4-induced binding to gp120 and neutralizes primary HIV-1 more efficiently than the prototypic CD4i antibodies. The E51 antibody blocks the interaction of gp120-CD4 complexes with CCR5 and binds to a highly conserved, basic gp120 element composed of the beta 19-strand and surrounding structures. Thus, on primary HIV-1 isolates, this gp120 region, which has been previously implicated in chemokine receptor binding, is accessible to a subset of CD4i antibodies.     

Role of maternal humoral immunity in vertical transmission of HIV-1 subtype E in Thailand.

The significance of the maternal humoral immune response in relation to vertical transmission of HIV-1 was investigated in 123 mothers infected with subtype E from Thailand. Antibody binding titers to HIV-1 env domains (monomeric gp120, the CD4/gp120 binding site [BS], V3 loop, and gp41) and antibody-mediated neutralization of primary and T-cell line-adapted (TCLA) subtypes B and E HIV-1 isolates were investigated. No correlation between maternal anti HIV-1 antibodies at delivery and vertical transmission of HIV-1 subtype E was found. However, a trend to higher titer antibody-mediated cross-neutralization of a heterologous subtype B TCLA isolate, HIV-1MN, was observed in nontransmitting mothers postpartum. The HIV-1-specific antibody titers in these infected mothers increased significantly from delivery to 6 months postpartum (p < .05), but this was only partially attributable to hemodilution and an additional factor or factors appear to affect humoral immunity to HIV-1 during late pregnancy.     

Conserved N-linked oligosaccharides of the C-terminal portion of human immunodeficiency virus type 1 gp120 and viral susceptibility to neutralizing antibodies

Summary We have constructed a mutated infectious HIV variant lacking the signals for addition of three N-linked glycans situated in the V4, C4 and V5 regions of HIV gp120. When comparing mutated virus with wildtype virus we found essentially no differences in the phenotypic characteristics of the two viruses except for the expected electrophoretic mobility shift of radioimmuno-precipitated mutated gp120, resulting from the missing N-glycans. Thus, the infectivity titer and the capacity to induce syncytia were similar for the two viruses. The sensitivity of mutant and wildtype virus to a number of neutralizing agents was determined. As expected, the mutant virus was significantly less sensitive to neutralization by Con A, with affinity for the N-glycans eliminated. We found, however, that antibodies to the V3 loop and sCD4 neutralized wildtype virus as efficiently as mutant virus, whereas 2G12, a monoclonal antibody, binding to a discontinuous neutralization epitope, and GP13, binding to the CD4-binding domain, neutralized wildtype virus better than mutant virus. Altogether the data suggest that the three conserved N-linked glycans, despite their location in immediate association with the CD4-binding domain, which is an important neutralization epitope, are not essential for virus replication in cell culture and they are not engaged in shielding neutralization epitopes of gp120 from neutralizing antibodies. However, the glycans evidently influence the three-dimensional conformation of gp120, since their presence increases the availability of the neutralization epitope of 2G12.     

N-linked glycosylation in C2 region of HIV-1 envelope reduces sensitivity to neutralizing antibodies

N-linked glycosylation at specific sites on human immunodeficiency virus (HIV)--1 gp120 envelope glycoprotein is believed to act as a glycan shield to protect the viral neutralizing epitopes. Various glycosylation sites have been shown to affect the sensitivity to antibody-mediated neutralization. These include sites on V1V2, C2, base of V3, V5 and C5. Among these, the sites around the base of V3 loop have been most consistently found to associate with neutralization sensitivity in subtype B viruses. In contrast, we found that N-linked glycosylation sites at the junction of V2--C2 and in the middle of C2 were responsible for the neutralization resistance in CRF01_A/E, whereas sites at the base of V3 loop and in V1 and V5 did not affect the neutralization phenotype.     

Sensitivity of HIV-1 to neutralization by antibodies against O-linked carbohydrate epitopes despite deletion of O-glycosylation signals in the V3 loop

Summary It has been suggested that threonine or serine residues in the V3 loop of HIV-1 gp120 are glycosylated with the short-chain O-linked oligosaccharides Tn or sialosyl-Tn that function as epitopes for broadly neutralizing carbohydrate specific antibodies. In this study we examined whether mutation of such threonine or serine residues could decrease the sensitivity to infectivity inhibition by Tn or sialosyl-Tn specific antibodies. All potentially O-glycosylated threonine and serine residues in the V3 loop of cloned HIV-1_BRU were mutagenized to alanine thus abrogating any O-glycosylation at these sites. Additionally, one of these T-A mutants (T308A) also abrogated the signal for N-glycosylation at N306 inside the V3-loop. The mutant clones were compared with the wild type virus as to sensitivity to neutralization with monoclonal and polyclonal antibodies specific for the tip of the V3 loop of BRU or for the O-linked oligosaccharides Tn or sialosyl-Tn. Deletion of the N-linked oligosaccharide at N306 increased the neutralization sensitivity to antibodies specific for the tip of the loop, which indicates that N-linked glycosylation modulates the accessibility to this immunodominant epitope. However, none of the mutants with deletions of O-glycosylation signals in the V3 loop displayed any decrease in sensitivity to anti-Tn or anti-sialosyl-Tn antibody. This indicates that these broadly specific neutralization epitopes are located outside the V3 loop of gp120.     

Selective recognition of oligomeric HIV-1 primary isolate envelope glycoproteins by potently neutralizing ligands requires efficient precursor cleavage

A critical component of an effective HIV vaccine will be the induction of broadly neutralizing antibodies. Comprising the HIV spike, the exterior envelope glycoprotein gp120 and the transmembrane glycoprotein gp41 mediate receptor binding, viral entry, and are the targets for neutralizing antibodies. The gp120 and gp41 glycoproteins are derived from the gp160 precursor glycoprotein and following gp160 glycosylation, oligomerization and cleavage in the endoplasmic reticulum and Golgi, remain as non-covalently associated trimers of heterodimers. Previously, using cell-surface envelope glycoproteins derived from infection of a laboratory-adapted HIV-1 strain, a correlation had been established between the binding of gp120-directed antibodies to the viral glycoprotein and the ability of the antibodies to neutralize laboratory-adapted isolates. However, this has been more difficult to demonstrate for glycoproteins derived from primary patient isolates. Here, using a FACS-based method, we report that only gp120-directed neutralizing antibodies and the neutralizing ligand soluble CD4 efficiently bind to glycoproteins derived from the JR-FL primary isolate provided that the gp160 precursor protein is efficiently cleaved. Precursor cleavage was demonstrated by cell-surface biotinylation and Western blotting. In stark contrast, both non-neutralizing and neutralizing antibodies bind non-cleaved envelope glycoproteins from JR-FL and YU2 isolates. These data imply that significant changes in Env spike structure are dependent upon precursor gp160 cleavage and are consistent with a restricted-binding-to-Env model of neutralization. The data also have implications in regards to the use and design of non-cleaved envelope glycoprotein trimeric immunogens as a means to selectively and preferentially present neutralizing epitopes to the host immune system.     

Analysis of the neutralizing antibody response elicited in rabbits by repeated inoculation with trimeric HIV-1 envelope glycoproteins

The elicitation of broadly neutralizing antibodies directed against the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins, gp120 and gp41, remains a major challenge. Attempts to utilize monomeric gp120 as an immunogen to elicit high titers of neutralizing antibodies have been disappointing. Envelope glycoprotein constructs that better reflect the trimeric structure of the functional envelope spike have exhibited improved immunogenicity compared with monomeric gp120. We have described soluble gp140 ectodomain constructs with a heterologous trimerization motif; these have previously been shown to elicit antibodies in mice that were able to neutralize a number of HIV-1 isolates, among them primary isolate viruses. Recently, solid-phase proteoliposomes retaining the envelope glycoproteins as trimeric spikes in a physiologic membrane setting have been described. Here, we compare the immunogenic properties of these two trimeric envelope glycoprotein formulations and monomeric gp120 in rabbits. Both trimeric envelope glycoprotein preparations generated neutralizing antibodies more effectively than gp120. In contrast to monomeric gp120, the trimeric envelope glycoproteins elicited neutralizing antibodies with some breadth of neutralization. Furthermore, repeated boosting with the soluble trimeric formulations resulted in an increase in potency that allowed neutralization of a subset of neutralization-resistant HIV-1 primary isolates. We demonstrate that the neutralization is concentration-dependent, is mediated by serum IgG and that the major portion of the neutralizing activity is not directed against the gp120 V3 loop. Thus, mimics of the trimeric envelope glycoprotein spike described here elicit HIV-1-neutralizing antibodies that could contribute to a protective immune response and provide platforms for further modifications to improve the efficiency of this process.     

Synergistic interaction between ligands binding to the CD4 binding site and V3 domain of human immunodeficiency virus type I gp120.

We demonstrate that soluble CD4 (sCD4) or a monoclonal antibody (mAb), 39.13g, binding to a conformational epitope of gp120 involved in CD4 binding, and mAbs binding to the V3 domain of gp120, can synergistically neutralize human immunodeficiency virus type I (HIV-1). In contrast, a neutralizing mAb binding to a linear epitope within the CD4 binding domain was unable to exert a synergistic effect in combination with V3 mAbs, suggesting that synergism is dependent on ligands binding to the critical, discontinuous, gp120 residues constituting the CD4 binding site. A number of V3 mAbs showed increased binding to virion gp120 in the presence of sCD4, suggesting a mechanism for the synergistic neutralization. This effect was not observed with recombinant or detergent solubilized viral gp120, suggesting that the oligomeric structure of gp120 on viral particles affects V3 epitope exposure. This hypothesis is supported by the ability of two new V3 mAbs, 8/38c and 8/64b, to only neutralize HIV-1 in the presence of sCD4 or mAb 39.13g; binding studies demonstrate that these mAbs only bind to virion gp120 in the presence of sCD4. Thus, V3 epitope exposure is modulated by the interaction of virion gp120 with ligands specific for the CD4 binding domain and results in enhanced antibody-mediated neutralization.     

The prolonged culture of human immunodeficiency virus type 1 in primary lymphocytes increases its sensitivity to neutralization by soluble CD4

Primary strains of human immunodeficiency virus type 1 (HIV-1) are known to adapt to replication in cell lines in vitro by becoming sensitive to soluble CD4 (sCD4) and neutralizing antibodies (NAb). T-cell lines favor isolation of variants that use CXCR4 as a co-receptor, while primary isolates predominantly use CCR5. We have now studied how a primary R5 isolate, CC1/85, adapts to prolonged replication in primary human peripheral blood mononuclear cells (PBMC). After 19 passages, a variant virus, CCcon.19, had increased sensitivity to both sCD4 and NAb b12 that binds to a CD4-binding site (CD4BS)-associated epitope, but decreased sensitivity to anti-CD4 antibodies. CCcon.19 retains the R5 phenotype, its resistance to other NAbs was unaltered, its sensitivity to various entry inhibitors was unchanged, and its ability to replicate in macrophages was modestly increased. We define CCcon.19 as a primary T-cell adapted (PTCA) variant. Genetic sequence analysis combined with mutagenesis studies on clonal, chimeric viruses derived from CC1/85 and the PTCA variant showed that the most important changes were in the V1/V2 loop structure, one of them involving the loss of an N-linked glycosylation site. Monomeric gp120 proteins expressed from CC1/85 and the PTCA variant did not differ in their affinities for sCD4, suggesting that the structural consequences of the sequence changes were manifested at the level of the native, trimeric Env complex. Overall, the adaptation process probably involves selection for variants with higher CD4 affinity and hence greater fusion efficiency, but this also involves the loss of some resistance to neutralization by agents directed at or near to the CD4BS. The loss of neutralization resistance is of no relevance under in vitro conditions, but NAbs would presumably be a counter-selection pressure against such adaptive changes in vivo, at least when the humoral immune response is intact.     

Analysis of HIV-1 subtype B third variable region peptide motifs for induction of neutralizing antibodies against HIV-1 primary isolates

The HIV-1 gp120 V3 loop is a potent inducer of neutralizing antibodies for T cell line adapted-HIV-1, but less so for primary isolates. We hypothesized that peptides representative of the diversity of natural HIV-1 V3 loop variants might capture elements of conserved higher order structures and so stimulate broadly reactive neutralizing antibodies. We designed a panel of 29 subtype B V3 sequences postulated to reflect the range of V3 diversity. These peptides were used to immunize guinea pigs. The most effective peptide (62.19) clustered around the subtype B consensus sequence and induced antibodies that reproducibly neutralized 31% of the subtype B HIV-1 primary isolates evaluated, but exhibited limited cross-neutralization of non-subtype B HIV-1 strains. Taken together, these data demonstrated that the limited neutralization profile of antibodies induced by optimal subtype B V3 motifs likely represents the maximum breadth of neutralization of subtype B HIV-1 primary isolates attainable by anti-V3 peptide antibodies.     

Potent broad cross-neutralizing sera inhibit attachment of primary HIV-1 isolates (groups M and O) to peripheral blood mononuclear cells

Gp160-directed antibody-mediated neutralization is thought to function by at least two different mechanisms that impair virus entry into the host cell: inhibition of virus attachment and inhibition of virus-cell membrane fusion. Previously, the neutralization spectra of sera derived from human immunodeficiency virus type 1 (HIV-1) infected patients were determined using 17 primary isolates belonging to HIV-1 group M (env clades A-H) and group O. The sera could be categorized as potent broad cross-neutralizing, limited cross-neutralizing, and nonneutralizing sera. The aim of this study was to examine whether the neutralizing capacity of polyclonal human sera correlates with their capacity to inhibit the attachment of infectious virions to the surface of peripheral blood mononuclear cells. A 100% correlation was found between the broad cross-neutralizing capacity and the ability to inhibit binding of primary isolates belonging to different genetic clades and groups to peripheral blood mononuclear cells. These results may indicate that broad cross-neutralizing antibodies are directed against those conserved regions on gp120 that interact with the cell receptor(s) and that those antibodies can therefore interfere with the binding of virus to the host cell.     

The HIV-1 V3 domain on field isolates: participation in generation of escape virus in vivo and accessibility to neutralizing antibodies

Summary The V3 domain is highly variable and induces HIV neutralizing antibodies (NA). Here we addressed the issues of 1) the participation of mutations in V3 in generation of neutralization resistant escape virus in vivo and 2) the applicability of synthetic V3 peptides corresponding to field isolates to induce neutralizing immune sera. Seven peptides corresponding to the V3 region of primary and escape virus from 3 HIV-1 infected patients were synthesized and used for antibody (Abs) studies and immunizations. The anti-V3 Abs titre in patient serum was generally low against peptides corresponding to autologous virus isolated later than the serum sample in contrast to the titre against peptides corresponding to virus isolated earlier than the serum sample. Furthermore, neutralizing anti-V3 monoclonal antibodies (MAbs) raised against V3 peptides from laboratory strains of HIV-1 showed distinct binding patterns against V3 peptides corresponding to sequential primary and escape field isolates, with the strongest reactivity against late isolated escape virus. These observations suggest that the neutralization epitope was influenced by the appearance of mutations. When used as immunogen in rabbits, V3 peptides corresponding to field isolates were highly immunogenic but failed to induce neutralizing or gp120-precipitating Abs. On the contrary, V3 peptide corresponding to the laboratory strain HXB2 induced HIV neutralizing, gp120-precipitating immune serum. In conclusion, these data suggest a participation of the V3 domain in the immunoselection of escape virus, and that V3 on early field virus is less accessible to NA than that on laboratory strains.     

Structural elements of primary CCR5-using HIV-1 gp120 proteins influencing sensitivity and resistance to the broadly neutralizing monoclonal antibody b12

Structure-guided approaches to HIV-1 vaccine design depend on knowledge of the presentation of neutralizing epitopes on gp120, such as the epitope for the broadly neutralizing mAb b12. Here, we characterized predicted three-dimensional structures of functionally diverse gp120 proteins in their b12-bound conformation, to better understand the gp120 determinants that expose or occlude the b12 epitope. Mapping the gp120-b12 binding interface identified amino acid polymorphisms within the C2, C3, C4 and V5 regions of gp120 associated with augmented b12 binding, and importantly, identified residues in the b12-exclusive binding domain of gp120 that are important for b12 neutralization resistance. Structural studies suggest that these b12 resistance variants promote reduced conformational flexibility in the b12 recognition site, which we show involves structural alterations within the gp120 CD4 binding loop and the V4 loop. Together, our studies provide new mechanistic insights into the gp120 determinants influencing sensitivity and resistance to HIV-1 neutralization by b12.     

Detection of orientation-specific anti-gp120 antibodies by a new N-glycanase protection assay

Several functions have been assigned to the extensive glycosylation of HIV-1 envelope glycoprotein gp120, especially immune escape mechanisms, but the intramolecular interactions between gp120 and its carbohydrate complement are not well understood. To analyse this phenomenon we established a new microwell deglycosylation assay for determining N-linked glycan accessibility after binding of gp120-specific agents. Orientation-specific exposition of gp120 in ELISA microplates was achieved by catching with either anti-C5 antibody D7324 or anti-V3 antibody NEA-9205. We found that soluble CD4 inhibited the deglycosylation of gp120 only when gp120 was caught by D7324 and not by NEA9205. In contrast, antibodies from HIV-infected individuals inhibited the deglycosylation best when gp120 was caught by NEA9205. These results demonstrated that both the CD4-binding site and the epitopes recognised by antibodies from HIV-infected individuals have N-glycans in the close vicinity. However, the difference in gp120 orientation indicates that antibodies in HIV-infected individuals, at least partly, bind to epitopes different from the CD4-binding site. Finally, we determined the structural class of the glycan of one V1 glycosylation site of prototype HIV-1 LAI gp120, which remained unsolved from previous studies, and found that it belonged to the complex type of glycans.     

Improving on nature: focusing the immune response on the V3 loop

The conventional wisdom suggests that "constant" rather than "variable" regions of the HIV envelope (Env) glycoproteins would induce the most broadly reactive antibodies (Abs). However, of the several epitopes in the conserved regions of gp120 and gp41 that induce neutralizing Abs, all are well-protected by protein folding, glycosylation, and/or oligomerization of the Env proteins on the virus surface; most are only transiently exposed during the process of infection or are poorly immunogenic. In contrast, the third variable region (V3) of gp120 appears to be at least partially exposed during various stages of the infectious process, is immunogenic in essentially all HIV+ subjects, and is capable of inducing Abs able to neutralize a broad array of primary isolates. While these Abs were originally thought to be isolate-specific, a large body of data now shows that anti-V3 Abs from HIV-infected individuals indeed show intra- and inter-clade cross-reactivity with respect to both binding to diverse gp120 molecules and neutralization of many primary isolates. This cross-reactivity of anti-V3 Abs is counter-intuitive if one focuses on the sequence variability rather than on the conserved V3 structures which must be present in order to allow this region of the virus envelope to mediate selection of and interaction with chemokine receptors. Current data, summarized here, support the hypothesis that the V3 region of gp120 can induce broadly-reactive, cross-neutralizing Abs and as such should constitute a prominent target of the immune response induced with an HIV vaccine.     

Temporal development and prognostic value of antibody response to the major neutralizing epitopes of gp120 during HIV-1 infection

Our objective was to analyse the humoral response to the major neutralizing epitopes of gp120. The kinetics of the appearance of antibodies directed to the V3 region (V3 Abs) and antibodies directed to the CD4 binding site (CD4BS Abs) were compared in sequential sera from 20 seroconverters. V3 Abs were titrated using 2 different indirect EIAs with synthetic oligopeptides coated on the solid phase. The sequences of the oligopeptides used were those of the MN isolate or a mixture of the consensus sequences of the 5 major HIV-1 subtypes (A–E). CD4BS Abs titers were determined using an EIA in which serum antibodies compete with a labeled human monoclonal antibody, F105, whose corresponding epitope overlaps the conformation-dependent CD4BS, for binding to purified recombinant gp120 coated on a solid phase. The prognostic value of both antibodies was analyzed in a longitudinal study of 60 HIV-1 infected patients (17 nonprogressors and 43 progressors). Eighty-five percent and 70% of HIV seroconverters were positive for V3 Abs and CD4BS Abs, respectively, during the observation period. V3 Abs were detected first in the majority of the patients (mean delay of appearance, 1.22 ± 0.96 months vs. 4.81 ± 2.05 months for CD4BS Abs). Both categories of antibodies appeared simultaneously in 4 patients (20%). No prognostic value could be attributed to these antibodies. Our data confirm that V3 Abs and CD4BS Abs appear with some delay after primary infection, suggesting that they do not play a large or early role in the rapid clearance of viremia in primary HIV-1 infection. These antibodies were not associated with progression to symptomatic infection and are thus of no value for surveillance in HIV-1 infected patients. J. Med. Virol. 52:309–315, 1997. © 1997 Wiley-Liss, Inc.     

A comparative immunogenicity study in rabbits of disulfide-stabilized, proteolytically cleaved, soluble trimeric human immunodeficiency virus type 1 gp140, trimeric cleavage-defective gp140 and monomeric gp120

The human immunodeficiency virus type 1 (HIV-1) surface envelope glycoprotein (Env) complex, a homotrimer containing gp120 surface glycoprotein and gp41 transmembrane glycoprotein subunits, mediates the binding and fusion of the virus with susceptible target cells. The Env complex is the target for neutralizing antibodies (NAbs) and is the basis for vaccines intended to induce NAbs. Early generation vaccines based on monomeric gp120 subunits did not confer protection from infection; one alternative approach is therefore to make and evaluate soluble forms of the trimeric Env complex. We have directly compared the immunogenicity in rabbits of two forms of soluble trimeric Env and monomeric gp120 based on the sequence of HIV-1(JR-FL). Both protein-only and DNA-prime, protein-boost immunization formats were evaluated, DNA-priming having little or no influence on the outcome. One form of trimeric Env was made by disrupting the gp120-gp41 cleavage site by mutagenesis (gp140(UNC)), the other contains an intramolecular disulfide bond to stabilize the cleaved gp120 and gp41 moieties (SOSIP.R6 gp140). Among the three immunogens, SOSIP.R6 gp140 most frequently elicited neutralizing antibodies against the homologous, neutralization-resistant strain, HIV-1(JR-FL). All three proteins induced NAbs against more sensitive strains, but the breadth of activity against heterologous primary isolates was limited. When antibodies able to neutralize HIV-1(JR-FL) were detected, antigen depletion studies showed they were not directed at the V3 region but were targeted at other, undefined gp120 and also non-gp120 epitopes.     

Characterization of neutralizing sites in the second variable and fourth variable region in gp125 and a conserved region in gp36 of human immunodeficiency virus type 2.

Several determinants of human immunodeficiency virus (HIV) have been suggested to harbor sites important for neutralization. The third variable region (V3) of the envelope glycoprotein (gp) is an important neutralizing determinant for both serotypes of HIV. The localization of additional neutralizing regions is an urgent task because the virus appears to mutate to phenotypes that escape neutralizing antibodies. Therefore, we have focused on the possibility of finding other immunodominant regions in the envelope glycoproteins of human immunodeficiency virus type 2 (HIV-2). By immunization of guinea pigs with peptides corresponding to different selected regions of gp125 and gp36, we have found three antigenic determinants located in the V2 and V4 regions of the envelope protein gp125, and one region in the glycoprotein gp36, which are important for human antibody binding and also as targets for neutralization. The peptide representing the V2 region had the most pronounced capacity to induce neutralizing anti-HIV-2 antibodies in guinea pigs. Neutralizing activity was also detected in an antipeptide guinea pig sera representing a linear site in gp36, amino acids 644-658. A substitution set of peptides representing the conserved antigenic site in the central part of gp36 was used to identify the role of individual amino acids important for human antibody binding.