Human anti-V2 monoclonal antibody that neutralizes primary but not laboratory isolates of human immunodeficiency virus type 1.

A human immunoglobulin G1 lambda monoclonal antibody (MAb), 697-D, was developed that recognizes the V2 region of human immunodeficiency virus type 1 (HIV-1) gp120. Substitutions at amino acid positions 176/177, 179/180, 183/184, and 192 to 194 in the V2 loop of gp120 each completely abolished the binding capacity of 697-D in an enzyme-linked immunosorbent assay format. Competition analysis with three different neutralizing murine anti-V2 MAbs confirmed the specificity of 697-D. The 697-D epitope is primarily conformation dependent, although there was weak reactivity of the MAb with a V2 peptide spanning residues 161 to 180. Treatment of recombinant gp120 HIVIIIB with sodium metaperiodate, which oxidizes carbohydrates, abolished the binding of the MAb, showing the dependence of the epitope on intact carbohydrates. The broad reactivity of 697-D was displayed by its binding to the gp120 molecules from four of four laboratory isolates and five of five primary isolates. The MAb 697-D neutralized three out of four primary isolates but failed to neutralize any of four laboratory strains of HIV-1. 697-D and a human anti-V3 MAb, 447-52-D, displayed similar potency in neutralizing primary isolates, indicating that the V2 region of gp120, like the V3 region and the CD4-binding domain, can induce potent neutralizing antibodies against HIV-1 in humans.     

Human immunodeficiency virus type 1 mutants that escape neutralization by human monoclonal antibody IgG1b12. off.

IgG1b12, a human monoclonal antibody (MAb) to an epitope overlapping the CD4-binding site on gp120, has broad and potent neutralizing activity against most primary human immunodeficiency virus type 1 (HIV-1) isolates. To assess whether and how escape mutants resistant to IgG1b12 can be generated, we cultured primary HIV-1 strain JRCSF in its presence. An escape mutant emerged which was approximately 100-fold more resistant to neutralization by IgG1b12. Both virion-associated and solubilized gp120 from this variant had a reduced affinity for IgG1b12, and sequencing of its env gene showed that amino acid substitutions had occurred at three positions within gp120. Two (D164N and D182N) were located in V2, and one (P365L) was in C3. By site-directed mutagenesis, we demonstrated that the D182N and P365L mutations, but not D164N, contribute to the IgG1b12-resistant phenotype. However, the former two substitutions, individually or in combination, hinder the replication of the neutralization-resistant virus. Introduction of the D164N substitution into the P365L variant results in a nonviable virus (D164N/P365L). In contrast, addition of D164N to the D182N or D182N/P365L mutant partially restored replicative function to near wild-type levels. Furthermore, we found that all of the IgG1b12-resistant mutant viruses remained sensitive to other human MAbs, such as 2G12 and 2F5, and to the CD4-IgG molecule, except that the P365L-containing mutant was slightly resistant to CD4-IgG. These results suggest that escape from IgG1b12 neutralization is due to a local rather than a global modification of the gp120 structure. Our findings have implications for the therapeutic and prophylactic applications of antibodies for HIV-1 infection.     

Immunization with a soluble CD4-gp120 complex preferentially induces neutralizing anti-human immunodeficiency virus type 1 antibodies directed to conformation-dependent epitopes of gp120.

Preservation of the conformation of recombinant gp120 in an adjuvant, enabling it to elicit conformation-dependent, epitope-specific, broadly neutralizing antibodies, may be critical for the development of any gp120-based human immunodeficiency virus type 1 (HIV-1) vaccine. It was hypothesized that recombinant gp120 complexed with recombinant CD4 could stabilize the conformation-dependent neutralizing epitopes and effectively deliver them to the immune system. Therefore, a soluble CD4-gp120 complex in Syntex adjuvant formulation was tested with mice for its ability to induce neutralizing anti-gp120 antibody responses. Seventeen monoclonal antibodies (MAbs) were generated and characterized. Immunochemical studies, neutralization assays, and mapping studies with gp120 mutants indicated that the 17 MAbs fell into three groups. Four of them were directed to what is probably a conformational epitope involving the C1 domain and did not possess virus-neutralizing activities. Another four MAbs bound to V3 peptide 302-321 and exhibited cross-reactive gp120 binding and relatively weak virus-neutralizing activities. These MAbs were very sensitive to amino acid substitutions, not only in the V3 regions but also in the base of the V1/V2 loop, implying a conformational constraint on the epitope. The last group of nine MAbs recognized conformation-dependent epitopes near the CD4 binding site of gp120 and inhibited the gp120-soluble CD4 interaction. Four of these nine MAbs showed broadly neutralizing activities against multiple laboratory-adapted strains of HIV-1, three of them neutralized only HIVIIIB, and the two lower-affinity MAbs did not neutralize any strain tested. Collectively, the results from this study indicate that immunization with the CD4-gp120 complex can elicit antibodies to conformationally sensitive gp120 epitopes, with some of the antibodies having broadly neutralizing activities. We suggest that immunization with CD4-gp120 complexes may be worth evaluating further for the development of an AIDS vaccine.     

Immune escape by human immunodeficiency virus type 1 from neutralizing antibodies: evidence for multiple pathways.

Sera from many HIV-1-infected individuals contain broadly reactive, specific neutralizing antibodies. Despite their broad reactivity, variant viruses, resistant to neutralization, can be selected in vitro in the presence of such antisera. We have previously shown that neutralization resistance of an escape mutant with an amino acid substitution in the transmembrane protein (A582T) occurs because of alteration of a conformational epitope that is recognized by neutralizing antibodies directed against the CD4 binding site. In this report we demonstrate that immune escape via a single-amino-acid substitution (A281V) within a conserved region of the envelope glycoprotein gp120 confers neutralization resistance against a broadly reactive neutralizing antiserum from a seropositive individual. We show this alteration affects V3 and additional regions unrelated to V3 or the CD4 binding site. Together with previous studies on escape mutants selected in vitro, our findings suggest that immune-selective pressure can arise by multiple pathways.     

Identification and characterization of monoclonal antibodies specific for polymorphic antigenic determinants within the V2 region of the human immunodeficiency virus type 1 envelope glycoprotein.

We have identified six monoclonal antibodies (MAbs) mapping to both linear and conformation-dependent epitopes within the V2 region of the human immunodeficiency virus type 1 clone HXB10. Three of the MAbs (12b, 66c, and 66a) were able to neutralize the molecular clones HXB10 and HXB2, with titers in the range of 9.5 to 20.0 micrograms/ml. MAbs mapping to the crown of the V2 loop (12b, 60b, and 74) bound poorly to cell surface-expressed oligomeric gp120, suggesting an explanation for the poor or negligible neutralizing activity of MAbs to this region. In contrast, MAbs 12b and 60b demonstrated good reactivity with recombinant gp120 in an enzyme-linked immunosorbent assay format, suggesting differential epitope exposure between the recombinant and native forms of gp120. Cross-competition analysis of these MAbs and additional V1V2 MAbs for gp120 binding enabled us to assign the MAbs to six groups (A to F). Selection of neutralization escape mutants with MAbs 10/76b and 11/68b, belonging to nonoverlapping competition groups, identified amino acid changes at residues 165 (I to T) and 185 (D to N), respectively. Interestingly, these escape variants remained sensitive to neutralization by the nonselecting V2 MAbs. All MAbs demonstrated good recognition of IIIB viral gp120 yet failed to neutralize nonclonal stocks of IIIB. In addition, MAbs 12b and 62c bound MN and RF viral gp120, respectively, yet failed to neutralize the respective isolates. Cloning and expression of a library of gp120 and V1V2 fragments from IIIB-, MN-, and RF-infected H9 cultures identified a number of polymorphic sites, resulting in antigenic variation and subsequent loss of V2 MAb recognition. In contrast, the V3 region from the clones of the same isolates showed no amino acid changes, suggesting that the V2 region is polymorphic in long-term-passaged laboratory isolates and may account for the reduced antibody recognition observed.     

Identification and characterization of a neutralization site within the second variable region of human immunodeficiency virus type 1 gp120.

Two monoclonal antibodies designated BAT085 and G3-136 were raised by immunizing BALB/c mice with gp120 purified from human immunodeficiency virus type 1 (HIV-1) IIIB-infected H9 cell extracts. Among three HIV-1 laboratory isolates (IIIB, MN, and RF), BAT085 neutralized only IIIB infection of CEM-SS cells, whereas G3-136 neutralized both IIIB and RF. These antibodies also neutralized a few primary HIV-1 isolates in the infection of activated human peripheral blood mononuclear cells. In indirect immunofluorescence assays, BAT085 bound to H9 cells infected with IIIB or MN, while G3-136 bound to H9 cells infected with IIIB or RF, but not MN. Using sequence-overlapping synthetic peptides of HIV-1 IIIB gp120, the binding site of BAT085 and G3-136 was mapped to a peptidic segment in the V2 region (amino acid residues 169 to 183). The binding of these antibodies to immobilized gp120 was not inhibited by the antibodies directed to the principal neutralization determinant in the V3 region or to the CD4-binding domain of gp120. In a competition enzyme-linked immunosorbent assay, soluble CD4 inhibited G3-136 but not BAT085 from binding to gp120. Deglycosylation of gp120 by endo-beta-N-acetylglucosaminidase H or reduction of gp120 by dithiothreitol diminished its reactivity with G3-136 but not with BAT085. These results indicate that the V2 region of gp120 contains multiple neutralization determinants recognized by antibodies in both a conformation-dependent and -independent manner.     

Synergistic neutralization of human immunodeficiency virus type 1 by a chimpanzee monoclonal antibody against the V2 domain of gp120 in combination with monoclonal antibodies against the V3 loop and the CD4-binding site.

Synergistic neutralization of human immunodeficiency virus type 1 (HIV-1) was observed in studies using a chimpanzee anti-V2 monoclonal antibody (MAb), C108G, in combination with anti-V3 loop and anti-CD4 binding-site (bs) MAbs of different epitope specificities. C108G paired with either of two anti-V3 loop MAbs or either of two anti-CD4 bs MAbs synergistically neutralized both the uncloned IIIB and clonal HXB2 strains of virus in H9 target cells. Synergism was quantitated by calculation of combination indices. Significant synergy with a given MAb pair was seen over a range of MAb ratios, with the optimal effect centering around the ratio at which the MAbs were equipotent for a given HIV-1 strain (on the basis of the 50% neutralization titer). In preliminary experiments with monocytotropic strains of HIV-1 in peripheral blood mononuclear cell targets, significant synergism was also observed between anti-V2-anti-V3 and anti-V2-anti-CD4 bs MAb pairs. Synergism by all MAb pairs tested was greater against heterogeneous isolates of HIV-1 (IIIB and Ba-L) than against clonal isolates (HXB2 and NLHXADA), suggesting that strain broadening may be a component of the synergism observed against the heterogeneous isolates. In addition, conformational changes in gp120 upon binding of one or both MAbs may result in increased affinity or exposure of the epitope of one or both MAbs. Finally, a three-MAb combination of C108G, an anti-V3 MAb, and an anti-CD4 bs MAb was more effective in neutralizing the HXB2 strain of HIV-1 than any of the three two-MAb combinations within this trio, as determined by the dose reduction indices of each MAb required to achieve a given level of neutralization. This is the first report of synergistic neutralization of HIV-1 by a three-MAb combination composed of MAbs directed against the three major neutralization epitope clusters in gp120. Implications for vaccine design and for immunoprophylaxis and immunotherapy with a combination of MAbs are discussed.     

Impact of V2 mutations on escape from a potent neutralizing anti-V3 monoclonal antibody during in vitro selection of a primary human immunodeficiency virus type 1 isolate

KD-247, a humanized monoclonal antibody to an epitope of gp120-V3 tip, has potent cross-neutralizing activity against subtype B primary human immunodeficiency virus type 1 (HIV-1) isolates. To assess how KD-247 escape mutants can be generated, we induced escape variants by exposing bulked primary R5 virus, MOKW, to increasing concentrations of KD-247 in vitro. In the presence of relatively low concentrations of KD-247, viruses with two amino acid mutations (R166K/D167N) in V2 expanded, and under high KD-247 pressure, a V3 tip substitution (P313L) emerged in addition to the V2 mutations. However, a virus with a V2 175P mutation dominated during passaging in the absence of KD-247. Using domain swapping analysis, we demonstrated that the V2 mutations and the P313L mutation in V3 contribute to partial and complete resistance phenotypes against KD-247, respectively. To identify the V2 mutation responsible for the resistance to KD-247, we constructed pseudoviruses with single or double amino acid mutations in V2 and measured their sensitivity to neutralization. Interestingly, the neutralization phenotypes were switched, so that amino acid residue 175 (Pro or Leu) located in the center of V2 was exchanged, indicating that the amino acid at position 175 has a crucial role, dramatically changing the Env oligomeric state on the membrane surface and affecting the neutralization phenotype against not only anti-V3 antibody but also recombinant soluble CD4. These data suggested that HIV-1 can escape from anti-V3 antibody attack by changing the conformation of the functional envelope oligomer by acquiring mutations in the V2 region in environments with relatively low antibody concentrations.     

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.     

epitopes immediately below the base of the V3 loop of gp120 as targets for the initial autologous neutralizing antibody response in two HIV-1 subtype B-infected individuals

Epitopes that drive the initial autologous neutralizing antibody response in HIV-1-infected individuals could provide insights for vaccine design. Although highly strain specific, these epitopes are immunogenic, vulnerable to antibody attack on infectious virus, and could be involved in the ontogeny of broadly neutralizing antibody responses. To delineate such epitopes, we used site-directed mutagenesis, autologous plasma samples, and autologous monoclonal antibodies to map the amino acid changes that led to escape from the initial autologous neutralizing antibody response in two HIV-1 subtype B-infected individuals. Additional mapping of the epitopes was accomplished by using alanine scanning mutagenesis. Escape in the two individuals occurred by different pathways, but the responses in both cases appeared to be directed against the same region of gp120. In total, three amino acid positions were identified that were independently associated with autologous neutralization. Positions 295 and 332 are located immediately before and after the N- and C-terminal cysteines of the V3 loop, respectively, the latter of which affected an N-linked glycan that was critical to the neutralization epitope. Position 415 affected an N-linked glycan at position 413 in the C terminus of V4 that might mask epitopes near the base of V3. All three sites lie in close proximity on a four-stranded antiparallel sheet on the outer domain of gp120. We conclude that a region just below the base of the V3 loop, near the coreceptor binding domain of gp120, can be a target for autologous neutralization.     

A novel human antibody against human immunodeficiency virus type 1 gp120 is V1, V2, and V3 loop dependent and helps delimit the epitope of the broadly neutralizing antibody immunoglobulin G1 b12

The V1/V2 and V3 loops are proximal to the CD4 binding site (CD4bs) of human immunodeficiency virus type 1 (HIV-1) gp120 and undergo conformational change upon CD4 receptor engagement by the HIV-1 envelope spike. Nearly all of the reported monoclonal antibodies (MAbs) against the CD4bs exhibit a very limited capacity to neutralize HIV-1. However, one such human MAb, immunoglobulin G1 (IgG1) b12, is uniquely able to neutralize primary isolates across subtypes with considerable potency. The molecular basis for the anti-HIV-1 activity of b12 is not fully understood but is relevant to vaccine design. Here we describe a novel human MAb, 4KG5, whose binding to monomeric gp120 is moderately enhanced by IgG1 b12. In sharp contrast, 4KG5 binding to gp120 is inhibited by soluble CD4 (sCD4) and by all other (n = 14) anti-CD4bs MAbs tested. 4KG5 is unable to recognize gp120 in which either V1, V2, or V3 has been deleted, and MAbs against the V2 or V3 loops inhibit the binding of 4KG5 to gp120. Moreover, 4KG5 is able to inhibit the binding of the CD4-induced MAbs 17b and X5 in the absence of sCD4, whereas 17b and X5 only weakly inhibit the binding of 4KG5 to gp120. Mutagenesis of gp120 provides further evidence of a discontinuous epitope of 4KG5 that is formed by the V1/V2 loop, the V3 loop, and a portion of the bridging sheet (C4). 4KG5 was isolated as a single-chain Fv from a phage display library constructed from the bone marrow of an HIV-1-seropositive subject (FDA2) whose serum neutralizes HIV-1 across subtypes. Despite its source, we observed no significant neutralization with 4KG5 against the autologous (R2) virus and several other strains of HIV-1. The results suggest a model in which antibody access to the CD4bs on the envelope spike of HIV-1 is restricted by the orientation and/or dynamics of the V1/V2 and V3 loops, and b12 avoids these restrictions.     

Cryptic nature of a conserved, CD4-inducible V3 loop neutralization epitope in the native envelope glycoprotein oligomer of CCR5-restricted, but not CXCR4-using, primary human immunodeficiency virus type 1 strains

The external subunit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env), gp120, contains conserved regions that mediate sequential interactions with two cellular receptor molecules, CD4 and a chemokine receptor, most commonly CCR5 or CXCR4. However, antibody accessibility to such regions is hindered by diverse protective mechanisms, including shielding by variable loops, conformational flexibility and extensive glycosylation. For the conserved neutralization epitopes hitherto described, antibody accessibility is reportedly unrelated to the viral coreceptor usage phenotype. Here, we characterize a novel, conserved gp120 neutralization epitope, recognized by a murine monoclonal antibody (MAb), D19, which is differentially accessible in the native HIV-1 Env according to its coreceptor specificity. The D19 epitope is contained within the third variable (V3) domain of gp120 and is distinct from those recognized by other V3-specific MAbs. To study the reactivity of MAb D19 with the native oligomeric Env, we generated a panel of PM1 cells persistently infected with diverse primary HIV-1 strains. The D19 epitope was conserved in the majority (23/29; 79.3%) of the subtype-B strains tested, as well as in selected strains from other genetic subtypes. Strikingly, in CCR5-restricted (R5) isolates, the D19 epitope was invariably cryptic, although it could be exposed by addition of soluble CD4 (sCD4); epitope masking was dependent on the native oligomeric structure of Env, since it was not observed with the corresponding monomeric gp120 molecules. By contrast, in CXCR4-using strains (X4 and R5X4), the epitope was constitutively accessible. In accordance with these results, R5 isolates were resistant to neutralization by MAb D19, becoming sensitive only upon addition of sCD4, whereas CXCR4-using isolates were neutralized regardless of the presence of sCD4. Other V3 epitopes examined did not display a similar divergence in accessibility based on coreceptor usage phenotype. These results provide the first evidence of a correlation between HIV-1 biological phenotype and neutralization sensitivity, raising the possibility that the in vivo evolution of HIV-1 coreceptor usage may be influenced by the selective pressure of specific host antibodies.     

Isolation of a monoclonal antibody that targets the alpha-2 helix of gp120 and represents the initial autologous neutralizing-antibody response in an HIV-1 subtype C-infected individual

The C3-V4 region is a major target of autologous neutralizing antibodies in HIV-1 subtype C infection. We previously identified a Center for AIDS Program of Research in South Africa (CAPRISA) participant, CAP88, who developed a potent neutralizing-antibody response within 3 months of infection that targeted an epitope in the C3 region of the HIV-1 envelope (P. L. Moore et al., PLoS Pathog. 5:e1000598, 2009). Here we showed that these type-specific antibodies could be adsorbed using recombinant gp120 from the transmitted/founder virus from CAP88 but not by gp120 made from other isolates. Furthermore, this activity could be depleted using a chimeric gp120 protein that contained only the C3 region from the CAP88 viral envelope engrafted onto the unrelated CAP63 viral envelope (called 63-88C3). On the basis of this, a differential sorting of memory B cells was performed using gp120s made from 63-88C3 and CAP63 labeled with different fluorochromes as positive and negative probes, respectively. This strategy resulted in the isolation of a highly specific monoclonal antibody (MAb), called CAP88-CH06, that neutralized the CAP88 transmitted/founder virus and viruses from acute infection but was unable to neutralize CAP88 viruses isolated at 6 and 12 months postinfection. The latter viruses contained 2 amino acid changes in the alpha-2 helix of C3 that mediated escape from this MAb. One of these changes involved the introduction of an N-linked glycan at position 339 that occluded the epitope, while the other mutation (either E343K or E350K) was a charge change. Our data validate the use of differential sorting to isolate a MAb targeting a specific epitope in the envelope glycoprotein and provided insights into the mechanisms of autologous neutralization escape.     

Probing the structure of the V2 domain of human immunodeficiency virus type 1 surface glycoprotein gp120 with a panel of eight monoclonal antibodies: human immune response to the V1 and V2 domains.

We have analyzed a panel of eight murine monoclonal antibodies (MAbs) that depend on the V2 domain for binding to human immunodeficiency virus type 1 (HIV-1) gp120. Each MAb is sensitive to amino acid changes within V2, and some are affected by substitutions elsewhere. With one exception, the MAbs were not reactive with peptides from the V2 region, or only poorly so. Hence their ability to bind recombinant strain IIIB gp120 depended on the preservation of native structure. Three MAbs cross-reacted with strain RF gp120, but only one cross-reacted with MN gp120, and none bound SF-2 gp120. Four MAbs neutralized HIV-1 IIIB with various potencies, and the one able to bind MN gp120 neutralized that virus. Peptide serology indicated that antibodies cross-reactive with the HxB2 V1 and V2 regions are rarely present in HIV-1-positive sera, but the relatively conserved segment between the V1 and V2 loops was recognized by antibodies in a significant fraction of sera. Antibodies able to block the binding of V2 MAbs to IIIB or MN gp120 rarely exist in sera from HIV-1-infected humans; more common in these sera are antibodies that enhance the binding of V2 MAbs to gp120. This enhancement effect of HIV-1-positive sera can be mimicked by several human MAbs to different discontinuous gp120 epitopes. Soluble CD4 enhanced binding of one V2 MAb to oligomeric gp120 but not to monomeric gp120, perhaps by inducing conformational changes in the oligomer.     

Comprehensive cross-clade neutralization analysis of a panel of anti-human immunodeficiency virus type 1 monoclonal antibodies

Broadly neutralizing monoclonal antibodies (MAbs) are potentially important tools in human immunodeficiency virus type 1 (HIV-1) vaccine design. A few rare MAbs have been intensively studied, but we still have a limited appreciation of their neutralization breadth. Using a pseudovirus assay, we evaluated MAbs from clade B-infected donors and a clade B HIV(+) plasma against 93 viruses from diverse backgrounds. Anti-gp120 MAbs exhibited greater activity against clade B than non-B viruses, whereas anti-gp41 MAbs exhibited broad interclade activity. Unexpectedly, MAb 4E10 (directed against the C terminus of the gp41 ectodomain) neutralized all 90 viruses with moderate potency. MAb 2F5 (directed against an epitope adjacent to that of 4E10) neutralized 67% of isolates, but none from clade C. Anti-gp120 MAb b12 (directed against an epitope overlapping the CD4 binding site) neutralized 50% of viruses, including some from almost every clade. 2G12 (directed against a high-mannose epitope on gp120) neutralized 41% of the viruses, but none from clades C or E. MAbs to the gp120 V3 loop, including 447-52D, neutralized a subset of clade B viruses (up to 45%) but infrequently neutralized other clades (相似文献   

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.     

The C108g epitope in the V2 domain of gp120 functions as a potent neutralization target when introduced into envelope proteins derived from human immunodeficiency virus type 1 primary isolates

Monoclonal antibodies (MAbs) directed against epitopes in the V2 domain of human immunodeficiency virus type 1 gp120 often possess neutralizing activity, but these generally are highly type specific, neutralize only laboratory isolates, or have low potency. The most potent of these is C108g, directed against a type-specific epitope in HXB2 and BaL gp120s, which is glycan dependent and, in contrast to previous reports, dependent on intact disulfide bonds. This epitope was introduced into two primary Envs, derived from a neutralization-sensitive (SF162) and a neutralization-resistant (JR-FL) isolate, by substitution of two residues and, for SF162, addition of an N-linked glycosylation site. C108g effectively neutralized both variant Envs with considerably higher potency than standard MAbs against the V3 and CD4-binding domains and the broadly neutralizing MAbs 2G12 and 2F5. These amino acid substitutions also introduced the epitope recognized by a second V2-specific MAb, 10/76b, but this MAb possessed potent neutralizing activity only in the absence of the glycan required for C108g reactivity. In contrast to other gp120-specific neutralizing MAbs, C108g did not block binding of soluble Env proteins to either the CD4 or the CCR5 receptor, but studies with a fusion-arrested Env indicated that C108g neutralized at a step preceding the one blocked by the gp41-specific MAb, 2F5. These results indicate that the V1/V2 domain possesses targets that mediate potent neutralization of primary viral isolates via a novel mechanism and suggest that inclusion of carbohydrate determinants into these epitopes may help overcome the indirect masking effects that limit the neutralizing potency of antibodies commonly produced after infection.     

A global neutralization resistance phenotype of human immunodeficiency virus type 1 is determined by distinct mechanisms mediating enhanced infectivity and conformational change of the envelope complex

We have described previously genetic characterization of neutralization-resistant, high-infectivity, and neutralization-sensitive, low-infectivity mutants of human immunodeficiency virus type 1 (HIV-1) MN envelope. The distinct phenotypes of these clones are attributable to six mutations affecting functional interactions between the gp120 C4-V5 regions and the gp41 leucine zipper. In the present study we examined mechanisms responsible for the phenotypic differences between these envelopes using neutralization and immunofluorescence assays (IFA). Most monoclonal antibodies (MAbs) tested against gp120 epitopes (V3, CD4 binding site, and CD4-induced) were 20 to 100 times more efficient at neutralizing pseudovirus expressing sensitive rather than resistant envelope. By IFA cells expressing neutralization sensitive envelope bound MAbs to gp120 epitopes more, but gp41 epitopes less, than neutralization-resistant envelope. This binding difference appeared to reflect conformational change, since it did not correlate with the level of protein expression or gp120-gp41 dissociation. This conformational change was mostly attributable to one mutation, L544P, which contributes to neutralization resistance but not to infectivity enhancement. The V420I mutation, which contributes a major effect to both high infectivity and neutralization resistance, had no apparent effect on conformation. Notably, a conformation-dependent V3 neutralization epitope remained sensitive to neutralization and accessible to binding by MAbs on neutralization-resistant HIV-1 envelope. Sensitivity to sCD4 did not distinguish the clones, suggesting that the phenotypes may be related to post-CD4-binding effects. The results demonstrate that neutralization resistance can be determined by distinguishable effects of mutations, which cause changes in envelope conformation and/or function(s) related to infectivity. A conformation-dependent V3 epitope may be an important target for neutralization of resistant strains of HIV-1.     

Infectivity and neutralization of simian immunodeficiency virus with FLAG epitope insertion in gp120 variable loops

A FLAG epitope tag was substituted within variable loop 1 (V1), 2 (V2), or 4 (V4) of the gp120 envelope glycoprotein of simian immunodeficiency virus strain 239 (SIV239) to evaluate the extent to which each variable loop may serve as a target for antibody-mediated neutralization. Two sites within each variable loop of SIV239 were chosen for individual epitope tag insertions. FLAG epitope substitutions were also made in the V1, V2, and V4 loops of a neutralization-sensitive derivative of SIV239, SIV316. Of the 10 FLAG-tagged recombinant viruses analyzed, three (SIV239FV1b, SIV239FV2b, and SIV239FV4a) replicated with kinetics similar to those of the parental strain, SIV239, in both CEMx174 cells and the immortalized rhesus monkey T-cell line 221. The SIV316FV1b and SIV316FV4a FLAG variants replicated with a substantial lag, and the five remaining recombinants did not replicate detectably. Both gp160 and gp120 from replication-competent FLAG variants could be immunoprecipitated from transfected 293T cells by the anti-gp120 rhesus monoclonal antibody (RhMAb) 3.11H, the anti-FLAG MAb M2, and CD4-immunoglobulin, whereas only unprocessed gp160 was detected in 293T cells transfected with replication-defective variants. Furthermore, gp120 was detectably incorporated only into virions that were infectious. SIV239FV1b was sensitive to neutralization by MAb M2, with a 50% inhibitory concentration of 1 mug/ml. Neither SIV239FV2b nor SIV239FV4a was sensitive to M2 neutralization. The ability of the M2 antibody to neutralize SIV239FV1b infectivity was associated with an increased ability of the M2 antibody to detect native, oligomeric SIV239FV1b envelope protein on the surfaces of cells relative to that for the other SIV FLAG variants. Furthermore, SIV239FV1b was globally more sensitive to antibody-mediated neutralization than was parental SIV239 when these strains were screened with a panel of anti-SIV MAbs of various specificities. These results indicate that the V1 loop can serve as an effective target for neutralization on SIV239FV1b. However, antibody-mediated neutralization of this variant, similar to that of other SIV239 variants that have been studied previously, was associated with a global increase in neutralization sensitivity. These results suggest that the variable loops on the neutralization-resistant SIV239 strain are difficult for antibodies to access effectively and that mutations that allow neutralization have global effects on the trimeric envelope glycoprotein structure and accessibility.     

Relationship of the human immunodeficiency virus type 1 gp120 third variable loop to a component of the CD4 binding site in the fourth conserved region.

Neutralizing antibodies that recognize the human immunodeficiency virus gp120 exterior envelope glycoprotein and are directed against either the third variable (V3) loop or conserved, discontinuous epitopes overlapping the CD4 binding region have been described. Here we report several observations that suggest a structural relationship between the V3 loop and amino acids in the fourth conserved (C4) gp120 region that constitute part of the CD4 binding site and the conserved neutralization epitopes. Treatment of the gp120 glycoprotein with ionic detergents resulted in a V3 loop-dependent masking of both linear C4 epitopes and discontinuous neutralization epitopes overlapping the CD4 binding site. Increased recognition of the native gp120 glycoprotein by an anti-V3 loop monoclonal antibody, 9284, resulted from from single amino acid changes either in the base of the V3 loop or in the gp120 C4 region. These amino acid changes also resulted in increased exposure of conserved epitopes overlapping the CD4 binding region. The replication-competent subset of these mutants exhibited increased sensitivity to neutralization by antibody 9284 and anti-CD4 binding site antibodies. The implied relationship of the V3 loop, which mediates post-receptor binding steps in virus entry, and components of the CD4 binding region may be important for the interaction of these functional gp120 domains and for the observed cooperativity of neutralizing antibodies directed against these regions.