Antibody binding and neutralization of primary and T-cell line-adapted isolates of human immunodeficiency virus type 1

The relative resistance of human immunodeficiency virus type 1 (HIV-1) primary isolates (PIs) to neutralization by a wide range of antibodies remains a theoretical and practical barrier to the development of an effective HIV vaccine. One model to account for the differential neutralization sensitivity between Pls and laboratory (or T-cell line-adapted [TCLA]) strains of HIV suggests that the envelope protein (Env) complex is made more accessible to antibody binding as a consequence of adaptation to growth in established cell lines. Here, we revisit this question using genetically related PI and TCLA viruses and molecularly cloned env genes. By using complementary techniques of flow cytometry and virion binding assays, we show that monoclonal antibodies targeting the V3 loop, CD4-binding site, CD4-induced determinant of gp120, or the ectodomain of gp41 bind equally well to PI and TCLA Env complexes, despite large differences in neutralization outcome. The data suggest that the differential neutralization sensitivity of PI and TCLA viruses may derive not from differences in the initial antibody binding event but rather from differences in the subsequent functioning of the PI and TCLA Envs during virus entry. An understanding of these as yet undefined differences may enhance our ability to generate broadly neutralizing HIV vaccine immunogens.     

Expression and characterization of glycophospholipid-anchored human immunodeficiency virus type 1 envelope glycoproteins.

Four chimeric human immunodeficiency virus type 1 (HIV-1) env genes were constructed which encoded the extracellular domain of either the wild-type or a cleavage-defective HIV-1 envelope glycoprotein (gp160) fused at one of two different positions in env to a C-terminal glycosyl-phosphatidylinositol (GPI) attachment signal from the mouse Thy-1.1 glycoprotein. All four of the constructs encoded glycoproteins that were efficiently expressed when Rev was supplied in trans, and the two cleavable forms were processed normally to gp120 and a chimeric "gp41." The chimeric glycoproteins, in contrast to the wild-type glycoprotein, could be cleaved from the surface of transfected cells by treatment with phosphatidylinositol-specific phospholipase C, indicating that they were anchored in the plasma membrane by a GPI moiety. These GPI-anchored glycoproteins were transported intracellularly at a rate only slightly lower than that of the full-length HIV-1 glycoprotein and were present on the cell surface in equivalent amounts. Nevertheless, all four glycoproteins were defective in mediating both cell-cell and virus-cell fusion as determined by syncytium formation in COS-1-HeLa-T4 cell mixtures and trans complementation of an env-defective HIV-1 genome.     

Enhancing the proteolytic maturation of human immunodeficiency virus type 1 envelope glycoproteins

In virus-infected cells, the envelope glycoprotein (Env) precursor, gp160, of human immunodeficiency virus type 1 is cleaved by cellular proteases into a fusion-competent gp120-gp41 heterodimer in which the two subunits are noncovalently associated. However, cleavage can be inefficient when recombinant Env is expressed at high levels, either as a full-length gp160 or as a soluble gp140 truncated immediately N-terminal to the transmembrane domain. We have explored several methods for obtaining fully cleaved Env for use as a vaccine antigen. We tested whether purified Env could be enzymatically digested with purified protease in vitro. Plasmin efficiently cleaved the Env precursor but also cut at a second site in gp120, most probably the V3 loop. In contrast, a soluble form of furin was specific for the gp120-gp41 cleavage site but cleaved inefficiently. Coexpression of Env with the full-length or soluble form of furin enhanced Env cleavage but also reduced Env expression. When the Env cleavage site (REKR) was mutated in order to see if its use by cellular proteases could be enhanced, several mutants were found to be processed more efficiently than the wild-type protein. The optimal cleavage site sequences were RRRRRR, RRRRKR, and RRRKKR. These mutations did not significantly alter the capacity of the Env protein to mediate fusion, so they have not radically perturbed Env structure. Furthermore, unlike that of wild-type Env, expression of the cleavage site mutants was not significantly reduced by furin coexpression. Coexpression of Env cleavage site mutants and furin is therefore a useful method for obtaining high-level expression of processed Env.     

Cryptic nature of envelope V3 region epitopes protects primary monocytotropic human immunodeficiency virus type 1 from antibody neutralization.

Characterization of biological and immunological properties of human immunodeficiency virus type 1 (HIV-1) is critical to developing effective therapies and vaccines for AIDS. With the use of a novel CD4+ T-cell line (PM-1) permissive to infection by both monocytotropic (MT) and T-cell-tropic virus types, we present a comparative analysis of the immunological properties of a prototypic primary MT isolate of HIV-1 strain JR-CSF (MT-CSF) with those of a T-cell-tropic variant (T-CSF) of the same virus, which emerged spontaneously in vitro. The parental MT-CSF infected only PM-1 cells and was markedly resistant to neutralization by sera from HIV-1-infected individuals, rabbit antiserum to recombinant MT-CSF gp120, and anti-V3 monoclonal antibodies. The T-CSF variant infected a variety of CD4+ T-cell lines, contained positively charged amino acid substitutions in the gp120 V3 region, and was highly sensitive to antibody neutralization. Neutralization and antibody staining of T-CSF-expressing cells were significantly inhibited by HIV-1 V3 peptides; in contrast, the MT strain showed only weak V3-specific binding of polyclonal and monoclonal antibodies. Exposure of PM-1 cells to a mixture of both viruses in the presence of human anti-HIV-1 neutralizing antiserum resulted in infection with only MT-CSF. These results demonstrate that although the V3 region of MT viruses is immunogenic, the target epitopes in the V3 principal neutralizing domain on the membrane form of the MT envelope appear to be cryptic or hidden from blocking antibodies.     

Quantitative model of antibody- and soluble CD4-mediated neutralization of primary isolates and T-cell line-adapted strains of human immunodeficiency virus type 1.

Primary isolates (PI) of human immunodeficiency virus type 1 (HIV-1) are considerably less sensitive than T-cell line-adapted strains to neutralization by soluble CD4 and by most cross-reactive monoclonal antibodies to the viral envelope (Env) glycoprotein, as well as by postinfection and postvaccination sera (J. P. Moore and D. D. Ho, AIDS 9 [suppl. A]:5117-5136, 1995). We developed a quantitative model to explain the neutralization resistance of PI. The factors incorporated into the model are the dissociation constants for the binding of the neutralizing agent to native Env oligomers, the number of outer Env molecules on the viral surface (which decreases by shedding), and the minimum number of Env molecules required for attachment and fusion. We conclude that modest differences in all these factors can, when combined, explain a relative neutralization resistance of PI versus T-cell line-adapted strains that sometimes amounts to several orders of magnitude. The hypothesis that neutralization of HIV is due to the reduction below a minimum number of the Env molecules on a virion available for attachment and fusion is at odds with single- and few-hit neutralization theories. Our analysis of these ideas favors the hypothesis that neutralization of HIV is instead a competitive blocking of interactions with cellular factors, including adsorption receptors.     

Differential loss of envelope glycoprotein gp120 from virions of human immunodeficiency virus type 1 isolates: effects on infectivity and neutralization.

Several parameters which may affect the infectivity of human immunodeficiency virus type 1 in tissue culture were analyzed. In particular, we used gel exclusion chromatography to investigate how the loss of the surface glycoprotein gp120 from virions of the HTLV-IIIB (IIIB), HTLV-IIIRF (RF), and SF-2 isolates modulates infectivity. In IIIB and RF cultures, a high proportion of the total gp120 was virion bound initially but was gradually lost from the virions over time. In contrast, most of the gp120 (and p24) in SF-2-infected cultures was soluble and the few particles present had a fivefold-lower level of virus-bound gp120. However, this reduced level of virion-bound gp120 was more resistant to shedding. Loss of a major proportion of gp120 from IIIB and RF virions resulted in reduced infectivities, and in addition, the resulting accumulation of soluble gp120 in the cultures could competitively inhibit viral infection, especially with SF-2. Increased shedding of virion gp120 also affected the neutralization of IIIB and RF particles. However, the high sensitivity to human serum neutralization characteristic of SF-2 was unaffected by soluble gp120 in cultures, suggesting that the epitopes responsible are not present on soluble gp120.     

Carbohydrate binding properties of the envelope glycoproteins of human immunodeficiency virus type 1

Here, we confirm and extend our previous findings on human immunodeficiency virus type 1 (HIV-1) envelope glycoproteinN-acetylglucosaminyl binding properties. We show the occurrence of saturable, temperature, pH, and calcium dependent carbohydrate-specific interactions between recombinant precursor gp160 (rgp160) and two affinity matrices:d-mannose-divinylsulfone-agarose, and natural glycoprotein, fetuin, also coupled to agarose. Binding of rgp160 to the matrices was inhibited by soluble mannosyl derivatives, -d-Man₁₇-BSA and mannan, by -d-GlcNAc₄₇-BSA and by glycopeptides from Pronase-treated porcine thyroglobulin, which produces oligomannose and complex N-linked glycans. Glycopeptides from Endoglycosidase H-treated thyroglobulin partially inhibited rgp160 binding, as did the asialo-agalacto-tetraantennary precursor oligosaccharide of human ₁-acid glycoprotein for binding to fetuin-agarose. -d-Glucan and -d-Gal₁₇-BSA had no or only limited effect. Also, surface unit rgp120 specifically interacted with fetuin-agarose and soluble fetuin, but in the latter case with a twofold reduced affinity relative to rgp160. After affinity chromatography, rgp160 was specifically retained by the two matrices and eluted by mannan in both cases, while rgp120 was not retained by fetuin-agarose but only eluted as a significantly retarded peak, which confirms its specific but weak interaction. Thus, rgp160 interacts with both oligomannose type, and the mannosyl core of complex type N-linked glycans, and its gp120 region plays a role in this interaction. Because fetuin and asialofetuin inhibit to nearly the same extent, the binding of rgp160 or rgp120 to fetuin-agarose, interaction with sialic acid or -d-galactosyl structures of complex N- or O-linked glycans can be ruled out. Specific rgp160 and rgp120 binding to ap-aminophenyl--d-GlcNAc-agarose matrix, which was inhibited by -d-GlcNAc₄₇-BSA and by fetuin, confirms that HIV-1 envelope glycoproteins can also specifically interact with theN-acetylglucosaminyl core of oligosaccharide structures.     

Utility of human immunodeficiency virus type 1 envelope as a T-cell immunogen

Human immunodeficiency virus (HIV)-specific CD8 T lymphocytes are important for the control of viremia, but the relative utility of responses to the various HIV proteins is controversial. Immune responses that force escape mutations that exact a significant fitness cost from the mutating virus would help slow progression to AIDS. The HIV envelope (Env) protein is subject to both humoral and cellular immune responses, suggesting that multiple rounds of mutation are needed to facilitate viral escape. The Gag protein, however, has recently been shown to elicit a more effective CD8 T-cell immune response in humans. We studied 30 pigtail macaques for their CD8 T-lymphocyte responses to HIV-1 Env and simian immunodeficiency virus (SIV) Gag following prime/boost vaccination and intrarectal challenge with simian-human immunodeficiency virus SHIV_mn229. Eight CD8 Env-specific T-cell epitopes were identified and mapped in 10 animals. Animals that generated Env-specific CD8 T-cell responses had equivalent viral loads and only a modest advantage in retention of peripheral CD4 T lymphocytes compared to those animals without responses to Env. This contrasts with animals that generated CD8 T-cell responses to SIV Gag in the same trial, demonstrating superior control of viral load and a larger advantage in retention of peripheral CD4 T cells than Gag nonresponders. Mutational escape was common in Env but, in contrast to mutations in Gag, did not result in the rapid emergence of dominant escape motifs, suggesting modest selective pressure from Env-specific T cells. These results suggest that Env may have limited utility as a CD8 T-cell immunogen.     

Cross-clade neutralization of primary isolates of human immunodeficiency virus type 1 by human monoclonal antibodies and tetrameric CD4-IgG.

We have tested three human monoclonal antibodies (MAbs) IgG1b12, 2G12, and 2F5) to the envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1), and a tetrameric CD4-IgG molecule (CD4-IgG2), for the ability to neutralize primary HIV-1 isolates from the genetic clades A through F and from group O. Each of the reagents broadly and potently neutralized B-clade isolates. The 2F5 MAb and the CD4-IgG2 molecule also neutralized strains from outside the B clade, with the same breadth and potency that they showed against B-clade strains. The other two MAbs were able to neutralize a significant proportion of strains from outside the B clade, although there was a reduction in their efficacy compared with their activity against B-clade isolates. Neutralization of isolates by 2F5 correlated with their possession of the LDKW motif in a segment of gp41 near the membrane-spanning domain. The other two MAbs and CD4-IgG2 recognize discontinuous binding sites on gp120, and so no comparison between genetic sequence and virus neutralization was possible. Our data show that a vaccine based on the induction of humoral immunity that is broadly active across the genetic clades is not impossible if immunogens that express the epitopes for MAbs such as 2F5, 2G12, and IgG1b12 in immunogenic configurations can be created. Furthermore, if the three MAbs and CD4-IgG2 produce clinical benefit in immunotherapeutic trials in the United States or Europe, they may also do so elsewhere in the world.     

Purified envelope glycoproteins from human immunodeficiency virus type 1 variants induce individual, type-specific neutralizing antibodies.

Repeated immunizations of goats, horses, or chimpanzees with envelope glycoprotein gp120 isolated from human immunodeficiency virus type 1 (HIV-1) resulted in type-specific neutralizing-antibody responses, which began to decay approximately 20 days following the administration of antigen. This was true repeatedly for serum samples from animals hyperimmunized with gp120s from either the HTLV-IIIB (IIIB) or the envelope-divergent HTLV-IIIRF (RF) HIV-1 isolates. Animals previously immunized with the IIIB gp120 were then inoculated with purified RF gp120. The first response in these animals was an anamnestic resurgence of neutralizing antibody to IIIB without detectable neutralizing antibody for RF. However, with later RF gp120 boosts, the IIIB neutralizing-antibody titers fell and an RF type-specific neutralizing-antibody response developed. When assessed with other HIV-1 variants, no group-specific neutralizing antibody was seen in any of the vaccination protocols evaluated. These results will pose real obstacles in the development of an effective vaccine for HIV.     

Antibody-mediated neutralization of primary human immunodeficiency virus type 1 isolates: investigation of the mechanism of inhibition

Human immunodeficiency virus type 1 (HIV-1) neutralization occurs when specific antibodies, mainly those directed against the envelope glycoproteins, inhibit infection, most frequently by preventing the entry of the virus into target cells. However, the precise mechanisms of neutralization remain unclear. Previous studies, mostly with cell lines, have produced conflicting results involving either the inhibition of virus attachment or interference with postbinding events. In this study, we investigated the mechanisms of neutralization by immune sera and compared the inhibition of peripheral blood mononuclear cells (PBMC) infection by HIV-1 primary isolates (PI) with the inhibition of T-cell line infection by T-cell line-adapted (TCLA) strains. We followed the kinetics of neutralization to determine at which step of the viral cycle the antibodies act. We found that neutralization of the TCLA strain HIV-1MN/MT-4 required an interaction between antibodies and cell-free virions before the addition of MT-4 cells, whereas PI were neutralized even after adsorption onto PBMC. In addition, the dose-dependent inhibition of HIV-1MN binding to MT-4 cells was strongly correlated with serum-induced neutralization. In contrast, neutralizing sera did not reduce the adhesion of PI to PBMC. Postbinding inhibition was also detected for HIV-1MN produced by and infecting PBMC, demonstrating that the mechanism of neutralization depends on the target cell used in the assay. Finally, we considered whether the different mechanisms of neutralization may reflect the recognition of qualitatively different epitopes on the surface of PI and HIV-1MN or whether they reflect differences in virus attachment to PBMC and MT-4 cells.     

Regional clustering of shared neutralization determinants on primary isolates of clade C human immunodeficiency virus type 1 from South Africa

Clade C is one of the most prevalent genetic subtypes of human immunodeficiency virus type 1 (HIV-1) in the world today and one of the least studied with respect to neutralizing antibodies. Most information on HIV-1 serology as it relates to neutralization is derived from clade B. Clade C primary isolates of HIV-1 from South Africa and Malawi were shown here to resemble clade B isolates in their resistance to inhibition by soluble CD4 and their sensitivity to neutralization by human monoclonal antibody immunoglobulin G1b12 and, to a lesser extent, 2F5. Unlike clade B isolates, however, all 16 clade C isolates examined resisted neutralization by 2G12. Infection with clade C HIV-1 in a cohort of female sex workers in South Africa generated antibodies that neutralized the autologous clade C isolate and T-cell-line-adapted (TCLA) strains of clade B. Neutralization of clade B TCLA strains was much more sensitive to the presence of autologous gp120 V3 loop peptides compared to the neutralization of clade C isolates in most cases. Thus, the native structure of gp120 on primary isolates of clade C will likely pose a challenge for neutralizing antibody induction by candidate HIV-1 vaccines much the same as it has for clade B. The autologous neutralizing antibody response following primary infection with clade C HIV-1 in South Africa matured slowly, requiring at least 4 to 5 months to become detectable. Once detectable, extensive cross-neutralization of heterologous clade C isolates from South Africa was observed, suggesting an unusual degree of shared neutralization determinants at a regional level. This high frequency of cross-neutralization differed significantly from the ability of South African clade C serum samples to neutralize clade B isolates but did not differ significantly from results of other combinations of clade B and C reagents tested in checkerboard assays. Notably, two clade C serum samples obtained after less than 2 years of infection neutralized a broad spectrum of clade B and C isolates. Other individual serum samples showed a significant clade preference in their neutralizing activity. Our results suggest that clades B and C are each comprised of multiple neutralization serotypes, some of which are more clade specific than others. The clustering of shared neutralization determinants on clade C primary HIV-1 isolates from South Africa suggests that neutralizing antibodies induced by vaccines will have less epitope diversity to overcome at a regional level.     

Role of N-linked glycans of envelope glycoproteins in infectivity of human immunodeficiency virus type 1.

We have shown that enzymatic removal of N-linked glycans from human immunodeficiency virus type 1 (HIV-1) recombinant envelope glycoproteins gp160 and gp120 produced in BHK-21 cells did not significantly reduce their ability to bind to CD4, the cellular receptor for the virus. Because recombinant proteins may behave differently from proteins present on virions, we investigated whether such viral envelope glycoproteins either in a purified form or present on viral particles could be deglycosylated by treatment with an endoglycosidase F-N-glycanase mixture which cleaves all accessible glycan moieties. Endoglycosidase analysis of the carbohydrate composition of purified viral gp120 (vgp120) indicated a glycosylation pattern similar to that for recombinant gp120 (rgp120), and treatment with endoglycosidase F-N-glycanase resulted in comparable molecular weight (MW) reduction for both molecules. Similarly, after immunoblotting of the deglycosylated viral preparation, the characteristic 160- and 120-kilodalton (kDa) bands were replaced by 90- and 60-kDa bands, respectively. The apparent MW of gp41 shifted to 35 kDa. These results are consistent with complete deglycosylation. The immunoreactive conformation of envelope glycoproteins remained unaltered after deglycosylation: they were recognized to the same extent by specific human polyclonal or mouse monoclonal antibodies, and no proteolysis of viral proteins occurred during enzymatic treatment. Deglycosylation of vgp120 resulted in a less than 10-fold reduction of the ability to bind to CD4, presented either in a soluble form or at the cell membrane. In addition, deglycosylation significantly reduced, but did not abolish, HIV-1 binding to and infectivity of CD4+ cells as determined, respectively, by an indirect immunofluorescence assay and a quantitative dose-response infection assay. Taken together, these results indicate that removal of glycans present on mature envelope glycoproteins of HIV-1 diminishes but does not abolish either virus binding to CD4 or its capacity to infect CD4+ cells.     

Transmembrane envelope glycoproteins of human immunodeficiency virus type 2 and simian immunodeficiency virus SIV-mac exist as homodimers.

An 80-kilodalton glycoprotein (gp80) was produced in human immunodeficiency virus type 2 (HIV-2)-infected cells along with three envelope glycoproteins that we have recently reported: the extracellular glycoprotein (gp125), the envelope glycoprotein precursor (gp140), and the transient dimeric form of the precursor (gp300). gp125 and gp80 were detectable after the synthesis of gp140 and the formation of gp300. Using a specific monoclonal antibody, we showed here that gp80 is a dimeric form of the transmembrane glycoprotein gp36 of HIV-2. Dimerization of the envelope glycoprotein precursor and dimeric forms of the transmembrane glycoproteins were also observed in cells infected with simian immunodeficiency virus (SIV-mac), a virus closely related to HIV-2. Under routine conditions of our experiments (i.e., extraction by 1% Triton X-100 before polyacrylamide gel electrophoresis in sodium dodecyl sulfate [SDS]), monomeric forms of the transmembrane glycoprotein of HIV-2 and SIV-mac were only seldomly observed. Dimeric forms of the envelope precursors and the transmembrane glycoproteins are probably stabilized by extraction in the nonionic detergent Triton X-100 since such dimeric forms resist dissociation during subsequent electrophoresis in the presence of the ionic detergent SDS. However, the dissociation of these dimeric forms might occur when samples are prepared by extraction directly in 1% SDS or by incubation of the purified dimers at acidic pH. Dimerization of the envelope precursor might be required for its processing to give the mature envelope proteins, whereas the transmembrane dimer might be essential for optimal structure of the virion and thus its infectivity.     

Enfuvirtide resistance mutations: impact on human immunodeficiency virus envelope function, entry inhibitor sensitivity, and virus neutralization

Enfuvirtide (ENF/T-20/Fuzeon), the first human immunodeficiency virus (HIV) entry inhibitor to be licensed, targets a structural intermediate of the entry process. ENF binds the HR1 domain in gp41 after Env has bound CD4, preventing conformational changes needed for membrane fusion. Mutations in HR1 that confer ENF resistance can arise following ENF therapy. ENF resistance mutations were introduced into an R5- and X4-tropic Env to examine their impact on fusion, infection, and sensitivity to different classes of entry inhibitors and neutralizing antibodies. HR1 mutations could reduce infection and fusion efficiency and also delay fusion kinetics, likely accounting for their negative impact on viral fitness. HR1 mutations had minimal effect on virus sensitivity to other classes of entry inhibitors, including those targeting CD4 binding (BMS-806 and a CD4-specific monoclonal antibody [MAb]), coreceptor binding (CXCR4 inhibitor AMD3100 and CCR5 inhibitor TAK-779), or fusion (T-1249), indicating that ENF-resistant viruses can remain sensitive to other entry inhibitors in vivo. Some HR1 mutations conferred increased sensitivity to a subset of neutralizing MAbs that likely target fusion intermediates or with epitopes preferentially exposed following receptor interactions (17b, 48D, 2F5, 4E10, and IgGb12), as well as sera from some HIV-positive individuals. Mechanistically, enhanced neutralization correlated with reduced fusion kinetics, indicating that, in addition to steric constraints, kinetics may also limit virus neutralization by some antibodies. Therefore, escape from ENF comes at a cost to viral fitness and may confer enhanced sensitivity to humoral immunity due to prolonged exposure of epitopes that are not readily accessible in the native Env trimer. Resistance to other entry inhibitors was not observed.     

Cytotoxic T-cell abundance and virus load in human immunodeficiency virus type 1 and human T-cell leukaemia virus type 1.

The correlation between virus load and specific cytotoxic T-lymphocyte (CTL) frequency during the chronic phase in human immunodeficiency virus type 1 (HIV-1) infection has been found to be negative in cross-sectional studies. We report here that, in infection with the related retrovirus human T-cell leukaemia virus type 1 (HTLV-1), the correlation is positive in asymptomatic carriers and zero in patients with the associated inflammatory disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). We demonstrate that the direction of the correlation may depend on the efficacy of the CTL response using mathematical models. We conclude that the CTL response is effective in asymptomatic carriers of HTLV-1, but ineffective in patients with HAM/TSP. Virus-mediated impairment of specific CTL production in HIV-1 infection can account for the negative correlation observed.     

Human immunodeficiency virus type 1 and 2 envelope glycoproteins oligomerize through conserved sequences.

Hetero-oligomerization between human immunodeficiency virus type 2 (HIV-2) envelope glycoprotein (Env) truncation mutants and epitope-tagged gp160 is dependent on the presence of gp41 transmembrane protein (TM) amino acids 552 to 589, a putative amphipathic alpha-helical sequence. HIV-2 Env truncation mutants containing this sequence were also able to form cross-type hetero-oligomers with HIV-1 Env. HIV-2/HIV-1 hetero-oligomerization was, however, more sensitive to disruption by mutagenesis or increased temperature. The conservation of the Env oligomerization function of the HIV-1 and HIV-2 alpha-helical sequences suggests that retroviral TM alpha-helical motifs may have a universal role in oligomerization.     

Multivariate analysis of human immunodeficiency virus type 1 neutralization data.

We report on the use of spectral map analysis of the inter- and intraclade neutralization data of 14 sera of human immunodeficiency virus type 1 (HIV-1)-infected individuals and 16 primary isolates, representing genetic clades A to H in group M and group O. This multivariate analysis has been used previously to study the interaction between drugs and receptors and between viruses and antiviral compounds. The analysis reveals the existence of neutralization clusters, not correlated with the known genetic clades. The structural factors that have been identified may correlate with the most important neutralization epitopes. Three key primary HIV-1 isolates, which allow discrimination of sera that are likely or unlikely to neutralize primary isolates from most of the genetic clades, were identified. Our method of analysis will facilitate the evaluation as well as the design of suitable HIV-1 vaccines, which induce high-titer interclade cross-neutralizing antibodies.     

Adaptation of the human immunodeficiency virus type 1 envelope glycoproteins to new world monkey receptors

Human immunodeficiency virus type 1 (HIV-1) infection encounters an early block in the cells of New World monkeys because the CD4 receptor does not efficiently support HIV-1 entry. We adapted HIV-1(NL4-3) and HIV-1(KB9), two HIV-1 variants with different envelope glycoproteins, to replicate efficiently in cells expressing the CD4 and CXCR4 proteins of the common marmoset, a New World monkey. The HIV-1(NL4-3) adaptation involves three gp120 changes that result in a specific increase in affinity for the marmoset CD4 glycoprotein. The already high affinity of the HIV-1(KB9) envelope glycoproteins for marmoset CD4 did not significantly change as a result of the adaptation. Instead, changes in the gp120 variable loops and gp41 ectodomain resulted in improved replication in cells expressing the marmoset receptors. HIV-1(KB9) became relatively sensitive to neutralization by soluble CD4 and antibodies as a result of the adaptation. These results demonstrate the distinct mechanistic pathways by which the HIV-1 envelope glycoproteins can adapt to less-than-optimal CD4 molecules and provide HIV-1 variants that can overcome some of the early blocks in New World monkey cells.