Addition of a single gp120 glycan confers increased binding to dendritic cell-specific ICAM-3-grabbing nonintegrin and neutralization escape to human immunodeficiency virus type 1

The potential role of dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) binding in human immunodeficiency virus transmission across the mucosal barrier was investigated by assessing the ability of simian-human immunodeficiency chimeric viruses (SHIVs) showing varying degrees of mucosal transmissibility to bind the DC-SIGN expressed on the surface of transfected cells. We found that gp120 of the highly transmissible, pathogenic CCR5-tropic SHIV(SF162P3) bound human and rhesus DC-SIGN with an efficiency threefold or greater than that of gp120 of the nonpathogenic, poorly transmissible parental SHIV(SF162), and this increase in binding to the DC-SIGN of the SHIV(SF162P3) envelope gp120 translated into an enhancement of T-cell infection in trans. The presence of an additional glycan at the N-terminal base of the V2 loop of SHIV(SF162P3) gp120 compared to that of the parental virus was shown to be responsible for the increase in binding to DC-SIGN. Interestingly, this glycan also conferred escape from autologous neutralization, raising the possibility that the modification occurred as a result of immune selection. Our data suggest that more-efficient binding of envelope gp120 to DC-SIGN could be relevant to the enhanced mucosal transmissibility of SHIV(SF162P3) compared to that of parental SHIV(SF162).     

Properties of the Surface Envelope Glycoprotein Associated with Virulence of Simian-Human Immunodeficiency Virus SHIVSF33A Molecular Clones

In vivo adaptation of simian-human immunodeficiency virus (SHIV) clone SHIV(SF33) resulted in the emergence of pathogenic isolate SHIV(SF33A), which caused a rapid and severe CD4(+) T-cell depletion when inoculated into rhesus macaques. Two molecular clones generated by inserting the env V1-to-V5 region amplified from SHIV(SF33A)-infected animals into the parental SHIV(SF33) genome retained a pathogenic phenotype. The gp120 envelope glycoproteins of pathogenic clones SHIV(SF33A2) and SHIV(SF33A5) conferred a threefold increase in viral entry and fusogenicity compared to the parental glycoprotein. Changes in gp120 were also responsible for a higher replication capacity and cytopathicity in primary CD4(+) T-cell cultures. Last, gp120 carried the determinants of SHIV(SF33A) neutralization resistance. Thus, changes in SHIV(SF33A) gp120 produced a set of properties that could account for the pathogenic phenotype observed in vivo. Measurement of antibody binding to SHIV(SF33A) viral particles revealed an increased exposure of the CD4-induced epitope recognized by the 17b monoclonal antibody in a region that was shown to contribute to coreceptor binding. Exposure of this epitope occurred in the absence of CD4 binding, suggesting that the envelope glycoprotein of pathogenic SHIV(SF33A) clones folded in a conformation that was primed for interaction with CXCR4 or for the subsequent step of fusion.     

Determinants of Neutralization Resistance in the Envelope Glycoproteins of a Simian-Human Immunodeficiency Virus Passaged In Vivo

In vivo passage of a simian-human immunodeficiency virus (SHIV-89.6) generated a virus, SHIV-89.6P, that exhibited increased resistance to some neutralizing antibodies (G. B. Karlsson et al., J. Exp. Med. 188:1159-1171, 1998). Here we examine the range of human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies to which the passaged virus became resistant and identify envelope glycoprotein determinants of antibody resistance. Compared with the envelope glycoproteins derived from the parental SHIV-89.6, the envelope glycoproteins of the passaged virus were resistant to antibodies directed against the gp120 V3 variable loop and the CD4 binding site. By contrast, both viral envelope glycoproteins were equally sensitive to neutralization by two antibodies, 2G12 and 2F5, that recognize poorly immunogenic structures on gp120 and gp41, respectively. Changes in the V2 and V3 variable loops of gp120 were necessary and sufficient for full resistance to the IgG1b12 antibody, which is directed against the CD4 binding site. Changes in the V3 loop specified complete resistance to a V3 loop-directed antibody, while changes in the V1/V2 loops conferred partial resistance to this antibody. The epitopes of the neutralizing antibodies were not disrupted by the resistance-associated changes. These results indicate that in vivo selection occurs for HIV-1 envelope glycoproteins with variable loop conformations that restrict the access of antibodies to immunogenic neutralization epitopes.     

Length polymorphism within the second variable region of the human immunodeficiency virus type 1 envelope glycoprotein affects accessibility of the receptor binding site.

Sequential mutations were introduced into the V2 region of human immunodeficiency virus (HIV) type 1 HXB2, affecting the length, charge, and number of potential glycosylation sites. The insertions had no effect on cytopathicity or on the ability of virus to replicate in peripheral blood mononuclear cells and established T-cell lines. However, deletion of amino acids 186 to 188, encoding a conserved glycosylation site, resulted in a nonviable virus, suggesting a minimal length requirement of 40 amino acids for a functional V2 loop. However, all amino acid insertions affected the sensitivity of the variants to neutralization by soluble CD4 and monoclonal antibodies specific for epitopes in the V3 and CD4 binding site regions. Furthermore, these mutant viruses showed resistance to neutralization by HIV-positive human sera. Soluble gp120 mutant glycoproteins showed increased affinities for soluble CD4 and monoclonal antibodies specific for a number of epitopes overlapping the CD4 binding site, confirming that length increases in V2 affect exposure of the CD4 binding site. In summary, these data demonstrate that differences in V2 length modulate immunoreactivity of the envelope glycoprotein and support an association between the V2 and CD4 binding site regions.     

Importance of the V1/V2 loop region of simian-human immunodeficiency virus envelope glycoprotein gp120 in determining the strain specificity of the neutralizing antibody response

Plasma samples from individuals infected with human immunodeficiency virus type 1 (HIV-1) are known to be highly strain specific in their ability to neutralize HIV-1 infectivity. Such plasma samples exhibit significant neutralizing activity against autologous HIV-1 isolates but typically exhibit little or no activity against heterologous strains, although some cross-neutralizing activity can develop late in infection. Monkeys infected with the simian-human immunodeficiency virus (SHIV) clone DH12 generated antibodies that neutralized SHIV DH12, but not SHIV KB9. Conversely, antibodies from monkeys infected with the SHIV clone KB9 neutralized SHIV KB9, but not SHIV DH12. To investigate the role of the variable loops of the HIV-1 envelope glycoprotein gp120 in determining this strain specificity, variable loops 1 and 2 (V1/V2), V3, or V4 were exchanged individually or in combination between SHIV DH12 and SHIV KB9. Despite the fact that both parental viruses exhibited significant infectivity and good replication in the cell lines examined, 3 of the 10 variable-loop chimeras exhibited such poor infectivity that they could not be used further for neutralization assays. These results indicate that a variable loop that is functional in the context of one particular envelope background will not necessarily function within another. The remaining seven replication-competent chimeras allowed unambiguous assignment of the sequences principally responsible for the strain specificity of the neutralizing activity present in SHIV-positive plasma. Exchange of the V1/V2 loop sequences conferred a dominant loss of sensitivity to neutralization by autologous plasma and a gain of sensitivity to neutralization by heterologous plasma. Substitution of V3 or V4 had little or no effect on the sensitivity to neutralization. These data demonstrate that the V1/V2 region of HIV-1 gp120 is principally responsible for the strain specificity of the neutralizing antibody response in monkeys infected with these prototypic SHIVs.     

N-linked glycosylation of the V3 loop and the immunologically silent face of gp120 protects human immunodeficiency virus type 1 SF162 from neutralization by anti-gp120 and anti-gp41 antibodies

We examined how asparagine-linked glycans within and adjacent to the V3 loop (C2 and C3 regions) and within the immunologically silent face (V4, C4, and V5 regions) of the human immunodeficiency virus (HIV) SF612 envelope affect the viral phenotype. Five of seven potential glycosylation sites are utilized when the virus is grown in human peripheral blood mononuclear cells, with the nonutilized sites lying within the V4 loop. Elimination of glycans within and adjacent to the V3 loop renders SF162 more susceptible to neutralization by polyclonal HIV(+)-positive and simian/human immunodeficiency virus-positive sera and by monoclonal antibodies (MAbs) recognizing the V3 loop, the CD4- and CCR5-binding sites, and the extracellular region of gp41. Importantly, our studies also indicate that glycans located within the immunologically silent face of gp120, specifically the C4 and V5 regions, also conferred on SF162 resistance to neutralization by anti-V3 loop, anti-CD4 binding site, and anti-gp41 MAbs but not by antibodies targeting the coreceptor binding site. We also observed that the amino acid composition of the V4 region contributes to the neutralization phenotype of SF162 by anti-V3 loop and anti-CD4 binding site MAbs. Collectively, our data support the proposal that the glycosylation and structure of the immunologically silent face of the HIV envelope plays an important role in defining the neutralization phenotype of HIV type 1.     

Neutralizing Antibody Escape during HIV-1 Mother-to-Child Transmission Involves Conformational Masking of Distal Epitopes in Envelope

HIV-1 variants transmitted to infants are often resistant to maternal neutralizing antibodies (NAbs), suggesting that they have escaped maternal NAb pressure. To define the molecular basis of NAb escape that contributes to selection of transmitted variants, we analyzed 5 viruses from 2 mother-to-child transmission pairs, in which the infant virus, but not the maternal virus, was resistant to neutralization by maternal plasma near transmission. We generated chimeric viruses between maternal and infant envelope clones obtained near transmission and examined neutralization by maternal plasma. The molecular determinants of NAb escape were distinct, even when comparing two maternal variants to the transmitted infant virus within one pair, in which insertions in V4 of gp120 and substitutions in HR2 of gp41 conferred neutralization resistance. In another pair, deletions and substitutions in V1 to V3 conferred resistance, but neither V1/V2 nor V3 alone was sufficient. Although the sequence determinants of escape were distinct, all of them involved modifications of potential N-linked glycosylation sites. None of the regions that mediated escape were major linear targets of maternal NAbs because corresponding peptides failed to compete for neutralization. Instead, these regions disrupted multiple distal epitopes targeted by HIV-1-specific monoclonal antibodies, suggesting that escape from maternal NAbs occurred through conformational masking of distal epitopes. This strategy likely allows HIV-1 to utilize relatively limited changes in the envelope to preserve the ability to infect a new host while simultaneously evading multiple NAb specificities present in maternal plasma.     

Characterization of Simian-Human Immunodeficiency Virus Envelope Glycoprotein Epitopes Recognized by Neutralizing Antibodies from Infected Monkeys

We characterized human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein epitopes recognized by neutralizing antibodies from monkeys recently infected by molecularly cloned simian-human immunodeficiency virus (SHIV) variants. The early neutralizing antibody response in each infected animal was directed mainly against a single epitope. This primary neutralizing epitope, however, differed among individual monkeys infected by identical viruses. Two such neutralization epitopes were determined by sequences in the V2 and V3 loops of the gp120 envelope glycoprotein, while a third neutralization epitope, apparently discontinuous, was determined by both V2 and V3 sequences. These results indicate that the early neutralizing antibody response in SHIV-infected monkeys is monospecific and directed against epitopes composed of the gp120 V2 and V3 variable loops.     

Envelope glycoprotein determinants of neutralization resistance in a simian-human immunodeficiency virus (SHIV-HXBc2P 3.2) derived by passage in monkeys

The simian-human immunodeficiency virus SHIV-HXBc2 contains the envelope glycoproteins of a laboratory-adapted, neutralization-sensitive human immunodeficiency virus type 1 variant, HXBc2. Serial in vivo passage of the nonpathogenic SHIV-HXBc2 generated SHIV KU-1, which causes rapid CD4(+) T-cell depletion and an AIDS-like illness in monkeys. A molecularly cloned pathogenic SHIV, SHIV-HXBc2P 3.2, was derived from the SHIV KU-1 isolate and differs from the parental SHIV-HXBc2 by only 12 envelope glycoprotein amino acid residues. Relative to SHIV-HXBc2, SHIV-HXBc2P 3.2 was resistant to neutralization by all of the antibodies tested with the exception of the 2G12 antibody. The sequence changes responsible for neutralization resistance were located in variable regions of the gp120 exterior envelope glycoprotein and in the gp41 transmembrane envelope glycoprotein. The 2G12 antibody, which neutralized SHIV-HXBc2 and SHIV-HXBc2P 3.2 equally, bound the HXBc2 and HXBc2P 3.2 envelope glycoproteins on the cell surface comparably. The ability of the other tested antibodies to achieve saturation was less for the HXBc2P 3.2 envelope glycoproteins than for the HXBc2 envelope glycoproteins, even though the affinity of the antibodies for the two envelope glycoproteins was similar. Thus, a highly neutralization-sensitive SHIV, by modifying both gp120 and gp41 glycoproteins, apparently achieves a neutralization-resistant state by decreasing the saturability of its envelope glycoproteins by antibodies.     

Antibody neutralization escape mediated by point mutations in the intracytoplasmic tail of human immunodeficiency virus type 1 gp41

The persistence of human immunodeficiency virus type 1 (HIV-1) infection in the presence of robust host immunity has been associated in part with variation in viral envelope proteins leading to antigenic variation and escape from neutralizing antibodies. Previous studies of natural neutralization escape mutants have predominantly focused on gp120 and gp41 ectodomain sequence variations that alter antibody binding via changes in conformation or glycosylation pattern of the Env, likely due to the immune pressure exerted on the exposed ectodomain component of the glycoprotein. Here, we show for the first time a novel mechanism by which point mutations in the intracytoplasmic tail of the transmembrane component (gp41) of envelope can render the virus resistant to neutralization by monoclonal antibodies and broadly neutralizing polyclonal serum antibodies. Point mutations in a highly conserved structural motif within the intracytoplasmic tail resulted in decreased binding of neutralizing antibodies to the Env ectodomain, evidently due to allosteric changes both in the gp41 ectodomain and in gp120. While receptor binding and infectivity of the mutant virus remained unaltered, the changes in Env antigenicity were associated with an increase in neutralization resistance of the mutant virus. These studies demonstrate the structurally integrated nature of gp120 and gp41 and underscore a previously unrecognized potentially critical role for even minor sequence variation of the intracytoplasmic tail in modulating the antigenicity of the ectodomain of HIV-1 envelope glycoprotein complex.     

Macaques infected with a CCR5-tropic simian/human immunodeficiency virus (SHIV) develop broadly reactive anti-HIV neutralizing antibodies

The development of anti-human immunodeficiency virus (anti-HIV) neutralizing antibodies and the evolution of the viral envelope glycoprotein were monitored in rhesus macaques infected with a CCR5-tropic simian/human immunodeficiency virus (SHIV), SHIVSF162P4. Homologous neutralizing antibodies developed within the first month of infection in the majority of animals, and their titers were independent of the extent and duration of viral replication during chronic infection. The appearance of homologous neutralizing antibody responses was preceded by the appearance of amino acid changes in specific variable and conserved regions of gp120. Amino acid changes first appeared in the V1, V2, C2, and V3 regions and subsequently in the C3, V4, and V5 regions. Heterologous neutralizing antibody responses developed over time only in animals with sustained plasma viremia. Within 2 years postinfection the breadth of these responses was as broad as that observed in certain patients infected with HIV type 1 (HIV-1) for over a decade. Despite the development of broad anti-HIV-1 neutralizing antibody responses, viral replication persisted in these animals due to viral escape. Our studies indicate that cross-reactive neutralizing antibodies are elicited in a subset of SHIVSF162P4 infected macaques and that their development requires continuous viral replication for extended periods of time. More importantly, their late appearance does not prevent progression to disease. The availability of an animal model where cross-reactive anti-HIV neutralizing antibodies are developed may facilitate the identification of virologic and immunologic factors conducive to the development of such antibodies.     

V2 loop glycosylation of the human immunodeficiency virus type 1 SF162 envelope facilitates interaction of this protein with CD4 and CCR5 receptors and protects the virus from neutralization by anti-V3 loop and anti-CD4 binding site antibodies

We examined the role of asparagine-linked glycosylation of the V2 loop of the human immunodeficiency virus (HIV) SF162 envelope on viral replication potential and neutralization susceptibility. We report that the asparagines located at the amino- and carboxy-terminal sites (at positions 154 and 195, respectively), as well as within the V2 loop of the SF162 envelope (at position 186), are glycosylated during in vitro replication of this virus in human peripheral blood mononuclear cells. Our studies indicate that glycosylation of the V2 loop, in particular at its base, facilitates the interaction of the HIV envelope with the CD4 and CCR5 receptor molecules present on the surface of target cells and affects viral replication kinetics in a cell type-dependent manner. In cells expressing high numbers of receptor molecules on their surfaces, the SF162-derived V2 loop-deglycosylated mutant viruses replicate as efficiently as the parental SF162 virus, while in cells expressing small numbers of receptor molecules, the mutant viruses replicate with markedly reduced efficiency. In addition to expanding the viral tropism, V2 loop glycosylation at the three sites examined prevents neutralization by anti-CD4 binding site antibodies. In contrast, glycosylation at the amino- and carboxy-terminal sites of the V2 loop but not within the loop itself offers protection against anti-V3 loop antibodies. Thus, the epitopes masked by the sugar molecules present on the three glycosylation sites examined are not identical but overlap.     

R5X4 viruses are evolutionary, functional, and antigenic intermediates in the pathway of a simian-human immunodeficiency virus coreceptor switch

To examine the pathway of the coreceptor switching of CCR5-using (R5) virus to CXCR4-using (X4) virus in simian-human immunodeficiency virus SHIV(SF162P3N)-infected rhesus macaque BR24, analysis was performed on variants present at 20 weeks postinfection, the time when the signature gp120 V3 loop sequence of the X4 switch variant was first detected by PCR. Unexpectedly, circulating and tissue variants with His/Ile instead of the signature X4 V3 His/Arg insertions predominated at this time point. Phylogenetic analysis of the sequences of the C2 conserved region to the V5 variable loop of the envelope (Env) protein showed that viruses bearing HI insertions represented evolutionary intermediates between the parental SHIV(SF162P3N) and the final X4 HR switch variant. Functional analyses demonstrated that the HI variants were phenotypic intermediates as well, capable of using both CCR5 and CXCR4 for entry. However, the R5X4 intermediate virus entered CCR5-expressing target cells less efficiently than the parental R5 strain and was more sensitive to both CCR5 and CXCR4 inhibitors than either the parental R5 or the final X4 virus. It was also more sensitive than the parental R5 virus to antibody neutralization, especially to agents directed against the CD4 binding site, but not as sensitive as the late X4 virus. Significantly, the V3 loop sequence that determined CXCR4 use also conferred soluble CD4 neutralization sensitivity. Collectively, the data illustrate that, similar to human immunodeficiency virus type 1 (HIV-1) infection in individuals, the evolution from CCR5 to CXCR4 usage in BR24 transitions through an intermediate phase with reduced virus entry and coreceptor usage efficiencies. The data further support a model linking an open envelope gp120 conformation, better CD4 binding, and expansion to CXCR4 usage.     

Mutations in both gp120 and gp41 Are Responsible for the Broad Neutralization Resistance of Variant Human Immunodeficiency Virus Type 1 MN to Antibodies Directed at V3 and Non-V3 Epitopes

The escape of human immunodeficiency virus type 1 from effects of neutralizing antibodies was studied by using neutralization-resistant (NR) variants generated by growing the neutralization-sensitive (NS) wild-type MN virus in the presence of human serum with neutralizing antibodies, more than 99% of which were directed at the V3 region of gp120. The variants obtained had broad neutralization resistance to human sera, without limitation with respect to the V3 specificity of the sera. The molecular basis for the resistance was evaluated with molecularly cloned viruses, as well as with pseudoviruses expressing envelope glycoproteins of the NS and NR phenotypes. Nucleotide sequence analyses comparing NS and NR clones revealed a number of polymorphisms, including six in the V1/V2 region, two in C4/V5 of gp120, three in the leucine zipper (LZ) domain of gp41, and two in the second external putative α-helix region of gp41. A series of chimeras from NS and NR env genes was constructed, and each was presented on pseudoviruses to locate the domain(s) which conferred the phenotypic changes. The neutralization phenotypes of the chimeric clones were found to be dependent on mutations in both the C4/V5 region of gp120 and the LZ region of gp41. Additionally, interaction between mutations in gp120 and gp41 was demonstrated in that a chimeric env gene consisting of a gp120 coding sequence from an NS clone and a gp41 sequence from an NR clone yielded a pseudovirus with minimal infectivity. The possible significance of predicted amino acid changes in these domains is discussed. The results indicate that polyvalent antibodies predominantly directed against V3 can induce NR through selection for mutations that alter interactions of other domains in the envelope complex.     

Removal of N-linked glycosylation sites in the V1 region of simian immunodeficiency virus gp120 results in redirection of B-cell responses to V3

One mechanism of immune evasion utilized by human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) envelope glycoproteins is the presence of a dense carbohydrate shield. Accumulating evidence from in vitro and in vivo experiments suggests that alterations in N-linked glycosylation of SIV gp120 can enhance host humoral immune responses that may be involved in immune control. The present study was designed to determine the ability of glycosylation mutant viruses to redirect antibody responses to shielded envelope epitopes. The influence of glycosylation on the maturation and specificity of antibody responses elicited by glycosylation mutant viruses containing mutations of specific N-linked sites in and near the V1 and V2 regions of SIVmac239 gp120 was determined. Results from these studies demonstrated a remarkably similar maturation of antibody responses to native, fully glycosylated envelope proteins. However, analyses of antibodies to defined envelope domains revealed that mutation of glycosylation sites in V1 resulted in increased antibody recognition to epitopes in V1. In addition, we demonstrated for the first time that mutation of glycosylation sites in V1 resulted in a redirection of antibody responses to the V3 loop. Taken together, these results demonstrate that N-linked glycosylation is a determinant of SIV envelope B-cell immunogenicity in addition to in vitro antigenicity. In addition, our results demonstrate that the absence of N-linked carbohydrates at specific sites can influence the exposure of epitopes quite distant in the linear sequence.     

A single injection of recombinant measles virus vaccines expressing human immunodeficiency virus (HIV) type 1 clade B envelope glycoproteins induces neutralizing antibodies and cellular immune responses to HIV

The anchored and secreted forms of the human immunodeficiency virus type 1 (HIV-1) 89.6 envelope glycoprotein, either complete or after deletion of the V3 loop, were expressed in a cloned attenuated measles virus (MV) vector. The recombinant viruses grew as efficiently as the parental virus and expressed high levels of the HIV protein. Expression was stable during serial passages. The immunogenicity of these recombinant vectors was tested in mice susceptible to MV and in macaques. High titers of antibodies to both MV and HIV-Env were obtained after a single injection in susceptible mice. These antibodies neutralized homologous SHIV89.6p virus, as well as several heterologous HIV-1 primary isolates. A gp160 mutant in which the V3 loop was deleted induced antibodies that neutralized heterologous viruses more efficiently than antibodies induced by the native envelope protein. A high level of CD8+ and CD4+ cells specific for HIV gp120 was also detected in MV-susceptible mice. Furthermore, recombinant MV was able to raise immune responses against HIV in mice and macaques with a preexisting anti-MV immunity. Therefore, recombinant MV vaccines inducing anti-HIV neutralizing antibodies and specific T lymphocytes responses deserve to be tested as a candidate AIDS vaccine.     

Macrophage-tropic and T-cell line-adapted chimeric strains of human immunodeficiency virus type 1 differ in their susceptibilities to neutralization by soluble CD4 at different temperatures.

Molecular clones of three macrophage-tropic and three T-cell line-adapted strains of human immunodeficiency virus type 1 (HIV-1) were used to explore the mechanism of HIV-1 resistance to neutralization by soluble CD4 (sCD4). The three macrophage-tropic viruses, each possessing the V3 and flanking regions of JR-FL, were all resistant to sCD4 neutralization under the standard conditions of a short preincubation of the virus and sCD4 at 37 degrees C prior to inoculation of peripheral blood mononuclear cells. In contrast, the three T-cell line-adapted viruses, NL4-3 and two chimeras possessing the V3 and flanking regions of NL4-3 in the envelope background of JR-FL, were all sCD4 sensitive under these conditions. Sensitivity to sCD4 neutralization at 37 degrees C corresponded with rapid, sCD4-induced gp120 shedding from the viruses. However, when the incubation temperature of the sCD4 and virus was reduced to 4 degrees C, the three macrophage-tropic viruses shed gp120 and became more sensitive to sCD4 neutralization. In contrast, the rates of sCD4-induced gp120 shedding and virus neutralization were reduced for the three T-cell line-adapted viruses at 4 degrees C. Thus, HIV resistance to sCD4 is a conditional phenomenon; macrophage-tropic and T-cell line-adapted strains can be distinguished by the temperature dependencies of their neutralization by sCD4. The average density of gp120 molecules on the macrophage-tropic viruses exceeded by about fourfold that on the T-cell line-adapted viruses, suggesting that HIV growth in T-cell lines may select for a destabilized envelope glycoprotein complex. Further studies of early events in HIV-1 infection should focus on primary virus strains.     

Glycans Flanking the Hypervariable Connecting Peptide between the A and B Strands of the V1/V2 Domain of HIV-1 gp120 Confer Resistance to Antibodies That Neutralize CRF01_AE Viruses

Understanding the molecular determinants of sensitivity and resistance to neutralizing antibodies is critical for the development of vaccines designed to prevent HIV infection. In this study, we used a genetic approach to characterize naturally occurring polymorphisms in the HIV envelope protein that conferred neutralization sensitivity or resistance. Libraries of closely related envelope genes, derived from virus quasi-species, were constructed from individuals infected with CRF01_AE viruses. The libraries were screened with plasma containing broadly neutralizing antibodies, and neutralization sensitive and resistant variants were selected for sequence analysis. In vitro mutagenesis allowed us to identify single amino acid changes in three individuals that conferred resistance to neutralization by these antibodies. All three mutations created N-linked glycosylation sites (two at N136 and one at N149) proximal to the hypervariable connecting peptide between the C-terminus of the A strand and the N-terminus of the B strand in the four-stranded V1/V2 domain β-sheet structure. Although N136 has previously been implicated in the binding of broadly neutralizing monoclonal antibodies, this glycosylation site appears to inhibit the binding of neutralizing antibodies in plasma from HIV-1 infected subjects. Previous studies have reported that the length of the V1/V2 domain in transmitted founder viruses is shorter and possesses fewer glycosylation sites compared to viruses isolated from chronic infections. Our results suggest that vaccine immunogens based on recombinant envelope proteins from clade CRF01_AE viruses might be improved by inclusion of envelope proteins that lack these glycosylation sites. This strategy might improve the efficacy of the vaccines used in the partially successful RV144 HIV vaccine trial, where the two CRF01_AE immunogens (derived from the A244 and TH023 isolates) both possessed glycosylation sites at N136 and N149.     

Glycosylation of gp41 of simian immunodeficiency virus shields epitopes that can be targets for neutralizing antibodies

Human immunodeficiency virus type 1 and simian immunodeficiency virus possess three closely spaced, highly conserved sites for N-linked carbohydrate attachment in the extracellular domain of the transmembrane protein gp41. We infected rhesus monkeys with a variant of cloned SIVmac239 lacking the second and third sites or with a variant strain lacking all three of SIVmac239's glycosylation sites in gp41. For each mutation, asparagine (N) in the canonical N-X-S/T recognition sequence for carbohydrate attachment was changed to the structurally similar glutamine such that two nucleotide changes would be required for a reversion of the mutated codon. By 16 weeks, experimentally infected monkeys made antibodies that neutralized the mutant viruses to high titers. Such antibodies were not observed in monkeys infected with the parental virus. Thus, new specificities were revealed as a result of the carbohydrate attachment mutations, and antibodies of these specificities had neutralizing activity. Unlike monkeys infected with the parental virus, monkeys infected with the mutant viruses made antibodies that reacted with peptides corresponding to the sequences in this region. Furthermore, there was strong selective pressure for the emergence of variant sequences in this region during the course of infection. By analyzing the neutralization profiles of sequence variants, we were able to define three mutations (Q625R, K631N, and Q634H) in the region of the glycosylation site mutations that conferred resistance to neutralization by plasma from the monkeys infected with mutant virus. Based on the reactivity of antibodies to peptides in this region and the colocalization of neutralization escape mutations, we conclude that N-linked carbohydrates in the ectodomain of the transmembrane protein shield underlying epitopes that would otherwise be the direct targets of neutralizing antibodies.     

Human immunodeficiency virus type 1 V1-V2 envelope loop sequences expand and add glycosylation sites over the course of infection, and these modifications affect antibody neutralization sensitivity

Over the course of infection, human immunodeficiency virus type 1 (HIV-1) continuously adapts to evade the evolving host neutralizing antibody responses. Changes in the envelope variable loop sequences, particularly the extent of glycosylation, have been implicated in antibody escape. To document modifications that potentially influence antibody susceptibility, we compared envelope variable loops 1 and 2 (V1-V2) from multiple sequences isolated at the primary phase of infection to those isolated around 2 to 3 years into the chronic phase of infection in nine women with HIV-1 subtype A. HIV-1 sequences isolated during chronic infection had significantly longer V1-V2 loops, with a significantly higher number of potential N-linked glycosylation sites, than the sequences isolated early in infection. To assess the effects of these V1-V2 changes on antibody neutralization and infectivity, we created chimeric envelope sequences, which incorporated a subject's V1-V2 sequences into a common subtype A envelope backbone and then used them to generate pseudotyped viruses. Compared to the parent virus, the introduction of a subject's early-infection V1-V2 envelope variable loops rendered the chimeric envelope more sensitive to that subject's plasma samples but only to plasma samples collected >6 months after the sequences were isolated. Neutralization was not detected with the same plasma when the early-infection V1-V2 sequences were replaced with chronic-infection V1-V2 sequences, suggesting that changes in V1-V2 contribute to antibody escape. Pseudotyped viruses with V1-V2 segments from different times in infection, however, showed no significant difference in neutralization sensitivity to heterologous pooled plasma, suggesting that viruses with V1-V2 loops from early in infection were not inherently more neutralization sensitive. Pseudotyped viruses bearing chimeric envelopes with early-infection V1-V2 sequences showed a trend in infecting cells with low CD4 concentrations more efficiently, while engineered viruses with V1-V2 sequences isolated during chronic infection were moderately better at infecting cells with low CCR5 concentrations. These studies suggest that changes within the V1-V2 envelope domains over the course of an infection influence sensitivity to autologous neutralizing antibodies and may also impact host receptor/coreceptor interactions.