Unique mutational patterns in the envelope alpha 2 amphipathic helix and acquisition of length in gp120 hypervariable domains are associated with resistance to autologous neutralization of subtype C human immunodeficiency virus type 1

Autologous neutralizing antibodies (NAb) against human immunodeficiency virus type 1 generate viral escape variants; however, the mechanisms of escape are not clearly defined. In a previous study, we determined the susceptibilities of 48 donor and 25 recipient envelope (Env) glycoproteins from five subtype C heterosexual transmission pairs to NAb in donor plasma by using a virus pseudotyping assay, thereby providing an ideal setting to probe the determinants of susceptibility to neutralization. In the present study, acquisition of length in the Env gp120 hypervariable domains was shown to correlate with resistance to NAb in donor plasma (P = 0.01; Kendall's tau test) but not in heterologous plasma. Sequence divergence in the gp120 V1-to-V4 region also correlated with resistance to donor (P = 0.0002) and heterologous (P = 0.001) NAb. A mutual information analysis suggested possible associations of nine amino acid positions in V1 to V4 with NAb resistance to the donor's antibodies, and five of these were located within an 18-residue amphipathic helix (alpha2) located on the gp120 outer domain. High nonsynonymous-to-synonymous substitution (dN/dS) ratios, indicative of positive selection, were also found at these five positions in subtype C sequences in the database. Nevertheless, exchange of the entire alpha2 helix between resistant donor Envs and sensitive recipient Envs did not alter the NAb phenotype. The combined mutual information and dN/dS analyses suggest that unique mutational patterns in alpha2 and insertions in the V1-to-V4 region are associated with NAb resistance during subtype C infection but that the selected positions within the alpha2 helix must be linked to still other changes in Env to confer antibody escape. These findings suggest that subtype C viruses utilize mutations in the alpha2 helix for efficient viral replication and immune avoidance.     

Longer V1V2 region with increased number of potential N-linked glycosylation sites in the HIV-1 envelope glycoprotein protects against HIV-specific neutralizing antibodies

Human immunodeficiency virus type 1 (HIV-1) has the ability to adapt to the host environment by escaping from host immune responses. We previously observed that escape from humoral immunity, both at the individual and at a population level, coincided with longer variable loops and an increased number of potential N-linked glycosylation sites (PNGS) in the viral envelope glycoprotein (Env) and, in particular, in variable regions 1 and 2 (V1V2). Here, we provide several lines of evidence for the role of V1V2 in the resistance of HIV-1 to neutralizing antibodies. First, we determined that the increasing neutralization resistance of a reference panel of tier-categorized neutralization-sensitive and -resistant HIV-1 variants coincided with a longer V1V2 loop containing more PNGS. Second, an exchange of the different variable regions of Env from a neutralization-sensitive HIV-1 variant into a neutralization-resistant escape variant from the same individual revealed that the V1V2 loop is a strong determinant for sensitivity to autologous-serum neutralization. Third, exchange of the V1V2 loop of neutralization-sensitive HIV-1 variants from historical seroconverters with the V1V2 loop of neutralization-resistant HIV-1 variants from contemporary seroconverters decreased the neutralization sensitivity to CD4-binding site-directed antibodies. Overall, we demonstrate that an increase in the length of the V1V2 loop and/or the number of PNGS in that same region of the HIV-1 envelope glycoprotein is directly involved in the protection of HIV-1 against HIV-specific neutralizing antibodies, possibly by shielding underlying epitopes in the envelope glycoprotein from antibody recognition.     

Rapid Escape from Preserved Cross-Reactive Neutralizing Humoral Immunity without Loss of Viral Fitness in HIV-1-Infected Progressors and Long-Term Nonprogressors

A substantial proportion of human immunodeficiency virus type 1 (HIV-1)-infected individuals has cross-reactive neutralizing activity in serum, with a similar prevalence in progressors and long-term nonprogressors (LTNP). We studied whether disease progression in the face of cross-reactive neutralizing serum activity is due to fading neutralizing humoral immunity over time or to viral escape. In three LTNP and three progressors, high-titer cross-reactive HIV-1-specific neutralizing activity in serum against a multiclade pseudovirus panel was preserved during the entire clinical course of infection, even after AIDS diagnosis in progressors. However, while early HIV-1 variants from all six individuals could be neutralized by autologous serum, the autologous neutralizing activity declined during chronic infection. This could be attributed to viral escape and the apparent inability of the host to elicit neutralizing antibodies to the newly emerging viral escape variants. Escape from autologous neutralizing activity was not associated with a reduction in the viral replication rate in vitro. Escape from autologous serum with cross-reactive neutralizing activity coincided with an increase in the length of the variable loops and in the number of potential N-linked glycosylation sites in the viral envelope. Positive selection pressure was observed in the variable regions in envelope, suggesting that, at least in these individuals, these regions are targeted by humoral immunity with cross-reactive potential. Our results may imply that the ability of HIV-1 to rapidly escape cross-reactive autologous neutralizing antibody responses without the loss of viral fitness is the underlying explanation for the absent effect of potent cross-reactive neutralizing humoral immunity on the clinical course of infection.The need for an effective vaccine to prevent the global spread of human immunodeficiency virus type 1 (HIV-1) is well recognized. The ability to elicit broadly neutralizing antibodies (BrNAbs) is believed to be crucial to developing a successful vaccine, ideally to acquire protective immunity or, alternatively, to achieve a nonprogressive infection with viral loads sufficiently low to limit HIV-1 transmission (1, 39).During natural infection, antibodies that are able to neutralize autologous virus variants are elicited in the majority of HIV-1-infected individuals. Early in infection, these neutralizing antibodies (NAbs) are mainly type specific, due to the fact that they are primarily directed against the variable domains in the viral envelope, and allow for the rapid escape of HIV-1 from antibody neutralization (8, 9, 14, 15, 20, 28, 41). Escape from type-specific neutralizing humoral immunity has been associated with enormous sequence variation, particularly in variable loops 1 and 2 (V1V2) of the envelope protein where large insertions and deletions are observed, as well as with changes in the number of potential N-linked glycosylation sites (PNGS) in the envelope protein (8, 15, 19, 22, 25, 27-31, 41). The rapid escape of HIV-1 from autologous type-specific NAbs seems to be the underlying explanation for the absent correlation between autologous humoral immunity and HIV-1 disease course. Furthermore, we recently observed that the changes in envelope that are associated with escape from autologous neutralizing humoral immunity do not coincide with a loss of viral fitness (7), providing an additional explanation for the lack of protection from disease progression by the autologous type-specific NAb response.In the last couple of years, the focus of research has shifted toward neutralizing humoral immunity with cross-reactive activity, defined as the ability to neutralize a range of heterologous HIV-1 variants from different subtypes. It has become apparent that about one-third of HIV-1-infected individuals develop cross-reactive neutralizing activity in serum. However, the prevalence of cross-reactive neutralizing activity in serum was similar for HIV-infected individuals with a progressive disease course and long-term nonprogressors (LTNP) (11, 12, 34, 37).We studied the underlying explanation for this observation in three LTNP and three progressors who all had high-titer cross-reactive neutralizing activity in serum within 2 to 4 years after seroconversion (SC). In all individuals, we observed that the potent and cross-reactive neutralizing immunity was preserved during the entire course of infection. However, the presence of cross-reactive neutralizing activity in serum did not prevent rapid viral escape from humoral immunity, which coincided with changes in envelope similar to those described for escape from type-specific autologous humoral immunity. Although broadly neutralizing antibodies are assumed to target the more conserved epitopes that may lie in crucial parts of the viral envelope, escape from cross-reactive neutralizing activity did not coincide with a loss in viral fitness. Our findings underscore that vaccine-elicited cross-reactive neutralizing immunity should protect against HIV-1 acquisition, since protection from disease progression, even by humoral immunity with strong cross-reactivity, may be an unachievable goal.     

Analysis of Neutralization Specificities in Polyclonal Sera Derived from Human Immunodeficiency Virus Type 1-Infected Individuals

During human immunodeficiency virus type 1 (HIV-1) infection, patients develop various levels of neutralizing antibody (NAb) responses. In some cases, patient sera can potently neutralize diverse strains of HIV-1, but the antibody specificities that mediate this broad neutralization are not known, and their elucidation remains a formidable challenge. Due to variable and nonneutralizing determinants on the exterior envelope glycoprotein (Env), nonnative Env protein released from cells, and the glycan shielding that assembles in the context of the quaternary structure of the functional spike, HIV-1 Env elicits a myriad of binding antibodies. However, few of these antibodies can neutralize circulating viruses. We present a systematic analysis of the NAb specificities of a panel of HIV-1-positive sera, using methodologies that identify both conformational and continuous neutralization determinants on the HIV-1 Env protein. Characterization of sera included selective adsorption with native gp120 and specific point mutant variants, chimeric virus analysis, and peptide inhibition of viral neutralization. The gp120 protein was the major neutralizing determinant for most sera, although not all neutralization activity against all viruses could be identified. In some broadly neutralizing sera, the gp120-directed neutralization mapped to the CD4 binding region of gp120. In addition, we found evidence that regions of the gp120 coreceptor binding site may also be a target of neutralizing activity. Sera displaying limited neutralization breadth were mapped to the immunogenic V3 region of gp120. In a subset of sera, we also identified NAbs directed against the conserved, membrane-proximal external region of gp41. These data allow a more detailed understanding of the humoral responses to the HIV-1 Env protein and provide insights regarding the most relevant targets for HIV-1 vaccine design.     

Evidence for potent autologous neutralizing antibody titers and compact envelopes in early infection with subtype C human immunodeficiency virus type 1

Information about neutralizing antibody responses in subtype C-infected individuals is limited, even though this viral subtype causes the majority of AIDS cases worldwide. Here we compared the course and magnitude of the autologous neutralizing antibody (NAb) response against viral envelope (Env) glycoproteins present during acute and early infection with subtypes B and C human immunodeficiency virus type 1 (HIV-1). NAb responses were evaluated in 6 subtype B-infected and 11 subtype C-infected subjects over a mean evaluation period of 25 months using a pseudovirus reporter gene assay. All subjects in the C cohort were infected through heterosexual contact, while five of the six subjects in the B cohort were infected via male-to-male contact. The kinetics and magnitude of the NAb responses varied among subjects in the B and C cohorts; however, the median 50% inhibitory concentration (IC(50) titer) reached by antibody in the plasma of subtype C-infected subjects, overall, was 3.5-fold higher than in the subtype B-infected subjects (P = 0.06). The higher titers of NAbs in the C cohort were associated with viruses having significantly shorter amino acid length (P = 0.002) in the V1 to V4 region of the surface Env glycoprotein, gp120, compared to the B cohort. Despite the potency of the autologous subtype C NAb response, it was not directed against cross-neutralizing epitopes. These data demonstrate that subtype C Envs elicit a potent yet restricted NAb response early in infection that frequently reaches IC(50) titers in excess of 1:1,000 and suggest that clade-specific differences may exist in Env immunogenicity or susceptibility to neutralization.     

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.     

Escape from Autologous Neutralizing Antibodies in Acute/Early Subtype C HIV-1 Infection Requires Multiple Pathways

One aim for an HIV vaccine is to elicit neutralizing antibodies (Nab) that can limit replication of genetically diverse viruses and prevent establishment of a new infection. Thus, identifying the strengths and weaknesses of Nab during the early stages of natural infection could prove useful in achieving this goal. Here we demonstrate that viral escape readily occurred despite the development of high titer autologous Nab in two subjects with acute/early subtype C infection. To provide a detailed portrayal of the escape pathways, Nab resistant variants identified at multiple time points were used to create a series of envelope (Env) glycoprotein chimeras and mutants within the background of a corresponding newly transmitted Env. In one subject, Nab escape was driven predominantly by changes in the region of gp120 that extends from the beginning of the V3 domain to the end of the V5 domain (V3V5). However, Nab escape pathways in this subject oscillated and at times required cooperation between V1V2 and the gp41 ectodomain. In the second subject, escape was driven by changes in V1V2. This V1V2-dependent escape pathway was retained over time, and its utility was reflected in the virus''s ability to escape from two distinct monoclonal antibodies (Mabs) derived from this same patient via introduction of a single potential N-linked glycosylation site in V2. Spatial representation of the sequence changes in gp120 suggested that selective pressure acted upon the same regions of Env in these two subjects, even though the Env domains that drove escape were different. Together the findings argue that a single mutational pathway is not sufficient to confer escape in early subtype C HIV-1 infection, and support a model in which multiple strategies, including potential glycan shifts, direct alteration of an epitope sequence, and cooperative Env domain conformational masking, are used to evade neutralization.     

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.     

Sequential evolution and escape from neutralization of simian immunodeficiency virus SIVsmE660 clones in rhesus macaques

Simian immunodeficiency virus (SIV) infection of rhesus macaques has become an important surrogate model for evaluating HIV vaccine strategies. The extreme resistance to neutralizing antibody (NAb) of many commonly used strains, such as SIVmac251/239 and SIVsmE543-3, limits their potential relevance for evaluating the role of NAb in vaccine protection. In contrast, SIVsmE660 is an uncloned virus that appears to be more sensitive to neutralizing antibody. To evaluate the role of NAb in this model, we generated full-length neutralization-sensitive molecular clones of SIVsmE660 and evaluated two of these by intravenous inoculation of rhesus macaques. All animals became infected and maintained persistent viremia that was accompanied by a decline in memory CD4(+) T cells in blood and bronchoalveolar lavage fluid. High titers of autologous NAb developed by 4 weeks postinoculation but were not associated with control of viremia, and neutralization escape variants were detected concurrently with the generation of NAb. Neutralization escape was associated with substitutions and insertion/deletion polymorphisms in the V1 and V4 domains of envelope. Analysis of representative variants revealed that escape variants also induced NAbs within a few weeks of their appearance in plasma, in a pattern that is reminiscent of the escape of human immunodeficiency virus type 1 (HIV-1) isolates in humans. Although early variants maintained a neutralization-sensitive phenotype, viruses obtained later in infection were significantly less sensitive to neutralization than the parental viruses. These results indicate that NAbs exert selective pressure that drives the evolution of the SIV envelope and that this model will be useful for evaluating the role of NAb in vaccine-mediated protection.     

Frequency and phenotype of human immunodeficiency virus envelope-specific B cells from patients with broadly cross-neutralizing antibodies

Induction of broadly cross-reactive neutralizing antibodies (NAb) is an important goal for a prophylactic human immunodeficiency virus type 1 (HIV-1) vaccine. Some HIV-infected patients make a NAb response that reacts with diverse strains of HIV-1, but most candidate vaccines have induced NAb only against a subset of highly sensitive isolates. To better understand the nature of broad NAb responses that arise during natural infection, we screened patients for sera able to neutralize diverse HIV strains and explored the frequency and phenotype of their peripheral Envelope-specific B cells. We screened 113 HIV-infected patients of various clinical statuses for the prevalence of broad NAb. Sera able to neutralize at least four of five viral isolates were found in over one-third of progressors and slow progressors, but much less frequently in aviremic long-term nonprogressors. Most Env-specific antibody-secreting B cells were CD27^hi CD38^hi plasmablasts, and the total plasmablast frequency was higher in HIV-infected patients than in uninfected donors. We found that 0.0031% of B cells and 0.047% of plasmablasts secreted Env-specific immunoglobulin G (IgG) in an enzyme-linked immunospot (ELISPOT) assay. We developed a novel staining protocol to label HIV-specific B cells with Env gp140 protein. A total of 0.09% of B cells were found to be Env-specific by this method, a frequency far higher than that indicated by ELISPOT assay. gp140-labeled B cells were predominantly CD27⁺ and surface IgG⁺. These data describe the breadth and titer of serum NAb and the frequency and phenotype of HIV-specific B cells in a cohort of patients with broad cross-neutralizing antibody responses that are potential goals for vaccines for HIV.     

Selection pressure on HIV-1 envelope by broadly neutralizing antibodies to the conserved CD4-binding site

The monoclonal antibody (MAb) VRC01 was isolated from a slowly progressing HIV-1-infected donor and was shown to neutralize diverse HIV-1 strains by binding to the conserved CD4 binding site (CD4bs) of gp120. To better understand the virologic factors associated with such antibody development, we characterized HIV-1 envelope (Env) variants from this donor and five other donors who developed broadly neutralizing antibodies. A total of 473 env sequences were obtained by single-genome amplification, and 100 representative env clones were expressed and tested for entry and neutralization sensitivity. While VRC01 neutralizes about 90% of the genetically diverse heterologous HIV-1 strains tested, only selective archival Env variants from the VRC01 donor were sensitive to VRC01 and all of the Env variants derived from the donor plasma were resistant, indicating strong antibody-based selection pressure. Despite their resistance to this broadly reactive MAb that partially mimics CD4, all Env variants required CD4 for entry. Three other CD4bs MAbs from the same donor were able to neutralize some VRC01 escape variants, suggesting that CD4bs antibodies continued to evolve in response to viral escape. We also observed a relatively high percentage of VRC01-resistant Env clones in the plasma of four of five additional broadly neutralizing donors, suggesting the presence of CD4bs-directed neutralizing antibodies in these donors. In total, these data indicate that the CD4bs-directed neutralizing antibodies exert ongoing selection pressure on the conserved CD4bs epitope of HIV-1 Env.     

Envelope variable region 4 is the first target of neutralizing antibodies in early simian immunodeficiency virus mac251 infection of rhesus monkeys

A major goal of AIDS vaccine development is to design vaccination strategies that can elicit broad and potent protective antibodies. The initial viral targets of neutralizing antibodies (NAbs) early after human or simian immunodeficiency virus (HIV/SIV) infection are not known. The identification of early NAb epitopes that induce protective immunity or retard the progression of disease is important for AIDS vaccine development. The aim of this study was to determine the Env residues targeted by early SIV NAbs and to assess the influence of prior vaccination on neutralizing antibody kinetics and specificity during early infection. We previously described stereotypic env sequence variations in SIVmac251-infected rhesus monkeys that resulted in viral escape from NAbs. Here, we defined the early viral targets of neutralization and determined whether the ability of serum antibody from infected monkeys to neutralize SIV was altered in the setting of prior vaccination. To localize the viral determinants recognized by early NAbs, a panel of mutant pseudoviruses was assessed in a TZM-bl reporter gene neutralization assay to define the precise changes that eliminate recognition by SIV Env-specific NAbs in 16 rhesus monkeys. Changing R420 to G or R424 to Q in V4 of Env resulted in the loss of recognition by NAbs in vaccinated monkeys. In contrast, mutations in the V1 region of Env did not alter the NAb profile. These findings indicate that early NAbs are directed toward SIVmac251 Env V4 but not the V1 region, and that this env vaccination regimen did not alter the kinetics or the breadth of NAbs during early infection.     

Lack of Viral Escape and Defective In Vivo Activation of Human Immunodeficiency Virus Type 1-Specific Cytotoxic T Lymphocytes in Rapidly Progressive Infection

Human immunodeficiency virus type 1 (HIV-1)-specific immune responses over the course of rapidly progressive infection are not well defined. Detailed longitudinal analyses of neutralizing antibodies, lymphocyte proliferation, in vivo-activated and memory cytotoxic T-lymphocyte (CTL) responses, and viral sequence variation were performed on a patient who presented with acute HIV-1 infection, developed an AIDS-defining illness 13 months later, and died 45 months after presentation. Neutralizing-antibody responses remained weak throughout, and no HIV-1-specific lymphocyte proliferative responses were seen even early in the disease course. Strong in vivo-activated CTL directed against Env and Pol epitopes were present at the time of the initial drop in viremia but were quickly lost. Memory CTL against Env and Pol epitopes were detected throughout the course of infection; however, these CTL were not activated in vivo. Despite an initially narrow CTL response, new epitopes were not targeted as the disease progressed. Viral sequencing showed the emergence of variants within the two targeted CTL epitopes; however, viral variants within the immunodominant Env epitope were well recognized by CTL, and there was no evidence of viral escape from immune system detection within this epitope. These data demonstrate a narrowly directed, static CTL response in a patient with rapidly progressive disease. We also show that disease progression can occur in the presence of persistent memory CTL recognition of autologous epitopes and in the absence of detectable escape from CTL responses, consistent with an in vivo defect in activation of CTL.     

Mutations in envelope gp120 can impact proteolytic processing of the gp160 precursor and thereby affect neutralization sensitivity of human immunodeficiency virus type 1 pseudoviruses

The design of an efficient human immunodeficiency virus (HIV) immunogen able to generate broad neutralizing antibodies (NAbs) remains an elusive goal. As more data emerge, it is becoming apparent that one important aspect of such an immunogen will be the proper representation of the envelope protein (Env) as it exists on native virions. Important questions that are yet to be fully addressed include what factors dictate Env processing, how different Env forms are represented on the virion, and ultimately how these issues influence the development and efficacy of NAbs. Recent data have begun to illuminate the extent to which changes in gp41 can impact the overall structure and neutralizing sensitivity of Env. Here, we present evidence to suggest that minor mutations in gp120 can significantly impact Env processing. We analyzed the gp120 sequences of 20 env variants that evolved in multiple macaques over 8 months of infection with simian/human immunodeficiency virus 89.6P. Variant gp120 sequences were subcloned into gp160 expression plasmids with identical cleavage motifs and gp41 sequences. Cells cotransfected with these plasmids and Δenv genomes were able to produce competent virus. The resulting pseudoviruses incorporated high levels of Env onto virions that exhibited a range of degrees of virion-associated Env cleavage (15 to 40%). Higher levels of cleavage correlated with increased infectivity and increased resistance to macaque plasma, HIV immunoglobulin, soluble CD4, and human monoclonal antibodies 4E10, 2F5, and b12. Based on these data, we discuss a model whereby changes in gp120 of 89.6P impact Env processing and thereby mediate escape from a range of neutralizing agents.     

Potent autologous and heterologous neutralizing antibody responses occur in HIV-2 infection across a broad range of infection outcomes

Few studies have explored the role of neutralizing antibody (NAb) responses in controlling HIV-2 viremia and disease progression. Using a TZM-bl neutralization assay, we assessed heterologous and autologous NAb responses from a community cohort of HIV-2-infected individuals with a broad range of disease outcomes in rural Guinea-Bissau. All subjects (n = 40) displayed exceptionally high heterologous NAb titers (50% inhibitory plasma dilution or 50% inhibitory concentration [IC(50)], 1:7,000 to 1:1,000,000) against 5 novel primary HIV-2 envelopes and HIV-2 7312A, whereas ROD A and 3 primary envelopes were relatively resistant to neutralization. Most individuals also showed high autologous NAb against contemporaneous envelopes (78% of plasma-envelope combinations in 69 envelopes from 21 subjects), with IC(50)s above 1:10,000. No association between heterologous or autologous NAb titer and greater control of HIV-2 was found. A subset of envelopes was found to be more resistant to neutralization (by plasma and HIV-2 monoclonal antibodies). These envelopes were isolated from individuals with greater intrapatient sequence diversity and were associated with changes in potential N-linked glycosylation sites but not CD4 independence or CXCR4 use. Plasma collected from up to 15 years previously was able to potently neutralize recent autologous envelopes, suggesting a lack of escape from NAb and the persistence of neutralization-sensitive variants over time, despite significant NAb pressure. We conclude that despite the presence of broad and potent NAb responses in HIV-2-infected individuals, these are not the primary forces behind the dichotomous outcomes observed but reveal a limited capacity for adaptive selection and escape from host immunity in HIV-2 infection.     

Neutralizing antibodies do not mediate suppression of human immunodeficiency virus type 1 in elite suppressors or selection of plasma virus variants in patients on highly active antiretroviral therapy

Neutralizing antibodies (NAb) against autologous virus can reach high titers in human immunodeficiency virus type 1 (HIV-1)-infected patients with progressive disease. Less is known about the role of NAb in HIV-1-infected patients with viral loads of <50 copies/ml of plasma, including patients on effective highly active antiretroviral therapy (HAART) and elite suppressors, who control HIV-1 replication without antiretroviral therapy. In this study, we analyzed full-length env sequences from plasma viruses and proviruses in resting CD4(+) T cells of HAART-treated patients, elite suppressors, and untreated HIV-1-infected patients with progressive disease. For each patient group, we assessed plasma virus neutralization by autologous, contemporaneous plasma. The degree of env diversity, the number of N-linked glycosylation sites, and the lengths of variable loops were all lower in elite suppressors than in HAART-treated and untreated viremic patients. Both elite suppressors and HAART-treated patients had lower titers of NAb against HIV-1 lab strains than those of untreated viremic patients. Surprisingly, titers of NAb against autologous, contemporaneous plasma viruses were similarly low in chronic progressors, elite suppressors, and HAART-treated patients. In elite suppressors and HAART-treated patients, titers of NAb against autologous plasma viruses also did not differ significantly from titers against autologous proviruses from resting CD4(+) T cells. These results suggest that high-titer NAb are not required for maintenance of viral suppression in elite suppressors and that NAb do not select plasma virus variants in most HAART-treated patients. Both drug-mediated and natural suppression of HIV-1 replication to levels below 50 copies/ml may limit the stimulation and maintenance of effective NAb responses.     

Human immunodeficiency virus (HIV)-positive sera obtained shortly after seroconversion neutralize autologous HIV type 1 isolates on primary macrophages but not on lymphocytes

The aim of this study was to analyze the role of humoral immunity in early human immunodeficiency virus (HIV) infection. As neutralizing activities in HIV-positive sera are rarely detectable earlier than 9 to 12 months after infection using primary lymphocytes as target cells in neutralization assays, humoral immunity is generally thought not to contribute significantly to early virus control in the patients. Besides lymphocytes, cells of the monocyte/macrophage lineage are known to be important target cells for HIV in vivo during the establishment of the infection. Therefore, we studied the neutralization of early primary HIV isolates by autologous serum samples using primary macrophages as target cells in the neutralization assays. We analyzed neutralizing activities against the autologous HIV-1 isolates in 10 patients' sera taken shortly after seroconversion, both on primary macrophages and, for comparison, on lymphocytes. Viruses were isolated and expanded in primary mixed cultures containing macrophages and lymphocytes in order to avoid selection for one particular cell type. All viruses replicated to different degrees in macrophages and lymphocytes; nine had a nonsyncytium-inducing phenotype, and one was syncytium inducing. The detection of neutralizing antibodies in acute primary HIV infection depended on the target cells used. Confirming previous studies, we did not find neutralizing activities on lymphocytes at this early time point. In contrast, neutralizing activities were detectable in the same sera if primary macrophages were used as target cells. Differences in neutralizing activities on macrophages and lymphocytes were not due to different virus variants being present in the different cell systems, as gp120 sequences derived from both cell types were homogeneous. Neutralization activities on macrophages did not correlate with the amount of beta-chemokines in the sera. As affinity-purified immunoglobulin G preparations from an early patient serum also exhibited neutralization of the autologous virus isolate on primary macrophages, but not on lymphocytes, neutralization is very likely due to antibodies against viral epitopes necessary for infection of macrophages but not for infection of lymphocytes. Our data suggest that, along with cell-mediated immunity, humoral immunity may contribute to the reduction of primary viremia in the patient. This was further supported by a certain association between neutralizing antibody titers on macrophages and viral load in the patients.     

Continuous viral escape and selection by autologous neutralizing antibodies in drug-naive human immunodeficiency virus controllers

We assessed differences in the character and specificity of autologous neutralizing antibodies (ANAbs) against individual viral variants of the quasispecies in a cohort of drug-naïve subjects with long-term controlled human immunodeficiency virus type 1 (HIV-1) infection and moderate levels of broad heterologous neutralizing antibodies (HNAb). Functional plasma virus showed continuous env evolution despite a short time frame and low levels of viral replication. Neutralization-sensitive variants dominated in subjects with intermittent viral blips, while neutralization-resistant variants predominated in elite controllers. By sequence analysis of this panel of autologous variants with various sensitivities to neutralization, we identified more than 30 residues in envelope proteins (Env) associated with resistance or sensitivity to ANAbs. The appearance of new sensitive variants is consistent with a model of continuous selection and turnover. Strong ANAb responses directed against autologous Env variants are present in long-term chronically infected individuals, suggesting a role for these responses in contributing to the durable control of HIV replication.Antibodies capable of neutralizing a subject''s own virus, called autologous neutralizing antibodies (ANAbs), have been the subject of recent studies redefining the timing and character of this response. ANAbs develop early in essentially all seropositive subjects and increase in titer during the first few months and years of infection (15, 30). Previously published data were obtained using an assay that measures ANAbs against the complete quasispecies without an analysis of the individual envelope protein (Env) sequences to which these ANAb responses were directed (10). The contemporaneous virus pool was poorly neutralized, leading to an assumption that contemporaneous ANAbs are ineffective in controlling viremia. In chronic infection, ANAbs generally have been difficult to detect (3, 29, 31, 40), but there is ample evidence for selection by NAb and resulting virus env evolution in the host (12, 30, 38). The titers of ANAbs measured against clinical or autologous isolates cultured in peripheral blood mononuclear cells typically have been low in chronic infection (31, 40), while other studies indicated the presence of strong ANAbs (2). Although ANAbs may be ineffective in subjects with high virus loads due to the continuous generation of escape variants, their role in maintaining low viral loads in human immunodeficiency virus (HIV) controllers is not known.NAbs that recognize heterologous isolates to which the subject has never been exposed, called heterologous NAbs (HNAbs), are found later in infection, and not all subjects develop this broadening of the response (5). In studies that utilized easy-to-neutralize laboratory or primary viruses, titers of HNAbs can be high (5, 6, 26, 29). Early work had shown that polyclonal HNAbs in HIV-infected subjects are directed to conserved conformational determinants on gp120 (32), including the CD4-binding site (CD4bs) (22). Several human neutralizing monoclonal antibodies with broad activity also are directed to conserved conformational determinants on Env proteins, such as the CD4bs (4) and V3 (17). However, the mechanisms that lead to the development of broad HNAbs are unknown. Their development likely is dependent upon the specific autologous Env proteins to which the subject is exposed, and these proteins are variants of the original infection in these subjects, except for cases of superinfection. Thus, we reasoned that a detailed analysis of the neutralization of individual autologous variants in subjects with broad responses and viral control could be informative.The purpose of this study was to examine the autologous neutralizing responses against autologous viral variants in the plasma of HIV-positive subjects that were controlling infection for many years. These subjects have moderate HNAbs against the quasispecies of other subjects (27). We compared longitudinal samples from five chronically infected, antiretroviral treatment-naive adults late in infection. Despite the short time frame between the sample time points, the amount of env variation was surprisingly high, indicating continuous viral evolution in controllers; contemporaneous ANAbs were present and maintained in all except one elite controller. We cloned individual env gp160 plasma variants and analyzed sequence changes related to the autologous neutralization sensitivity or resistance. We systematically examined the ANAb response directed to individual variants using contemporaneous and noncontemporaneous plasma samples and observed patterns that have not been previously reported. Mutations that were significantly associated with sensitivity or resistance to ANAbs were found on parts of the envelope that are exposed and thus may be accessible to antibodies, consistently with a role in escape and containment by NAbs.     

A neutralizing antibody target in early HIV-1 infection was recapitulated in rhesus macaques immunized with the transmitted/founder envelope sequence

Transmitted/founder (T/F) HIV-1 envelope proteins (Envs) from infected individuals that developed neutralization breadth are likely to possess inherent features desirable for vaccine immunogen design. To explore this premise, we conducted an immunization study in rhesus macaques (RM) using T/F Env sequences from two human subjects, one of whom developed potent and broad neutralizing antibodies (Z1800M) while the other developed little to no neutralizing antibody responses (R66M) during HIV-1 infection. Using a DNA/MVA/protein immunization protocol, 10 RM were immunized with each T/F Env. Within each T/F Env group, the protein boosts were administered as either monomeric gp120 or stabilized trimeric gp140 protein. All vaccination regimens elicited high titers of antigen-specific IgG, and two animals that received monomeric Z1800M Env gp120 developed autologous neutralizing activity. Using early Env escape variants isolated from subject Z1800M as guides, the serum neutralizing activity of the two immunized RM was found to be dependent on the gp120 V5 region. Interestingly, the exact same residues of V5 were also targeted by a neutralizing monoclonal antibody (nmAb) isolated from the subject Z1800M early in infection. Glycan profiling and computational modeling of the Z1800M Env gp120 immunogen provided further evidence that the V5 loop is exposed in this T/F Env and was a dominant feature that drove neutralizing antibody targeting during infection and immunization. An expanded B cell clonotype was isolated from one of the neutralization-positive RM and nmAbs corresponding to this group demonstrated V5-dependent neutralization similar to both the RM serum and the human Z1800M nmAb. The results demonstrate that neutralizing antibody responses elicited by the Z1800M T/F Env in RM converged with those in the HIV-1 infected human subject, illustrating the potential of using immunogens based on this or other T/F Envs with well-defined immunogenicity as a starting point to drive breadth.     

Broad and potent neutralizing antibody responses elicited in natural HIV-2 infection

Compared with human immunodeficiency virus type 1 (HIV-1), little is known about the susceptibility of HIV-2 to antibody neutralization. We characterized the potency and breadth of neutralizing antibody (NAb) responses in 64 subjects chronically infected with HIV-2 against three primary HIV-2 strains: HIV-2(7312A), HIV-2(ST), and HIV-2(UC1). Surprisingly, we observed in a single-cycle JC53bl-13/TZM-bl virus entry assay median reciprocal 50% inhibitory concentration (IC(50)) NAb titers of 1.7 × 10(5), 2.8 × 10(4), and 3.3 × 10(4), respectively. A subset of 5 patient plasma samples tested against a larger panel of 17 HIV-2 strains where the extracellular gp160 domain was substituted into the HIV-2(7312A) proviral backbone showed potent neutralization of all but 4 viruses. The specificity of antibody neutralization was confirmed using IgG purified from patient plasma, HIV-2 Envs cloned by single-genome amplification, viruses grown in human CD4(+) T cells and tested for neutralization sensitivity on human CD4(+) T target cells, and, as negative controls, env-minus viruses pseudotyped with HIV-1, vesicular stomatitis virus, or murine leukemia virus Env glycoproteins. Human monoclonal antibodies (MAbs) specific for HIV-2 V3 (6.10F), V4 (1.7A), CD4 binding site (CD4bs; 6.10B), CD4 induced (CD4i; 1.4H), and membrane-proximal external region (MPER; 4E10) epitopes potently neutralized the majority of 32 HIV-2 strains bearing Envs from 13 subjects. Patient antibodies competed with V3, V4, and CD4bs MAbs for binding to monomeric HIV-2 gp120 at titers that correlated significantly with NAb titers. HIV-2 MPER antibodies did not contribute to neutralization breadth or potency. These findings indicate that HIV-2 Env is highly immunogenic in natural infection, that high-titer broadly neutralizing antibodies are commonly elicited, and that unlike HIV-1, native HIV-2 Env trimers expose multiple broadly cross-reactive epitopes readily accessible to NAbs.