Multiple V1/V2 env variants are frequently present during primary infection with human immunodeficiency virus type 1

Human immunodeficiency virus type 1 (HIV-1) exists as a complex population of multiple genotypic variants in persons with chronic infection. However, acute HIV-1 infection via sexual transmission is a low-probability event in which there is thought to be low genetic complexity in the initial inoculum. In order to assess the viral complexity present during primary HIV-1 infection, the V1/V2 and V3 variable regions of the env gene were examined by using a heteroduplex tracking assay (HTA) capable of resolving these genotypic variants. Blood plasma samples from 26 primary HIV-1-infected subjects were analyzed for their level of diversity. Half of the subjects had more than one V1/V2 viral variant during primary infection, indicating the frequent transmission of multiple variants. This observation is inconsistent with the idea of infrequent transmission based on a small transmitting inoculum of cell-free virus. In chronically infected subjects, the complexity of the viral populations was even greater in both the V1/V2 and the V3 regions than in acutely infected subjects, indicating that in spite of the presence of multiple variants in acute infection, the virus does pass through a genetic bottleneck during transmission. We also examined how well the infecting virus penetrated different anatomical compartments by using the HTA. Viral variants detected in blood plasma were compared to those detected in seminal plasma and/or cerebral spinal fluid of six individuals. The virus in each of these compartments was to a large extent identical to virus in blood plasma, a finding consistent with rapid penetration of the infecting variant(s). The low-probability transmission of multiple variants could be the result of transient periods of hyperinfectiousness or hypersusceptibility. Alternatively, the inefficient transfer of a multiply infected cell could account for both the low probability of transmission and the transfer of multiple variants.     

Turnover of env variable region 1 and 2 genotypes in subjects with late-stage human immunodeficiency virus type 1 infection

The env gene of human immunodeficiency virus type 1 (HIV-1) includes some of the most genetically diverse regions of the viral genome, which are called variable regions 1 through 5 (V1 through V5). We have developed a heteroduplex tracking assay to detect changes in variable regions 1 and 2 of env (V1/V2-HTA). Using sequences from two molecular clones as probes, we have studied the nature of longitudinal virus population changes in a cohort of HIV-1-infected subjects. Viral sequences present in 21 subjects with late-stage HIV-1 infection were initially screened for stability of the virus population by V1/V2-HTA. The virus populations at entry comprised an average of five coexisting V1/V2 genotypic variants (as identified by HTA). Eight of the 21 subjects were examined in detail because of the dynamic behavior of their env variants over an approximately 9-month period. In each of these cases we detected a single discrete transition of V1/V2 genotypes based on monthly sampling. The major V1/V2 genotypes (those present at >10% abundance) from the eight subjects were cloned and sequenced to define the nature of V1/V2 variability associated with a discrete transition. Based on a comparison of V1/V2 genotypic variants present at entry with the newly emerged variants we categorized the newly emerged variants into two groups: variants without length differences and variants with length differences. Variants without length differences had fewer nucleotide substitutions, with the changes biased to either V1 or V2, suggestive of recent evolutionary events. Variants with length differences included ones with larger numbers of changes that were distributed, suggestive of recall of older genotypes. Most length differences were located in domains where the codon motif AVT (V = A, G, C) had become enriched and fixed. Finally, recombination events were detected in two subjects, one of which resulted in the reassortment of V1 and V2 regions. We suggest that turnover in V1/V2 populations was largely driven by selection on either V1 or V2 and that escape was accomplished either through changes focused in the region under selection or by the appearance of a highly divergent variant.     

Evolution of human immunodeficiency virus type 1 env sequence variation in patients with diverse rates of disease progression and T-cell function.

We examined the relationship between env sequence variation and disease progression in 10 human immunodeficiency virus type 1 (HIV-1)-seropositive subjects selected from a longitudinal cohort receiving zidovudine therapy. Five subjects were chosen for stable clinical status and CD4 counts (slow progressors), and five were selected for rapid clinical deterioration and CD4 count decline (rapid progressors). The slow progressors had significantly lower plasma viral RNA loads and greater lymphoproliferative responses to mitogens than the rapid progressors. DNA sequences representing the C1 through C3 regions of env were amplified from two peripheral blood mononuclear cell DNA samples from each subject separated by an average of 2.5 years. Molecular clones of these amplicons were then sequenced, and DNA sequence and deduced amino acid sequence distances were compared. Inter-time point sequence comparison showed a higher rate of sequence evolution for the rapid progressors in three of five matched pairs of rapid progressors and slow progressors and for the slow progressors in the remaining two subject pairs. However, intra-time point sequence comparisons showed that four of five slow progressors developed a more diverse quasispecies over time and one showed no change. In contrast, four of five rapid progressors showed no change in quasispecies diversity over time and one showed a significant decrease in diversity. The overall C1 through C3 region quasispecies diversity in the slow progressors at baseline was lower than that for the rapid progressors, but this difference was not significant at the follow-up time points. These diversity relationships were obscured if sequence analyses were limited to the 300-bp C2 to V3 region. Thus, HIV-1 quasispecies diversity increased over time in subjects with more functional immune systems.     

Consistent viral evolutionary changes associated with the progression of human immunodeficiency virus type 1 infection

To understand the high variability of the asymptomatic interval between primary human immunodeficiency virus type 1 (HIV-1) infection and the development of AIDS, we studied the evolution of the C2-V5 region of the HIV-1 env gene and of T-cell subsets in nine men with a moderate or slow rate of disease progression. They were monitored from the time of seroconversion for a period of 6 to 12 years until the development of advanced disease in seven men. Based on the analysis of viral divergence from the founder strain, viral population diversity within sequential time points, and the outgrowth of viruses capable of utilizing the CXCR4 receptor (X4 viruses), the existence of three distinct phases within the asymptomatic interval is suggested: an early phase of variable duration during which linear increases ( approximately 1% per year) in both divergence and diversity were observed; an intermediate phase lasting an average of 1.8 years, characterized by a continued increase in divergence but with stabilization or decline in diversity; and a late phase characterized by a slowdown or stabilization of divergence and continued stability or decline in diversity. X4 variants emerged around the time of the early- to intermediate-phase transition and then achieved peak representation and began a decline around the transition between the intermediate and late phases. The late-phase transition was also associated with failure of T-cell homeostasis (defined by a downward inflection in CD3(+) T cells) and decline of CD4(+) T cells to 相似文献   

Enhancement of human immunodeficiency virus type 1 infectivity by replacing the region including Env derived from defective particles with an ability to form particle-mediated syncytia in CD4+T cells

The infection and subsequent replication rates of human immunodeficiency virus type 1 (HIV-1) affect the pathogenicity. The initial stage of HIV-1 infection is largely regulated by viral envelope sequence. We previously reported that the defective doughnut-shaped particles produced from a persistently infected cell clone, named L-2, obtained from human CD4+ T-cell line MT-4 that was persistently infected with HIV-1 LAI strain, efficiently form particle-mediated syncytia with uninfected human CD4+ T-cell line, MOLT-4. Here, we prepared a molecular clone (pL2) containing the L-2 provirus to characterize the viral genetic region contributing to this activity to form particle-mediated syncytia. Several recombinants were constructed with pNL4-3 by replacing the pL2-derived region including full-length env. Characterization of the particles obtained by transfection with these recombinant clones confirmed that pL2-derived env carried the particle-mediated syncytia formation activity. It is noteworthy that the pL2-derived env region could also contribute to enhancement of infectivity in CD4+ T-cell lines as well as primary peripheral blood mononuclear cells (PBMCs). Thus, the HIV-1 particle-mediated syncytium formation activity could also contribute to the enhancement of HIV-1 infectivity.     

Host-specific modulation of the selective constraints driving human immunodeficiency virus type 1 env gene evolution

To address the evolution of human immunodeficiency virus type 1 (HIV-1) within a single host, we analyzed the HIV-1 C2-V5 env regions of both cell-free genomic-RNA- and proviral-DNA-derived clones. Sequential samples were collected over a period of 3 years from six untreated subjects (three typical progressors [TPs] and three slow progressors [SPs], all with a comparable length of infection except one. The evolutionary analysis of the C2-V5 env sequences performed on 506 molecular clones (253 RNA- and 253 DNA-derived sequences) highlighted a series of differences between TPs and SPs. In particular, (i) clonal sequences from SPs (DNA and RNA) showed lower nucleotide similarity than those from TPs (P = 0. 0001), (ii) DNA clones from SPs showed higher intra- and intersample nucleotide divergence than those from TPs (P < 0.05), (iii) higher host-selective pressure was generally detectable in SPs (DNA and RNA sequences), and (iv) the increase in the genetic distance of DNA and RNA sequences over time was paralleled by an increase in both synonymous (Ks) and nonsynonymous (Ka) substitutions in TPs but only in nonsynonymous substitutions in SPs. Several individual peculiarities of the HIV-1 evolutionary dynamics emerged when the V3, V4, and V5 env regions of both TPs and SPs were evaluated separately. These peculiarities, probably reflecting host-specific features of selective constraints and their continuous modulation, are documented by the dynamics of Ka/Ks ratios of hypervariable env domains.     

The envelope gp120 gene of human immunodeficiency virus type 1 determines the rate of CD4-positive T-cell depletion in SCID mice engrafted with human peripheral blood leukocytes.

We have used envelope recombinant viruses generated between two molecular clones of human immunodeficiency virus type 1 (HIV-1), T-cell-tropic HIV-1SF2 and macrophage-tropic HIV-1SF162, to assess pathogenic potential in the human peripheral blood leukocyte-reconstituted severe combined immune deficiency mouse model. Recombinant HIV-1SF2 viruses expressing the envelope gp120 gene of HIV-ISF162 caused as rapid a CD4+ T-cell depletion as did HIV-1SF162. The reciprocal HIV-1SF162 recombinant virus with the HIV-1SF2 envelope caused slower CD4+ T-cell loss. Although changing the V3 loop sequence of HIV-1SF162 to that of HIV-1SF2 did not change the rate of CD4+ T-cell depletion, replacing the V3 of HIV-1SF2 with the sequence of HIV-1SF162 resulted in virus that was poorly infectious in vivo but not in vitro. These studies suggest that the envelope gene determines properties important for pathogenesis in vivo as well as for cell tropism in vitro. HIV-1 infection in vivo may have more stringent requirements for envelope conformation.     

Compartmentalization of human immunodeficiency virus type 1 between blood monocytes and CD4+ T cells during infection

Distinct sequences of human immunodeficiency virus type 1 (HIV-1) have been found between different tissue compartments or subcompartments within a given tissue. Whether such compartmentalization of HIV-1 occurs between different cell populations is still unknown. Here we address this issue by comparing HIV-1 sequences in the second constant region through the fifth hypervariable region (C2 to V5) of the surface envelope glycoprotein (Env) between viruses in purified blood CD14(+) monocytes and CD4(+) T cells obtained longitudinally from five infected patients over a time period ranging from 117 to 3,409 days postseroconversion. Viral populations in both cell types at early infection time points appeared relatively homogeneous. However, later in infections, all five patients showed heterogeneous populations in both CD14(+) monocytes and CD4(+) T cells. Three of the five patients had CD14(+) monocyte populations with significantly more genetic diversity than the CD4(+) T-cell population, while the other two patients had more genetic diversity in CD4(+) T cells. The cellular compartmentalization of HIV-1 between CD14(+) monocytes and CD4(+) T cells was not seen early during infections but was evident at the later time points for all five patients, indicating an association of viral compartmentalization with the time course of HIV-1 infection. The majority of HIV-1 V3 sequences indicated a macrophage-tropic phenotype, while a V3 sequence-predicted T-cell tropic virus was found in the CD4(+) T cells and CD14(+) monocytes of two patients. These findings suggest that HIV-1 in CD14(+) monocytes could disseminate and evolve independently from that in CD4(+) T cells over the course of HIV-1 infection, which may have implications on the development of new therapeutic strategies.     

Slower evolution of human immunodeficiency virus type 1 quasispecies during progression to AIDS.

The evolution of human immunodeficiency virus type 1 (HIV-1) quasispecies at the envelope gene was studied from the time of infection in 11 men who experienced different rates of CD4+ cell count decline and 6 men with unknown dates of infection by using DNA heteroduplex mobility assays. Quasispecies were genetically homogeneous near the time of seroconversion. Subsequently, slower proviral genetic diversification and higher plasma viremia correlated with rapid CD4+ cell count decline. Except for the fastest progressors to AIDS, highly diverse quasispecies developed in all subjects within 3 to 4 years. High quasispecies diversity was then maintained for years until again becoming more homogeneous in a subset of late-stage AIDS patients. Individuals who maintained high CD4+ cell counts showed continuous genetic turnover of their complex proviral quasispecies, while more closely related sets of variants were found in longitudinal samples of severely immunocompromised patients. The limited number of variants that grew out in short-term PBMC cocultures were rare in the uncultured proviral quasispecies of healthy, long-term infected individuals but more common in vivo in patients with low CD4+ cell counts. The slower evolution of HIV-1 observed during rapid progression to AIDS and in advanced patients may reflect ineffective host-mediated selection pressures on replicating quasispecies.     

Efficient thymopoiesis contributes to the maintenance of peripheral CD4 T cells during chronic human immunodeficiency virus type 2 infection

Human immunodeficiency virus type 2 (HIV-2) infection leads to a lifelong asymptomatic period in the majority of patients. Even in patients with progressive disease, a slow CD4 count decline characterizes the chronic phase of HIV-2 infection, suggesting that peripheral T-cell homeostasis is controlled better following HIV-2 infection than following HIV-1 infection. Herein we showed that, in contrast to HIV-1-infected patients, HIV-2-infected patients demonstrate enhanced thymic function compared to age-matched healthy individuals. The correlation between higher thymic production and lower CD4 T-cell loss in these patients suggests that efficient thymopoiesis is implicated in the long-lasting maintenance of CD4 T-cell counts in HIV-2 disease.     

Are Long-Term Non-Progressors Very Slow Progressors? Insights from the Chelsea and Westminster HIV Cohort, 1988–2010

Define and identify long-term non-progressors (LTNP) and HIV controllers (HIC), and estimate time until disease progression. LTNP are HIV-1⁺ patients who maintain stable CD4⁺ T-cell counts, with no history of opportunistic infection or antiretroviral therapy (ART). HIC are a subset of LTNP who additionally have undetectable viraemia. These individuals may provide insights for prophylactic and therapeutic development. Records of HIV-1⁺ individuals attending Chelsea and Westminster Hospital (1988–2010), were analysed. LTNP were defined as: HIV-1⁺ for >7 years; ART-naïve; no history of opportunistic infection and normal, stable CD4⁺ T-cell counts. MIXED procedure in SAS using random intercept model identified long-term stable CD4⁺ T-cell counts. Survival analysis estimated time since diagnosis until disease progression. Subjects exhibiting long-term stable CD4⁺ T-cell counts with history below the normal range (<450 cells/µl blood) were compared to LTNP whose CD4⁺ T-cell count always remained normal. Within these two groups subjects with HIV-1 RNA load below limit of detection (BLD) were identified. Of 14,227 patients, 1,204 were diagnosed HIV-1⁺ over 7 years ago and were ART-naïve. Estimated time until disease progression for the 20% (239) whose CD4⁺ T-cell counts remained within the normal range, was 6.2 years (IQR: 2.0 to 9.6); significantly longer than 4.0 years (IQR: 1.0 to 7.3) for patients with historical CD4⁺ T-cell count below normal (Logrank chi-squared = 21.26; p<0.001). Within a subpopulation of 312 asymptomatic patients, 50 exhibited long-term stable CD4⁺ T-cell counts. Of these, 13 were LTNP, one of whom met HIC criteria. Of the remaining 37 patients with long-term stable low CD4⁺ T-cell counts, 3 controlled HIV-1 RNA load BLD. Individuals with stable, normal CD4⁺ T-cell counts progressed less rapidly than those with low CD4⁺ T-cell counts. Few LTNP and HIC identified in this and other studies, endorse the need for universal definitions to facilitate comparison.     

Natural killer cell function is well preserved in asymptomatic human immunodeficiency virus type 2 (HIV-2) infection but similar to that of HIV-1 infection when CD4 T-cell counts fall

Natural killer (NK) cells are potent effectors of natural immunity and their activity prevents human immunodeficiency virus type 1 (HIV-1) viral entry and viral replication. We sought to determine whether NK immune responses are associated with different clinical course of HIV-1 and HIV-2 infections. A cross-sectional analysis of NK cell responses was undertaken in 30 HIV-1 and 30 HIV-2 subjects in each of three categories of CD4(+)-T-cell counts (>500, 200 to 500, and <200 cells/microl) and in 50 HIV-uninfected control subjects. Lytic activity and gamma interferon (IFN-gamma) secretion were measured by chromium release and enzyme-linked immunospot assays, respectively. Flow cytometry was used to assess intracellular cytokines and chemokines. Levels of NK cytotoxicity were significantly higher in HIV-2 than in HIV-1 infections in subjects with high CD4(+)-T-cell counts and were similar to that of the healthy controls. In these HIV-2 subjects, cytolytic activity was positively correlated to NK cell count and inversely related to plasma viremia. Levels of intracellular MIP-1beta, RANTES, tumor necrosis factor alpha, and IFN-gamma produced by NK CD56(bright) cells were significantly higher in HIV-2- than HIV-1-infected subjects with high CD4(+)-T-cell counts but fell to similar levels as CD4 counts dropped. The data suggest efficient cytolytic and chemokine-suppressive activity of NK cells early in HIV-2 infection, which is associated with high CD4(+) T-cell counts. Enhancement of these functions may be important in immune-based therapy to control HIV disease.     

Designed cyclic permutants of HIV-1 gp120: implications for envelope trimer structure and immunogen design

Most HIV-1 broadly neutralizing antibodies are directed against the gp120 subunit of the env surface protein. Native env consists of a trimer of gp120-gp41 heterodimers, and in contrast to monomeric gp120, preferentially binds CD4 binding site (CD4bs)-directed neutralizing antibodies over non-neutralizing ones. Some cryo-electron tomography studies have suggested that the V1V2 loop regions of gp120 are located close to the trimer interface. We have therefore designed cyclically permuted variants of gp120 with and without the h-CMP and SUMO2a trimerization domains inserted into the V1V2 loop. h-CMP-V1cyc is one such variant in which residues 153 and 142 are the N- and C-terminal residues, respectively, of cyclically permuted gp120 and h-CMP is fused to the N-terminus. This molecule forms a trimer under native conditions and binds CD4 and the neutralizing CD4bs antibodies b12 with significantly higher affinity than wild-type gp120. It binds non-neutralizing CD4bs antibody F105 with lower affinity than gp120. A similar derivative, h-CMP-V1cyc1, bound the V1V2 loop-directed broadly neutralizing antibodies PG9 and PG16 with ～20-fold higher affinity than wild-type JRCSF gp120. These cyclic permutants of gp120 are properly folded and are potential immunogens. The data also support env models in which the V1V2 loops are proximal to the trimer interface.     

The viral envelope gene is involved in macrophage tropism of a human immunodeficiency virus type 1 strain isolated from brain tissue.

Human immunodeficiency virus type 1 (HIV-1) strains isolated from the central nervous system (CNS) may represent a subgroup that displays a host cell tropism different from those isolated from peripheral blood and lymph nodes. One CNS-derived isolate, HIV-1SF128A, which can be propagated efficiently in primary macrophage culture but not in any T-cell lines, was molecularly cloned and characterized. Recombinant viruses between HIV-1SF128A and the peripheral blood isolate HIV-1SF2 were generated in order to map the viral gene(s) responsible for the macrophage tropism. The env gene sequences of the two isolates are about 91.1% homologous, with variations scattered mainly in the hypervariable regions of gp120. Recombinant viruses that have acquired the HIV-1SF128A env gene display HIV-1SF128A tropism for macrophages. Furthermore, the gp120 variable domains, V1, V2, V4, and V5, the CD4-binding domain, and the gp41 fusion domain are not directly involved in determining macrophage tropism.     

Virus population homogenization following acute human immunodeficiency virus type 1 infection

Understanding the properties of human immunodeficiency virus type 1 (HIV-1) variants capable of establishing infection is critical to the development of a vaccine against AIDS. Previous studies of men have shown that the HIV-1 env gene is homogeneous early in infection, leading to the suggestion that infection is established by a single transmitted variant. However, we report here that all of eight homosexual men evaluated beginning 3.7 to 9 weeks following onset of symptoms of acute infection harbored diverse virus populations in their blood, with median genetic distances averaging 1.08% in the env C2V5 region and 0.81% in the gag p17 gene. Within another 4.7 to 11 weeks, the variant lineage in env became more homogeneous, while gag sequences continued to diversify. Thus, the homogenization that has been reported to characterize acute infection is actually preceded by the replication of multiple virus variants. This early selective process focuses on viral properties within Env but not Gag p17. Hence, the viral homogeneity observed early in HIV-1 infection results from a selective process that occurs during the establishment of infection.     

Human immunodeficiency virus type 1 (HIV-1)-specific CD4+ T cells that proliferate in vitro detected in samples from most viremic subjects and inversely associated with plasma HIV-1 levels

Diminished in vitro proliferation of human immunodeficiency virus type 1 (HIV-1)-specific CD4+T cells has been associated with HIV-1 viremia and declining CD4+ T-cell counts during chronic infection. To better understand this phenomenon, we examined whether HIV-1 Gag p24 antigen-induced CD4+ T-cell proliferation might recover in vitro in a group of subjects with chronic HIV-1 viremia and no history of antiretroviral therapy (ART). We found that depletion of CD8+ cells from peripheral blood mononuclear cells (PBMC) before antigen stimulation was associated with a 6.5-fold increase in the median p24-induced CD4+ T-cell proliferative response and a 57% increase in the number of subjects with positive responses. These p24-induced CD4+ T-cell proliferative responses from CD8-depleted PBMC were associated with expansion of the numbers of p24-specific, gamma interferon (IFN-gamma)-producing CD4+ T cells. Among the 20 viremic, treatment-naive subjects studied, the only 5 subjects lacking proliferation-competent, p24-specific CD4+ T-cell responses from CD8-depleted PBMC showed plasma HIV-1 RNA levels > 100,000 copies/ml. Furthermore, both the magnitude of p24-induced CD4+ T-cell proliferative responses from CD8-depleted PBMC and the frequency of p24-specific, IFN-gamma-producing CD4+ T cells expanded from CD8-depleted PBMC were associated inversely with plasma HIV-1 RNA levels. Therefore, proliferation-competent, HIV-1-specific CD4+ T cells that might help control HIV-1 disease may persist during chronic, progressive HIV-1 disease except at very high levels of in vivo HIV-1 replication.     

De novo generation of escape variant-specific CD8+ T-cell responses following cytotoxic T-lymphocyte escape in chronic human immunodeficiency virus type 1 infection

Human immunodeficiency virus type 1 (HIV-1) evades CD8(+) T-cell responses through mutations within targeted epitopes, but little is known regarding its ability to generate de novo CD8(+) T-cell responses to such mutants. Here we examined gamma interferon-positive, HIV-1-specific CD8(+) T-cell responses and autologous viral sequences in an HIV-1-infected individual for more than 6 years following acute infection. Fourteen optimal HIV-1 T-cell epitopes were targeted by CD8(+) T cells, four of which underwent mutation associated with dramatic loss of the original CD8(+) response. However, following the G(357)S escape in the HLA-A11-restricted Gag(349-359) epitope and the decline of wild-type-specific CD8(+) T-cell responses, a novel CD8(+) T-cell response equal in magnitude to the original response was generated against the variant epitope. CD8(+) T cells targeting the variant epitope did not exhibit cross-reactivity against the wild-type epitope but rather utilized a distinct T-cell receptor Vbeta repertoire. Additional studies of chronically HIV-1-infected individuals expressing HLA-A11 demonstrated that the majority of the subjects targeted the G(357)S escape variant of the Gag(349-359) epitope, while the wild-type consensus sequence was significantly less frequently recognized. These data demonstrate that de novo responses against escape variants of CD8(+) T-cell epitopes can be generated in chronic HIV-1 infection and provide the rationale for developing vaccines to induce CD8(+) T-cell responses directed against both the wild-type and variant forms of CD8 epitopes to prevent the emergence of cytotoxic T-lymphocyte escape variants.     

Induction of antibodies in guinea pigs and rhesus monkeys against the human immunodeficiency virus type 1 envelope: neutralization of nonpathogenic and pathogenic primary isolate simian/human immunodeficiency virus strains

We have compared the abilities of human immunodeficiency virus type 1 (HIV-1) envelope V3 peptides and recombinant gp120 to induce antibodies that neutralize simian/human immunodeficiency viruses (SHIVs). SHIV-89.6 is a nonpathogenic SHIV that expresses the envelope protein of primary HIV-1 isolate 89.6. SHIV-89.6P, clone KB9, is a pathogenic SHIV variant derived from SHIV-89.6. Infection of rhesus monkeys with these SHIVs rarely induces anti-V3 region antibodies. To determine the availability of the gp120 V3 loop for neutralizing antibody binding on SHIV-89.6 and KB9 virions, we have constructed immunogenic C4-V3 peptides from these SHIVs and induced anti-V3 antibodies in guinea pigs and rhesus monkeys. We found that both SHIV-89.6 and KB9 C4-V3 peptides induced antibodies that neutralized SHIV-89.6 but that only SHIV-KB9 C4-V3 peptide induced antibodies that neutralized SHIV-KB9. Immunoprecipitation assays demonstrated that SHIV-KB9 C4-V3 peptide-induced antibodies had a greater ability to bind SHIV-KB9 envelope proteins than did antibodies raised against SHIV-89.6 C4-V3 peptide. We have used a series of mutant HIV-1 envelope constructs to map the gp120 determinants that affect neutralization by anti-V3 antibodies. The residue change at position 305 of arginine (in SHIV-89.6) to glutamic acid (in SHIV-KB9) played a central role in determining the ability of peptide-induced anti-V3 antiserum to neutralize primary isolate SHIVs. Moreover, residue changes in the SHIV-89.6 V1/V2 loops also played roles in regulating the availability of the V3 neutralizing epitope on SHIV-89.6 and -KB9. Thus, SHIV-89.6 and -KB9 V3 region peptides are capable of inducing neutralizing antibodies against these primary isolate SHIVs, although the pathogenic SHIV-KB9 is less easily neutralized than its nonpathogenic variant SHIV-89.6. In contrast to natural infection with SHIV-89.6, in which few animals make anti-V3 antibodies, C4-V3 peptides frequently induced anti-V3 antibodies that neutralized primary isolate SHIV strains.