The Association of HIV-1 Neutralization in Aviremic Children and Adults with Time to ART Initiation and CD4+/CD8+ Ratios.

Broadly neutralizing antibodies (bnAbs) bind and neutralize diverse HIV isolates and demonstrate protective effects in primate models and humans against specific isolates. To develop an effective HIV vaccine, it is widely believed that inducing these antibodies is crucial. However, the high somatic hypermutation in bnAbs and the limited affinity of HIV Env proteins for bnAb germline precursors suggest that extended antigen exposure is necessary for their production. Consequently, HIV vaccine research is exploring complex sequential vaccination strategies to guide the immune response through maturation stages. In this context, the exploration of the factors linked to the generation of these antibodies across diverse age groups becomes critical. In this study, we assessed the anti-HIV-1 neutralization potency and breadth in 108 aviremic adults and 109 aviremic children under 15 years of age who were receiving ART. We used a previously described minipanel of recombinant viruses and investigated the factors associated with neutralization in these individuals. We identified individuals in both groups who were capable of neutralizing viruses from three different subtypes, with greater cross-neutralization observed in the adult group (49.0% vs. 9.2%). In both groups, we observed an inverse association between neutralization breadth and the CD4+/CD8+ ratio, as well as a direct association with the time to ART initiation. However, we found no association with time post-infection, cumulative ART duration, or CD8+ cell levels. The present study demonstrates that children receiving antiretroviral therapy generate broadly neutralizing responses to HIV-1, albeit with lower magnitude compared to adults. We also observed that neutralization breadth is associated with CD4+/CD8+ levels and time to treatment initiation in both children and adults living with HIV-1. Our interpretation of these results is that a delay in ART initiation could have prolonged the antigenic stimulation associated with viral replication and thus facilitate the capacity to elicit long-lasting broadly neutralizing responses. These results corroborate prior findings that show that HIV-1-neutralizing responses can persist for years, even at low antigen levels, implying an HIV-1 vaccine may induce lasting neutralizing antibody response.

Evaluation of the Thermal Stability of a Vaccine Prototype Based on Virus-like Particle Formulated HIV-1 Envelope.

The long-term storage stability of vaccines has a major impact on the roll-out and success of global immunization programs. For the Human Immunodeficiency Virus type 1 (HIV-1) virus-like particle (VLP) vaccine prototype evaluated here, nanoparticle tracking analysis (NTA), and enzyme-linked immunoabsorbent assay (ELISA) results demonstrated a remarkable structural stability. VLPs maintained their integrity and the recognition of relevant B-cell epitopes for three months at 4 and -20 °C. Interestingly, most particles remained intact and preserved the recognition of relevant epitopes even after a week of storage at room temperature.

Potent Induction of Envelope-Specific Antibody Responses by Virus-Like Particle Immunogens Based on HIV-1 Envelopes from Patients with Early Broadly Neutralizing Responses.

Longitudinal studies in HIV-1-infected individuals have indicated that 2 to 3 years of infection are required to develop broadly neutralizing antibodies. However, we have previously identified individuals with broadly neutralizing activity (bNA) in early HIV-1 infection, indicating that a vaccine may be capable of bNA induction after short periods of antigen exposure. Here, we describe 5 HIV-1 envelope sequences from individuals who have developed bNA within the first 100 days of infection (early neutralizers) and selected two of them to design immunogens based on HIV-1-Gag virus-like particles (VLPs). These VLPs were homogeneous and incorporated the corresponding envelopes (7 to 9 µg of gp120 in 1010 VLPs). Both envelopes (Envs) bound to well-characterized broadly neutralizing antibodies (bNAbs), including trimer-specific antibodies (PGT145, VRC01, and 35022). For immunogenicity testing, we immunized rabbits with the Env-VLPs or with the corresponding stabilized soluble envelope trimers. A short immunization protocol (105 days) was used to recapitulate the early nAb induction observed after HIV-1 infection in these two individuals. All VLP and trimeric envelope immunogens induced a comparably strong anti-gp120 response despite having immunized rabbits with 30 times less gp120 in the case of the Env-VLPs. In addition, animals immunized with VLP-formulated Envs induced antibodies that cross-recognized the corresponding soluble stabilized trimer and vice versa, even though no neutralizing activity was observed. Nevertheless, our data may provide a new platform of immunogens, based on HIV-1 envelopes from patients with early broadly neutralizing responses, with the potential to generate protective immune responses using vaccination protocols similar to those used in classical preventive vaccines. IMPORTANCE It is generally accepted that an effective HIV-1 vaccine should be able to induce broad-spectrum neutralizing antibodies. Since most of these antibodies require long periods of somatic maturation in vivo, several groups are developing immunogens, based on the HIV envelope protein, that require complex and lengthy immunization protocols that would be difficult to implement in the general population. Here, we show that rabbits immunized with new envelopes (VLP formulated) from two individuals who demonstrated broadly neutralizing activity very early after infection induced specific HIV-1 antibodies after a short immunization protocol. This evidence provides the basis for generating protective immune responses with classic vaccination protocols with vaccine prototypes based on HIV envelope sequences from individuals who have developed early broadly neutralizing responses.

Permanent control of HIV-1 pathogenesis in exceptional elite controllers: a model of spontaneous cure.

Elite controllers (EC) represent a small subset of HIV-1-infected people that spontaneously control viral replication. However, natural virological suppression and absence of immune dysfunction are not always long-term sustained. We define exceptional EC (EEC) as HIV-1 subjects who maintain the EC characteristics without disease progression for more than 25 years. We analyzed three EEC, diagnosed between 1988 and 1992, who never showed signs of clinical disease progression in absence of any antiretroviral treatment. A comprehensive clinical, virological, and immunological study was performed. The individuals simultaneously exhibited ≥3 described host protective alleles, low levels of total HIV-1 DNA (<20 copies/106 CD4+ T-cells) without evidence of replication-competent viruses (<0.025 IUPM), consistent with high levels of defective genomes, strong cellular HIV-1-specific immune response, and a high poly-functionality index (>0.50). Inflammation levels of EEC were similar to HIV-1 negative donors. Remarkably, they showed an exceptional lack of viral evolution and 8-fold lower genetic diversity (<0.01 s/n) in env gene than other EC. We postulate that these EEC represent cases of spontaneous functional HIV-1 cure. A non-functional and non-genetically evolving viral reservoir along with an HIV-1-specific immune response seems to be key for the spontaneous functional cure.

Guiding the humoral response against HIV-1 toward a MPER adjacent region by immunization with a VLP-formulated antibody-selected envelope variant.

Preventive HIV-1 vaccine strategies rely on the elicitation of broadly neutralizing antibody (bNAb) responses, but their induction in vivo by vaccination remains challenging. Considering that the ability of an epitope to elicit effective humoral immunity depends on its exposure on the virion, we have used a reverse genetics approach to select variants from an HIV-1 AC10_29 randomly mutated envelope library that showed increased affinity for a selected bNAb (4E10 bNAb targeting the HIV-1 MPER region). Isolated envelope sequences were analyzed by deep-sequencing showing a small number of dominant changes, including the loss of four potential N-linked glycosylation sites and disruption of the V1/V2 loop. Accordingly, the dominant variant (LR1-C1), showed not only increased affinity for MPER bNAbs 4E10 and 2F5, but also higher affinity for an additional antibody targeting the V3 loop (447-52D) that could be a consequence of an open conformation tier 1-like Env. Furthermore, the amino acids specific for the selected variant are associated with an increased sensitivity for 4E10 and 2F5 antibodies. In vivo studies showed that sera from mice immunized with LR1-C1 viruses possessed an improved neutralizing activity compared to the wild-type AC10_29 env. While Virus Like Particles (VLPs) carrying this envelope were unable to induce detectable neutralizing activity in immunized rabbits, one animal showed antibody response to the 4E10-proximal region. Our data establish a novel approach that has the potential to yield HIV envelope immunogen sequences that direct antibody responses to specific envelope regions.

Lipid raft-like liposomes used for targeted delivery of a chimeric entry-inhibitor peptide with anti-HIV-1 activity.

The work reports the design and synthesis of a chimeric peptide that is composed of the peptide sequences of two entry inhibitors which target different sites of HIV-1 gp41. The chimeric peptide offers the advantage of targeting two gp41 regions simultaneously: the fusion peptide and the loop both of which are membrane active and participate in the membrane fusion process. We therefore use lipid raft-like liposomes as a tool to specifically direct the chimeric inhibitor peptide to the membrane domains where the HIV-1 envelope protein is located. Moreover, the liposomes that mimic the viral membrane composition protect the chimeric peptide against proteolytic digestion thereby increasing the stability of the peptide. The described liposome preparations are suitable nanosystems for managing hydrophobic entry-inhibitor peptides as putative therapeutics.

Antibody-Based Preventive and Therapeutic Strategies Against HIV.

BACKGROUND: Over the years, numerous studies have been carried out demonstrating the role of antibodies in HIV control leading to the development of antibody-based therapeutic and prophylactic strategies. OBJECTIVE: The objective of this review is to provide updated information on the role of antibodies in the prevention and control of HIV infection and the strategies against HIV that have been designed based on this information. RESULTS: Passive transfer of anti-HIV antibodies in animal models has proven the efficacy of certain antibodies in the prevention and treatment of infection. The capacity of antibodies to control the virus was first attributed to their neutralizing capacity. However, we now know that there are other Fc-mediated antibody activities associated with virus protection. When it comes to better understanding protection against HIV, we ought to pay particular attention to mucosal immune responses. The evidence accumulated so far indicates that an effective vaccine against HIV should generate both mucosal IgAs and systemic IgGs. Due to the problematic induction of protective anti-HIV antibodies, several groups have developed alternative approaches based on antibody delivery via gene therapy vectors. Experiments in animal models with these vectors have shown impressive protection levels and this strategy is now being clinically trialed. CONCLUSION: Taking into account all the information included in this review, it seems evident that anti-HIV-1 antibodies play an important role in virus control and prevention. This review aims to give an overview of the strategies used and the advances in antibody-based preventive and therapeutic strategies against HIV-1.

HIV-1 Dual Infected LTNP-EC Patients Developed an Unexpected Antibody Cross-Neutralizing Activity.

This study evaluated the neutralization breadth in dually infected (DI) HIV-1 long-term non-progressor elite controller patients (LTNP-EC) using a representative minipanel of 6 viruses from 5 different subtypes. Our results showed an improved neutralization breadth in DI LTNP-EC patients when compared with matched LTNP single-infected patients. The role of viral diversity in neutralization was estimated with the Shannon Entropy and the p-distance in viral quasispecies. We found a positive correlation between neutralization breadth and diversity within the viral quasispecies. This correlation could explain why a group of LTNP-EC patients developed a broad neutralizing response despite having undetectable levels of viremia.

HIV-1 inhibiting capacity of novel forms of presentation of GB virus C peptide domains is enhanced by coordination to gold compounds.

Following the report of beneficial effects of co-infection by GB virus C (GBV-C) for HIV-infected patients, we have studied synthetic GBV-C peptides and their relationship with HIV type-1. This paper reports the design and synthesis of new forms of presentation of two peptide inhibitors corresponding to the envelope proteins E1 and E2 of GBV-C, together with a study of their anti-HIV-1 activity. Homogeneous and heterogeneous multiple antigenic peptides (MAPs), lipophilic derivatizations, cyclization and peptide-gold conjugations are the chemical design strategies adopted. Our aim is to enhance the anti-viral potency of the GBV-C peptide domains. Of all the GBV-C peptide derivatives studied, peptide-gold complexes derived from the (22-39) sequence of the GBV-C E1 protein were the most active entry inhibitors. These results support the putative modulation of HIV-1 infection by the GBV-C E1 protein and open new perspectives for the development of novel peptide-derived HIV-1 entry inhibitors.

Evolution of broadly cross-reactive HIV-1-neutralizing activity: therapy-associated decline, positive association with detectable viremia, and partial restoration of B-cell subpopulations.

Little is known about the stability of HIV-1 cross-neutralizing responses. Taking into account the fact that neutralization breadth has been positively associated with plasma viral load, there is no explanation for the presence of broadly neutralizing responses in a group of patients on treatment with undetectable viremia. In addition, the B-cell profile responsible for broadly cross-neutralizing responses is unknown. Here we studied the evolution of neutralizing responses and the B-cell subpopulation distribution in a group of patients with broadly cross-reactive HIV-1-neutralizing activity. We studied neutralization breadth evolution in a group of six previously identified broadly cross-neutralizing patients and six control patients during a 6-year period with a previously described minipanel of recombinant viruses from five different subtypes. B-cell subpopulation distribution during the study was also determined by multiparametric flow cytometry. Broadly cross-neutralizing activity was transient in four broad cross-neutralizers and stable, up to 4.6 years, in the other two. In four out of five broad cross-neutralizers who initiated treatment, a neutralization breadth loss occurred after viremia had been suppressed for as much as 20 months. B-cell subpopulation analyses revealed a significant increase in the frequency of naive B cells in broadly cross-reactive samples, compared with samples with less neutralization breadth (increased from 44% to 62%). We also observed a significant decrease in tissue-like and activated memory B cells (decreased from 19% to 12% and from 17% to 9%, respectively). Our data suggest that HIV-1 broadly cross-neutralizing activity is variable over time and associated with detectable viremia and partial B-cell restoration.

Influenza, but not HIV-specific CTL epitopes, elicits delayed-type hypersensitivity (DTH) reactions in HIV-infected patients.

The induction of cytotoxic T lymphocytes (CTLs) is believed to be an important defense mechanism against viral infections. The availability of simple, sensitive, specific and physiologically informative in vivo tests, applicable to humans, would greatly elucidate the nature of protective immune responses and facilitate immune monitoring in large vaccine trials. Here we studied the possibility of using defined HLA-A*02:01-restricted CTL epitopes from influenza matrix protein (GL9, GILGFVFTL) and HIV Gag p17 (SL9, SLYNTVATL) to elicit a cutaneous delayed-type hypersensitivity (DTH) reaction. Our results show that the GL9 but not the SL9 epitope was able to induce a DTH reaction. HIV infection status, HIV RNA level and CD4(+) T-cell counts were not predictive of the extent of DTH reactions. However, a markedly reduced expression of skin homing markers CD103 and cutaneous lymphocyte associated Ag (CLA) on epitope-specific CTL populations was associated with a lack of SL9 DTH reactivity. These data demonstrate that DTH reactions can be elicited by optimally defined CTL epitopes per se and point towards specific homing markers that are required for such reactions. These data may offer new insights into the immune pathogenesis of HIV infection and provide the basis of novel immune monitoring approaches for large-scale HIV vaccine trials.

Low-replicating viruses and strong anti-viral immune response associated with prolonged disease control in a superinfected HIV-1 LTNP elite controller.

OBJECTIVE: To study the causes for the lack of clinical progression in a superinfected HIV-1 LTNP elite controller patient. METHODOLOGY AND PRINCIPAL FINDINGS: We studied host genetic, virological and immunological factors associated with viral control in a SI long term non progressor elite controller (LTNP-EC). The individual contained both viruses and maintained undetectable viral loads for >20 years and he did not express any of the described host genetic polymorphisms associated with viral control. None of four full-length gp160 recombinants derived from the LTNP-EC replicated in heterologous peripheral blood mononuclear cells. CTL responses after SI were maintained in two samples separated by 9 years and they were higher in breadth and magnitude than responses seen in most of 250 treatment naïve patients and also 25 controller subjects. The LTNP-EC showed a neutralization response, against 4 of the 6 viruses analyzed, superior to other ECs. CONCLUSIONS: The study demonstrated that a strong and sustained cellular and humoral immune response and low replicating viruses are associated with viral control in the superinfected LTNP-EC.

Definition of the viral targets of protective HIV-1-specific T cell responses.

BACKGROUND: The efficacy of the CTL component of a future HIV-1 vaccine will depend on the induction of responses with the most potent antiviral activity and broad HLA class I restriction. However, current HIV vaccine designs are largely based on viral sequence alignments only, not incorporating experimental data on T cell function and specificity. METHODS: Here, 950 untreated HIV-1 clade B or -C infected individuals were tested for responses to sets of 410 overlapping peptides (OLP) spanning the entire HIV-1 proteome. For each OLP, a "protective ratio" (PR) was calculated as the ratio of median viral loads (VL) between OLP non-responders and responders. RESULTS: For both clades, there was a negative relationship between the PR and the entropy of the OLP sequence. There was also a significant additive effect of multiple responses to beneficial OLP. Responses to beneficial OLP were of significantly higher functional avidity than responses to non-beneficial OLP. They also had superior in-vitro antiviral activities and, importantly, were at least as predictive of individuals' viral loads than their HLA class I genotypes. CONCLUSIONS: The data thus identify immunogen sequence candidates for HIV and provide an approach for T cell immunogen design applicable to other viral infections.

Human immunodeficiency virus type 1 and related primate lentiviruses engage clathrin through Gag-Pol or Gag.

The Gag-Pol polyprotein of human immunodeficiency virus type 1 (HIV-1) is not required for efficient viral particle production. However, premature termination codons in pol, particularly in the integrase (IN)-coding region, can markedly impair HIV-1 particle formation, apparently due to the premature activation of the viral protease (PR). We now report that the IN domain of Gag-Pol is required for the incorporation of clathrin into HIV-1 virions. Significantly, PR-dependent effects of point mutations in IN on particle production correlated strictly with their effects on clathrin incorporation. A possible interpretation of these findings is that certain IN mutations impair particle production in a PR-dependent manner by promoting Gag-Pol dimerization, which also occludes a binding site for clathrin. Consistently with this model, the reverse transcriptase (RT) inhibitor efavirenz, which is thought to promote Gag-Pol dimerization, inhibited the incorporation of clathrin into HIV-1 virions. Clathrin-depleted cells produced normal amounts of HIV-1 virions; however, their infectivity was reduced. We also observed that HIV-2 and the simian immunodeficiency virus SIVmac interact with clathrin through one or two copies of a peptide motif in the p6 domain of Gag that resembles the clathrin box of cellular adaptor proteins. Furthermore, the substitution of the hydrophobic residues in the single clathrin box motif of SIVmac caused a replication defect in primary cells. Taken together, our results indicate that primate lentiviruses from two different subgroups functionally interact with clathrin during assembly.

Programmed Death-1 expression on Epstein Barr virus specific CD8+ T cells varies by stage of infection, epitope specificity, and T-cell receptor usage.

BACKGROUND: Programmed Death-1 (PD-1) is an inhibitory member of the CD28 family of molecules expressed on CD8+ T cells in response to antigenic stimulation. To better understand the role of PD-1 in antiviral immunity we examined the expression of PD-1 on Epstein-Barr virus (EBV) epitope-specific CD8+ T cells during acute infectious mononucleosis (AIM) and convalescence. METHODOLOGY/PRINCIPAL FINDINGS: Using flow cytometry, we observed higher frequencies of EBV-specific CD8+ T cells and higher intensity of PD-1 expression on EBV-specific CD8+ T cells during AIM than during convalescence. PD-1 expression during AIM directly correlated with viral load and with the subsequent degree of CD8+ T cell contraction in convalescence. Consistent differences in PD-1 expression were observed between CD8+ T cells with specificity for two different EBV lytic antigen epitopes. Similar differences were observed in the degree to which PD-1 was upregulated on these epitope-specific CD8+ T cells following peptide stimulation in vitro. EBV epitope-specific CD8+ T cell proliferative responses to peptide stimulation were diminished during AIM regardless of PD-1 expression and were unaffected by blocking PD-1 interactions with PD-L1. Significant variability in PD-1 expression was observed on EBV epitope-specific CD8+ T cell subsets defined by V-beta usage. CONCLUSIONS/SIGNIFICANCE: These observations suggest that PD-1 expression is not only dependent on the degree of antigen presentation, but also on undefined characteristics of the responding cell that segregate with epitope specificity and V-beta usage.

Identification of ongoing human immunodeficiency virus type 1 (HIV-1) replication in residual viremia during recombinant HIV-1 poxvirus immunizations in patients with clinically undetectable viral loads on durable suppressive highly active antiretroviral therapy.

In most human immunodeficiency virus type 1 (HIV-1)-infected individuals who achieve viral loads of <50 copies/ml during highly active antiretroviral therapy (HAART), low levels of plasma virus remain detectable for years by ultrasensitive methods. The relative contributions of ongoing virus replication and virus production from HIV-1 reservoirs to persistent low-level viremia during HAART remain controversial. HIV-1 vaccination of HAART-treated individuals provides a model for examining low-level viremia, as immunizations may facilitate virus replication and sequence evolution. In a phase 1 trial of modified vaccinia virus Ankara/fowlpox virus-based HIV-1 vaccines in 20 HIV-infected young adults receiving HAART, we assessed the prevalence of low-level viremia and sequence evolution, using ultrasensitive viral load (<6.5 copies/ml) and genotyping (five-copy sensitivity) assays. Viral evolution, consisting of new drug resistance mutations and novel amino acid changes within a relevant HLA-restricted allele (e.g., methionine, isoleucine, glutamine, or arginine for leucine at position 205 of RT), was found in 1 and 3 of 20 subjects, respectively. Sequence evolution was significantly correlated with levels of viremia of between 6.5 and <50 copies/ml (P = 0.03) and was more likely to occur within epitopes presented by relevant HLA alleles (P < 0.001). These findings suggest that ongoing virus replication contributes to low-level viremia in patients on HAART and that this ongoing replication is subject to CD8(+) T-cell selective pressures.

Identification and characterization of HIV-1 CD8+ T cell escape variants with impaired fitness.

In this study, amino acid sequence variation in human immunodeficiency virus (HIV)-1 Gag CD8(+) T cell epitopes was examined in untreated mother-infant pairs. Several HIV-1 CD8(+) T cell escape variants were identified within maternal plasma viral p17 and p24 sequences that were either not detected or did not persist in the plasma of their non-HLA-matched HIV-1-infected infants. Viruses constructed with each of these mutations demonstrated reduced viral replication in vitro and reduced expression of p17 and p24 proteins compared with wild type. Reduced recognition of the variant sequences compared with wild-type sequence was also demonstrated by enzyme-linked immunospot assays. Nontransmission or reversion after transmission was thus associated with reduced viral fitness cost in vivo. Better understanding of the balance between CD8(+) T cell selective pressures and viral fitness cost may facilitate the identification of optimal viral sequences for inclusion in HIV-1 vaccines.

Genetic changes associated with distinct patterns of HIV type 1 persistence in chronically infected cell lines.

Three persistently infected cell lines (H61, M61, and U61) were established by infection with an HIV-1 isolate (s61) of two T cell lines, H9 and MT-4, and the promonocytic U937-2. In H61, 35% of cells expressed viral antigens yielding low virus titers and a majority of mature particles. M61 showed viral expression in every cell but with the frequent generation of immature particles. In U61, 1% of cells displayed viral expression, which increased after cell activation, indicating a latent infection. Nucleotide sequences of the complete provirus from the persistent cell lines revealed extremely high mutation rates in accessory genes and non-coding regions from 1.1 to 2.8 x 10(-2), whereas in structural genes they ranged from 3.2 to 9.8 x 10(-3). Ten nonsynonymous mutations were shared by all persistent proviruses including five strong amino acid changes in the env gene (related to the NSI phenotype) and in vpr and tat genes; other alterations were in accessory genes and two in the USF and c-Myb motifs in LTR. Truncated vpr and vpu proteins were found specifically in H61 and in vif in M61. This comprehensive study disclosed the role of the cell on the HIV-1 persistence pattern as well as common and specific mutations in the virus.

HIV-1-specific CD8+ T cell responses and viral evolution in women and infants.

CD8+ T lymphocyte responses play an important role in controlling HIV-1 replication but escape from CD8+ T cell surveillance may limit the effectiveness of these responses. Mother-to-child transmission of CD8+ T cell escape variants may particularly affect CD8+ T cell recognition of infant HIV-1 epitopes. In this study, amino acid sequence variation in HIV-1 gag and nef was examined in five untreated mother-infant pairs to evaluate the potential role of CD8+ T cell responses in the evolution of the viral quasispecies. Several CD8+ T cell escape variants were detected in maternal plasma. Evaluation of infant plasma viruses at 1-3 mo documented heterogeneity of gag and nef gene sequences and mother-to-child transmission of CD8+ T cell escape variants. Infant HLA haplotype and viral fitness appeared to determine the stability of the escape mutants in the infant over time. Changes in CD8+ T cell epitope sequences were detected in infants' sequential plasma specimens, suggesting that infants are capable of generating virus-specific CD8+ T cell responses that exert selective pressures in vivo. Altogether, these studies document that HIV-1-specific CD8+ T cell responses contribute to the evolution of the viral quasispecies in HIV-1-infected women and their infants and may have important implications for vaccine design.

Genetic analysis of culture-negative UNAIDS subtype C samples.

To help in the vaccine development, WHO-UNAIDS launched a program for the isolation and characterization of subtype C viruses, the most prevalent HIV-1 subtype in the world. Isolates were obtained from Brazil, China, India, Israel, and South Africa, countries in which these strains are circulating. In this study we genetically characterized a set of samples displaying the culture-negative phenotype by sequencing the nucleotides of three genomic regions: the p17 region of the gag gene, the C2V3C3 fragment of the env gene, and the nef gene. The association of the culture-negative phenotype with the nef gene was studied, and we found a significant accumulation of gene alterations. Except for one B/C recombinant from India, the samples studied formed a monophylogenetic subtype C clade, although samples from Brazil formed a statistically significant, independent subcluster in two of the three genes analyzed.