ABSTRACT RC-101 is a synthetic microbicide analog of retrocyclin, which has shown in vitro activity against X4 and R5 HIV-1. In an effort to develop a safe and effective RC-101 vaginal microbicide product, we assessed safety in ex vivo macaque and human models and efficacy using in vitro and ex vivo models. A polyvinyl-alcohol vaginal film containing RC-101 (100 μg/film) was developed. Formulation assessment was conducted by evaluating disintegration, drug content, mechanical properties, and stability. Efficacy was evaluated by in vitro peripheral blood mononuclear cells (PBMC) assay and ex vivo human ectocervical tissue explant model. Ex vivo safety studies were conducted by exposing RC-101 to an excised monkey reproductive tract and excised human ectocervical tissue. RC-101 100 μg films were shown to be safe to human and monkey tissue and effective against HIV-1 in vitro and ex vivo in human ectocervical tissue. The 90% inhibitory concentration (IC90) for RC-101 films at 2,000 μg (IC90 = 57.5 μM) using an ex vivo model was 10-fold higher than the IC90 observed using an in vitro model (IC90 = 5.0 μM). RC-101 films were stable for 1 month at 25°C, with in vitro bioactivity maintained for up to 6 months. RC-101 was developed in a quick-dissolve film formulation that was shown to be safe in an ex vivo model and effective in in vitro and ex vivo models. RC-101 film formulations were shown to maintain bioactivity for a period of 6 months. Findings from the present study contribute to the development of a safe and effective topical microbicide product.

Seminal HIV-1-specific cytotoxic T lymphocytes (CTLs) could provide an important immune defense against local HIV-1 infection, and be important in impeding the spread of HIV-1 infection. In this study, we demonstrate that autologous blood-derived dendritic cells (DCs) loaded in vitro with synthetic HIV-1 peptides representing known CTL epitopes activated HLA class I restricted, anti-HIV-1 CTLs and interferon gamma responses in seminal CD8+ T cells from subjects with chronic HIV-1 infection on antiretroviral therapy. CTLs specific for the same HLA-restricted epitopes were detected in semen and blood of the same individuals by stimulation with peptide-loaded DCs. Anti-HIV-1 CTL responses from semen were enhanced by stimulation with DCs loaded with HIV-1 peptides and interleukin 12. Our results suggest that blood-derived DCs have HIV-1 antigen-presenting capacity for seminal CTL in HIV-1-infected subjects. The DC-T cell system can serve as a model for immunotherapy of HIV-1 infection in the local genital tract as well as systemic blood circulation.

ABSTRACT 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.

HIV-1 RNA levels in semen and blood compartments decrease below detection limits during highly active antiretroviral therapy. Despite these therapeutic effects, it is clear that persistent, latent HIV-1 reservoirs are capable of rebounding in the absence of drug treatment or by evolution of escape mutants remain. The current study was designed to examine the presence of latent virus in semen and blood compartments and its evolution following potent combination therapy with indinavir (protease inhibitor) and efavirenz [nonnucleoside reverse transcriptase (RT) inhibitor]. Using an ultrasensitive in situ hybridization assay HIV-1 mRNA was detected in cultured seminal and blood mononuclear cells in all patients up to 1789 days posttherapy. Higher levels of HIV-1 mRNA were consistently detected in seminal mononuclear cells as compared to peripheral blood mononuclear cells (PBMC) in all time points analyzed posttherapy. Analysis of viral RNA from cultured PBMC before and after therapy displayed no evidence of therapy-induced drug resistance in the viral polymerase gene in the majority of patients. However, distinct envelope populations were detected in these viral RNA populations following therapy, indicating possible selection of quasispecies. The observed ongoing replication and evolution in the PBMC viral envelope sequences likely occurred in the seminal compartment HIV populations, given that the seminal cells showed the ability to express HIV-1 mRNA following cultivation. This together with our previous studies (Gupta P, et al.: J Infect Dis 2000;182:79-87) suggest that the genital and blood compartments likely serve as distinct reservoirs harboring latent HIV-1 during prolonged drug therapy.

Two highly conserved cationic amphipathic alpha-helical motifs, designated lentivirus lytic peptides 1 and 2 (LLP-1 and LLP-2), have been characterized in the carboxyl terminus of the transmembrane (TM) envelope glycoprotein (Env) of lentiviruses. Although various properties have been attributed to these domains, their structural and functional significance is not clearly understood. To determine the specific contributions of the Env LLP domains to Env expression, processing, and incorporation and to viral replication and syncytium induction, site-directed LLP mutants of a primary dualtropic infectious human immunodeficiency virus type 1 (HIV-1) isolate (ME46) were examined. Substitutions were made for highly conserved arginine residues in either the LLP-1 or LLP-2 domain (MX1 or MX2, respectively) or in both domains (MX4). The HIV-1 mutants with altered LLP domains demonstrated distinct phenotypes. The LLP-1 mutants (MX1 and MX4) were replication defective and showed an average of 85% decrease in infectivity, which was associated with an evident decrease in gp41 incorporation into virions without a significant decrease in Env expression or processing in transfected 293T cells. In contrast, MX2 virus was replication competent and incorporated a full complement of Env into its virions, indicating a differential role for the LLP-1 domain in Env incorporation. Interestingly, the replication-competent MX2 virus was impaired in its ability to induce syncytia in T-cell lines. This defect in cell-cell fusion did not correlate with apparent defects in the levels of cell surface Env expression, oligomerization, or conformation. The lack of syncytium formation, however, correlated with a decrease of about 90% in MX2 Env fusogenicity compared to that of wild-type Env in quantitative luciferase-based cell-cell fusion assays. The LLP-1 mutant MX1 and MX4 Envs also exhibited an average of 80% decrease in fusogenicity. Altogether, these results demonstrate for the first time that the highly conserved LLP domains perform critical but distinct functions in Env incorporation and fusogenicity.

The virologic and cellular factors that are involved in transmission of human immunodeficiency virus type 1 (HIV-1) across the female genital tissue are poorly understood. We have recently developed a human cervical tissue-derived organ culture model to study heterosexual transmission of HIV-1 that mimics the in vivo situation. Using this model we investigated the role of phenotypic characteristics of HIV-1 and identified the cell types that are first infected during transmission. Our data indicate that the cell-free R5 HIV-1 was more efficiently transmitted than cell-free X4 HIV-1. Cell-free and cell-associated HIV-1 had comparable transmission efficiency regardless of whether the virus was of R5 or X4 type. We have demonstrated that memory CD4(+) T cells and not Langerhans cells were the first HIV-1 RNA-positive cells detected at the epithelial-submucosal junction 6 h after virus exposure. Multicolor laser confocal microscopy demonstrated a globular distribution of HIV-1 gag-pol mRNA in the cytoplasm, and the distribution of CD4 and the CD45RO isoform was irregular on the cellular membrane. At 96 h postinoculation, in addition to memory CD4(+) T cells, HIV-1 RNA-positive Langerhans cells and macrophages were also detected. The identification of CD4(+) T cells in the tissue at 6 h was confirmed by flow cytometric simultaneous immunophenotyping and ultrasensitive fluorescence in situ hybridization assay on immune cells isolated from disaggregated tissue. Furthermore, PMPA {9-[2-(phosphonomethoxy)propyl] adenine}, an antiretroviral compound, and UC781, a microbicide, inhibited HIV-1 transmission across the mucosa, indicating the utility of the organ culture to screen topical microbicides for their ability to block sexual transmission of HIV-1.

India is experiencing a rapid spread of human immunodeficiency virus type 1 (HIV-1), primarily through heterosexual transmission of subtype C viruses. To delineate the molecular features of HIV-1 circulating in India, we sequenced the V3-V4 region of viral env from 21 individuals attending an HIV clinic in Calcutta, the most populous city in the eastern part of the country, and analyzed these and the other Indian sequences in the HIV database. Twenty individuals were infected with viruses having a subtype C env, and one had viruses with a subtype A env. Analyses of 192 subtype C sequences that included one sequence for each subject from this study and from the HIV database revealed that almost all sequences from India, along with a small number from other countries, form a phylogenetically distinct lineage within subtype C, which we designate C-IN. Overall, C-IN lineage sequences were more closely related to each other (level of diversity, 10.2%) than to subtype C sequences from Botswana, Burundi, South Africa, Tanzania, and Zimbabwe (range, 15.3 to 20.7%). Of the three positions identified as signature amino acid substitution sites for C-IN sequences (K340E, K350A, and G429E), 56% of the C-IN sequences contained all three amino acids while 87% of the sequences contained at least two of these substitutions. Among the non-C-IN sequences, all three amino acids were present in 2%, while 22% contained two or more of these amino acids. These results suggest that much of the current Indian epidemic is descended from a single introduction into the country. Identification of conserved signature amino acid positions could assist epidemiologic tracking and has implications for the development of a vaccine against subtype C HIV-1 in India.

High levels of human immunodeficiency virus (HIV) type 1 have been detected in semen at all stages of disease. However, it is not clear whether HIV-1 is shed in semen continuously or intermittently. In a prospective longitudinal study viral RNA was measured weekly for 10 weeks in semen and blood of HIV-seropositive subjects, Results showed three different patterns of HIV-1 shedding in semen: none (28%), continuous (28%), and intermittent (44%). In contrast, there was no change in blood plasma virus load during the study period, Phylogenetic analysis of the envelope sequences of HIV-1 RNA in semen and blood revealed distinct virus populations in semen and blood of intermittent shedders but similar virus populations in the semen and blood of continuous shedder. These results indicate for the first time that HIV-1 is shed primarily in an intermittent manner and that shedding patterns of HIV-1 in semen are related to compartmentalization of HIV-1 between semen and blood.

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 </=200 cells/microliter. The strength of these temporal associations between viral divergence and diversity, viral coreceptor specificity, and T-cell homeostasis and subset composition supports the concept that the phases described represent a consistent pattern of viral evolution during the course of HIV-1 infection in moderate progressors. Recognition of this pattern may help explain previous conflicting data on the relationship between viral evolution and disease progression and may provide a useful framework for evaluating immune damage and recovery in untreated and treated HIV-1 infections.

ABSTRACT We conducted two studies to determine the potential influence of delays in blood processing, type of anticoagulant, and assay method on human immunodeficiency virus type 1 (HIV-1) RNA levels in plasma. The first was an experimental study in which heparin- and EDTA-anticoagulated blood samples were collected from 101 HIV-positive individuals and processed to plasma after delays of 2, 6, and 18 h. HIV-1 RNA levels in each sample were then measured by both branched-DNA (bDNA) and reverse transcriptase PCR (RT-PCR) assays. Compared to samples processed within 2 h, the loss (decay) of HIV-1 RNA in heparinized blood was significant ( P < 0.05) but small after 6 h (bDNA assay, −0.12 log 10 copies/ml; RT-PCR, −0.05 log 10 copies/ml) and after 18 h (bDNA assay, −0.27 log 10 copies/ml; RT-PCR, −0.15 log 10 copies/ml). Decay in EDTA-anticoagulated blood was not significant after 6 h (bDNA assay, −0.002 log 10 copies/ml; RT-PCR, −0.02 log 10 copies/ml), but it was after 18 h (bDNA assay, −0.09 log 10 copies/ml; RT-PCR, −0.09 log 10 copies/ml). Only 4% of samples processed after 6 h lost more than 50% (≥0.3 log 10 copies/ml) of the HIV-1 RNA, regardless of the anticoagulant or the assay that was used. The second study compared HIV-1 RNA levels in samples from the Multicenter AIDS Cohort Study (MACS; samples were collected in heparin-containing tubes in 1985, had a 6-h average processing delay, and were assayed by bDNA assay) and the British Columbia Drug Treatment Program (BCDTP) (collected in EDTA- or acid citrate dextrose-containing tubes in 1996 and 1997, had a 2-h maximum processing delay, and were assayed by RT-PCR). HIV-1 RNA levels in samples from the two cohorts were not significantly different after adjusting for CD4 + -cell count and converting bDNA assay values to those corresponding to the RT-PCR results. In summary, the decay of HIV-1 RNA measured in heparinized blood after 6 h was small (−0.05 to −0.12 log 10 copies/ml), and the minor impact of this decay on HIV-1 RNA concentrations in archived plasma samples of the MACS was confirmed by the similarity of CD4 + -cell counts and assay-adjusted HIV-1 RNA concentrations in the MACS and BCDTP.

ABSTRACT Transmission of human immunodeficiency virus type 1 (HIV-1) usually results in outgrowth of viruses with macrophage-tropic phenotype and consensus non-syncytium-inducing (NSI) V3 loop sequences, despite the presence of virus with broader host range and the syncytium-inducing (SI) phenotype in the blood of many donors. We examined proviruses in contemporaneous peripheral blood mononuclear cells (PBMC) and nonspermatozoal semen mononuclear cells (NSMC) of five HIV-1-infected individuals to determine if this preferential outgrowth could be due to compartmentalization and thus preferential transmission of viruses of the NSI phenotype from the male genital tract. Phylogenetic reconstructions of ∼700-bp sequences covering the second constant region through the fifth variable region (C2 to V5) of the viral envelope gene revealed distinct variant populations in the blood versus the semen in two patients with AIDS and in one asymptomatic individual (patient 613), whereas similar variant populations were found in both compartments in two other asymptomatic individuals. Variants with amino acids in the V3 loop that predict the SI phenotype were found in both AIDS patients and in patient 613; however, the distribution of these variants between the two compartments was not consistent. SI variants were found only in the PBMC of one AIDS patient but only in the NSMC of the other, while they were found in both compartments in patient 613. It is therefore unlikely that restriction of SI variants from the male genital tract accounts for the observed NSI transmission bias. Furthermore, no evidence for a semen-specific signature amino acid sequence was detected.

Sequence variation displayed by the human immunodeficiency virus type 1 (HIV-1) has been proposed to be linked to the pathogenesis of acquired immunodeficiency syndrome (AIDS). To assess viral evolution during the course of infection, we evaluated sequence variability in theenvvariable domains in four HIV-1-infected individuals exhibiting differing profiles of CD4+T cell decline when followed from seroconversion until the development of AIDS or loss of followup. Proviral sequences encoding the V3–V5 region of gp120 were obtained following PCR amplification of peripheral blood mononuclear cell DNA and cloning. Virus in each patient was relatively homogeneous early in infection and then diverged with time, more consistently at its nonsynonymous sites. Just prior to or coincident with a rapid decline in CD4+T cell numbers, sequences were found with basic amino acid substitutions clustered within and downstream of the gp120 V3 domain. Within the constraints of the current dataset, we conclude that the virus appears to continually accumulate changes in its amino acid sequences well into the time of marked CD4+T cell decline.

We investigated memory cytotoxic T lymphocyte (CTLm) responses to HIV-1 as a determinant of HIV-1 disease progression, in relation to plasma HIV-1 load and T lymphocyte numbers in a longitudinal study of 14 homosexual men with incident HIV-1 infection. Study participants were selected who exhibited failure of T cell homeostasis, i.e., a downward inflection in CD3+ T cells that occurs in >75% of persons 1.5 to 2.5 years before development of AIDS, and compared with participants who developed low CD4+ T cell counts associated with possible T cell homeostasis failure, a subject who progressed rapidly to AIDS without well-defined T cell inflection, and subjects who had long-term preservation of T cell homeostasis (nonprogressors). High CTLm responses against Gag, but not Pol or Env, soon after seroconversion were associated with a slower loss of CD4+ T cells 1-4 years after seroconversion. Anti-Env CTLm responses decreased in most subjects around the time that T cell homeostasis failed. Plasma HIV-1 RNA increased exponentially (1.59-fold per year) over the 5 years preceding failure of T cell homeostasis, and there was a shift from a non-syncytium-inducing/CCR5 coreceptor phenotype of HIV-1 to a syncytium-inducing/CXCR4 phenotype, regardless of high or increasing levels of anti-HIV-1 CTLm during this time. These observations suggest that decreases in CTLm and increasing virus load are independent factors contributing to HIV-1 disease progression.