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Objective(s) The glycan shield of the HIV-1 envelope glycoprotein complex (Env), in particular the glycan at position 332 in gp120, is frequently targeted by broadly neutralizing antibodies (bNAbs) isolated from HIV-1-infected individuals. We investigated the role of the second amino acid position of the N332 glycosylation motif Asn-X-Ser in HIV-1 evolution and neutralization sensitivity. Design and methods Viral variants harbouring glycosylation motifs with different probabilities of glycan occupancy were tested for their sensitivity to a subset of N332-dependent bNAbs. Furthermore, longitudinal Env sequences of 37 HIV-1 infected individuals were used to analyse the evolution of the N332 glycosylation motif within these individuals. Finally, early Env sequences from 31 historical and 21 contemporaneous seroconverters were compared to analyse this evolution on a population level. Results Viral variants with a higher probability of N332 occupancy were more sensitive to neutralization by some N332-dependent bNAbs. Furthermore, the longitudinal analyses revealed an increase in probability of glycan occupancy of the N332 site over time, both within patients, and at the population level over the course of 20 years of HIV-1 epidemic. Conclusions These observations suggest that modulation of N332 glycan occupancy by the second amino acid position of the canonical glycosylation motif Asn-X-Ser plays a previously unappreciated role in viral escape from immune responses, and should be considered in Env-based vaccine design.

Objective: Heterozygosity for a 32 base pair deletion in the CCR5 gene (CCR5wt/Δ32) and the minor alleles of a single-nucleotide polymorphism in the HCP5 gene (rs2395029) and in the HLA-C gene region (−35HLA-C; rs9264942) has been associated with a lower viral load set point. Recent studies have shown that over calendar time, viral load set point has significantly increased at a population level. Here we studied whether this increase coincides with a fading impact of above-mentioned host genetic markers on HIV-1 control. Methods: We compared the association between viral load set point and HCP5 rs2395029, −35HLA-C rs9264942, and the CCR5wt/Δ32 genotype in HIV-1-infected individuals in the Netherlands who had seroconverted between 1982 and 2002 (pre-2003 seroconverters, n = 459) or between 2003 and 2009 (post-2003 seroconverters, n = 231). Results: Viral load set point in post-2003 seroconverters was significantly higher than in pre-2003 seroconverters (P = 4.5 × 10−5). The minor alleles for HCP5 rs2395029, −35HLA-C rs9264942 and CCR5wt/Δ32 had a similar prevalence in both groups and were all individually associated with a significantly lower viral load set point in pre-2003 seroconverters. In post-2003 seroconverters, this association was no longer observed for HCP5 rs2395029 and CCR5wt/Δ32. The association between viral load set point and HCP5 rs2395029 had significantly changed over time, whereas the change in impact of the CCR5wt/Δ32 genotype over calendar time was not independent from the other markers under study. Conclusion: The increased viral load set point at a population level coincides with a lost impact of certain host genetic factors on HIV-1 control.

Objective: An important pending question is whether temporary highly active antiretroviral therapy during primary HIV infection can influence viral rebound dynamics and the subsequently established viral setpoint, through preservation and enhancement of HIV-1-specific immune responses, or through other mechanisms. Methods: We included all patients from two prospective studies who underwent a single treatment interruption while being well suppressed on highly active antiretroviral therapy. One group started highly active antiretroviral therapy during primary HIV infection, and the other group started it during chronic HIV infection with CD4 cell counts above 350 cells/ml. Data were collected up to 48 weeks from treatment interruption.Themediantimetoviralreboundwasanalysedforthreelevelsofviraemia: 50, 500 and 5000 copies HIV-RNA/ml plasma. Results: The median time to viral rebound was significantly longer in primary HIV infection patients (n ¼24) than in chronic HIV infection patients (n ¼46): 8 versus 4 weeks (P

OBJECTIVE: To assess syncytium-inducing (SI) and non-syncytium-inducing (NSI) frequencies, coreceptor usage and gp120 V3 sequences of HIV-1 isolates from Ethiopian AIDS patients. PATIENTS: Cross-sectional study on 48 hospitalized AIDS patients (CD4 T cells < 200 x 10(6) cell/l) with stage III or IV of the WHO staging system for HIV-1 infection and disease. METHODS: Peripheral blood mononuclear cells (PBMC) from all 48 patients were tested by MT-2 assay to determine SI/NSI phenotypes. Lymphocyte subsets were enumerated using Coulter counting and FACScan analysis. Viral load determination used a nucleic acid sequence-based amplification assay (NASBA). Coreceptor usage of HIV-1 biological clones was measured using U87 CD4/chemokine receptor transfectants and phytohemagglutinin-stimulated PBMC of healthy donors with wild-type CCR5 and homozygous mutation CCR5delta32 (a 32 base-pair deletion in CCR5). Reverse transcriptase polymerase chain reaction sequencing was performed on the third variable region (V3) of the HIV-1 gene gp120. Sequence alignments were done manually; phylogenetic analyses used PHYLIP software packages. RESULTS: SI viruses were detected for 3/48 (6%) AIDS patients only. Lower mean absolute CD4 counts were determined in patients with SI virus compared with NSI (P = 0.04), but no differences in viral load were observed. All patients were found to be infected with HIV-1 subtype C, based on V3 sequencing. NSI biological clones used CCR5 as coreceptor; SI biological clones used CXCR4 and/or CCR5 and/or CCR3. CONCLUSIONS: Ethiopian patients with HIV-1 C-subtype AIDS harbour a remarkably low frequency of SI phenotype viruses. Coreceptor usage of these viruses correlates with their biological phenotypes

Background A stable reservoir of latently infected, resting CD4 T cells has been demonstrated in HIV-1-infected patients despite prolonged antiretroviral treatment. This is a major barrier for the eradication of HIV by antiretroviral agents alone. Activation of these cells in the presence of antiretroviral therapy might be a strategy to increase the turnover rate of this reservoir. Methods Three HIV-1-positive patients on potent antiretroviral therapy, in whom plasma viremia had been suppressed to below 5 copies/ml for at least 26 weeks, were treated with a combination of OKT3 (days 1-5) and recombinant human IL-2 (days 2 6). Results The side-effects were fever, headache, nausea, diarrhea, and in one of the patients transient renal failure and seizures. The regimen resulted in profound T cell activation. In one patient plasma HIV-1 RNA transiently increased with a peak at 1500 copies/ml. In the other two patients plasma HIV-1 RNA levels remained below the detection limit, but HIV-1 RNA levels in the lymph nodes increased two- to threefold. All patients developed antibodies against OKT3. Conclusion OKT3/IL-2 resulted in T cell activation and proliferation, and could stimulate HIV replication in patients having achieved prolonged suppression of plasma viremia. OKT3/IL-2 therapy was toxic and rapidly induced antibodies against OKT3.

Objective: To investigate the relationship between exposure to antiretroviral drugs and the initial decline of plasma HIV-1 RNA. Design: Open-label study in antiretroviral-naive HIV-1 infected patients using a quadruple drug regimen [nelfinavir (NFV), saquinavir (SQV), stavudine, and lamivudine]. Methods: The elimination rate constant (k) for HIV-1 clearance was calculated during the first 2 weeks of treatment in 29 patients. Exposure to NFV and SQV was quantified on each study visit. Observed NFV and SQV concentrations were related to those expected in a reference population and a concentration ratio was calculated. The median concentration ratios for NFV and SQV, the baseline CD4+ lymphocyte count and baseline log10 HIV-1 RNA were correlated with k. Results: A significant positive correlation was observed between k and the median NFV (P = 0.001) or SQV concentration ratio (P = 0.016) in univariate analysis. In multivariate analyses, the median NFV concentration ratio remained significantly correlated with k. Conclusions: The variation in the rate of decline of plasma HIV-1 RNA between patients after the initiation of a quadruple drug regimen could be explained by differences in exposure to NFV or SQV. Determination of k could be used to optimise further antiretroviral drug therapy and may be a first tool to assess antiretroviral activities of new or increasing doses of drugs administered in combination regimens. Furthermore, our data suggest that exposure to antiretroviral drugs should be incorporated in mathematical models to describe HIV-1 dynamics in more detail.

A polymorphism in CX3CR1, a co-receptor for some HIV-1 variants, has been associated with a strong acceleration of HIV-1 disease progression. In the Amsterdam Cohort of Homosexual Men with HIV and AIDS, we were unable to confirm these findings. In addition, we did not find an effect of this polymorphism on the acquisition of syncytium-inducing/X4 HIV variants.

Objective: Recent studies have suggested that the dynamics of HIV-1 evolutionary rate reflect the rate of disease progression. We wished to determine whether viral diversity early in infection is predictive of the subsequent disease course. Design: HIV-1 envelope diversity at seroconversion and 1 year thereafter from 89 homosexual participants of the Amsterdam Cohort Studies on HIV infection and AIDS was correlated with clinical endpoints and markers of disease progression. Methods: Heteroduplex mobility assay (HMA) and sequencing followed by calculation of pairwise genetic distances were applied to determine HIV-1 envelope diversity. The HMA pattern (presence or absence of heteroduplexes) and sequence diversity were each tested for correlation with the clinical course of infection. Results: HMA pattern at 1-year postseroconversion was significantly associated with progression to AIDS and AIDS-related death, with presence of heteroduplexes associated with accelerated disease progression. Moreover, not only this dichotomous measure of viral diversity (absence or presence of heteroduplexes), but also genetic diversity itself was associated with disease course. HMA pattern was an independent predictor of accelerated disease progression, also when CCR5 genotype, human leukocyte antigen (HLA)-type, viral load, CD4(+) T-cell counts, and coreceptor use at viral load set point were included in the analysis. Conclusion: Viral diversity early in HIV-1 infection is predictive of the subsequent disease progression. It remains to be established whether viral diversity itself plays a causal role in the increased damage to the immune system or whether it is a reflection of immune pressure or other selective forces. (C) 2012 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins

Objective To monitor a patient who presented with symptomatic HIV-1 infection for virological and immunological parameters in relation to the clinical course. Methods Virological studies included determination of frequency of productively HIV-1-infected peripheral blood mononuclear cells (PBMC) and viral RNA load in plasma and p24 antigenaemia. Immunological studies included the analysis of T-cell subsets, the expression of activation markers, CD45RO and CD45RA antigens, the frequency of cells programmed for death, and T-cell function. Results During the first week post onset of primary HIV-1 infection symptoms high plasma titres of p24 and HIV-1 RNA were observed. The number of productively HIV-1-infected PBMC peaked, coinciding with CD4+ T lymphocytopaenia, during week 2 when clinical improvement started. CD8+ T lymphocytosis was observed 10 days post onset of clinical symptoms, the expanded cell population being of the CD8+CD38+, CD8+CD27+ and CD8+CD28-phenotype. CD8+ T lymphocytosis was paralleled by a high percentage of cells undergoing programmed cell death on in vitro culture. In vitro T-cell function was severely depressed during the first 10 days post onset of clinical symptoms. Within about 3 weeks, following resolution of clinical symptoms, phytohaemagglutinin-induced proliferation was restored to normal levels while responses to the CD3 monoclonal antibody only showed a partial restoration. During follow-up, concomitant with the rise of activated CD8+ T cells, p24 antigen levels and viral RNA load in serum as well as the number of HIV-producing PBMC steeply declined after 2 weeks. Conclusion These findings demonstrate HIV-1-induced abnormalities during severe clinical symptoms of primary HIV-1 infection. The subsequent strong immune response, which is believed to be responsible for efficient control of viral replication, appears to precede clinical improvement.

To examine temporal variation in the effects of CCR5-Delta32 and CCR2-64I chemokine receptor gene polymorphisms on HIV-1 disease progression.Pooled analysis of individual patient data from 10 cohorts of HIV-1 seroconverters from the United States, Europe, and Australia.We studied HIV-1 seroconverters of European (n = 1635) or African (n = 215) ancestry who had been genotyped for CCR5-Delta32 and CCR2-64I. We used Cox proportional hazards models with time-varying coefficients to determine whether the genetic protection against AIDS (1987 case definition) and death varied with time since seroconversion.Protection against AIDS conferred by CCR5-Delta32 held constant at a 31% (RH 0.69, 95% CI 0.54, 0.88) reduction in risk over the course of HIV-1 infection, whereas protection against death held constant at a 39% reduction in risk (RH 0.61, 95% CI 0.45, 0.88). When the period from AIDS to death was isolated, the survival benefit of CCR5-Delta32 diminished 2 years after AIDS. Protection against AIDS conferred by CCR2-64I was greatest early in the disease course. Compared with individuals without CCR5-Delta32 or CCR2-64I, individuals with one or two copies of CCR2-64I had a 58% lower risk of AIDS during the first 4 years after seroconversion (RH 0.42, 95% CI 0.23, 0.76), a 19% lower risk during the subsequent 4 years (RH 0.81, 95% CI 0.59, 1.12), and no significant protection thereafter.The protection against AIDS provided by CCR5-Delta32 is continuous during the course of infection. In contrast, the protection provided by CCR2-64I is greatest early in the course of infection.

OBJECTIVE: To study the relationship between toxicity and the exposure to nelfinavir and saquinavir as part of a quadruple drug regimen. DESIGN: The ADAM study is a randomized study to investigate the feasibility of induction-maintenance therapy in HIV-1 infection. METHODS: HIV-1-infected patients with no prior use of antiretroviral treatment started induction therapy consisting of stavudine + lamivudine + nelfinavir + saquinavir for a period of 26 weeks. Data regarding toxicity of the quadruple regimen and exposure to the protease inhibitors were collected. RESULTS: Seven of the 65 patients enrolled had to switch therapy for reasons of toxicity within the first 26 weeks. Diarrhoea was frequently reported (49 of 65, one discontinuation), but could be relieved by using antidiarrhoeal agents. Laboratory monitoring revealed elevated liver enzymes (leading to four discontinuations) and mild to moderate elevations of triglycerides and cholesterol (nine and 23 of 65, respectively). The exposure to saquinavir and nelfinavir was lower than expected. Abdominal pain was associated with a higher exposure to nelfinavir or saquinavir. The association of nausea and abdominal distension with drug exposure appeared to vary over time. CONCLUSIONS: The quadruple drug regimen was quite well tolerated. Diarrhoea was frequently reported but could be relieved by the use of antidiarrhoeal agents. In comparison with other protease inhibitor combinations, lipid abnormalities in plasma were infrequent and mild. With the exception of diarrhoea, all gastrointestinal complaints observed were found to be associated with the level of exposure to nelfinavir or saquinavir. The exposure to the protease inhibitors was relatively low, although the virologic efficacy of the regimen used was satisfactory