AIDS Research and Human Retroviruses

A theoretical concern exists that atazanavir (ATV) use during pregnancy may exacerbate physiologic neonatal hyperbilirubinemia. The aim of this substudy was to evaluate patterns of neonatal bilirubin following ATV/ritonavir (RTV) treatment of pregnant mothers and clinical and pharmacogenetic factors that may correlate. The design involved a subanalysis of study AI424182, a multicenter, open-label, prospective, single-arm Phase I study. The study had two treatment arms: (1) ATV/RTV 300/100 mg once daily or (2) ATV/RTV 400/100 mg once daily, both in combination with zidovudine/lamivudine 300/150 mg twice daily. Total bilirubin was assessed at baseline, each visit, and delivery day for mothers and on days 1 (delivery day), 3, 5, and 7 and weeks 2 and 6 for neonates. Blood samples were obtained for UGT1A1 genotyping and ATV cord blood concentration. Bilirubin elevation of any grade occurred in 14/40 neonates (35%). All Grade 3 to 4 bilirubin abnormalities (n=7) occurred after day 14. The pattern of neonatal bilirubin levels reported was consistent with neonatal physiologic elevations of bilirubin. Little correlation was observed between either maternal bilirubin levels over the last 4 weeks of pregnancy (including delivery) or ATV cord concentration and neonatal bilirubin. There was a significant association between UGT1A1 genotype and bilirubin grade in the maternal population (p=0.0006) but not neonates (p=0.49). Neither neonatal UGT1A1 genotype nor cord blood ATV concentration is a good predictor of neonatal hyperbilirubinemia. ATV/RTV treatment of mothers does not appear to exacerbate neonatal physiologic hyperbilirubinemia.

Studies in persons of European descent have suggested that mitochondrial DNA (mtDNA) haplogroups influence antiretroviral therapy (ART) toxicity. We explored associations between mtDNA variants and changes in endothelial function and biomarkers among non-Hispanic white, ART-naive subjects starting ART. A5152s was a substudy of A5142, a randomized trial of initial class-sparing ART regimens that included efavirenz or lopinavir/ritonavir with nucleoside reverse transcriptase inhibitors (NRTIs), or both without NRTIs. Brachial artery flow-mediated dilation (FMD) and cardiovascular biomarker assessments were performed at baseline and at weeks 4 and 24. Ten haplogroup-defining mtDNA polymorphisms were determined. FMD and biomarker changes from baseline to week 24 by mtDNA variant were assessed using Wilcoxon rank-sum tests. Thirty-nine non-Hispanic white participants had DNA and 24-week data. The nonsynonymous m.10398A>G mtDNA polymorphism (N=8) was associated with higher median baseline adiponectin (5.0 vs. 4.2 μg/ml; p=0.003), greater absolute (−1.9 vs. −0.2 μg/ml) and relative (−33% vs. −3%) adiponectin decreases (p<0.001 for both), and lower week 24 brachial artery FMD (3.6% vs. 5.4%; p=0.04). Individual mtDNA haplogroups, including haplogroups H (N=13) and U (N=6), were not associated with adiponectin or FMD changes. In this small pilot study, adiponectin and brachial artery FMD on ART differed in non-Hispanic whites with a nonsynonymous mtDNA variant associated with several human diseases. These preliminary findings support the hypothesis that mtDNA variation influences metabolic ART effects. Validation studies in larger populations and in different racial/ethnic groups that include m.10398G carriers are needed.

We estimated the impact of loss to follow-up (LTFU) on the mortality rate among HIV-1-infected patients in Curaçao. A total of 214 therapy-naive HIV-1-infected patients aged 15 years or older upon entering into HIV care between January 2005 and July 2009 were included. Persons who discontinued follow-up for more than 365 days were defined as LTFU and traced with the aim of registering their vital status. If no personal contact could be made, data were matched with the Curaçao National Death Registry. Mortality rates were estimated before and after starting combination antiretroviral therapy (cART). We used log-rank tests to compare survival rates among patients LTFU and patients who experienced continuous follow-up. Pre-cART mortality in patients LTFU was similar to pre-cART mortality in those with continuous follow-up (p=0.79). All pre-cART deaths occurred within 6 months after entry. Low CD4 cell count was predictive of a shorter time to death after entry. Adjusting for those who were LTFU, the mortality rate after starting cART increased from 4.3 to 5.5 per 100 person years of observation (p=0.06). Mortality after starting cART was highest in the first 2 months after starting cART, especially for those who had late disease stage. Mortality rates were lower in patients with continuous follow-up compared to LTFUs (p<0.001). Mortality rates in HIV-1-infected patients who have started cART in Curaçao are underestimated as a result of inefficient patient administration combined with people starting cART at a very late disease stage. Monitoring HIV treatment could help in reducing the risk of LTFU and may improve the effect of treatment.

Transmitted drug resistance (TDR) influencing nonnucleoside reverse transcriptase inhibitor (NNRTI) activity is increasing among new HIV-1 patients in several countries. As we recently observed an increase of K103N prevalence among new diagnoses in Belgium, we mined the Belgian national sequence database for homologous sequences. The earliest reverse transcriptase (RT) sequences available for drug-naive patients as well as sequences related to treatment failure were included. Fifty-five sequences were aligned and subjected to phylogenetic analysis, revealing the presence of a cluster of 29 virus sequences. All except one of those sequences were from antiretroviral (ARV)-naive patients at the time of sampling, and 22 had the K103N mutation. Epidemiological data of clustered patients were collected through the Institute of Public Health. Seventy-two percent of the clustered patients were infected through homosexual or bisexual contacts while the others reported heterosexual contacts only. All patients reside and were infected in Belgium. Sixteen were diagnosed between January 2011 and June 2012; 14 were aged between 18 and 29 years at the time of diagnosis. Nearly 60% of the clustered patients live close to the city of Namur, where HIV incidence substantially increased in the past 2 years. The identification of this transmission network advocates for local prevention reinforcement and underscores the need for continuous TDR monitoring. The spread of NNRTI TDR could affect ARV initiation schemes and prophylaxis strategies.

The U.S. military represents a unique population within the human immunodeficiency virus 1 (HIV-1) pandemic. The last comprehensive study of HIV-1 in members of the U.S. Navy and Marine Corps (Sea Services) was completed in 2000, before large-scale combat operations were taking place. Here, we present molecular characterization of HIV-1 from 40 Sea Services personnel who were identified during their seroconversion window and initially classified as HIV-1 negative during screening. Protease/reverse transcriptase (pro/rt) and envelope (env) sequences were obtained from each member of the cohort. Phylogenetic analyses were carried out on these regions to determine relatedness within the cohort and calculate the most recent common ancestor for the related sequences. We identified 39 individuals infected with subtype B and one infected with CRF01_AE. Comparison of the pairwise genetic distance of Sea Service sequences and reference sequences in the env and pro/rt regions showed that five samples were part of molecular clusters, a group of two and a group of three, confirmed by single genome amplification. Real-time molecular monitoring of new HIV-1 acquisitions in the Sea Services may have a role in facilitating public health interventions at sites where related HIV-1 infections are identified.

The potential role of conventional and regulatory T cells (Tregs) in protection from HIV-1 infection remains unclear. To address this question, we analyzed samples from 129 HIV-1-exposed seronegative individuals (HESN) from an HIV-1-serodiscordant couples cohort. To assess the presence of HIV-specific T cell responses and Treg function, we measured the proliferation of T cells in response to HIV-1 peptide pools in peripheral blood mononuclear cells (PBMCs) and PBMCs depleted of Tregs. We identified HIV-specific CD4⁺ and CD8⁺ T cell responses and, surprisingly, the overall CD4⁺ and CD8⁺ T cell response rate was not increased when Tregs were removed from cell preparations. Of the 20 individuals that had HIV-1-specific CD4⁺ T cell responses, only eight had Tregs that could suppress this proliferation. When compared with individuals whose Tregs could suppress HIV-1-specific CD4⁺ T cell proliferation, individuals with Tregs unable to suppress showed a trend toward increased T cell activation and Treg frequency and a significant increase in HIV-1-specific production of microphage inflammatory protein-1β (MIP-1β) by CD4⁺ T cells, autocrine production of which has been shown to be protective in terms of HIV-1 infection of CD4⁺ T cells.

Memory CD4⁺ T lymphocytes in peripheral blood that express integrins α4ß7 preferentially recirculate through gut-associated lymphoid tissue (GALT), a proposed site of significant HIV-1 replication. Tregs and activated CD4⁺ T cells in GALT could also be particularly susceptible to infection. We therefore hypothesized that infection of these subsets of memory CD4⁺ T cells may contribute disproportionately to the HIV-1 reservoir. A cross-sectional study of CD4⁺ T cell subsets of memory CD45RO⁺ cells in peripheral blood mononuclear cells (PBMCs) was conducted using leukapheresis from eight subjects with untreated chronic HIV-1 infection. Real-time polymerase chain reaction (PCR) was used to quantify total and integrated HIV-1 DNA levels from memory CD4⁺ T cells sorted into integrin β7⁺ vs. β7⁻, CD25⁺CD127^low Treg vs. CD127^high, and activated CD38⁺ vs. CD38⁻. More than 80% of total HIV-1 DNA was found to reside in the integrin β7-negative non-gut-homing subset of CD45RO⁺ memory CD4⁺ T cells. Less than 10% was found in highly purified Tregs or CD38⁺ activated memory cells. Similarly, integrated HIV-1 DNA copies were found to be more abundant in resting non-gut-homing memory CD4⁺ T cells (76%) than in their activated counterparts (23%). Our investigations showed that the majority of both total and integrated HIV-1 DNA was found within non-gut-homing resting CD4⁺ T cells.

Allogeneic hematopoietic stem cell transplantation (Allo-HSCT) is a well-established therapeutic option for hematological malignancies. Combination antiretroviral therapy (cART) has enabled the treatment of medical conditions in patients infected with the human immunodeficiency virus (HIV) in the same way as in the general population. Moreover, improvements in supportive care have allowed HIV-infected patients with life-threatening hematological disorders to be treated with Allo-HSCT. We report on four HIV-infected patients with hematological malignancies receiving an Allo-HSCT in our institution, and on the use of donor lymphocyte infusions to successfully treat post-Allo-HSCT relapse. Of note, one of them is the first HIV⁺ patient to receive a “dual transplant” (unrelated umbilical cord blood stem cells combined with mobilized T cell-depleted CD34⁺ stem cells from a mismatched third party donor). cART drugs interactions were satisfactorily managed. This approach provided long-term control of the hematological disease. Nevertheless, despite adequate immune reconstitution, infections were the main cause of morbidity and mortality after Allo-HSCT.

In the context of HIV, the initiation of effective antiretroviral therapy (ART) has been found to increase the risk of dyslipidemia in HIV-infected individuals, and dyslipidemia has been found to be a risk factor for kidney disease in the general population. Therefore, we examined changes in lipid profiles in HIV-infected men following ART initiation and the association with future kidney dysfunction. HIV-infected men from the Multicenter AIDS Cohort Study initiating ART between December 31, 1995 and September 30, 2011 with measured lipid and serum creatinine values pre-ART and post-ART were selected. The associations between changes in total cholesterol or high-density lipoprotein following ART initiation and the estimated change in glomerular filtration rate (eGFR) over time were assessed using piecewise linear mixed effects models. There were 365 HIV-infected men who contributed to the analysis. In the adjusted models, at 3 years post-ART, those with changes in total cholesterol >50 mg/dl had an average decrease in eGFR of 2.6 ml/min/1.73 m² per year (p<0.001) and at 5 years post-ART, the average decrease was 2.4 ml/min/1.73 m² per year (p=0.008). This decline contrasted with the estimates for those with changes in total cholesterol ≤50 mg/dl: 1.4 ml/min/1.73 m² decrease per year (p<0.001) and 0.1 ml/min/1.73 m² decrease per year (p=0.594) for the same time periods, respectively. Large decreases in high-density lipoprotein (a decline of greater than 5 mg/dl) were not associated with declines in eGFR. These results indicate that large ART-related increases in total cholesterol may be a risk factor for kidney function decline in HIV-infected men. Should these results be generalizable to the broader HIV population, monitoring cholesterol changes following the initiation of ART may be important in identifying HIV-infected persons at risk for kidney disease.

The mechanisms underlying B cell activation that persists during antiretroviral therapy (ART) are unknown. Toll-like receptor (TLR) signaling is a critical mediator of innate cell activation and though B cells express TLRs, few studies have investigated a role for TLR signaling in B cell activation during HIV infection. We addressed this question by assessing the activated phenotype and TLR expression/responsiveness of B cells from ART-treated HIV-infected subjects (HIV_ART ⁺). We evaluated activation markers implicated in B cell-mediated T cell trans infection during HIV pathogenesis. We found no significant difference in TLR expression between B cells of HIV_ART ⁺ and HIV⁻ subjects. However, B cells of HIV_ART ⁺ subjects exhibited heightened endogenous expression levels of IL-6 (p=0.0051), T cell cognate ligands CD40 (p=0.0475), CD54 (p=0.0229), and phosphorylated p38 (p<0.0001), a marker of TLR signaling. In vitro, B cells of HIV_ART ⁺ individuals were less responsive to TLR stimulation compared to B cells of HIV⁻ subjects. The activated phenotype of in vitro TLR-stimulated B cells of HIV⁻ subjects was similar to ex vivo B cells from HIV_ART ⁺ individuals. TLR2 stimulation was a potent mediator of B cell activation, whereas B cells were least responsive to TLR4 stimulation. Compared to HIV⁻ subjects, the serum level of lipoteichoic acid (TLR2 ligand) in HIV_ART ⁺ subjects was significantly higher (p=0.0207), correlating positively with viral load (p=0.0127, r=0.6453). Our data suggest that during HIV infection TLR-activated B cells may exert a pathogenic role and B cells from HIV_ART ⁺ subjects respond to in vitro TLR stimulation, yet exhibit a TLR tolerant phenotype suggesting prior in vivo TLR stimulation.

The advent of next generation sequencing technologies is providing new insight into HIV-1 diversity and evolution, which has created the need for bioinformatics tools that could be applied to the characterization of viral quasispecies. Here we present Nautilus, a bioinformatics package for the analysis of HIV-1 targeted deep sequencing data. The DeepHaplo module determines the nucleotide base frequency and read depth at each position and computes the haplotype frequencies based on the linkage among polymorphisms in the same next generation sequence read. The Motifs module computes the frequency of the variants in the setting of their sequence context and mapping orientation, which allows for the validation of polymorphisms and haplotypes when strand bias is suspected. Both modules are accessed through a user-friendly GUI, which runs on Mac OS X (version 10.7.4 or later), and are based on Python, JAVA, and R scripts. Nautilus is available from www.hivresearch.org/research.php?ServiceID=5&SubServiceID=6.

Although 2-long terminal repeat (2-LTR) circles are only a fraction of the total viral DNA in infected cells, sequence analysis of 2-LTR circles reveals critical information regarding viral DNA synthesis and the nature of actively replicating virus. It was observed that a large proportion of the 2-LTR circular molecules in the peripheral blood mononuclear cell (PBMC) DNA of infected individuals are mutated at the circle junction. The integrase inhibitor raltegravir (RAL) blocks the strand transfer step of the integration of HIV-1; as a consequence of abortive integration a significant increase of episomal 2-LTR circles is observed. Moreover, it was demonstrated that in patients treated with highly active retroviral therapy (HAART) changes in 2-LTR concentration did not affect junction sequences and flanking regions of 2-LTR. Here we evaluated whether RAL therapy could have a differential impact on the 2-LTR circle junctional sequences in patients with different virological profiles at the time of starting RAL therapy. Sequence analysis indicates that RAL acts differently in the two populations.

Human T cell leukemia virus type 1 (HTLV-1) is associated with a neurological syndrome named tropical spastic paraparesis/HTLV-associated myelopathy (TSP/HAM) and the disease progression involves viral factors. The gp21 glycoprotein is involved in envelope trafficking and membrane targeting while the bZIP protein is indispensable for cell growth and proliferation. This study aimed to assess the molecular diversity of gp21 and HBZ proteins in TSP/HAM and healthy carriers. DNA samples from HTLV-1-infected individuals were submitted to PCR and sequencing, and the molecular analyses were performed using bioinformatics tools. From eight gp21-analyzed sequences one amino acid change (Y477H) was associated with the switch of a helix to coil structure at secondary structure prediction. From 10 HBZ analyzed sequences, two amino acid changes were identified (S9P and T95I) at the activation domain. One mutation (R112C) located at the nuclear localization signal was present in 66.7% and 25% of healthy carriers (HC) and TSP/HAM groups, respectively. This is the first report of mutations in the HBZ region. These polymorphisms might be important for viral fitness.

The first line of defense protecting rhesus macaques from HIV-1 is the restriction factor rhTRIM5α, which recognizes the capsid core of the virus early after entry and normally blocks infection prior to reverse transcription. Cytoplasmic bodies containing rhTRIM5α have been implicated in the ubiquitin–proteasome pathway, but the specific roles these structures play remain uncharacterized. Here, we examine the ubiquitination status of cytoplasmic body proteins. Using antibodies specific for different forms of ubiquitin, we show that ubiquitinated proteins are present in cytoplasmic bodies, and that this localization is altered after proteasome inhibition. A decrease in polyubiquitinated proteins localizing to cytoplasmic bodies was apparent after 1 h of proteasome inhibition, and greater differences were seen after extended proteasome inhibition. The decrease in polyubiquitin conjugates within cytoplasmic bodies was also observed when deubiquitinating enzymes were inhibited, suggesting that the removal of ubiquitin moieties from polyubiquitinated cytoplasmic body proteins after extended proteasome inhibition is not responsible for this phenomenon. Superresolution structured illumination microscopy revealed finer details of rhTRIM5α cytoplasmic bodies and the polyubiquitin conjugates that localize to these structures. Finally, linkage-specific polyubiquitin antibodies revealed that K48-linked ubiquitin chains localize to rhTRIM5α cytoplasmic bodies, implicating these structures in proteasomal degradation. Differential staining of cytoplasmic bodies seen with different polyubiquitin antibodies suggests that structural changes occur during proteasome inhibition that alter epitope availability. Taken together, it is likely that rhTRIM5α cytoplasmic bodies are involved in recruiting components of the ubiquitin–proteasome system to coordinate proteasomal destruction of a viral or cellular protein(s) during restriction of HIV-1.

Despite its sequence variability and structural flexibility, the V3 loop of the HIV-1 envelope glycoprotein gp120 is capable of recognizing cell-bound coreceptors CCR5 and CXCR4 and infecting cells. Viral selection of CCR5 is associated with the early stages of infection, and transition to selection of CXCR4 indicates disease progression. We have developed a predictive statistical model for coreceptor selectivity that uses the discrete property of net charge and the binary coreceptor preference markers of the N⁶X⁷[T/S]⁸X⁹ glycosylation motif and 11/24/25 positive amino acid rule. The model is based on analysis of 2,054 V3 loop sequences from patient data and allows us to infer the most likely state of the disease from physicochemical characteristics of the sequences. The performance of the model is comparable to established sequence-based predictive methods, and may be used in combination with other methods as a supportive diagnostic for coreceptor selection. This model may be used for personalized medical decisions in administering coreceptor-specific therapies.