Intracellular cytokine staining and flow cytometry can be used to measure T-cell responses to defined antigens. Although CD8+ T-cell responses to soluble proteins are inefficiently detected by this approach, peptides can be used as antigens. Using overlapping peptides spanning an entire protein sequence, CD8+ T-cell responses can be detected to multiple epitopes, regardless of HLA type. In this study, overlapping peptide mixes of various lengths were compared and 15 amino acid peptides with 11 amino acid overlaps were found to stimulate both CD4+ and CD8+ T-cell responses. Such peptide mixes stimulated CD4+ T-cell responses equivalent to those observed with whole recombinant protein, while simultaneously stimulating CD8+ T-cell responses much higher than those observed with whole protein. Although 8-12 amino acid peptides produced the highest level of CD8+ T-cell responses, 15 amino acid peptides were still very effective. Peptides that were 20 amino acids in length, however, did not stimulate strong CD8+ T-cell responses at the same peptide dose. The cytokine responses to individual epitopes added up approximately to the response to the entire mix, demonstrating that large mixes can detect responses in a quantitative fashion. Unlike whole protein antigens, peptide mixes were effective at stimulating responses in both cryopreserved PBMC and blood stored for 24 h at room temperature. Thus, overlapping 15 amino acid peptide mixes may facilitate the analysis of antigen-specific CD4+ and CD8+ T-cell responses by cytokine flow cytometry, using clinical specimens that include shipped blood or cryopreserved PBMC.

OBJECTIVE

Early HIV infection is characterized by a dramatic depletion of CD4 T cells in the gastrointestinal tract and translocation of bacterial products from the gut into the blood. In this study, we evaluated if gut bacterial profiles were associated with immune status before and after starting antiretroviral therapy (ART).

METHODS

We evaluated the gut microbiota of men recently infected with HIV (n = 13) who were participating in a randomized, double-blind controlled trial of combination ART and maraviroc versus placebo and who were followed for 48 weeks.

METHODS

To evaluate the gut microbiota of participants, we pyrosequenced the bacterial populations from anal swabs collected before and longitudinally after the initiation of ART. Associations of the gut flora with clinical variables (lymphocyte profiles and viral loads), activation and proliferation markers in peripheral blood mononuclear cells and gut biopsies (measured by flow cytometry) and markers of microbial translocation (lipopolysaccharide and soluble CD14) were performed by regression analyses using R statistical software.

RESULTS

Using pyrosequencing, we identified that higher proportions of Lactobacillales in the distal gut of recently HIV-infected individuals were associated with lower markers of microbial translocation, higher CD4% and lower viral loads before ART was started. Similarly, during ART, higher proportions of gut Lactobacillales were associated with higher CD4%, less microbial translocation, less systemic immune activation, less gut T lymphocyte proliferation, and higher CD4% in the gut.

CONCLUSIONS

Shaping the gut microbiome, especially proportions of Lactobacillales, could help to preserve immune function during HIV infection.

Resistance to antiretroviral agents, and in particular the increasing levels of transmitted resistant virus could offset the substantial gains won with potent antiretroviral therapy. Primary and acquired antiretroviral resistance rates reflect the relative usage of different antiretroviral drugs in the population, as well as the inherent genetic barrier to the development of resistance associated with individual drugs. Data on antiretroviral resistance rates, gleaned from the growing HIV-1-infected population treated with a continuously increasing number of antiretroviral drugs and drug combinations, provide insights into patient management approaches for delaying the emergence of resistance and minimizing the degree of resistance. Evolving data suggest that the relative ease by which HIV-1 escapes the selective pressure of chronic drug exposure varies for the different antiretroviral drug classes and individual antiretroviral drugs. The development of resistance in vivo can be anticipated based on these data, in conjunction with the individuals treatment history and resistance testing results. These in turn can guide the judicious use of antiretroviral drugs to attain optimal treatment responses and to preserve therapeutic options for the time when antiretroviral-resistant strains emerge. The recent developments of new antiretroviral drugs, including the use of boosted protease inhibitors, suggest that treatment strategies can limit the development of resistance.

Computational docking is a useful tool for predicting macromolecular complexes, which are often difficult to determine experimentally. Here, we present the DOT2 software suite, an updated version of the DOT intermolecular docking program. DOT2 provides straightforward, automated construction of improved biophysical models based on molecular coordinates, offering checkpoints that guide the user to include critical features. DOT has been updated to run more quickly, allow flexibility in grid size and spacing, and generate an infinitive complete list of favorable candidate configurations. Output can be filtered by experimental data and rescored by the sum of electrostatic and atomic desolvation energies. We show that this rescoring method improves the ranking of correct complexes for a wide range of macromolecular interactions and demonstrate that biologically relevant models are essential for biologically relevant results. The flexibility and versatility of DOT2 accommodate realistic models of complex biological systems, improving the likelihood of a successful docking outcome.

We analyzed the evolution of the human immunodeficiency virus type 1 (HIV-1) env gene in 12 chronically infected individuals who underwent structured treatment interruptions (STIs). Analyses of length variation and of clonal sequences demonstrated highly unpredictable evolution, which may limit the strengthening of HIV-specific immune responses by STIs because of the variability in exposure to viral antigens.

Assays for classifying HIV infections as 'recent' or 'nonrecent' for incidence surveillance fail to simultaneously achieve large mean durations of 'recent' infection (MDRIs) and low 'false-recent' rates (FRRs), particularly in virally suppressed persons. The potential for optimizing recent infection testing algorithms (RITAs), by introducing viral load criteria and tuning thresholds used to dichotomize quantitative measures, is explored.

The Consortium for the Evaluation and Performance of HIV Incidence Assays characterized over 2000 possible RITAs constructed from seven assays (Limiting Antigen, BED, Less-sensitive Vitros, Vitros Avidity, BioRad Avidity, Architect Avidity, and Geenius) applied to 2500 diverse specimens.

MDRIs were estimated using regression, and FRRs as observed 'recent' proportions, in various specimen sets. Context-specific FRRs were estimated for hypothetical scenarios. FRRs were made directly comparable by constructing RITAs with the same MDRI through the tuning of thresholds. RITA utility was summarized by the precision of incidence estimation.

All assays produce high FRRs among treated patients and elite controllers (10-80%). Viral load testing reduces FRRs, but diminishes MDRIs. Context-specific FRRs vary substantially by scenario - BioRad Avidity and Limiting Antigen provided the lowest FRRs and highest incidence precision in scenarios considered.

The introduction of a low viral load threshold provides crucial improvements in RITAs. However, it does not eliminate nonzero FRRs, and MDRIs must be consistently estimated. The tuning of thresholds is essential for comparing and optimizing the use of assays. The translation of directly measured FRRs into context-specific FRRs critically affects their magnitudes and our understanding of the utility of assays.

The hydroxyethylurea human immunodeficiency virus type 1 (HIV-1) protease inhibitors SC-55389A and SC-52151 were used to select drug-resistant variants in vitro. One clinical HIV-1 strain (89-959) and one laboratory HIV-1 strain (LAI) were passaged in peripheral blood mononuclear cells or CEMT4 cells in the presence of SC-55389A. Resistant isolates from both strains consistently had a mutation to serine for asparagine at amino acid 88 (N88S) in the protease gene either alone or in combination with a change to phenylalanine at position 10. The N88S mutation, recreated by oligonucleotide-mediated site-directed mutagenesis in HXB2, was sufficient to confer resistance to SC-55389A. In contrast, SC-52151-resistant variants selected from the monocytotropic strain SF162 had multiple substitutions in the protease gene (I11V, M461, F53L, A71V, and N88D), and the N88D mutation, re-created by oligonucleotide-mediated site-directed mutagenesis in HXB2, did not confer resistance to SC-52151. The potencies of L735,524 and Ro31-8959 were not reduced when these compounds were assayed against variants with either the N88S or N88D substitution. Position 88 is in a helix that lies behind the substrate binding pocket and may indirectly influence inhibitor binding through interactions with the amino acid at position 31. The selected mutations were persistent in the viral populations after more than 20 passages in the absence of drugs. Passaging of virus first in SC-55389A alone and then in combination with SC-52151 resulted in the accumulation of more mutations in the protease gene (L10F, D35E, D37M, I47V, 154L, A71V, V82I, and S88D) and in the selection of a variant that was cross-resistant to multiple protease inhibitors. These results indicate that a mutation in the HIV-1 protease at a position that is located outside of the substrate binding pocket confers resistance to a protease inhibitor and that mutations in the protease gene accumulate with increasing selection pressure and can persist in the absence of selection pressure.

BACKGROUND

Coccidioidomycosis results from airborne infections caused by either Coccidioides immitis or C. posadasii. Both are pathogenic fungi that live in desert soil in the New World and can infect normal hosts, but most infections are self-limited. Disseminated infections occur in approximately 5% of cases and may prove fatal. Mouse models of the disease have identified strains that are resistant (e.g. DBA/2) or susceptible (e.g. C57BL/6) to these pathogens. However, the genetic and immunological basis for this difference has not been fully characterized.

RESULTS

Microarray technology was used to identify genes that were differentially expressed in lung tissue between resistant DBA/2 and sensitive C57BL/6 mice after infection with C. immitis. Differentially expressed genes were mapped onto biological pathways, gene ontologies, and protein interaction networks, which revealed that innate immune responses mediated by Type II interferon (i.e., IFNG) and the signal transducer and activator of transcription 1 (STAT1) contribute to the resistant phenotype. In addition, upregulation of hypoxia inducible factor 1A (HIF1A), possibly as part of a larger inflammatory response mediated by tumor necrosis factor alpha (TNFA), may also contribute to resistance. Microarray gene expression was confirmed by real-time quantitative PCR for a subset of 12 genes, which revealed that IFNG HIF1A and TNFA, among others, were significantly differentially expressed between the two strains at day 14 post-infection.

CONCLUSIONS

These results confirm the finding that DBA/2 mice express more Type II interferon and interferon stimulated genes than genetically susceptible strains and suggest that differential expression of HIF1A may also play a role in protection.

In Tijuana, Mexico, HIV is concentrated in sub-epidemics of key populations: persons who inject drugs (PWID), sex workers (SW), and men who have sex with men (MSM). To date, data on engagement in the HIV care continuum among these key populations, particularly in resource-constrained settings, are sparse. We pooled available epidemiological data from six studies (N = 3368) to examine HIV testing and treatment uptake in these key populations; finding an overall HIV prevalence of 5.7 %. Of the 191 identified HIV-positive persons, only 11.5 % knew their HIV-positive status and 3.7 % were on ART. Observed differences between these HIV-positive key populations suggest PWID (vs. non-PWID) were least likely to have previously tested or initiate HIV care. MSM (vs. non-MSM) were more likely to have previously tested but not more likely to know their HIV-positive status. Of persons aware of their HIV-positive status, SW (vs. non-SW) were more likely to initiate HIV care. Findings suggest engagement of key populations in HIV treatment is far below estimates observed for similarly resource-constrained generalized epidemics in sub-Saharan Africa. These data provide one of the first empirical-snapshots highlighting the extent of HIV treatment disparities in key populations.

Quantification of cell-associated replication-competent HIV, in blood samples from patients with undetectable plasma viremia, requires specialized culture conditions that include exogenous pan T cell stimulation. Different research groups have used several stimuli for this purpose; however, the relative efficacies of these T cell stimuli to induce productive HIV replication from latently infected cells ex vivo have not been systematically evaluated. To this end, we compared four commonly used T cell stimuli: 1) irradiated allogeneic cells plus phytohaemagglutinin (PHA); 2) PHA alone; 3) phorbol myristate acetate plus Ionomycin; and 4) immobilized αCD3 plus αCD28 antibodies. End-point dilutions of patient CD4 T cells were performed, using virion RNA production to quantify HIV induction. Our results demonstrated that these activation approaches were not equivalent and that antibody cross-linking of CD3 and CD28 membrane receptors was the most effective means to activate HIV replication from a resting cell state, closely followed by stimulation with irradiated allogeneic cells plus PHA.

Protein-DNA interactions are essential for many biological processes. X-ray crystallography can provide high-resolution structures, but protein-DNA complexes are difficult to crystallize and typically contain only small DNA fragments. Thus, there is a need for computational methods that can provide useful predictions to give insights into mechanisms and guide the design of new experiments. We used the program DOT, which performs an exhaustive, rigid-body search between two macromolecules, to investigate four diverse protein-DNA interactions. Here, we compare our computational results with subsequent experimental data on related systems. In all cases, the experimental data strongly supported our structural hypotheses from the docking calculations: a mechanism for weak, nonsequence-specific DNA binding by a transcription factor, a large DNA-binding footprint on the surface of the DNA-repair enzyme uracil-DNA glycosylase (UNG), viral and host DNA-binding sites on the catalytic domain of HIV integrase, and a three-DNA-contact model of the linker histone bound to the nucleosome. In the case of UNG, the experimental design was based on the DNA-binding surface found by docking, rather than the much smaller surface observed in the crystallographic structure. These comparisons demonstrate that the DOT electrostatic energy gives a good representation of the distinctive electrostatic properties of DNA and DNA-binding proteins. The large, favourably ranked clusters resulting from the dockings identify active sites, map out large DNA-binding sites, and reveal multiple DNA contacts with a protein. Thus, computational docking can not only help to identify protein-DNA interactions in the absence of a crystal structure, but also expand structural understanding beyond known crystallographic structures.

Rapid molecular diagnostics have great potential to limit the spread of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) (M/XDR-TB). These technologies detect mutations in the Mycobacterium tuberculosis genome that confer phenotypic drug resistance. However, there have been few data published regarding the relationships between the detected M. tuberculosis resistance mutations and M/XDR-TB treatment outcomes, limiting our current ability to exploit the full potential of molecular diagnostics. We analyzed clinical, microbiological, and sequencing data for 451 patients and their clinical isolates collected in a multinational, observational cohort study to determine if there was an association between M. tuberculosis resistance mutations and patient mortality. The presence of an rrs 1401G mutation was associated with significantly higher odds of patient mortality (adjusted odds ratio [OR] = 5.72; 95% confidence interval [CI], 1.65 to 19.84]) after adjusting for relevant patient clinical characteristics and all other resistance mutations. Further analysis of mutations, categorized by the associated resistance level, indicated that the detection of mutations associated with high-level fluoroquinolone (OR, 3.99 [95% CI, 1.10 to 14.40]) and kanamycin (OR, 5.47 [95% CI, 1.64 to 18.24]) resistance was also significantly associated with higher odds of patient mortality, even after accounting for clinical site, patient age, reported smoking history, body mass index (BMI), diabetes, HIV, and all other resistance mutations. Specific gyrA and rrs resistance mutations, associated with high-level resistance, were associated with patient mortality as identified in clinical M. tuberculosis isolates from a diverse M/XDR-TB patient population at three high-burden clinical sites. These results have important implications for the interpretation of molecular diagnostics, including identifying patients at increased risk for mortality during treatment. (This study has been registered at ClinicalTrials.gov under registration no. NCT02170441.).

Costs of care for persons living with HIV have been high historically. Cost estimates based on data from 1 health care site may underestimate total expenditures; using insurance claims avoids this limitation. We used Medicaid claims data to comprehensively assess payments for care for persons living with HIV between 2006 and 2010.

Five sites from the HIV Research Network (HIVRN) provided information on patients with Medicaid coverage. Medicaid data were obtained from the sites' states (MD, NY, and MA) and 3 surrounding states and matched to HIVRN medical record-based data. Individuals less than 18, those with Medicare, and those in Medicaid managed care plans were excluded. Medicaid and HIVRN data were compared to ascertain concordance in capturing any inpatient event and any antiretroviral (ART) medication use.

Of 6892 unique HIVRN identifiers, 6196 (90%) were linked to Medicaid data. The analytic sample included 11,341 person-years of Medicaid claims data from 3695 individuals in fee-for-service (FFS) programs. The mean annual FFS payment for all services was $47,434; mean annual FFS payment for only medical services was $38,311. Concordance between Medicaid and HIVRN data was excellent for ART use, but HIVRN data did not record a substantial proportion of years in which Medicaid recorded inpatient use.

Estimated Medicaid payment amounts in this study are higher than some previous estimates. More complete capture of expensive inpatient hospitalizations in Medicaid data may partially explain this finding. Although inpatient care and ART medications contribute the most, expenditures for nonmedical services are substantial.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) have been used widely for treating human immunodeficiency virus type 1 (HIV-1) infected patients as a component of highly active antiretroviral therapy (HAART) and for the prevention of mother-to-child transmission (MTCT). Cytochrome P450 (CYP) 2B6 is an important hepatic isoenzyme responsible for the metabolism of NNRTIs including efavirenz and nevirapine. Recent pharmacogenetic studies have shown that CYP2B6 genetic variants alter hepatic CYP2B6 protein expression and function, and the pharmacokinetics of several CYP2B6 substrates. In particular, the CYP2B6-G516T polymorphism in exon 4 affects the pharmacokinetics of efavirenz. Other studies have shown associations of the CYP2B6-G516T genotype with nevirapine pharmacokinetics and central nervous system adverse effects related to efavirenz use. In total, CYP2B6 genetic variants are important determinants of efavirenz and nevirapine pharmacokinetics . Further studies are needed to identify the associations of CYP2B6 genetic variants with the development of NNRTI resistant viruses.

Patients with HIV infection vaccinated early in the flu season are more likely to contract influenza or influenza-like illness (ILI) compared with those receiving late vaccination. It is known that antibody titres wane over time, particularly among individuals with HIV infection. It is unclear if the timing of influenza vaccination within the flu season affects the probability of an influenza infection later during the flu season. The aim of this study is to evaluate if there is a relationship between the timing of influenza vaccination and incidence of influenza or ILI in patients with HIV infection.

A retrospective cohort study of HIV-infected patients in care at the Department of Veterans Affairs San Diego Healthcare System was conducted from 1 September 2005 to 31 May 2013. Among these patients, we evaluated whether there was a relationship between the timing of influenza vaccination and the incidence of laboratory-confirmed influenza, influenza diagnosed by a physician, or ILI.

Patients who received influenza vaccine early in the season were more likely to contract influenza or ILI than patients who were vaccinated late (P < 0.01). Vaccinated patients who developed influenza or ILI were more likely to do so later in the season (P < 0.01).

HIV-infected patients vaccinated early in the flu season were more likely to contract influenza or ILI than those vaccinated later in the season.

Inflammation during HIV infection is associated with worse disease outcomes and progression. Many mechanisms have been indicted, including HIV itself, coinfections, and gut microbial translocation. Concerning microbial translocation, we hypothesized that adaptive immune responses to a specific bacterial species known to be present in gut-associated lymphoid tissue are higher among HIV-infected individuals than among HIV-uninfected controls and are associated with T cell activation and lower CD4 T cell counts. By characterizing the IgG response to Achromobacter xylosoxidans, we found that HIV-infected participants who were immunoresponsive (n = 48) had significantly lower CD4 percentages (P = 0.01), greater CD4 activation (percentages of RA(-) CD38(+)) (P = 0.03), and higher soluble CD14 (P = 0.01). HIV-positive individuals had higher anti-A. xylosoxidans IgG titers than HIV-uninfected individuals (P = 0.04). The results suggest an abnormal adaptive immune activation to gut microflora during HIV infection.

Genetic elements in HIV-1 subtype B tat and env are associated with neurotoxicity yet less is known about other subtypes. HIV-1 subtype C tat and env sequences were analyzed to determine viral genetic elements associated with neurocognitive impairment in a large Indian cohort. Population-based sequences of HIV-1 tat (exon 1) and env (C2-V3 coding region) were generated from blood plasma of HIV-infected patients in Pune, India. Participants were classified as cognitively normal or impaired based on neuropsychological assessment. Tests for signature residues, positive and negative selection, entropy, and ambiguous bases were performed using tools available through Los Alamos National Laboratory (http://www.hiv.lanl.gov) and Datamonkey (http://www.datamonkey.org). HIV-1 subtype C tat and env sequences were analyzed for 155 and 160 participants, of which 34-36% were impaired. Two signature residues were unique to impaired participants in exon 1 of tat at codons 29 (arginine) and 68 (proline). Positive selection was noted at codon 29 among normal participants and at codon 68 in both groups. The signature at codon 29 was also a signature for low CD4+ (<200 cells/mm(3)) counts but remained associated with impairment after exclusion of those with low CD4+ counts. No unique genetic signatures were noted in env. In conclusion, two signature residues were identified in exon 1 of HIV-1 subtype C tat that were associated with neurocognitive impairment in India and not completely accounted for by HIV disease progression. These signatures support a linkage between diversifying selection in HIV-1 subtype C tat and neurocognitive impairment.