Molecular mechanisms of HIV Type 1 prophylaxis failure revealed by Single-genome sequencing

Trials of HIV-1 pre- and post-exposure prophylaxis show promise. Here, we describe a novel strategy for deciphering mechanisms of prophylaxis failure that could improve therapeutic outcomes. A healthcare worker began antiretroviral prophylaxis immediately after a high-risk needlestick injury but nonetheless became viremic 11 weeks later. Single genome sequencing of plasma viral RNA identified 15 drug-sensitive transmitted/founder HIV-1 genomes responsible for productive infection. Sequences emanating from these genomes exhibited extremely low diversity, suggesting virus sequestration as opposed to low-level replication as the cause of breakthrough infection. Identification of transmitted/founder viruses allows for genome-wide assessment of molecular mechanisms of prophylaxis failure

Racial Disparities in Emergency General Surgery: Do Differences in Outcomes Persist Among Universally Insured Military Patients?

Research Objective: Described as one of the most serious health problems affecting the nation, racial disparities are estimated to account for \u3e83,000 deaths, \u3e$57 billion per year. They have been identified in multiple surgical settings, including differences in outcomes by race among emergency general surgery(EGS) patients. As many minority patients are uninsured, increasing access to care is thought to be a viable solution to mitigate inequities. The objectives of this study were to determine whether racial disparities in 30/90/180day outcomes exist within a universally-insured population of military/civilian-dependent EGS patients and whether differences in outcomes differentially persist in care received at military-vs-civilian hospitals and among sponsors who are enlisted-service members-vs-officers. It also considered longer-term outcomes of care. Study Design: Risk-adjusted survival analyses using Cox proportional-hazards models assessed race-based differences in mortality, major morbidity, and readmission from index-hospital admission (discharge for readmission) through 30/90/180days. Models accounted for hospital clustering and possible biases associated with missing race (reweighted-estimating equations). Sub-analyses considered effects restricted to operative interventions, stratified by 24 EGS-diagnostic categories defined by the American Association for the Surgery of Trauma(AAST), and effect modification related to rank (SES-proxy: officers-vs-enlisted-sponsors) and military-vs-civilian-hospital care. Population Studied: Five years of national TRICARE Prime/Prime-plus data, which provides insurance to active/reserve/retired members of the US Armed Services and dependents, were queried for adults (≥18y) with primary EGS conditions, defined by the AAST. Patients who did not have an index admission between 01/01/2006-01/07/2010 (minimum 180days follow-up) or who were not continuously enrolled in TRICARE for 180days were excluded. Non-surviving patients were retained while they survived. Principal Findings: A total of 101,011 patients were included: 73.5% White, 14.5% Black, 4.4% Asian, 7.7% other. Risk-adjusted analyses reported equivalent-or-better mortality and readmission outcomes among minority patients at 30/90/180days—even when restricted to civilian hospitals where studies suggest that EGS disparities are found. Readmissions within military hospitals were lower among minority patients. Major morbidity was higher among Black versus White patients (HR[95%CI]): 30day-1.23[1.13-1.35], 90day-1.18[1.09-1.28], 180day-1.15[1.07-1.24]—a finding driven by appendiceal disorders (HR:1.69-1.70). No other diagnostic category-based HR was significant. When considered by rank, significant effects were isolated to enlisted-service members. However, given the relatively small number of patients who were (dependents of) officers, it is difficult to determine whether rank-based findings are a result of social determinants or influenced by the limited number of minority patients. Conclusions: The first of its kind to examine racial disparities in longer-term outcomes of EGS care, this longitudinal analysis of military patients demonstrated apparent mitigation of racial disparities within a universally-insured health system when compared to the overall US health system. Efforts to explain findings based on consideration of care provided in military-vs-civilian hospitals, among specific EGS-diagnostic categories, and based on sponsor rank revealed modification of the association between race and outcomes to some extent for all three. Implications for Policy or Practice: The contrast between results for universally-insured military/civilian-dependent patients and reported disparities among all US civilian patients merits consideration. The data speak to the importance of insurance-coverage in the development of disparities interventions nationwide and will help to inform policy within the DoD

Contribution of proteasome-catalyzed peptide cis-splicing to viral targeting by CD8⁺ T cells in HIV-1 infection

Peptides generated by proteasome-catalyzed splicing of noncontiguous amino acid sequences have been shown to constitute a source of nontemplated human leukocyte antigen class I (HLA-I) epitopes, but their role in pathogen-specific immunity remains unknown. CD8⁺ T cells are key mediators of HIV type 1 (HIV-1) control, and identification of novel epitopes to enhance targeting of infected cells is a priority for prophylactic and therapeutic strategies. To explore the contribution of proteasome-catalyzed peptide splicing (PCPS) to HIV-1 epitope generation, we developed a broadly applicable mass spectrometry-based discovery workflow that we employed to identify spliced HLA-I–bound peptides on HIV-infected cells. We demonstrate that HIV-1–derived spliced peptides comprise a relatively minor component of the HLA-I–bound viral immunopeptidome. Although spliced HIV-1 peptides may elicit CD8⁺ T cell responses relatively infrequently during infection, CD8⁺ T cells primed by partially overlapping contiguous epitopes in HIV-infected individuals were able to cross-recognize spliced viral peptides, suggesting a potential role for PCPS in restricting HIV-1 escape pathways. Vaccine-mediated priming of responses to spliced HIV-1 epitopes could thus provide a novel means of exploiting epitope targets typically underutilized during natural infection

Use of Dried Blood Spots to Elucidate Full-Length Transmitted/Founder HIV-1 Genomes.

BACKGROUND: Identification of HIV-1 genomes responsible for establishing clinical infection in newly infected individuals is fundamental to prevention and pathogenesis research. Processing, storage, and transportation of the clinical samples required to perform these virologic assays in resource-limited settings requires challenging venipuncture and cold chain logistics. Here, we validate the use of dried-blood spots (DBS) as a simple and convenient alternative to collecting and storing frozen plasma. METHODS: We performed parallel nucleic acid extraction, single genome amplification (SGA), next generation sequencing (NGS), and phylogenetic analyses on plasma and DBS. RESULTS: We demonstrated the capacity to extract viral RNA from DBS and perform SGA to infer the complete nucleotide sequence of the transmitted/founder (TF) HIV-1 envelope gene and full-length genome in two acutely infected individuals. Using both SGA and NGS methodologies, we showed that sequences generated from DBS and plasma display comparable phylogenetic patterns in both acute and chronic infection. SGA was successful on samples with a range of plasma viremia, including samples as low as 1,700 copies/ml and an estimated ∼50 viral copies per blood spot. Further, we demonstrated reproducible efficiency in gp160 env sequencing in DBS stored at ambient temperature for up to three weeks or at -20°C for up to five months. CONCLUSIONS: These findings support the use of DBS as a practical and cost-effective alternative to frozen plasma for clinical trials and translational research conducted in resource-limited settings

High Multiplicity Infection by HIV-1 in Men Who Have Sex with Men

Elucidating virus-host interactions responsible for HIV-1 transmission is important for advancing HIV-1 prevention strategies. To this end, single genome amplification (SGA) and sequencing of HIV-1 within the context of a model of random virus evolution has made possible for the first time an unambiguous identification of transmitted/founder viruses and a precise estimation of their numbers. Here, we applied this approach to HIV-1 env analyses in a cohort of acutely infected men who have sex with men (MSM) and found that a high proportion (10 of 28; 36%) had been productively infected by more than one virus. In subjects with multivariant transmission, the minimum number of transmitted viruses ranged from 2 to 10 with viral recombination leading to rapid and extensive genetic shuffling among virus lineages. A combined analysis of these results, together with recently published findings based on identical SGA methods in largely heterosexual (HSX) cohorts, revealed a significantly higher frequency of multivariant transmission in MSM than in HSX [19 of 50 subjects (38%) versus 34 of 175 subjects (19%); Fisher's exact p = 0.008]. To further evaluate the SGA strategy for identifying transmitted/founder viruses, we analyzed 239 overlapping 5′ and 3′ half genome or env-only sequences from plasma viral RNA (vRNA) and blood mononuclear cell DNA in an MSM subject who had a particularly well-documented virus exposure history 3–6 days before symptom onset and 14–17 days before peak plasma viremia (47,600,000 vRNA molecules/ml). All 239 sequences coalesced to a single transmitted/founder virus genome in a time frame consistent with the clinical history, and a molecular clone of this genome encoded replication competent virus in accord with model predictions. Higher multiplicity of HIV-1 infection in MSM compared with HSX is consistent with the demonstrably higher epidemiological risk of virus acquisition in MSM and could indicate a greater challenge for HIV-1 vaccines than previously recognized

Local replication of simian immunodeficiency virus in the breast milk compartment of chronically-infected, lactating rhesus monkeys

Breast milk transmission remains a major mode of infant HIV acquisition, yet anatomic and immunologic forces shaping virus quasispecies in milk are not well characterized. In this study, phylogenic analysis of envelope sequences of milk SIV variants revealed groups of nearly identical viruses, indicating local virus production. However, comparison of the patterns and rates of CTL escape of blood and milk virus demonstrated only subtle differences between the compartments. These findings suggest that a substantial fraction of milk viruses are produced by locally-infected cells, but are shaped by cellular immune pressures similar to that in the blood

Phylogenetic Hypotheses for the Monocotyledons Constructed from rbcL Sequence Data

DNA sequences for the plastid locus that encodes the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (rbcL) were determined for 18 species of monocotyledons in 15 families. These data were analyzed together with sequences for 60 other monocot species in a total of 52 families by the maximum likelihood method producing one, presumably optimal, topology. An additional 26 species were added (104 total monocot species) and analyzed by the parsimony method with an outgroup of 18 dicot species producing 109 trees of 3,932 steps. The rbcL data show at least moderate support for seven lineages corresponding to the following orders, superorders, or combinations: Arecanae; Asparagales (excluding Hypoxidaceae) plus Iridaceae; Cyclanthanae plus Pandananae; Dioscoreales; Orchidales; Typhales; and Zingiberanae. Six clades corresponding to families or genera are well supported, including: Agavaceae, Asphodelaceae, Bromeliaceae, Hypoxidaceae, Poaceae, and Tradescantia. The two, earliest diverging multispecies clades in our rbcL phylogenies, Alismatanae and Aranae, are only weakly supported, and Bromelianae, Commelinanae, and Lilianae are paraphyletic. In all analyses Acorus calamus is phylogenetically isolated as the sister species to the remaining species of monocotyledons

HIV-1 Superinfection in the Antiretroviral Therapy Era: Are Seroconcordant Sexual Partners at Risk?

Acquisition of more than one strain of human immunodeficiency virus type 1 (HIV-1) has been reported to occur both during and after primary infection, but the risks and repercussions of dual and superinfection are incompletely understood. In this study, we evaluated a longitudinal cohort of chronically HIV-infected men who were sexual partners to determine if individuals acquired their partners' viral strains.Our cohort of HIV-positive men consisted of 8 couples that identified themselves as long-term sexual partners. Viral sequences were isolated from each subject and analyzed using phylogenetic methods. In addition, strain-specific PCR allowed us to search for partners' viruses present at low levels. Finally, we used computational algorithms to evaluate for recombination between partners' viral strains.All couples had at least one factor associated with increased risk for acquisition of new HIV strains during the study, including detectable plasma viral load, sexually transmitted infections, and unprotected sex. One subject was dually HIV-1 infected, but neither strain corresponded to that of his partner. Three couples' sequences formed monophyletic clusters at the entry visit, with phylogenetic analysis suggesting that one member of the couple had acquired an HIV strain from his identified partner or that both had acquired it from the same source outside their partnership. The 5 remaining couples initially displayed no evidence of dual infection, using phylogenetic analysis and strain-specific PCR. However, in 1 of these couples, further analysis revealed recombinant viral strains with segments of viral genomes in one subject that may have derived from the enrolled partner. Thus, chronically HIV-1 infected individuals may become superinfected with additional HIV strains from their seroconcordant sexual partners. In some cases, HIV-1 superinfection may become apparent when recombinant viral strains are detected