The widespread use of highly effective, combination antiretroviral therapy (cART) has led to a significant reduction in the incidence of HIV-associated dementia (HAD). Despite these advances, the prevalence of HIV-1 associated neurocognitive disorders (HANDs) has been estimated at approximately 40%-50%. In the cART era, the majority of this disease burden is represented by asymptomatic neurocognitive impairment and mild neurocognitive disorder (ANI and MND respectively). Although less severe than HAD, these diagnoses carry with them substantial morbidity.

An effective therapeutic vaccine that could augment immune control of HIV-1 replication may abrogate or delay the need for antiretroviral therapy. AIDS Clinical Trials Group (ACTG) A5187 was a phase I/II, randomized, placebo-controlled, double-blinded trial to evaluate the safety and immunogenicity of an HIV-1 DNA vaccine (VRC-HVDNA 009-00-VP) in subjects treated with antiretroviral therapy during acute/early HIV-1 infection. (clinicaltrials.gov NCT00125099)

Using 5' rapid amplification of cDNA ends, Illumina MiSeq, and basic flow cytometry, we systematically analyzed the expressed B cell receptor (BCR) repertoire in 14 healthy adult PBMCs, 5 HIV-1+ adult PBMCs, 5 cord blood samples, and 3 HIS-CD4/B mice, examining the full-length variable region of μ, γ, α, κ, and λ chains for V-gene usage, somatic hypermutation (SHM), and CDR3 length. Adding to the known repertoire of healthy adults, Illumina MiSeq consistently detected small fractions of reads with high mutation frequencies including hypermutated μ reads, and reads with long CDR3s. Additionally, the less studied IgA repertoire displayed similar characteristics to that of IgG. Compared to healthy adults, the five HIV-1 chronically infected adults displayed elevated mutation frequencies for all μ, γ, α, κ, and λ chains examined and slightly longer CDR3 lengths for γ, α, and λ. To evaluate the reconstituted human BCR sequences in a humanized mouse model, we analyzed cord blood and HIS-CD4/B mice, which all lacked the typical SHM seen in the adult reference. Furthermore, MiSeq revealed identical unmutated IgM sequences derived from separate cell aliquots, thus for the first time demonstrating rare clonal members of unmutated IgM B cells by sequencing.

Pattern recognition receptors (PRRs) protect against microbial invasion by detecting specific molecular patterns found in pathogens and initiating an immune response. Although microbial-derived PRR ligands have been extensively characterized, the contribution and relevance of endogenous ligands to PRR activation remains overlooked. Here, we characterize the landscape of endogenous ligands that engage RIG-I-like receptors (RLRs) upon infection by different RNA viruses. In each infection, several RNAs transcribed by RNA polymerase III (Pol3) specifically engaged RLRs, particularly the family of Y RNAs. Sensing of Y RNAs was dependent on their mimicking of viral secondary structure and their 5'-triphosphate extremity. Further, we found that HIV-1 triggered a VPR-dependent downregulation of RNA triphosphatase DUSP11 in vitro and in vivo, inducing a transcriptome-wide change of cellular RNA 5'-triphosphorylation that licenses Y RNA immunogenicity. Overall, our work uncovers the contribution of endogenous RNAs to antiviral immunity and demonstrates the importance of this pathway in HIV-1 infection.

The antibody response to HIV-1 does not appear in the plasma until approximately 2-5 weeks after transmission, and neutralizing antibodies to autologous HIV-1 generally do not become detectable until 12 weeks or more after transmission. Moreover, levels of HIV-1-specific antibodies decline on antiretroviral treatment. The mechanisms of this delay in the appearance of anti-HIV-1 antibodies and of their subsequent rapid decline are not known. While the effect of HIV-1 on depletion of gut CD4(+) T cells in acute HIV-1 infection is well described, we studied blood and tissue B cells soon after infection to determine the effect of early HIV-1 on these cells.

Objective: Toll-like receptor-3 agonist Poly-ICLC has been known to activate immune cells and induce HIV replication in pre-clinical experiments. In this study we investigated if Poly-ICLC could be used for disrupting HIV latency while simultaneously enhancing innate immune responses. Design: This was a randomized, placebo-controlled, double-blinded trial in aviremic, cART-treated HIV-infected subjects. Participants (n = 15) were randomized 3:1 to receive two consecutive daily doses of Poly-ICLC (1.4 mg subcutaneously) vs. placebo. Subjects were observed for adverse events, immune activation, and viral replication. Methods: Besides primary outcomes of safety and tolerability, several longitudinal immune parameters were evaluated including immune cell phenotype and function via flowcytometry, ELISA, and transcriptional profiling. PCR assays for plasma HIV-1 RNA, CD4+ T cell-associated HIV-1 RNA, and proviral DNA were performed to measure HIV reservoirs and latency. Results: Poly-ICLC was overall safe and well-tolerated. Poly-ICLC-related adverse events were Grade 1/2, with the exception of one Grade 3 neutropenia which was short-lived. Mild Injection site reactions were observed in nearly all participants in the Poly-ICLC arm. Transcriptional analyses revealed upregulation of innate immune pathways in PBMCs following Poly-ICLC treatment, including strong interferon signaling accompanied by transient increases in circulating IP-10 (CXCL10) levels. These responses generally peaked by 24-48 h after the first injection and returned to baseline by day 8. CD4+ T cell number and phenotype were unchanged, plasma viral control was maintained and no significant effect on HIV reservoirs was observed. Conclusions: These finding suggest that Poly-ICLC could be safely used for inducing transient innate immune responses in treated HIV+ subjects indicating promise as an adjuvant for HIV therapeutic vaccines. Trial Registration: www.ClinicalTrials.gov, identifier: NCT02071095.

Here we have identified HIV-1 B clade Envelope (Env) amino acid signatures from early in infection that may be favored at transmission, as well as patterns of recurrent mutation in chronic infection that may reflect common pathways of immune evasion. To accomplish this, we compared thousands of sequences derived by single genome amplification from several hundred individuals that were sampled either early in infection or were chronically infected. Samples were divided at the outset into hypothesis-forming and validation sets, and we used phylogenetically corrected statistical strategies to identify signatures, systematically scanning all of Env. Signatures included single amino acids, glycosylation motifs, and multi-site patterns based on functional or structural groupings of amino acids. We identified signatures near the CCR5 co-receptor-binding region, near the CD4 binding site, and in the signal peptide and cytoplasmic domain, which may influence Env expression and processing. Two signatures patterns associated with transmission were particularly interesting. The first was the most statistically robust signature, located in position 12 in the signal peptide. The second was the loss of an N-linked glycosylation site at positions 413-415; the presence of this site has been recently found to be associated with escape from potent and broad neutralizing antibodies, consistent with enabling a common pathway for immune escape during chronic infection. Its recurrent loss in early infection suggests it may impact fitness at the time of transmission or during early viral expansion. The signature patterns we identified implicate Env expression levels in selection at viral transmission or in early expansion, and suggest that immune evasion patterns that recur in many individuals during chronic infection when antibodies are present can be selected against when the infection is being established prior to the adaptive immune response.

Long-acting GSK1265744 (GSK744 LA) is a strand transfer inhibitor of the HIV/SIV (simian immunodeficiency virus) integrase and was shown to be an effective preexposure prophylaxis (PrEP) agent in a low-dose intrarectal SHIV (simian-human immunodeficiency virus) rhesus macaque challenge model. We examined the pharmacokinetics and efficacy of GSK744 LA as PrEP against repeat high-dose intravaginal SHIV challenge in female rhesus macaques treated with Depo-Provera (depot medroxyprogesterone acetate), which promotes viral transmission vaginally. When Depo-Provera-treated female rhesus macaques were dosed with GSK744 LA (50 mg/kg) monthly, systemic and tissue drug concentrations were lower than previously observed in male rhesus macaques. GSK744 concentrations were fivefold lower on average in cervical tissues than in rectal tissues. Eight female rhesus macaques were treated with GSK744 LA at week 0, and four female rhesus macaques served as controls. All animals received a high-dose challenge of SHIV162P3 at week 1. No infection was detected in GSK744 LA-treated rhesus macaques, whereas viremia was detected 1 to 2 weeks after SHIV challenge in all control animals. The GSK744 LA-treated rhesus macaques were given a second administration of drug at week 4 and further challenged at weeks 5 and 7. GSK744 LA treatment protected six of eight female rhesus macaques against three high-dose SHIV challenges, whereas all control animals became infected after the first challenge (P = 0.0003, log-rank test). These results support further clinical development of GSK744 LA for PrEP.

Mucosal mononuclear (MMC) CCR5+CD4+ T cells of the gastrointestinal (GI) tract are selectively infected and depleted during acute HIV-1 infection. Despite early initiation of combination antiretroviral therapy (cART), gut-associated lymphoid tissue (GALT) CD4+ T cell depletion and activation persist in the majority of HIV-1 positive individuals studied. This may result from ongoing HIV-1 replication and T-cell activation despite effective cART. We hypothesized that ongoing viral replication in the GI tract during cART would result in measurable viral evolution, with divergent populations emerging over time. Subjects treated during early HIV-1 infection underwent phlebotomy and flexible sigmoidoscopy with biopsies prior to and 15-24 months post initiation of cART. At the 2(nd) biopsy, three GALT phenotypes were noted, characterized by high, intermediate and low levels of immune activation. A representative case from each phenotype was analyzed. Each subject had plasma HIV-1 RNA levels <50 copies/ml at 2(nd) GI biopsy and CD4+ T cell reconstitution in the peripheral blood. Single genome amplification of full-length HIV-1 envelope was performed for each subject pre- and post-initiation of cART in GALT and PBMC. A total of 280 confirmed single genome sequences (SGS) were analyzed for experimental cases. For each subject, maximum likelihood phylogenetic trees derived from molecular sequence data showed no evidence of evolved forms in the GALT over the study period. During treatment, HIV-1 envelope diversity in GALT-derived SGS did not increase and post-treatment GALT-derived SGS showed no substantial genetic divergence from pre-treatment sequences within transmitted groups. Similar results were obtained from PBMC-derived SGS. Our results reveal that initiation of cART during acute/early HIV-1 infection can result in the interruption of measurable viral evolution in the GALT, suggesting the absence of de-novo rounds of HIV-1 replication in this compartment during suppressive cART.

We conducted a Phase I dose escalation trial of ADVAX, a DNA-based candidate HIV-1 vaccine expressing Clade C/B' env, gag, pol, nef, and tat genes. Sequences were derived from a prevalent circulating recombinant form in Yunnan, China, an area of high HIV-1 incidence. The objective was to evaluate the safety and immunogenicity of ADVAX in human volunteers.

The reproducible nature of HIV-1 escape from HLA-restricted CD8+ T-cell responses allows the identification of HLA-associated viral polymorphisms "at the population level" - that is, via analysis of cross-sectional, linked HLA/HIV-1 genotypes by statistical association. However, elucidating their timing of selection traditionally requires detailed longitudinal studies, which are challenging to undertake on a large scale. We investigate whether the extent and relative timecourse of immune-driven HIV adaptation can be inferred via comparative cross-sectional analysis of independent early and chronic infection cohorts.

Identification of full-length transmitted HIV-1 genomes could be instrumental in HIV-1 pathogenesis, microbicide, and vaccine research by enabling the direct analysis of those viruses actually responsible for productive clinical infection. We show in 12 acutely infected subjects (9 clade B and 3 clade C) that complete HIV-1 genomes of transmitted/founder viruses can be inferred by single genome amplification and sequencing of plasma virion RNA. This allowed for the molecular cloning and biological analysis of transmitted/founder viruses and a comprehensive genome-wide assessment of the genetic imprint left on the evolving virus quasispecies by a composite of host selection pressures. Transmitted viruses encoded intact canonical genes (gag-pol-vif-vpr-tat-rev-vpu-env-nef) and replicated efficiently in primary human CD4(+) T lymphocytes but much less so in monocyte-derived macrophages. Transmitted viruses were CD4 and CCR5 tropic and demonstrated concealment of coreceptor binding surfaces of the envelope bridging sheet and variable loop 3. 2 mo after infection, transmitted/founder viruses in three subjects were nearly completely replaced by viruses differing at two to five highly selected genomic loci; by 12-20 mo, viruses exhibited concentrated mutations at 17-34 discrete locations. These findings reveal viral properties associated with mucosal HIV-1 transmission and a limited set of rapidly evolving adaptive mutations driven primarily, but not exclusively, by early cytotoxic T cell responses.

GSK1265744 (GSK744) is an integrase strand-transfer inhibitor that has been formulated as a long-acting (LA) injectable suitable for monthly to quarterly clinical administration. GSK744 LA was administered at two time points 4 weeks apart beginning 1 week before virus administration, and macaques were challenged weekly for 8 weeks. GSK744 LA, at plasma concentrations achievable with quarterly injections in humans, protected all animals against repeated low-dose challenges. In a second experiment, macaques were given GSK744 LA 1 week before virus administration and challenged repeatedly until infection occurred. Protection decreased over time and correlated with the plasma drug levels. With a quarterly dosing schedule in humans, our results suggest that GSK744 LA could potentially decrease adherence problems associated with daily preexposure prophylaxis (PrEP).

HLA-restricted immune escape mutations that persist following HIV transmission could gradually spread through the viral population, thereby compromising host antiviral immunity as the epidemic progresses. To assess the extent and phenotypic impact of this phenomenon in an immunogenetically diverse population, we genotypically and functionally compared linked HLA and HIV (Gag/Nef) sequences from 358 historic (1979-1989) and 382 modern (2000-2011) specimens from four key cities in the North American epidemic (New York, Boston, San Francisco, Vancouver). Inferred HIV phylogenies were star-like, with approximately two-fold greater mean pairwise distances in modern versus historic sequences. The reconstructed epidemic ancestral (founder) HIV sequence was essentially identical to the North American subtype B consensus. Consistent with gradual diversification of a "consensus-like" founder virus, the median "background" frequencies of individual HLA-associated polymorphisms in HIV (in individuals lacking the restricting HLA[s]) were ∼ 2-fold higher in modern versus historic HIV sequences, though these remained notably low overall (e.g. in Gag, medians were 3.7% in the 2000s versus 2.0% in the 1980s). HIV polymorphisms exhibiting the greatest relative spread were those restricted by protective HLAs. Despite these increases, when HIV sequences were analyzed as a whole, their total average burden of polymorphisms that were "pre-adapted" to the average host HLA profile was only ∼ 2% greater in modern versus historic eras. Furthermore, HLA-associated polymorphisms identified in historic HIV sequences were consistent with those detectable today, with none identified that could explain the few HIV codons where the inferred epidemic ancestor differed from the modern consensus. Results are therefore consistent with slow HIV adaptation to HLA, but at a rate unlikely to yield imminent negative implications for cellular immunity, at least in North America. Intriguingly, temporal changes in protein activity of patient-derived Nef (though not Gag) sequences were observed, suggesting functional implications of population-level HIV evolution on certain viral proteins.

Injection drug use (IDU) remains a major risk factor for HIV-1 acquisition. The complex interplay between drug use, non-sterile injection, and Hepatitis C remains poorly understood. We conducted a pilot study to determine the effect of IDU on immune parameters among HIV-uninfected and -infected individuals. We hypothesized that IDU could further augment immunological changes associated with HIV-1 infection, which could in turn affect HIV pathogenesis.

The initial antibody response to HIV-1 is targeted to envelope (Env) gp41, and is nonneutralizing and ineffective in controlling viremia. To understand the origins and characteristics of gp41-binding antibodies produced shortly after HIV-1 transmission, we isolated and studied gp41-reactive plasma cells from subjects acutely infected with HIV-1. The frequencies of somatic mutations were relatively high in these gp41-reactive antibodies. Reverted unmutated ancestors of gp41-reactive antibodies derived from subjects acutely infected with HIV-1 frequently did not react with autologous HIV-1 Env; however, these antibodies were polyreactive and frequently bound to host or bacterial antigens. In one large clonal lineage of gp41-reactive antibodies, reactivity to HIV-1 Env was acquired only after somatic mutations. Polyreactive gp41-binding antibodies were also isolated from uninfected individuals. These data suggest that the majority of gp41-binding antibodies produced after acute HIV-1 infection are cross-reactive responses generated by stimulating memory B cells that have previously been activated by non-HIV-1 antigens.

We evaluated the effectiveness of cabotegravir (CAB; GSK1265744 or GSK744) long acting as preexposure prophylaxis (PrEP) against intravenous simian immunodeficiency virus (SIV) challenge in a model that mimics blood transfusions based on the per-act probability of infection.

Elite suppressors (ES) are a rare subset of HIV-1-infected individuals who are able to maintain HIV-1 viral loads below the limit of detection by ultra-sensitive clinical assays in the absence of antiretroviral therapy. Mechanism(s) responsible for this elite control are poorly understood but likely involve both host and viral factors. This study assesses ES plasma-derived envelope glycoprotein (env) fitness as a function of entry efficiency as a possible contributor to viral suppression. Fitness of virus entry was first evaluated using a novel inducible cell line with controlled surface expression levels of CD4 (receptor) and CCR5 (co-receptor). In the context of physiologic CCR5 and CD4 surface densities, ES envs exhibited significantly decreased entry efficiency relative to chronically infected viremic progressors. ES envs also demonstrated slow entry kinetics indicating the presence of virus with reduced entry fitness. Overall, ES env clones were less efficient at mediating entry than chronic progressor envs. Interestingly, acute infection envs exhibited an intermediate phenotypic pattern not distinctly different from ES or chronic progressor envs. These results imply that lower env fitness may be established early and may directly contribute to viral suppression in ES individuals.

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.