Analysis of SARS-CoV-2 Emergent Variants Following AZD7442 (Tixagevimab/Cilgavimab) for Early Outpatient Treatment of COVID-19 (TACKLE Trial).

INTRODUCTION: AZD7442 (tixagevimab/cilgavimab) comprises neutralising monoclonal antibodies (mAbs) that bind to distinct non-overlapping epitopes on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. Viral evolution during mAb therapy can select for variants with reduced neutralisation susceptibility. We examined treatment-emergent SARS-CoV-2 variants during TACKLE (NCT04723394), a phase 3 study of AZD7442 for early outpatient treatment of coronavirus disease 2019 (COVID-19). METHODS: Non-hospitalised adults with mild-to-moderate COVID-19 were randomised and dosed ≤ 7 days from symptom onset with AZD7442 (n = 452) or placebo (n = 451). Next-generation sequencing of the spike gene was performed on SARS-CoV-2 reverse-transcription polymerase chain reaction-positive nasopharyngeal swabs at baseline and study days 3, 6, and 15 post dosing. SARS-CoV-2 lineages were assigned using spike nucleotide sequences. Amino acid substitutions were analysed at allele fractions (AF; % of sequence reads represented by substitution) ≥ 25% and 3% to 25%. In vitro susceptibility to tixagevimab, cilgavimab, and AZD7442 was evaluated for all identified treatment-emergent variants using a pseudotyped microneutralisation assay. RESULTS: Longitudinal spike sequences were available for 461 participants (AZD7442, n = 235; placebo, n = 226) and showed that treatment-emergent variants at any time were rare, with 5 (2.1%) AZD7442 participants presenting ≥ 1 substitution in tixagevimab/cilgavimab binding sites at AF ≥ 25%. At AF 3% to 25%, treatment-emergent variants were observed in 15 (6.4%) AZD7442 and 12 (5.3%) placebo participants. All treatment-emergent variants showed in vitro susceptibility to AZD7442. CONCLUSION: These data indicate that AZD7442 creates a high genetic barrier for resistance and is a feasible option for COVID-19 treatment.

Breakthrough SARS-CoV-2 Infections in the PROVENT Prevention Trial Were Not Associated With AZD7442 (Tixagevimab/Cilgavimab) Resistant Variants.

BACKGROUND: We report spike protein-based lineage and AZD7442 (tixagevimab/cilgavimab) neutralizing activity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants identified from breakthrough infections in the PROVENT preexposure prophylaxis trial. METHODS: Variants identified from PROVENT participants with reverse-transcription polymerase chain reaction-positive symptomatic illness were phenotypically assessed to determine neutralization susceptibility of variant-specific pseudotyped virus-like particles. RESULTS: At completion of 6 months' follow-up, no AZD7442-resistant variants were observed in breakthrough coronavirus disease 2019 (COVID-19) cases. SARS-CoV-2 neutralizing antibody titers were similar in breakthrough and nonbreakthrough cases. CONCLUSIONS: Symptomatic COVID-19 breakthrough cases in PROVENT were not due to resistance-associated substitutions in AZD7442 binding sites or lack of AZD7442 exposure. CLINICAL TRIALS REGISTRATION: NCT04625725.

Molecular Characterization of AZD7442 (Tixagevimab-Cilgavimab) Neutralization of SARS-CoV-2 Omicron Subvariants.

Therapeutic anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) monoclonal antibodies (MAbs) provide immunosuppressed and vulnerable populations with prophylactic and treatment interventions against coronavirus disease 2019 (COVID-19). AZD7442 (tixagevimab-cilgavimab) is a combination of extended-half-life neutralizing MAbs that bind to distinct epitopes on the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. The Omicron variant of concern carries mutations at >35 positions in the spike protein and has undergone further genetic diversification since its emergence in November 2021. Here, we characterize the in vitro neutralization activity of AZD7442 toward major viral subvariants circulating worldwide during the first 9 months of the Omicron wave. BA.2 and its derived subvariants showed the highest susceptibility to AZD7442, while BA.1 and BA.1.1 showed a lower susceptibility. BA.4/BA.5 had a susceptibility level intermediate between BA.1 and BA.2. Mutagenesis of parental Omicron subvariant spike proteins was performed to establish a molecular model to describe the underlying determinants of neutralization by AZD7442 and its component MAbs. The concurrent mutation of residues at positions 446 and 493, located in the tixagevimab and cilgavimab binding sites, was sufficient to enhance in vitro susceptibility of BA.1 to AZD7442 and its component MAbs to levels similar to the Wuhan-Hu-1+D614G virus. AZD7442 maintained neutralization activity against all Omicron subvariants tested up to and including BA.5. The evolving nature of the SARS-CoV-2 pandemic warrants continuing real-time molecular surveillance and assessment of in vitro activity of MAbs used in prophylaxis against and the treatment of COVID-19. IMPORTANCE MAbs are key therapeutic options for COVID-19 prophylaxis and treatment in immunosuppressed and vulnerable populations. Due to the emergence of SARS-CoV-2 variants, including Omicron, it is vital to ensure that neutralization is maintained for MAb-based interventions. We studied the in vitro neutralization of AZD7442 (tixagevimab-cilgavimab), a cocktail of two long-acting MAbs targeting the SARS-CoV-2 spike protein, toward Omicron subvariants circulating from November 2021 to July 2022. AZD7442 neutralized major Omicron subvariants up to and including BA.5. The mechanism of action responsible for the lower in vitro susceptibility of BA.1 to AZD7442 was investigated using in vitro mutagenesis and molecular modeling. A combination of mutations at two spike protein positions, namely, 446 and 493, was sufficient to enhance BA.1 susceptibility to AZD7442 to levels similar to the Wuhan-Hu-1+D614G ancestral virus. The evolving nature of the SARS-CoV-2 pandemic warrants continuing real-time global molecular surveillance and mechanistic studies of therapeutic MAbs for COVID-19.

Terminal Effector CD8 T Cells Defined by an IKZF2+IL-7R- Transcriptional Signature Express FcγRIIIA, Expand in HIV Infection, and Mediate Potent HIV-Specific Antibody-Dependent Cellular Cytotoxicity.

HIV-1 infection expands large populations of late-stage differentiated CD8 T cells that may persist long after viral escape from TCR recognition. In this study, we investigated whether such CD8 T cell populations can perform unconventional innate-like antiviral effector functions. Chronic untreated HIV-1 infection was associated with elevated numbers of CD45RA+CD57+ terminal effector CD8 T cells expressing FcγRIIIA (CD16). The FcγRIIIA+ CD8 T cells displayed a distinctive transcriptional profile between conventional CD8 T cells and NK cells, characterized by high levels of IKZF2 and low expression of IL7R This transcriptional profile translated into a distinct NKp80+ IL-7Rα- surface phenotype with high expression of the Helios transcription factor. Interestingly, the FcγRIIIA+ CD8 T cells mediated HIV-specific Ab-dependent cellular cytotoxicity (ADCC) activity at levels comparable with NK cells on a per cell basis. The FcγRIIIA+ CD8 T cells were highly activated in a manner that correlated positively with expansion of the CD8 T cell compartment and with plasma levels of soluble mediators of antiviral immunity and inflammation such as IP-10, TNF, IL-6, and TNFRII. The frequency of FcγRIIIA+ CD8 T cells persisted as patients initiated suppressive antiretroviral therapy, although their activation levels declined. These data indicate that terminally differentiated effector CD8 T cells acquire enhanced innate cell-like characteristics during chronic viral infection and suggest that HIV-specific ADCC is a function CD8 T cells use to target HIV-infected cells. Furthermore, as the FcγRIIIA+ CD8 T cells persist in treatment, they contribute significantly to the ADCC-capable effector cell pool in patients on antiretroviral therapy.

Deep Sequencing Reveals Central Nervous System Compartmentalization in Multiple Transmitted/Founder Virus Acute HIV-1 Infection.

HIV-1 disseminates to a broad range of tissue compartments during acute HIV-1 infection (AHI). The central nervous system (CNS) can serve as an early and persistent site of viral replication, which poses a potential challenge for HIV-1 remission strategies that target the HIV reservoir. CNS compartmentalization is a key feature of HIV-1 neuropathogenesis. Thus far, the timing of how early CNS compartmentalization develops after infection is unknown. We examined whether HIV-1 transmitted/founder (T/F) viruses differ between CNS and blood during AHI using single-genome sequencing of envelope gene and further examined subregions in pol and env using next-generation sequencing in paired plasma and cerebrospinal fluid (CSF) from 18 individuals. Different proportions of mostly minor variants were found in six of the eight multiple T/F-infected individuals, indicating enrichment of some variants in CSF that may lead to significant compartmentalization in the later stages of infection. This study provides evidence for the first time that HIV-1 compartmentalization in the CNS can occur within days of HIV-1 exposure in multiple T/F infections. Further understanding of factors that determine enrichment of T/F variants in the CNS, as well as potential long-term implications of these findings for persistence of HIV-1 reservoirs and neurological impairment in HIV, is needed.

New Subtype B Containing HIV-1 Circulating Recombinant of sub-Saharan Africa Origin in Nigerian Men Who Have Sex With Men.

BACKGROUND: HIV-1 circulating recombinant forms (CRF) containing subtype B are uncommon in sub-Saharan Africa. Prevalent infections observed during enrollment of a prospective study of men who have sex with men (MSM) from Lagos, Nigeria, revealed the presence of a family of subtype B and CRF02_AG recombinants. This report describes the HIV-1 genetic diversity within a high-risk, high-prevalence, and previously undersampled cohort of Nigerian MSM. METHODS: Between 2013 and 2016, 672 MSM were enrolled at the Lagos site of the TRUST/RV368 study. Prevalent HIV-1 infections were initially characterized by pol sequencing and phylogenetic subtyping analysis. Samples demonstrating the presence of subtype B were further characterized by near full-length sequencing, phylogenetic, and Bayesian analyses. RESULTS: Within this cohort, HIV-1 prevalence was 59%. The major subtype was CRF02_AG (57%), followed by CRF02/B recombinants (15%), subtype G (13%), and smaller amounts of A1, B, and other recombinants. Nine clusters of closely related pol sequences indicate ongoing transmission events within this cohort. Among the CRF02_AG/B, a new CRF was identified and termed CRF95_02B. Shared risk factors and Bayesian phylogenetic inference of the new CRF95_02B and the similarly structured CRF56_cpx indicate a Nigerian or West African origin of CRF56_cpx before its observation in France. CONCLUSION: With high HIV-1 prevalence, new strains, and multiple transmission networks, this cohort of Nigerian MSM represents a previously hidden reservoir of HIV-1 strains, including the newly identified CRF95_02B and closely related CRF56_cpx. These strains will need to be considered during vaccine selection and development to optimize the design of a globally effective HIV-1 vaccine.

HIV-1 genetic diversity and demographic characteristics in Bulgaria.

HIV-1 strain diversity in Bulgaria is extensive and includes contributions from nearly all major subtypes and the Circulating Recombinant Forms (CRF): 01_AE, 02_AG, and 05_DF. Prior to this study, HIV-1 sequence information from Bulgaria has been based solely on the pro-RT gene, which represent less than 15% of the viral genome. To further characterize HIV-1 in Bulgaria, assess participant risk behaviors, and strengthen knowledge of circulating strains in the region, the study "Genetic Subtypes of HIV-1 in Bulgaria (RV240)" was conducted. This study employed the real time-PCR based Multi-region Hybridization Assay (MHA) B/non-B and HIV-1 sequencing to survey 215 of the approximately 1100 known HIV-1 infected Bulgarian adults (2008-2009) and determine if they were infected with subtype B HIV-1. The results indicated a subtype B prevalence of 40% and demonstrate the application of the MHA B/non-B in an area containing broad HIV-1 strain diversity. Within the assessed risk behaviors, the proportion of subtype B infection was greatest in men who have sex with men and lowest among those with drug use risk factors. During this study, 15 near full-length genomes and 22 envelope sequences were isolated from study participants. Phylogenetic analysis shows the presence of subtypes A1, B, C, F1, and G, CRF01_AE, CRF02_AG, CRF05_DF, and one unique recombinant form (URF). These sequences also show the presence of two strain groups containing participants with similar risk factors. Previous studies in African and Asian cohorts have shown that co-circulation of multiple subtypes can lead to viral recombination within super-infected individuals and the emergence of new URFs. The low prevalence of URFs in the presence of high subtype diversity in this study, may be the result of successful infection prevention and control programs. Continued epidemiological monitoring and support of infection prevention programs will help maintain control of the HIV-1 epidemic in Bulgaria.

Next-generation sequencing of HIV-1 single genome amplicons.

The analysis of HIV-1 sequences has helped understand the viral molecular epidemiology, monitor the development of antiretroviral drug resistance, and design candidate vaccines. The introduction of single genome amplification (SGA) has been a major advancement in the field, allowing for the characterization of multiple sequences per patient while preserving linkage among polymorphisms in the same viral genome copy. Sequencing of SGA amplicons is performed by capillary Sanger sequencing, which presents low throughput, requires a high amount of template, and is highly sensitive to template/primer mismatching. In order to meet the increasing demand for HIV-1 SGA amplicon sequencing, we have developed a platform based on benchtop next-generation sequencing (NGS) (IonTorrent) accompanied by a bioinformatics pipeline capable of running on computer resources commonly available at research laboratories. During assay validation, the NGS-based sequencing of 10 HIV-1 env SGA amplicons was fully concordant with Sanger sequencing. The field test was conducted on plasma samples from 10 US Navy and Marine service members with recent HIV-1 infection (sampling interval: 2005-2010; plasma viral load: 5,884-194,984 copies/ml). The NGS analysis of 101 SGA amplicons (median: 10 amplicons/individual) showed within-individual viral sequence profiles expected in individuals at this disease stage, including individuals with highly homogeneous quasispecies, individuals with two highly homogeneous viral lineages, and individuals with heterogeneous viral populations. In a scalability assessment using the Ion Chef automated system, 41/43 tested env SGA amplicons (95%) multiplexed on a single Ion 318 chip showed consistent gene-wide coverage >50×. With lower sample requirements and higher throughput, this approach is suitable to support the increasing demand for high-quality and cost-effective HIV-1 sequences in fields such as molecular epidemiology, and development of preventive and therapeutic strategies.

Transmitted, pre-treatment and acquired antiretroviral drug resistance among men who have sex with men and transgender women living with HIV in Nigeria.

BACKGROUND: Across sub-Saharan Africa, men who have sex with men (MSM) and transgender women (TGW) have disproportionately poor HIV treatment outcomes. Stigma and criminalization create barriers to health-care engagement and adherence to antiretroviral therapy (ART), potentially promoting the development of HIV drug resistance (HIVDR). We evaluated transmitted, pre-treatment and acquired HIVDR among MSM and TGW in Lagos and Abuja, Nigeria. METHODS: Adults with HIV RNA ≥1,000 copies/ml in the TRUST/RV368 cohort, including incident cases diagnosed via 3-monthly screening, underwent HIVDR testing using the Sanger sequencing method. Major mutations conferring resistance to nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs) were identified from the 2017 IAS-USA list. World Health Organization surveillance drug resistance mutations (SDRMs) were identified in ART-naive participants. RESULTS: From March 2013 to June 2017, 415 participants with median age 24 (interquartile range [IQR] 21-27) years, CD4+ T-cell count 370 (IQR 272-502) cells/mm3, and HIV RNA 4.73 (IQR 4.26-5.15) log10 copies/ml underwent HIVDR testing. SDRMs were observed in 36 of 373 ART-naive participants (9.7%, 95% confidence interval [95% CI 6.8, 13.1%]), including 8 of 39 incident cases (20.5%, [95% CI] 9.3, 36.5%). Among 42 ART-experienced participants, NNRTI resistance was detected in 18 (42.9%, 95% CI 27.7, 59.0%) and NRTI resistance in 10 (23.8%, 95% CI 12.0, 39.4%). No PI resistance was detected. CONCLUSIONS: The high prevalence of transmitted and acquired drug resistance among Nigerian MSM and TGW living with HIV suggests the need for programmatic solutions to improve uninterrupted access to ART and timely switch to second-line regimens in cases of viral failure.

Transmission dynamics among participants initiating antiretroviral therapy upon diagnosis of early acute HIV-1 infection in Thailand.

OBJECTIVE: To assess transmission characteristics in a predominantly MSM cohort initiating antiretroviral therapy (ART) immediately following diagnosis of acute HIV-1infection (AHI). METHODS: A longitudinal study (2009-2017) was performed in participants with AHI (n = 439) attending a single clinic in Bangkok. Plasma samples obtained prior to ART were used to obtain HIV-1 pol sequences and combined with clinical and epidemiologic data to assess transmission dynamics (cluster formation and size) using phylogenetic analysis. Clusters were estimated using maximum likelihood, genetic distance of 1.5% and visual inspection. The potential transmitter(s) in a cluster was determined using time to viral suppression and interview data. RESULTS: The cohort was predominantly MSM (93%) and infected with HIV-1 CRF01_AE (87%). Medians (ranges) for age and viral load prior to ART were 26 (18-70) years and 5.9 (2.5-8.2) log10 HIV-1 RNA copies/ml. Median time from history of HIV-1 exposure to diagnosis was 19 (3-61) days. Viral suppression was observed in 388 of 412 (94%) participants at a median time of 12 weeks following ART. Twenty-six clusters with median cluster size of 2 (2-5) representing 62 of 439 (14%) participants were observed. Younger age was associated with cluster formation: median 28 versus 30 years for unique infections (P = 0.01). A potential transmitter was identified in 11 of 26 (42%) clusters. CONCLUSION: Despite high rates of viral suppression following diagnosis and treatment of AHI within a cohort of young Thai MSM, HIV-1 transmission continued, reflecting the need to expand awareness and treatment access to the entire MSM population.

Neutralization Sensitivity of a Novel HIV-1 CRF01_AE Panel of Infectious Molecular Clones.

BACKGROUND: HIV-1 CRF01_AE is dominant in Thailand where RV144 vaccine trial was conducted. To study immune correlates of protection in ongoing trials, CRF01_AE-derived reagents are essential. Here, we present a panel of 14 HIV-1 infectious molecular clones (IMCs) identified from different stages of infection and characterization of their neutralization sensitivity using 2 standard assays. METHODS: One full-length IMC was constructed using a transmitted-founder virus to express Renilla luciferase (LucR) reporter gene and full-length envelopes (envs) of exogenous HIV-1. A panel of IMCs was generated, expressing envs of viruses from acute (Fiebig stages I/II and I-IV) and chronic (>Fiebig VI) infection. Neutralization assays were performed using TZM-bl or A3R5 cell lines, and sera or monoclonal antibodies (mAbs). Wilcoxon matched-paired test was used to assess neutralization differences between assays and reagents; correlation coefficients were evaluated by linear regression. RESULTS: Neutralization potency observed was significantly higher in the A3R5 assay when testing mAbs and serum pools (P < 0.0001); the stage of infection from which env was derived did not associate with IMC neutralization sensitivity. Neutralization values from A3R5 and TZM-bl assays were strongly correlated when mAbs were tested (R = 0.7, P < 0.0001), but a weaker association was seen with serum pools (R = 0.17, P = 0.03). CONCLUSIONS: This novel panel of CRF01_AE reporter IMC is useful for assessing vaccine-induced neutralizing antibodies in multiple assays, including those using primary cell targets. The significant differences in TZM-bl and A3R5 neutralization sensitivity, as well as the poor association when using polyclonal sera indicates the need for caution in choosing one specific platform.

Correction: Rare HIV-1 transmitted/founder lineages identified by deep viral sequencing contribute to rapid shifts in dominant quasispecies during acute and early infection.

[This corrects the article DOI: 10.1371/journal.ppat.1006510.].

Phylodynamic analysis to inform prevention efforts in mixed HIV epidemics.

In HIV epidemics of Sub Saharan Africa, the utility of HIV prevention efforts focused on key populations at higher risk of HIV infection and transmission is unclear. We conducted a phylodynamic analysis of HIV-1 pol sequences from four different risk groups in Abuja, Nigeria to estimate transmission patterns between men who have sex with men (MSM) and a representative sample of newly enrolled treatment naive HIV clients without clearly recorded HIV acquisition risks. We develop a realistic dynamical infectious disease model which was fitted to time-scaled phylogenies for subtypes G and CRF02_AG using a structured-coalescent approach. We compare the infectious disease model and structured coalescent to commonly used genetic clustering methods. We estimate HIV incidence among MSM of 7.9% (95%CI, 7.0-10.4) per susceptible person-year, and the population attributable fraction of HIV transmissions from MSM to reproductive age females to be 9.1% (95%CI, 3.8-18.6), and from the reproductive age women to MSM as 0.2% (95%CI, 0.06-0.3). Applying these parameter estimates to evaluate a test-and-treat HIV strategy that target MSM reduces the total HIV infections averted by half with a 2.5-fold saving. These results suggest the importance of addressing the HIV treatment needs of MSM in addition to cost-effectiveness of specific scale-up of treatment for MSM in the context of the mixed HIV epidemic observed in Nigeria.

Characteristics of HIV-infected U.S. Army soldiers linked in molecular transmission clusters, 2001-2012.

OBJECTIVE: Recent surveillance data suggests the United States (U.S.) Army HIV epidemic is concentrated among men who have sex with men. To identify potential targets for HIV prevention strategies, the relationship between demographic and clinical factors and membership within transmission clusters based on baseline pol sequences of HIV-infected Soldiers from 2001 through 2012 were analyzed. METHODS: We conducted a retrospective analysis of baseline partial pol sequences, demographic and clinical characteristics available for all Soldiers in active service and newly-diagnosed with HIV-1 infection from January 1, 2001 through December 31, 2012. HIV-1 subtype designations and transmission clusters were identified from phylogenetic analysis of sequences. Univariate and multivariate logistic regression models were used to evaluate and adjust for the association between characteristics and cluster membership. RESULTS: Among 518 of 995 HIV-infected Soldiers with available partial pol sequences, 29% were members of a transmission cluster. Assignment to a southern U.S. region at diagnosis and year of diagnosis were independently associated with cluster membership after adjustment for other significant characteristics (p<0.10) of age, race, year of diagnosis, region of duty assignment, sexually transmitted infections, last negative HIV test, antiretroviral therapy, and transmitted drug resistance. Subtyping of the pol fragment indicated HIV-1 subtype B infection predominated (94%) among HIV-infected Soldiers. CONCLUSION: These findings identify areas to explore as HIV prevention targets in the U.S. Army. An increased frequency of current force testing may be justified, especially among Soldiers assigned to duty in installations with high local HIV prevalence such as southern U.S. states.

Rare HIV-1 transmitted/founder lineages identified by deep viral sequencing contribute to rapid shifts in dominant quasispecies during acute and early infection.

In order to inform the rational design of HIV-1 preventive and cure interventions it is critical to understand the events occurring during acute HIV-1 infection (AHI). Using viral deep sequencing on six participants from the early capture acute infection RV217 cohort, we have studied HIV-1 evolution in plasma collected twice weekly during the first weeks following the advent of viremia. The analysis of infections established by multiple transmitted/founder (T/F) viruses revealed novel viral profiles that included: a) the low-level persistence of minor T/F variants, b) the rapid replacement of the major T/F by a minor T/F, and c) an initial expansion of the minor T/F followed by a quick collapse of the same minor T/F to low frequency. In most participants, cytotoxic T-lymphocyte (CTL) escape was first detected at the end of peak viremia downslope, proceeded at higher rates than previously measured in HIV-1 infection, and usually occurred through the exploration of multiple mutational pathways within an epitope. The rapid emergence of CTL escape variants suggests a strong and early CTL response. Minor T/F viral strains can contribute to rapid and varied profiles of HIV-1 quasispecies evolution during AHI. Overall, our results demonstrate that early, deep, and frequent sampling is needed to investigate viral/host interaction during AHI, which could help identify prerequisites for prevention and cure of HIV-1 infection.

Antibody to HSV gD peptide induced by vaccination does not protect against HSV-2 infection in HSV-2 seronegative women.

BACKGROUND: In the HIV-1 vaccine trial RV144, ALVAC-HIV prime with an AIDSVAX® B/E boost reduced HIV-1 acquisition by 31% at 42 months post first vaccination. The bivalent AIDSVAX® B/E vaccine contains two gp120 envelope glycoproteins, one from the subtype B HIV-1 MN isolate and one from the subtype CRF01_AE A244 isolate. Each envelope glycoprotein harbors a highly conserved 27-amino acid HSV-1 glycoprotein D (gD) tag sequence that shares 93% sequence identity with the HSV-2 gD sequence. We assessed whether vaccine-induced anti-gD antibodies protected females against HSV-2 acquisition in RV144. METHODS: Of the women enrolled in RV144, 777 vaccine and 807 placebo recipients were eligible and randomly selected according to their pre-vaccination HSV-1 and HSV-2 serostatus for analysis. Immunoglobulin G (IgG) and IgA responses to gD were determined by a binding antibody multiplex assay and HSV-2 serostatus was determined by Western blot analysis. Ninety-three percent and 75% of the vaccine recipients had anti-gD IgG and IgA responses two weeks post last vaccination, respectively. There was no evidence of reduction in HSV-2 infection by vaccination compared to placebo recipients over 78 weeks of follow-up. The annual incidence of HSV-2 infection in individuals who were HSV-2 negative at baseline or HSV-1 positive and HSV-2 indeterminate at baseline were 4.38/100 person-years (py) and 3.28/100 py in the vaccine and placebo groups, respectively. Baseline HSV-1 status did not affect subsequent HSV-2 acquisition. Specifically, the estimated odds ratio of HSV-2 infection by Week 78 for female placebo recipients who were baseline HSV-1 positive (n = 422) vs. negative (n = 1120) was 1.14 [95% confidence interval 0.66 to 1.94, p = 0.64)]. No evidence of reduction in the incidence of HSV-2 infection by vaccination was detected. CONCLUSIONS: AIDSVAX® B/E containing gD did not confer protection from HSV-2 acquisition in HSV-2 seronegative women, despite eliciting anti-gD serum antibodies.

HIV-1 Genetic Diversity Among Incident Infections in Mbeya, Tanzania.

In preparation for vaccine trials, HIV-1 genetic diversity was surveyed between 2002 and 2006 through the Cohort Development study in the form of a retrospective and prospective observational study in and around the town of Mbeya in Tanzania's Southwest Highlands. This study describes the molecular epidemiology of HIV-1 strains obtained from 97 out of 106 incident HIV-1 infections identified in three subpopulations of participants (one rural, two urban) from the Mbeya area. Near full-genome or half-genome sequencing showed a subtype distribution of 40% C, 17% A1, 1% D, and 42% inter-subtype recombinants. Compared to viral subtyping results previously obtained from the retrospective phase of this study, the overall proportion of incident viral strains did not change greatly during the study course, suggesting maturity of the epidemic. A comparison to a current Phase I-II vaccine being tested in Africa shows ∼17% amino acid sequence difference between the gp120 of the vaccine and subtype C incident strains. Phylogenetic and recombinant breakpoint analysis of the incident strains revealed the emergence of CRF41_CD and many unique recombinants, as well as the presence of six local transmission networks most of which were confined to the rural subpopulation. In the context of vaccine cohort selection, these results suggest distinct infection transmission dynamics within these three geographically close subpopulations. The diversity and genetic sequences of the HIV-1 strains obtained during this study will greatly contribute to the planning, immunogen selection, and analysis of vaccine-induced immune responses observed during HIV-1 vaccine trials in Tanzania and neighboring countries.

Significant contribution of subtype G to HIV-1 genetic complexity in Nigeria identified by a newly developed subtyping assay specific for subtype G and CRF02_AG.

While abundant sequence information is available from human immunodeficiency virus type 1 (HIV-1) subtypes A, B, C and CRF01_AE for HIV-1 vaccine design, sequences from West Africa are less represented. We sought to augment our understanding of HIV-1 variants circulating in 6 Nigerian cities as a step to subsequent HIV-1 vaccine development.The G/CRF02_AG multi-region hybridization assay (MHA) was developed to differentiate subtype G, CRF02_AG and their recombinants from other subtypes based on 7 HIV-1 segments. Plasma from 224 HIV-1 infected volunteers enrolled in a cohort examining HIV-1 prevalence, risk factor, and subtype from Makurdi (30), Abuja (18), Enugu (11), Kaduna (12), Tafa (95), and Ojo/Lagos (58) was analyzed using MHA. HIV-1 genomes from 42 samples were sequenced to validate the MHA and fully explore the recombinant structure of G and CRF02_AG variants.The sensitivity and specificity of MHA varied between 73-100% and 90-100%, respectively. The subtype distribution as identified by MHA among 224 samples revealed 38% CRF02_AG, 28% G, and 26% G/CRF02_AG recombinants while 8% remained nontypeable strains. In envelope (env) gp120, 38.84% of the samples reacted to a G probe while 31.25% reacted to a CRF02 (subtype A) probe. Full genome characterization of 42 sequences revealed the complexity of Nigerian HIV-1 variants.CRF02_AG, subtype G, and their recombinants were the major circulating HIV-1 variants in 6 Nigerian cities. High proportions of samples reacted to a G probe in env gp120 confirms that subtype G infections are abundant and should be considered in strategies for global HIV-1 vaccine development.

Centralized HIV Program Oversight: An Investigation of a Case Series of New HIV Infections among US Army Soldiers, 2012 to 2013.

Centralized HIV program oversight and repeal of the Department of Defense policy "Don't Ask Don't Tell" permitted characterization of HIV transmission among soldiers assigned to a large US Army base continental United States from 2012 to 2013. An investigation of a greater than expected number of new HIV infections among soldiers was initiated to characterize transmission and identify opportunities to disrupt transmission and deliver services.All soldiers who were assigned to the base at the time of their first positive HIV test and who had their first positive HIV test in 2012 or in the first 6 months of 2013 and who had a clinical genotype available for analysis were eligible for inclusion in the investigation.All patients (n = 19) were men; most were black (52%) and less than 30 years old (64%). Fifteen of the 19 patients participated in in-depth interviews. Eighty percent were men who have sex with men who reported multiple sex partners having met through social and electronic networks. All were subtype B infections. Significant knowledge gaps and barriers to accessing testing and care in the military healthcare system were identified. Most (58%) belonged to transmission networks involving other soldiers.This investigation represents an important step forward in on-going efforts to develop a comprehensive understanding of transmission networks in the Army that can inform delivery of best practices combination prevention services. The Army is developing plans to directly engage individuals in key affected populations most at risk for HIV infection to identify and address unmet needs and expand delivery and uptake of prevention services. Further investigation is underway and will determine whether these findings are generalizable to the Army.

The Number and Complexity of Pure and Recombinant HIV-1 Strains Observed within Incident Infections during the HIV and Malaria Cohort Study Conducted in Kericho, Kenya, from 2003 to 2006.

Characterization of HIV-1 subtype diversity in regions where vaccine trials are conducted is critical for vaccine development and testing. This study describes the molecular epidemiology of HIV-1 within a tea-plantation community cohort in Kericho, Kenya. Sixty-three incident infections were ascertained in the HIV and Malaria Cohort Study conducted in Kericho from 2003 to 2006. HIV-1 strains from 58 of those individuals were full genome characterized and compared to two previous Kenyan studies describing 41 prevalent infections from a blood bank survey (1999-2000) and 21 infections from a higher-risk cohort containing a mix of incident and prevalent infections (2006). Among the 58 strains from the community cohort, 43.1% were pure subtypes (36.2% A1, 5.2% C, and 1.7% G) and 56.9% were inter-subtype recombinants (29.3% A1D, 8.6% A1CD, 6.9% A1A2D, 5.2% A1C, 3.4% A1A2CD, and 3.4% A2D). This diversity and the resulting genetic distance between the observed strains will need to be addressed when vaccine immunogens are chosen. In consideration of current vaccine development efforts, the strains from these three studies were compared to five candidate vaccines (each of which are viral vectored, carrying inserts corresponding to parts of gag, pol, and envelope), which have been developed for possible use in sub-Saharan Africa. The sequence comparison between the observed strains and the candidate vaccines indicates that in the presence of diverse recombinants, a bivalent vaccine is more likely to provide T-cell epitope coverage than monovalent vaccines even when the inserts of the bivalent vaccine are not subtype-matched to the local epidemic.