Developing a diversity, equity and inclusion compass to guide scientific capacity strengthening efforts in Africa.

Diversity, equity and inclusion (DEI) in science is vital to improve the scientific process and ensure societal uptake and application of scientific results. DEI challenges include a full spectrum of issues from the lack of, and promotion of, women in science, to the numerous barriers in place that limit representation of African scientists in global scientific efforts. DEI principles in African science remain relatively underdeveloped, with limited engagement and discussion among all stakeholders to ensure that initiatives are relevant to local environments. The Sub-Saharan African Network for TB/HIV research Excellence (SANTHE) is a network of African-led research in HIV, tuberculosis (TB), associated co-morbidities, and emerging pathogens, now based in eight African countries. Our aim, as a scientific capacity strengthening network, was to collaboratively produce a set of DEI guidelines and to represent them visually as a DEI compass. We implemented a consortium-wide survey, focus group discussions and a workshop where we were able to identify the key DEI challenges as viewed by scientists and support staff within the SANTHE network. Three thematic areas were identified: 1. Conquering Biases, 2. Respecting the Needs of a Diverse Workforce (including mental health challenges, physical disability, career stability issues, demands of parenthood, and female-specific challenges), and 3. Promotion of African Science. From this we constructed a compass that included proposed steps to start addressing these issues. The use of the compass metaphor allows 're-adjustment/re-positioning' making this a dynamic output. The compass can become a tool to establish an institution's DEI priorities and then to progress towards them.

How to improve research capacity strengthening efforts: learning from the monitoring and evaluation of four research consortia in Africa.

Recent efforts to shift the control and leadership of health research on African issues to Africa have led to increased investments for scientific research capacity strengthening (RCS) on the continent and a greater demand for accountability, value for money and demonstration of return on investment. There is limited literature on monitoring and evaluation (M&E) of RCS systems and there is a clear need to further explore whether the M&E frameworks and approaches that are currently used are fit for purpose. The M&E approaches taken by four African RCS consortia funded under the Developing Excellence in Leadership, Training and Science in Africa (DELTAS) I initiative were assessed using several methods, including a framework comparison of the M&E approaches, semi-structured interviews and facilitated discussion sessions. The findings revealed a wide range in the number of indicators used in the M&E plans of individual consortium, which were uniformly quantitative and at the output and outcome levels. Consortia revealed that additional information could have been captured to better evaluate the success of activities and measure the ripple effects of their efforts. While it is beneficial for RCS consortia to develop and implement their own M&E plans, this could be strengthened by routine engagement with funders/programme managers to further align efforts. It is also important for M&E plans to consider qualitative data capture for assessment of RCS efforts. Efforts could be further enhanced by supporting platforms for cross-consortia sharing, particularly when trying to assess more complex effects. Consortia should make sure that processes for developmental evaluation, and capturing and using the associated learning, are in place. Sharing the learning associated with M&E of RCS efforts is vital to improve future efforts. Investing and improving this aspect of RCS will help ensure tracking of progress and impact of future efforts, and ensure accountability and the return on investment. The findings are also likely applicable well beyond health research.

Subtle Longitudinal Alterations in Env Sequence Potentiate Differences in Sensitivity to Broadly Neutralizing Antibodies following Acute HIV-1 Subtype C Infection.

Broadly neutralizing antibodies (bNAbs) for HIV-1 prevention or cure strategies must inhibit transmitted/founder and reservoir viruses. Establishing sensitivity of circulating viruses to bNAbs and genetic patterns affecting neutralization variability may guide rational bNAbs selection for clinical development. We analyzed 326 single env genomes from nine individuals followed longitudinally following acute HIV-1 infection, with samples collected at ~1 week after the first detection of plasma viremia; 300 to 1,709 days postinfection but prior to initiating antiretroviral therapy (ART) (median = 724 days); and ~1 year post ART initiation. Sequences were assessed for phylogenetic relatedness, potential N- and O-linked glycosylation, and variable loop lengths (V1 to V5). A total of 43 env amplicons (median = 3 per patient per time point) were cloned into an expression vector and the TZM-bl assay was used to assess the neutralization profiles of 15 bNAbs targeting the CD4 binding site, V1/V2 region, V3 supersite, MPER, gp120/gp41 interface, and fusion peptide. At 1 µg/mL, the neutralization breadths were as follows: VRC07-LS and N6.LS (100%), VRC01 (86%), PGT151 (81%), 10-1074 and PGT121 (80%), and less than 70% for 10E8, 3BNC117, CAP256.VRC26, 4E10, PGDM1400, and N123-VRC34.01. Features associated with low sensitivity to V1/V2 and V3 bNAbs were higher potential glycosylation sites and/or relatively longer V1 and V4 domains, including known "signature" mutations. The study shows significant variability in the breadth and potency of bNAbs against circulating HIV-1 subtype C envelopes. VRC07-LS, N6.LS, VRC01, PGT151, 10-1074, and PGT121 display broad activity against subtype C variants, and major determinants of sensitivity to most bNAbs were within the V1/V4 domains. IMPORTANCE Broadly neutralizing antibodies (bNAbs) have potential clinical utility in HIV-1 prevention and cure strategies. However, bNAbs target diverse epitopes on the HIV-1 envelope and the virus may evolve to evade immune responses. It is therefore important to identify antibodies with broad activity in high prevalence settings, as well as the genetic patterns that may lead to neutralization escape. We investigated 15 bNAbs with diverse biophysical properties that target six epitopes of the HIV-1 Env glycoprotein for their ability to inhibit viruses that initiated infection, viruses circulating in plasma at chronic infection before antiretroviral treatment (ART), or viruses that were archived in the reservoir during ART in subtype C infected individuals in South Africa, a high burden country. We identify the antibodies most likely to be effective for clinical use in this setting and describe mutational patterns associated with neutralization escape from these antibodies.

crAssphage genomes identified in fecal samples of an adult and infants with evidence of positive genomic selective pressure within tail protein genes.

crAssphages are a broad group of diverse bacteriophages in the order Caudovirales that have been found to be highly abundant in the human gastrointestinal tract. Despite their high prevalence, we have an incomplete understanding of how crAssphages shape and respond to ecological and evolutionary dynamics in the gut. Here, we report genomes of crAssphages from feces of one South African woman and three infants. Across the complete genome sequences of the South African crAssphages described here, we identify particularly elevated positive selection in RNA polymerase and phage tail protein encoding genes, contrasted against purifying selection, genome-wide. We further validate these findings against a crAssphage genome from previous studies. Together, our results suggest hotspots of selection within crAssphage RNA polymerase and phage tail protein encoding genes are potentially mediated by interactions between crAssphages and their bacterial partners.

African-led health research and capacity building- is it working?

BACKGROUND: Africa bears a disproportionately high burden of globally significant disease but has lagged in knowledge production to address its health challenges. In this contribution, we discuss the challenges and approaches to health research capacity strengthening in sub-Saharan Africa and propose that the recent shift to an African-led approach is the most optimal. METHODS AND FINDINGS: We introduce several capacity building approaches and recent achievements, explore why African-led research on the continent is a potentially paradigm-shifting and innovative approach, and discuss the advantages and challenges thereof. We reflect on the approaches used by the African Academy of Sciences (AAS)-funded Sub-Saharan African Network for TB/HIV Research Excellence (SANTHE) consortium as an example of an effective African-led science and capacity building programme. We recommend the following as crucial components of future efforts: 1. Directly empowering African-based researchers, 2. Offering quality training and career development opportunities to large numbers of junior African scientists and support staff, and 3. Effective information exchange and collaboration. Furthermore, we argue that long-term investment from international donors and increasing funding commitments from African governments and philanthropies will be needed to realise a critical mass of local capacity and to create and sustain world-class research hubs that will be conducive to address Africa's intractable health challenges. CONCLUSIONS: Our experiences so far suggest that African-led research has the potential to overcome the vicious cycle of brain-drain and may ultimately lead to improvement of health and science-led economic transformation of Africa into a prosperous continent.

Combining Viral Genetics and Statistical Modeling to Improve HIV-1 Time-of-infection Estimation towards Enhanced Vaccine Efficacy Assessment.

Knowledge of the time of HIV-1 infection and the multiplicity of viruses that establish HIV-1 infection is crucial for the in-depth analysis of clinical prevention efficacy trial outcomes. Better estimation methods would improve the ability to characterize immunological and genetic sequence correlates of efficacy within preventive efficacy trials of HIV-1 vaccines and monoclonal antibodies. We developed new methods for infection timing and multiplicity estimation using maximum likelihood estimators that shift and scale (calibrate) estimates by fitting true infection times and founder virus multiplicities to a linear regression model with independent variables defined by data on HIV-1 sequences, viral load, diagnostics, and sequence alignment statistics. Using Poisson models of measured mutation counts and phylogenetic trees, we analyzed longitudinal HIV-1 sequence data together with diagnostic and viral load data from the RV217 and CAPRISA 002 acute HIV-1 infection cohort studies. We used leave-one-out cross validation to evaluate the prediction error of these calibrated estimators versus that of existing estimators and found that both infection time and founder multiplicity can be estimated with improved accuracy and precision by calibration. Calibration considerably improved all estimators of time since HIV-1 infection, in terms of reducing bias to near zero and reducing root mean squared error (RMSE) to 5-10 days for sequences collected 1-2 months after infection. The calibration of multiplicity assessments yielded strong improvements with accurate predictions (ROC-AUC above 0.85) in all cases. These results have not yet been validated on external data, and the best-fitting models are likely to be less robust than simpler models to variation in sequencing conditions. For all evaluated models, these results demonstrate the value of calibration for improved estimation of founder multiplicity and of time since HIV-1 infection.

Dual HLA B*42 and B*81-reactive T cell receptors recognize more diverse HIV-1 Gag escape variants.

Some closely related human leukocyte antigen (HLA) alleles are associated with variable clinical outcomes following HIV-1 infection despite presenting the same viral epitopes. Mechanisms underlying these differences remain unclear but may be due to intrinsic characteristics of the HLA alleles or responding T cell repertoires. Here we examine CD8+ T cell responses against the immunodominant HIV-1 Gag epitope TL9 (TPQDLNTML180-188) in the context of the protective allele B*81:01 and the less protective allele B*42:01. We observe a population of dual-reactive T cells that recognize TL9 presented by both B*81:01 and B*42:01 in individuals lacking one allele. The presence of dual-reactive T cells is associated with lower plasma viremia, suggesting a clinical benefit. In B*42:01 expressing individuals, the dual-reactive phenotype defines public T cell receptor (TCR) clones that recognize a wider range of TL9 escape variants, consistent with enhanced control of viral infection through containment of HIV-1 sequence adaptation.

Brief Report: Selection of HIV-1 Variants With Higher Transmission Potential by 1% Tenofovir Gel Microbicide.

BACKGROUND: Women in the CAPRISA 004 trial assigned to use 1% tenofovir (TFV) microbicide gel, who became HIV-1 infected, had higher viral load set-point and slower antibody avidity maturation compared with placebo participants. We investigated whether TFV gel was selected for viruses with altered genetic characteristics. SETTING: The participants of the CAPRISA 004 trial (n = 28 TFV and 43 placebo) were from KwaZulu-Natal Province, South Africa and were infected with HIV-1 subtype C. After HIV-1 diagnosis, they were recruited into the CAPRISA 002 cohort. METHODS: We analyzed gag sequences from the earliest time point post infection (within 3 months of estimated time of infection). Transmission index was measured using a model which predicts the likelihood of an amino acid to be transmitted. Phylogenetic distance from a regional consensus sequence was calculated from a maximum likelihood phylogenetic tree. RESULTS: Transmission index and distance from the most common (consensus) sequence have been shown to be markers of transmission fitness. We found that viruses infecting TFV gel recipients were closer to the consensus sequence of regional strains (P = 0.003) and had higher transmission index (P = 0.01). The transmission index was weakly correlated with concomitant viral load (Spearman r = 0.22, P = 0.06). CONCLUSION: Decreased acquisition risk may have increased the barrier to infection therefore selecting for fitter, more consensus-like viruses. Such virus fitness effects will need to be considered for future pre-exposure prophylaxis and vaccine trials.

Subtype-Specific Differences in Gag-Protease-Driven Replication Capacity Are Consistent with Intersubtype Differences in HIV-1 Disease Progression.

There are marked differences in the spread and prevalence of HIV-1 subtypes worldwide, and differences in clinical progression have been reported. However, the biological reasons underlying these differences are unknown. Gag-protease is essential for HIV-1 replication, and Gag-protease-driven replication capacity has previously been correlated with disease progression. We show that Gag-protease replication capacity correlates significantly with that of whole isolates (r = 0.51; P = 0.04), indicating that Gag-protease is a significant contributor to viral replication capacity. Furthermore, we investigated subtype-specific differences in Gag-protease-driven replication capacity using large well-characterized cohorts in Africa and the Americas. Patient-derived Gag-protease sequences were inserted into an HIV-1 NL4-3 backbone, and the replication capacities of the resulting recombinant viruses were measured in an HIV-1-inducible reporter T cell line by flow cytometry. Recombinant viruses expressing subtype C Gag-proteases exhibited substantially lower replication capacities than those expressing subtype B Gag-proteases (P < 0.0001); this observation remained consistent when representative Gag-protease sequences were engineered into an HIV-1 subtype C backbone. We identified Gag residues 483 and 484, located within the Alix-binding motif involved in virus budding, as major contributors to subtype-specific replicative differences. In East African cohorts, we observed a hierarchy of Gag-protease-driven replication capacities, i.e., subtypes A/C < D < intersubtype recombinants (P < 0.0029), which is consistent with reported intersubtype differences in disease progression. We thus hypothesize that the lower Gag-protease-driven replication capacity of subtypes A and C slows disease progression in individuals infected with these subtypes, which in turn leads to greater opportunity for transmission and thus increased prevalence of these subtypes.IMPORTANCE HIV-1 subtypes are unevenly distributed globally, and there are reported differences in their rates of disease progression and epidemic spread. The biological determinants underlying these differences have not been fully elucidated. Here, we show that HIV-1 Gag-protease-driven replication capacity correlates with the replication capacity of whole virus isolates. We further show that subtype B displays a significantly higher Gag-protease-mediated replication capacity than does subtype C, and we identify a major genetic determinant of these differences. Moreover, in two independent East African cohorts we demonstrate a reproducible hierarchy of Gag-protease-driven replicative capacity, whereby recombinants exhibit the greatest replication, followed by subtype D, followed by subtypes A and C. Our data identify Gag-protease as a major determinant of subtype differences in disease progression among HIV-1 subtypes; furthermore, we propose that the poorer viral replicative capacity of subtypes A and C may paradoxically contribute to their more efficient spread in sub-Saharan Africa.

Early evolution of human leucocyte antigen-associated escape mutations in variable Gag proteins predicts CD4+ decline in HIV-1 subtype C-infected women.

OBJECTIVE: HIV-1 escape from cytotoxic T-lymphocytes results in the accumulation of human leucocyte antigen (HLA)-associated mutations in the viral genome. To understand the contribution of early escape to disease progression, this study investigated the evolution and pathogenic implications of cytotoxic T-lymphocyte escape in a cohort followed from infection for 5 years. METHODS: Viral loads and CD4 cell counts were monitored in 78 subtype C-infected individuals from onset of infection until CD4 cell count decline to less than 350 cells/µl or 5 years postinfection. The gag gene was sequenced and HLA-associated changes between enrolment and 12 months postinfection were mapped. RESULTS: HLA-associated escape mutations were identified in 48 (62%) of the participants and were associated with CD4 decline to less than 350 cells/µl (P = 0.05). Escape mutations in variable Gag proteins (p17 and p7p6) had a greater impact on disease progression than escape in more conserved regions (p24) (P = 0.03). The association between HLA-associated escape mutations and CD4 decline was independent of protective HLA allele (B57, B58 : 01 and B81) expression. CONCLUSION: The high frequency of escape contributed to rapid disease progression in this cohort. Although HLA-adaption in both conserved and variable Gag domains in the first year of infection was detrimental to long-term clinical outcome, escape in variable domains had greater impact.

Fibroblast-Derived Extracellular Matrix Induces Chondrogenic Differentiation in Human Adipose-Derived Mesenchymal Stromal/Stem Cells in Vitro.

Mesenchymal stromal/stem cells (MSCs) represent an area being intensively researched for tissue engineering and regenerative medicine applications. MSCs may provide the opportunity to treat diseases and injuries that currently have limited therapeutic options, as well as enhance present strategies for tissue repair. The cellular environment has a significant role in cellular development and differentiation through cell-matrix interactions. The aim of this study was to investigate the behavior of adipose-derived MSCs (ad-MSCs) in the context of a cell-derived matrix so as to model the in vivo physiological microenvironment. The fibroblast-derived extracellular matrix (fd-ECM) did not affect ad-MSC morphology, but reduced ad-MSC proliferation. Ad-MSCs cultured on fd-ECM displayed decreased expression of integrins α2 and ß1 and subsequently lost their multipotency over time, as shown by the decrease in CD44, Octamer-binding transcription factor 4 (OCT4), SOX2, and NANOG gene expression. The fd-ECM induced chondrogenic differentiation in ad-MSCs compared to control ad-MSCs. Loss of function studies, through the use of siRNA and a mutant Notch1 construct, revealed that ECM-mediated ad-MSCs chondrogenesis requires Notch1 and ß-catenin signaling. The fd-ECM also showed anti-senescence effects on ad-MSCs. The fd-ECM is a promising approach for inducing chondrogenesis in ad-MSCs and chondrogenic differentiated ad-MSCs could be used in stem cell therapy procedures.

In Vitro Reversible and Time-Dependent CYP450 Inhibition Profiles of Medicinal Herbal Plant Extracts Newbouldia laevis and Cassia abbreviata: Implications for Herb-Drug Interactions.

This study evaluated the effects of Newbouldia laevis and Cassia abbreviata extracts on CYP450 enzyme activity. Recombinant CYP450 enzyme and fluorogenic substrates were used for evaluating inhibition, allowing the assessment of herb-drug interactions (HDI). Phytochemical fingerprinting was performed using UPLC-MS. The herbal extracts were risk ranked for HDI based on the IC50 values determined for each CYP enzyme. Newbouldia laevis inhibited CYP1A2, CYP2C9, and CYP2C19 enzyme activities with Ki of 2.84 µg/mL, 1.55 µg/mL, and 1.23 µg/mL, respectively. N. laevis exhibited a TDI (4.17) effect on CYP1A2 but not CYP2C9 and CYP2C19 enzyme activities. Cassia abbreviata inhibited CYP1A2, CYP2C9, and CYP2C19 enzyme activities showing a Ki of 4.86 µg/mL, 5.98 µg/mL, and 1.58 µg/mL, respectively. TDI potency assessment for Cassia abbreviata showed it as a potential TDI candidate (1.64) for CYP1A2 and CYP2C19 (1.72). UPLC-MS analysis showed that Newbouldia laevis and Cassia abbreviata possess polyphenols that likely give them their therapeutic properties; some of them are likely to be responsible for the observed inhibition. The observations made in this study suggest the potential for these herbal compounds to interact, especially when co-administered with other medications metabolized by these CYP450 enzymes.

Inhibition of CYP2B6 by Medicinal Plant Extracts: Implication for Use of Efavirenz and Nevirapine-Based Highly Active Anti-Retroviral Therapy (HAART) in Resource-Limited Settings.

Highly active antiretroviral therapy (HAART) has greatly improved health parameters of HIV infected individuals. However, there are several challenges associated with the chronic nature of HAART administration. For populations in health transition, dual use of medicinal plant extracts and conventional medicine poses a significant challenge. There is need to evaluate interactions between commonly used medicinal plant extracts and antiretroviral drugs used against HIV/AIDS. Efavirenz (EFV) and nevirapine (NVP) are the major components of HAART both metabolized by CYP2B6, an enzyme that can potentially be inhibited or induced by compounds found in medicinal plant extracts. The purpose of this study was to evaluate the effects of extracts of selected commonly used medicinal plants on CYP2B6 enzyme activity. Recombinant human CYP2B6 was used to evaluate inhibition, allowing the assessment of herb-drug interactions (HDI) of medicinal plants Hyptis suaveolens, Myrothamnus flabellifolius, Launaea taraxacifolia, Boerhavia diffusa and Newbouldia laevis. The potential of these medicinal extracts to cause HDI was ranked accordingly for reversible inhibition and also classified as potential time-dependent inhibitor (TDI) candidates. The most potent inhibitor for CYP2B6 was Hyptis suaveolens extract (IC50 = 19.09 ± 1.16 µg/mL), followed by Myrothamnus flabellifolius extract (IC50 = 23.66 ± 4.86 µg/mL), Launaea taraxacifolia extract (IC50 = 33.87 ± 1.54 µg/mL), and Boerhavia diffusa extract (IC50 = 34.93 ± 1.06 µg/mL). Newbouldia laevis extract, however, exhibited weak inhibitory effects (IC50 = 100 ± 8.71 µg/mL) on CYP2B6. Launaea taraxacifolia exhibited a TDI (3.17) effect on CYP2B6 and showed a high concentration of known CYP450 inhibitory phenolic compounds, chlorogenic acid and caffeic acid. The implication for these observations is that drugs that are metabolized by CYP2B6 when co-administered with these herbal medicines and when adequate amounts of the extracts reach the liver, there is a high likelihood of standard doses affecting drug plasma concentrations which could lead to toxicity.

Magnitude and Kinetics of CD8+ T Cell Activation during Hyperacute HIV Infection Impact Viral Set Point.

CD8(+) T cells contribute to the control of HIV, but it is not clear whether initial immune responses modulate the viral set point. We screened high-risk uninfected women twice a week for plasma HIV RNA and identified 12 hyperacute infections. Onset of viremia elicited a massive HIV-specific CD8(+) T cell response, with limited bystander activation of non-HIV memory CD8(+) T cells. HIV-specific CD8(+) T cells secreted little interferon-γ, underwent rapid apoptosis, and failed to upregulate the interleukin-7 receptor, known to be important for T cell survival. The rapidity to peak CD8(+) T cell activation and the absolute magnitude of activation induced by the exponential rise in viremia were inversely correlated with set point viremia. These data indicate that rapid, high magnitude HIV-induced CD8(+) T cell responses are crucial for subsequent immune control of acute infection, which has important implications for HIV vaccine design.

Pharmacogenomics Implications of Using Herbal Medicinal Plants on African Populations in Health Transition.

The most accessible points of call for most African populations with respect to primary health care are traditional health systems that include spiritual, religious, and herbal medicine. This review focusses only on the use of herbal medicines. Most African people accept herbal medicines as generally safe with no serious adverse effects. However, the overlap between conventional medicine and herbal medicine is a reality among countries in health systems transition. Patients often simultaneously seek treatment from both conventional and traditional health systems for the same condition. Commonly encountered conditions/diseases include malaria, HIV/AIDS, hypertension, tuberculosis, and bleeding disorders. It is therefore imperative to understand the modes of interaction between different drugs from conventional and traditional health care systems when used in treatment combinations. Both conventional and traditional drug entities are metabolized by the same enzyme systems in the human body, resulting in both pharmacokinetics and pharmacodynamics interactions, whose properties remain unknown/unquantified. Thus, it is important that profiles of interaction between different herbal and conventional medicines be evaluated. This review evaluates herbal and conventional drugs in a few African countries and their potential interaction at the pharmacogenomics level.

Identification of effective subdominant anti-HIV-1 CD8+ T cells within entire post-infection and post-vaccination immune responses.

Defining the components of an HIV immunogen that could induce effective CD8+ T cell responses is critical to vaccine development. We addressed this question by investigating the viral targets of CD8+ T cells that potently inhibit HIV replication in vitro, as this is highly predictive of virus control in vivo. We observed broad and potent ex vivo CD8+ T cell-mediated viral inhibitory activity against a panel of HIV isolates among viremic controllers (VC, viral loads <5000 copies/ml), in contrast to unselected HIV-infected HIV Vaccine trials Network (HVTN) participants. Viral inhibition of clade-matched HIV isolates was strongly correlated with the frequency of CD8+ T cells targeting vulnerable regions within Gag, Pol, Nef and Vif that had been identified in an independent study of nearly 1000 chronically infected individuals. These vulnerable and so-called "beneficial" regions were of low entropy overall, yet several were not predicted by stringent conservation algorithms. Consistent with this, stronger inhibition of clade-matched than mismatched viruses was observed in the majority of subjects, indicating better targeting of clade-specific than conserved epitopes. The magnitude of CD8+ T cell responses to beneficial regions, together with viral entropy and HLA class I genotype, explained up to 59% of the variation in viral inhibitory activity, with magnitude of the T cell response making the strongest unique contribution. However, beneficial regions were infrequently targeted by CD8+ T cells elicited by vaccines encoding full-length HIV proteins, when the latter were administered to healthy volunteers and HIV-positive ART-treated subjects, suggesting that immunodominance hierarchies undermine effective anti-HIV CD8+ T cell responses. Taken together, our data support HIV immunogen design that is based on systematic selection of empirically defined vulnerable regions within the viral proteome, with exclusion of immunodominant decoy epitopes that are irrelevant for HIV control.

Early immune adaptation in HIV-1 revealed by population-level approaches.

BACKGROUND: 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. RESULTS: Similarly-powered datasets of linked HLA/HIV-1 genotypes from individuals with early (median < 3 months) and chronic untreated HIV-1 subtype B infection, matched for size (N > 200/dataset), HLA class I and HIV-1 Gag/Pol/Nef diversity, were established. These datasets were first used to define a list of 162 known HLA-associated polymorphisms detectable at the population level in cohorts of the present size and host/viral genetic composition. Of these 162 known HLA-associated polymorphisms, 15% (occurring at 14 Gag, Pol and Nef codons) were already detectable via statistical association in the early infection dataset at p ≤ 0.01 (q < 0.2) - identifying them as the most consistently rapidly escaping sites in HIV-1. Among these were known rapidly-escaping sites (e.g. B*57-Gag-T242N) and others not previously appreciated to be reproducibly rapidly selected (e.g. A*31:01-associated adaptations at Gag codons 397, 401 and 403). Escape prevalence in early infection correlated strongly with first-year escape rates (Pearson's R = 0.68, p = 0.0001), supporting cross-sectional parameters as reliable indicators of longitudinally-derived measures. Comparative analysis of early and chronic datasets revealed that, on average, the prevalence of HLA-associated polymorphisms more than doubles between these two infection stages in persons harboring the relevant HLA (p < 0.0001, consistent with frequent and reproducible escape), but remains relatively stable in persons lacking the HLA (p = 0.15, consistent with slow reversion). Published HLA-specific Hazard Ratios for progression to AIDS correlated positively with average escape prevalence in early infection (Pearson's R = 0.53, p = 0.028), consistent with high early within-host HIV-1 adaptation (via rapid escape and/or frequent polymorphism transmission) as a correlate of progression. CONCLUSION: Cross-sectional host/viral genotype datasets represent an underutilized resource to identify reproducible early pathways of HIV-1 adaptation and identify correlates of protective immunity.

Nef-mediated down-regulation of CD4 and HLA class I in HIV-1 subtype C infection: association with disease progression and influence of immune pressure.

Nef plays a major role in HIV-1 pathogenicity. We studied HIV-1 subtype C infected individuals in acute/early (n = 120) or chronic (n = 207) infection to investigate the relationship between Nef-mediated CD4/HLA-I down-regulation activities and disease progression, and the influence of immune-driven sequence variation on these Nef functions. A single Nef sequence per individual was cloned into an expression plasmid, followed by transfection of a T cell line and measurement of CD4 and HLA-I expression. In early infection, a trend of higher CD4 down-regulation ability correlating with higher viral load set point was observed (r = 0.19, p = 0.05), and higher HLA-I down-regulation activity was significantly associated with faster rate of CD4 decline (p = 0.02). HLA-I down-regulation function correlated inversely with the number HLA-associated polymorphisms previously associated with reversion in the absence of the selecting HLA allele (r = -0.21, p = 0.0002). These data support consideration of certain Nef regions in HIV-1 vaccine strategies designed to attenuate the infection course.

Genotypic and functional impact of HIV-1 adaptation to its host population during the North American epidemic.

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.

No evidence for selection of HIV-1 with enhanced gag-protease or Nef function among breakthrough infections in the CAPRISA 004 tenofovir microbicide trial.

BACKGROUND: Use of antiretroviral-based microbicides for HIV-1 prophylaxis could introduce a transmission barrier that inadvertently facilitates the selection of fitter viral variants among incident infections. To investigate this, we assessed the in vitro function of gag-protease and nef sequences from participants who acquired HIV-1 during the CAPRISA 004 1% tenofovir microbicide gel trial. METHODS AND RESULTS: We isolated the earliest available gag-protease and nef gene sequences from 83 individuals and examined their in vitro function using recombinant viral replication capacity assays and surface protein downregulation assays, respectively. No major phylogenetic clustering and no significant differences in gag-protease or nef function were observed in participants who received tenofovir gel versus placebo gel prophylaxis. CONCLUSION: Results indicate that the partial protective effects of 1% tenofovir gel use in the CAPRISA 004 trial were not offset by selection of transmitted/early HIV-1 variants with enhanced Gag-Protease or Nef fitness.