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Abstract The primary obstacle to curing HIV-1 is a reservoir of CD4+ cells that contain stably integrated provirus. Previous studies characterizing the proviral landscape, which have been predominantly conducted in males in the United States and Europe living with HIV-1 subtype B, have revealed that most proviruses that persist during antiretroviral therapy (ART) are defective. In contrast, less is known about proviral landscapes in females with non-B subtypes, which represents the largest group of individuals living with HIV-1. Here, we analyze genomic DNA from resting CD4+ T-cells from 16 female and seven male Ugandans with HIV-1 receiving suppressive ART (n = 23). We perform near-full-length proviral sequencing at limiting dilution to examine the proviral genetic landscape, yielding 607 HIV-1 subtype A1, D, and recombinant proviral sequences (mean 26/person). We observe that intact genomes are relatively rare and clonal expansion occurs in both intact and defective genomes. Our modification of the primers and probes of the Intact Proviral DNA Assay (IPDA), developed for subtype B, rescues intact provirus detection in Ugandan samples for which the original IPDA fails. This work will facilitate research on HIV-1 persistence and cure strategies in Africa, where the burden of HIV-1 is heaviest.

HIV-1 group M (HIV-1M) lineages downregulate HLA-I and CD4 expression via their Nef proteins. We hypothesized that these Nef functions may be partially responsible for the differences in prevalence of viruses from different lineages that co-circulate within an epidemic. Here, we characterized these two Nef activities in HIV-1M isolates from Cameroon, where multiple variants have been circulating since the pandemic’s origin. Single HIV-1 Nef clones from 234 HIV-1-ART naïve individuals living in remote villages and two cosmopolitan cities of Cameroon, sampled between 2000 and 2013, were isolated from plasma HIV RNA and analyzed for their capacity to downregulate HLA-I and CD4 molecules. We found that, despite a large degree of within- and inter- lineage variation, the ability of Nef to downregulate HLA-I was similar across these different viruses. Moreover, Nef-mediated CD4 downregulation activity was also well conserved across the different lineages found in Cameroon. In addition, we observed a trend towards higher HLA-I downregulation activity of viruses circulating in the cosmopolitan cities versus the remote villages, whereas the CD4 downregulation activities were similar across the two settings. Furthermore, we noted a significant decline of HLA-I downregulation activity from 2000 to 2013, providing additional evidence supporting the attenuation of the global HIV-1M population over time. Finally, we identified 18 amino acids associated with differential HLA-I downregulation and 13 amino acids associated with differential CD4 downregulation within the dominant CRF02_AG lineage. Our lack of observation of HIV lineage-related differences in Nef-mediated HLA-I and CD4 downregulation function suggests that these activities do not substantively influence the prevalence of different HIV-1M lineages in Cameroon.

ABSTRACTIn order to cure human immunodeficiency virus (HIV), we need to better understand the within-host evolutionary origins of the small reservoir of genome-intact proviruses that persist within infected cells during antiretroviral therapy (ART). Most prior studies on reservoir evolutionary dynamics, however, did not discriminate genome-intact proviruses from the vast background of defective ones. We reconstructed within-host pre-ART HIV evolutionary histories in six individuals and leveraged this information to infer the ages of intact and defective proviruses sampled after an average of >9 years on ART, along with the ages of rebound and low-level/isolated viremia occurring during this time. We observed that the longest-lived proviruses persisting on ART were exclusively defective, usually due to large deletions. In contrast, intact proviruses and rebound HIV exclusively dated to the years immediately preceding ART. These observations are consistent with genome-intact proviruses having shorter lifespans, likely due to the cumulative risk of elimination following viral reactivation and protein production. Consistent with this, intact proviruses (and those with packaging signal defects) were three times more likely to be genetically identical compared to other proviral types, highlighting clonal expansion as particularly important in ensuring their survival. By contrast, low-level/isolated viremia sequences were heterogeneous in terms of age, with some potentially originating from defective proviruses. Results reveal that the HIV reservoir is dominated by clonally enriched and genetically younger sequences that date to the period of untreated infection when viral populations had been under within-host selection pressures for the longest duration. Knowledge of these qualities may help focus strategies for reservoir elimination.IMPORTANCECharacterizing the human immunodeficiency virus (HIV) reservoir that endures despite antiretroviral therapy (ART) is critical to cure efforts. We observed that the oldest proviruses persisting during ART were exclusively defective, while intact proviruses (and rebound HIV) dated to nearer ART initiation. This helps explain why studies that sampled sub-genomic proviruses on-ART (which are largely defective) routinely found sequences dating to early infection, whereas those that sampled replication-competent HIV found almost none. Together with our findings that intact proviruses were more likely to be clonal, and that on-ART low-level/isolated viremia originated from proviruses of varying ages (including possibly defective ones), our observations indicate that (i) on-ART and rebound viremia can have distinct within-host origins, (ii) intact proviruses have shorter lifespans than grossly defective ones and thus depend more heavily on clonal expansion for persistence, and (iii) an HIV reservoir predominantly “dating” to near ART initiation will be substantially adapted to within-host pressures, complicating immune-based cure strategies.

Abstract Background Nef performs multiple cellular activities that enhance HIV-1 pathogenesis. The role of Nef-mediated down-regulation of the host restriction factor SERINC5 in HIV-1 pathogenesis is not well-defined. We aimed to investigate if SERINC5 down-regulation activity contributes to HIV-1 subtype C disease progression, to assess the relative contribution of this activity to overall Nef function, and to identify amino acids required for optimal activity. We measured the SERINC5 down-regulation activity of 106 subtype C Nef clones, isolated from individuals in early infection, for which the Nef activities of CD4 and HLA-I down-regulation as well as alteration of TCR signalling were previously measured. The relationship between SERINC5 down-regulation and markers of disease progression, and the relative contribution of SERINC5 down-regulation to a Nef fitness model-derived E value (a proxy for overall Nef fitness in vivo), were assessed. Results No overall relationship was found between SERINC5 down-regulation and viral load set point (p = 0.28) or rate of CD4+ T cell decline (p = 0.45). CD4 down-regulation (p = 0.02) and SERINC5 down-regulation (p = 0.003) were significant determinants of E values in univariate analyses, with the greatest relative contribution for SERINC5 down-regulation, and only SERINC5 down-regulation remained significant in the multivariate analysis (p = 0.003). Using a codon-by-codon analysis, several amino acids were significantly associated with increased (10I, 11V, 38D, 51T, 65D, 101V, 188H and, 191H) or decreased (10K, 38E, 65E, 135F, 173T, 176T and, 191R) SERINC5 down-regulation activity. Site-directed mutagenesis experiments of selected mutants confirmed a substantial reduction in SERINC5 down-regulation activity associated with the mutation 173T, while mutations 10K, 135F, and 176T were associated with more modest reductions in activity that were not statistically significant. Conclusions These results suggest that SERINC5 down-regulation is a significant contributor to overall Nef function and identify potential genetic determinants of this Nef function that may have relevance for vaccines or therapeutics.

COVID-19 breakthrough infection (BTI) can occur despite vaccination. Using a multi-centre, prospective, observational Canadian cohort of people with HIV (PWH) receiving ≥2 COVID-19 vaccines, we compared the SARS-CoV-2 spike (S) and receptor-binding domain (RBD)-specific IgG levels 3 and 6 months post second dose, as well as 1 month post third dose, in PWH with and without BTI. BTI was defined as positivity based on self-report measures (data up to last study visit) or IgG data (up to 1 month post dose 3). The self-report measures were based on their symptoms and either a positive PCR or rapid antigen test. The analysis was restricted to persons without previous COVID-19 infection. Persons without BTI remained COVID-19-naïve until ≥3 months following the third dose. Of 289 participants, 92 developed BTI (31.5 infections per 100 person-years). The median days between last vaccination and BTI was 128 (IQR 67, 176), with the most cases occurring between the third and fourth dose (n = 59), corresponding to the Omicron wave. In analyses adjusted for age, sex, race, multimorbidity, hypertension, chronic kidney disease, diabetes and obesity, a lower IgG S/RBD (log10 BAU/mL) at 1 month post dose 3 was significantly associated with BTI, suggesting that a lower IgG level at this time point may predict BTI in this cohort of PWH.

People living with HIV (PLWH) can exhibit impaired immune responses to vaccines. Accumulating evidence indicates that PLWH, particularly those receiving antiretroviral therapy, mount strong antibody responses to COVID-19 vaccines, but fewer studies have examined cellular immune responses to the vaccinations. Here, we used an activation-induced marker (AIM) assay to quantify SARS-CoV-2 spike-specific CD4+ and CD8+ T cells generated by two and three doses of COVID-19 vaccines in 50 PLWH receiving antiretroviral therapy, compared to 87 control participants without HIV. In a subset of PLWH, T-cell responses were also assessed after post-vaccine breakthrough infections and/or receipt of a fourth vaccine dose. All participants remained SARS-CoV-2 infection-naive until at least one month after their third vaccine dose. SARS-CoV-2 infection was determined by seroconversion to a Nucleocapsid (N) antigen, which occurred in 21 PLWH and 38 control participants after the third vaccine dose. Multivariable regression analyses were used to investigate the relationships between sociodemographic, health- and vaccine-related variables, vaccine-induced T-cell responses, and breakthrough infection risk. We observed that a third vaccine dose boosted spike-specific CD4+ and CD8+ T-cell frequencies significantly above those measured after the second dose (all p < 0.0001). Median T-cell frequencies did not differ between PLWH and controls after the second dose (p > 0.1), but CD8+ T-cell responses were modestly lower in PLWH after the third dose (p = 0.02), an observation that remained significant after adjusting for sociodemographic, health- and vaccine-related variables (p = 0.045). In PLWH who experienced a breakthrough infection, median T-cell frequencies increased even higher than those observed after three vaccine doses (p < 0.03), and CD8+ T-cell responses in this group remained higher even after a fourth vaccine dose (p = 0.03). In multivariable analyses, the only factor associated with an increased breakthrough infection risk was younger age, which is consistent with the rapid increase in SARS-CoV-2 seropositivity that was seen among younger adults in Canada after the initial appearance of the Omicron variant. These results indicate that PLWH receiving antiretroviral therapy mount strong T-cell responses to COVID-19 vaccines that can be enhanced by booster doses or breakthrough infection.

Summary: HIV persists in tissues during antiretroviral therapy (ART), but the relative contribution of different anatomical compartments to the viral reservoir in humans remains unknown. We performed an extensive characterization of HIV reservoirs in two men who donated their bodies to HIV cure research and who had been on suppressive ART for years. HIV DNA is detected in all tissues, with large variations across anatomical compartments and between participants. Intact HIV genomes represent 2% and 25% of all proviruses in the two participants and are mainly detected in secondary lymphoid organs, with the spleen and mediastinal lymph nodes harboring intact viral genomes in both individuals. Multiple copies of identical HIV genomes are found in all tissues, indicating that clonal expansions are common in anatomical sites. The majority (>85%) of these expanded clones are shared across multiple tissues. These findings suggest that infected cells expand, migrate, and possibly circulate between anatomical sites.

Abstract Expression of the HIV-1 genome by RNA Polymerase II is regulated at multiple steps, as are most cellular genes, including recruitment of general transcription factors and control of transcriptional elongation from the core promoter. We recently discovered that tripartite motif protein TRIM24 is recruited to the HIV-1 Long Terminal Repeat (LTR) by interaction with TFII-I and causes transcriptional elongation by stimulating association of PTEF-b/ CDK9. Because TRIM24 is required for stimulation of transcription from the HIV-1 LTR, we were surprised to find that IACS-9571, a specific inhibitor of the TRIM24 C-terminal bromodomain, induces HIV-1 provirus expression in otherwise untreated cells. IACS-9571 reactivates HIV-1 in T cell lines bearing multiple different provirus models of HIV-1 latency. Additionally, treatment with this TRIM24 bromodomain inhibitor encourages productive HIV-1 expression in newly infected cells and inhibits formation of immediate latent transcriptionally repressed provirus. IACS-9571 synergizes with PMA, ionomycin, TNF-α and PEP005 to activate HIV-1 expression. Furthermore, co-treatment of CD4 + T cells from individuals with HIV-1 on antiretroviral therapy (ART) with PEP005 and IACS-9571 caused robust provirus expression. Notably, IACS-9571 did not cause global activation of T cells; rather, it inhibited induction of IL2 and CD69 expression in human PBMCs and Jurkat T cells treated with PEP005 or PMA. These observations indicate the TRIM24 bromodomain inhibitor IACS-9571 represents a novel HIV-1 latency reversing agent (LRA), and unlike other compounds with this activity, causes partial suppression of T cell activation while inducing expression of latent provirus.

Broadly neutralising anti-HIV-1 antibody (bNAb) administration in nonhuman primates has been shown to stimulate adaptive T cell-specific immunity, with infection prevention observed. In this work, the authors longitudinally analyse HIV-1 specific cellular immunity in HIV-1- infected individuals starting ART with or without adjunctive bNAb treatment.

Here, the authors show in a cohort of people with HIV, COVID mRNA vaccination is followed by a transient boost in a particular profile of HIV-specific T-cell responses and a corresponding decrease in residual HIV RNA – suggesting productive immune engagement with infected cells.

Abstract Humoral responses to COVID-19 vaccines in people living with HIV (PLWH) remain incompletely characterized. We measured circulating antibodies against the SARS-CoV-2 spike protein receptor-binding domain (RBD), ACE2 displacement and viral neutralization activities one month following the first and second COVID-19 vaccine doses, and again 3 months following the second dose, in 100 adult PLWH and 152 controls. All PLWH were receiving suppressive antiretroviral therapy, with median CD4+ T-cell counts of 710 (IQR 525–935) cells/mm3, though nadir CD4+ T-cell counts ranged as low as

SARS-CoV-2 Omicron infections are common among individuals who are vaccinated or have recovered from prior variant infection, but few reports have immunologically assessed serial Omicron infections. We characterized SARS-CoV-2 humoral responses in an individual who acquired laboratory-confirmed Omicron BA.1.15 ten weeks after a third dose of BNT162b2, and BA.2 thirteen weeks later. Responses were compared to 124 COVID-19-naive vaccinees. One month post-second and -third vaccine doses, the participant’s wild-type and BA.1-specific IgG, ACE2-displacement and virus neutralization activities were average for a COVID-19-naive triple-vaccinated individual. BA.1 infection boosted the participant’s responses to the cohort ≥95th percentile, but even this strong “hybrid” immunity failed to protect against BA.2. Reinfection increased BA.1 and BA.2-specific responses only modestly. Though vaccines clearly protect against severe disease, results highlight the continued importance of maintaining additional protective measures to counteract the immune-evasive Omicron variant, particularly as vaccine-induced immune responses naturally decline over time.

To screen all severe acute respiratory syndrome coronavirus 2–positive samples in Vancouver, British Columbia, Canada, and determine whether they represented variants of concern, we implemented a real-time reverse transcription PCR–based algorithm. We rapidly identified 77 samples with variants: 57 with B.1.1.7, 7 with B.1.351, and an epidemiologic cluster of 13 with B.1.1.28/P.1.

HIV-1 accessory proteins Nef and Vpu enhance viral pathogenesis through partially overlapping immune evasion activities. Attenuated Nef or Vpu functions have been reported in individuals who display slower disease progression, but few studies have assessed the relative impact of these proteins in non-B HIV-1 subtypes or examined paired proteins from the same individuals. Here, we examined the sequence and function of matched Nef and Vpu clones isolated from 29 long-term survivors (LTS) from Rwanda living with HIV-1 subtype A and compared our results to those of 104 Nef and 62 Vpu clones isolated from individuals living with chronic untreated HIV-1 subtype A from the same geographic area. Nef and vpu coding regions were amplified from plasma HIV RNA and cloned. The function of one intact, phylogenetically-validated Nef and Vpu clone per individual was then quantified by flow cytometry following transient expression in an immortalized CD4+ T-cell line. We measured the ability of each Nef clone to downregulate CD4 and HLA class I, and of each Vpu clone to downregulate CD4 and Tetherin, from the cell surface. Results were normalized to reference clones (Nef-SF2 and Vpu-NL4.3). We observed that Nef-mediated CD4 and HLA downregulation functions were lower in LTS compared to the control cohort (Mann-Whitney p=0.03 and p

The intact proviral DNA assay quantifies the genomically intact HIV reservoir, but assay failure due to HIV-1 polymorphism has been observed. Here, the authors report a 28% failure rate in a cohort of people with HIV-1, and note within-host HIV-1 diversity as a further challenge to IPDA accuracy.

Abstract The HIV accessory protein Nef downregulates the viral entry receptor CD4, the Human Leukocyte Antigen (HLA)-A and -B molecules, the Serine incorporator 5 (SERINC5) protein and other molecules from the infected cell surface, thereby promoting viral infectivity, replication and immune evasion. The nef locus also represents one of the most genetically variable regions in the HIV genome, and nef sequences undergo substantial evolution within a single individual over the course of infection. Few studies however have simultaneously characterized the impact of within-host nef sequence evolution on Nef protein function over prolonged timescales. Here, we isolated 50 unique Nef clones by single-genome amplification over an 11-year period from the plasma of an individual who was largely naïve to antiretroviral treatment during this time. Together, these clones harbored nonsynonymous substitutions at 13% of nef’s codons. We assessed their ability to downregulate cell-surface CD4, HLA and SERINC5 and observed that all three Nef functions declined modestly over time, where the reductions in CD4 and HLA downregulation (an average of 0.6% and 2.0% per year, respectively) achieved statistical significance. The results from this case study support all three Nef activities as being important to maintain throughout untreated HIV infection, but nevertheless suggest that, despite nef’s mutational plasticity, within-host viral evolution can compromise Nef function, albeit modestly, over prolonged periods.

People living with HIV (PLWH) may be at risk for poor immunogenicity to certain vaccines, including the ability to develop immunological memory. Here, we assessed T-cell immunogenicity following three SARS-CoV-2 vaccine doses in PLWH versus uninfected controls. Blood was collected from 38 PLWH on antiretroviral therapy and 24 age-matched HIV-negative controls, pre-vaccination and after 1st/2nd/3rd dose of SARS-CoV-2 vaccines, without prior SARS-CoV-2 infection. Flow cytometry was used to assess ex vivo T-cell immunophenotypes and intracellular Tumor necrosis factor (TNF)-α/interferon(IFN)-γ/interleukin(IL)-2 following SARS-CoV-2-Spike-peptide stimulation. Comparisons were made using Wilcoxon signed-rank test for paired variables and Mann–Whitney for unpaired. In PLWH, Spike-specific CD4 T-cell frequencies plateaued post-2nd dose, with no significant differences in polyfunctional SARS-CoV-2-specific T-cell proportions between PLWH and uninfected controls post-3rd dose. PLWH had higher frequencies of TNFα+CD4 T-cells and lower frequencies of IFNγ+CD8 T-cells than seronegative participants post-3rd dose. Regardless of HIV status, an increase in naive, regulatory, and PD1+ T-cell frequencies was observed post-3rd dose. In summary, two doses of SARS-CoV-2 vaccine induced a robust T-cell immune response in PLWH, which was maintained after the 3rd dose, with no significant differences in polyfunctional SARS-CoV-2-specific T-cell proportions between PLWH and uninfected controls post-3rd dose.

ABSTRACT Curing HIV will require eliminating the reservoir of integrated, replication-competent proviruses that persist despite antiretroviral therapy (ART). Understanding the burden, genetic diversity, and longevity of persisting proviruses in diverse individuals with HIV is critical to this goal, but these characteristics remain understudied in some groups. Among them are viremic controllers—individuals who naturally suppress HIV to low levels but for whom therapy is nevertheless recommended. We reconstructed within-host HIV evolutionary histories from longitudinal single-genome amplified viral sequences in four viremic controllers who eventually initiated ART and used this information to characterize the age and diversity of proviruses persisting on therapy. We further leveraged these within-host proviral age distributions to estimate rates of proviral turnover prior to ART. This is an important yet understudied metric, since pre-ART proviral turnover dictates reservoir composition at ART initiation (and thereafter), which is when curative interventions, once developed, would be administered. Despite natural viremic control, all participants displayed significant within-host HIV evolution pretherapy, where overall on-ART proviral burden and diversity broadly reflected the extent of viral replication and diversity pre-ART. Consistent with recent studies of noncontrollers, the proviral pools of two participants were skewed toward sequences that integrated near ART initiation, suggesting dynamic proviral turnover during untreated infection. In contrast, proviruses recovered from the other two participants dated to time points that were more evenly spread throughout infection, suggesting slow or negligible proviral decay following deposition. HIV cure strategies will need to overcome within-host proviral diversity, even in individuals who naturally controlled HIV replication before therapy. IMPORTANCE HIV therapy is lifelong because integrated, replication-competent viral copies persist within long-lived cells. To cure HIV, we need to understand when these viral reservoirs form, how large and genetically diverse they are, and how long they endure. Elite controllers—individuals who naturally suppress HIV to undetectable levels—are being intensely studied as models of HIV remission, but viremic controllers, individuals who naturally suppress HIV to low levels, remain understudied even though they too may hold valuable insights. We combined phylogenetics and mathematical modeling to reconstruct proviral seeding and decay from infection to therapy-mediated suppression in four viremic controllers. We recovered diverse proviruses persisting during therapy that broadly reflected HIV’s within-host evolutionary history, where the estimated half-lives of the persistent proviral pool during untreated infection ranged from

The greatest HIV-1 genetic diversity is found in West/Central Africa due to the pandemic’s origins in this region, but this diversity remains understudied. We characterized HIV-1 subtype diversity (from both sub-genomic and full-genome viral sequences), drug resistance and coreceptor usage in 103 predominantly (90%) antiretroviral-naive individuals living with HIV-1 in Ghana. Full-genome HIV-1 subtyping confirmed the circulating recombinant form CRF02_AG as the dominant (53.9%) subtype in the region, with the complex recombinant 06_cpx (4%) present as well. Unique recombinants, most of which were mosaics containing CRF02_AG and/or 06_cpx, made up 37% of sequences, while “pure” subtypes were rare (

Human immunodeficiency virus (HIV) can persist as an integrated provirus, in a transcriptionally repressed state, within infected cells. This small yet enduring pool of cellular reservoirs that harbor replication-competent HIV is the main barrier to cure. Entry of viral sequences into cellular reservoirs begins shortly after infection, and cells containing integrated proviral DNA are extremely stable once suppressive antiretroviral therapy (ART) is initiated. During untreated HIV infection however, reservoir turnover is likely to be more dynamic. Understanding these dynamics is important because the longevity of the persisting proviral pool during untreated infection dictates reservoir composition at ART initiation. If the persisting proviral pool turns over slowly pre-ART, then HIV sequences seeded into it during early infection would have a high likelihood of persisting for long periods. However, if pre-ART turnover was rapid, the persisting proviral pool would rapidly shift toward recently circulating HIV sequences. One-way to estimate this turnover rate is from the age distributions of proviruses sampled shortly after therapy initiation: this is because, at the time of sampling, the majority of proviral turnover would have already occurred prior to ART. Recently, methods to estimate a provirus’ age from its sequence have made this possible. Using data from 12 individuals with HIV subtype C for whom proviral ages had been determined phylogenetically, we estimated that the average proviral half-life during untreated infection was 0.78 (range 0.45–2.38) years, which is >15 times faster than that of proviral DNA during suppressive ART. We further show that proviral turnover during untreated infection correlates with both viral setpoint and rate of CD4+ T-cell decline during this period. Overall, our results support dynamic proviral turnover pre-ART in most individuals, which helps explain why many individuals’ reservoirs are skewed toward younger HIV sequences. Broadly, our findings are consistent with the notion that active viral replication creates an environment less favorable to proviral persistence, while viral suppression creates conditions more favorable to persistence, where ART stabilizes the proviral pool by dramatically slowing its rate of decay. Strategies to inhibit this stabilizing effect and/or to enhance reservoir turnover during ART could represent additional strategies to reduce the HIV reservoir.

Background: Identifying populations at high risk of HIV transmission is critical for prioritizing treatment and prevention resources and achieving the UNAIDS 90-90-90 Targets. Methods: HIV transmission rates can be estimated from phylogenetic trees as viral lineage-level diversification rates. To identify HIV-1 transmission foci in British Columbia, Canada, we inferred diversification rates from phylogenetic trees of 36 271 HIV-1 sequences from 9630 anonymized individuals. Diversification rates were combined with sociodemographic and clinical data, then aggregated by patients’ area of residence to predict the distribution of new HIV cases between 2008 and 2018. The predictive power of the model was compared with a phylogenetically uninformed model. Findings: Aggregated diversification rate measures were predictive of new HIV cases in the subsequent year after adjusting for prevalent and incident cases in the previous year. For every one-unit increase in the mean of the top five diversification rates, the number of new HIV cases increased by on average 1·38-fold (95% CI, 1·28–1·49). In a blind prediction of 2018 cases, diversification rate improved the model's specificity by 12%, accuracy by 9%, top 20 agreement by 100%, and correlation of predicted and observed values by 162% relative to a model that incorporated epidemiological data alone. Interpretation: By predicting the distribution of future HIV cases, a combined phylogenetic and epidemiological approach identifies hotspots where public health resources are needed most. Funding: Canadian Institutes of Health Research, University of British Columbia, Public Health Agency of Canada, Genome Canada, Genome BC, Michael Smith Foundation for Health Research, and BC Centre for Excellence in HIV/AIDS. Keywords: HIV, Phylogenetics, Epidemiology, Transmission, Predictive model

Summary: HIV-1 Nef enhances virion infectivity by counteracting host restriction factor SERINC5; however, the impact of natural Nef polymorphisms on this function is largely unknown. We characterize SERINC5 downregulation activity of 91 primary HIV-1 subtype B nef alleles, including isolates from 45 elite controllers and 46 chronic progressors. Controller-derived Nef clones display lower ability to downregulate SERINC5 (median 80% activity) compared with progressor-derived clones (median 96% activity) (p = 0.0005). We identify 18 Nef polymorphisms associated with differential function, including two CTL escape mutations that contribute to lower SERINC5 downregulation: K94E, driven by HLA-B∗08, and H116N, driven by the protective allele HLA-B∗57. HIV-1 strains encoding Nef K94E and/or H116N display lower infectivity and replication capacity in the presence of SERINC5. Our results demonstrate that natural polymorphisms in HIV-1 Nef can impair its ability to internalize SERINC5, indicating that variation in this recently described function may contribute to differences in viral pathogenesis. : HIV-1 Nef counteracts the cellular restriction factor SERINC5, but the importance of this for pathogenesis is unclear. Jin et al. show that Nef clones isolated from HIV controllers display lower ability to antagonize SERINC5, in part because of viral mutations that are selected to evade host T cells. Keywords: HIV-1 Nef, elite controllers, serine incorporator, host restriction, viral infectivity, viral pathogenesis

Genetic and immunologic analyses of epidemiologically-linked HIV transmission enable insights into the impact of immune responses on clinical outcomes. Human vaccine trials and animal studies of HIV-1 infection have suggested immune correlates of protection; however, their role in natural infection in terms of protection from disease progression is mostly unknown. Four HIV-1+ Cameroonian individuals, three of them epidemiologically-linked in a polygamous heterosexual relationship and one incidence-matched case, were studied over 15 years for heterologous and cross-neutralizing antibody responses, antibody binding, IgA/IgG levels, antibody-dependent cellular cytotoxicity (ADCC) against cells expressing wild-type or CD4-bound Env, viral evolution, Env epitopes, and host factors including HLA-I alleles. Despite viral infection with related strains, the members of the transmission cluster experienced contrasting clinical outcomes including cases of rapid progression and long-term non-progression in the absence of strongly protective HLA-I or CCR5Δ32 alleles. Slower progression and higher CD4/CD8 ratios were associated with enhanced IgG antibody binding to native Env and stronger V1V2 antibody binding responses in the presence of viruses with residue K169 in V2. ADCC against cells expressing Env in the CD4-bound conformation in combination with low Env-specific IgA/IgG ratios correlated with better clinical outcome. This data set highlights for the first time that V1V2-directed antibody responses and ADCC against cells expressing open, CD4-exposed Env, in the presence of low plasma IgA/IgG ratios, can correlate with clinical outcome in natural infection. These parameters are comparable to the major correlates of protection, identified post-hoc in the RV144 vaccine trial; thus, they may also modulate the rate of clinical progression once infected. The findings illustrate the potential of immune correlate analysis in natural infection to guide vaccine development.

Oncogenic “driver” mutations are theoretically attractive targets for the immunotherapy of lymphoid cancers, yet the proportion that can be recognized by T cells remains poorly defined. To address this issue without any confounding effects of the patient's immune system, we assessed T cells from 19 healthy donors for recognition of three common driver mutations in lymphoma: MYD88L265P, EZH2Y641F, and EZH2Y641N. Donors collectively expressed the 10 most prevalent HLA class I alleles, including HLA-A*02:01. Peripheral blood T cells were primed with peptide-loaded dendritic cells (DC), and reactive T cells were assessed for recognition of naturally processed mutant versus wild type full-length proteins. After screening three driver mutations across 17–26 HLA class I alleles and 3 × 106−3 × 107 T cells per donor, we identified CD4+ T cells against EFISENCGEII from EZH2Y641N (presented by HLA-DRB1*13:02) and CD8+ T cells against RPIPIKYKA from MYD88L265P (presented by HLA-B*07:02). We failed to detect RPIPIKYKA-specific T cells in seven other HLA-B*07:02-positive donors, including two lymphoma patients. Thus, healthy donors harbor T cells specific for common driver mutations in lymphoma. However, such responses appear to be rare due to the combined limitations of antigen processing, HLA restriction, and T cell repertoire size, highlighting the need for highly individualized approaches for selecting targets.

Pediatric human immunodeficiency virus (HIV) care in resource-limited settings remains a major challenge to achieving global HIV treatment and virologic suppression targets, in part because the administration of combination antiretroviral therapies (cART) is inherently complex in this population and because viral load and drug resistance genotyping are not routinely available in these settings. Children may also be at elevated risk of transmission of drug-resistant HIV as a result of suboptimal antiretroviral administration for prevention of mother-to-child transmission. We investigated the prevalence and the correlates of pretreatment HIV drug resistance (PDR) among HIV-infected, cART-naive children in Ethiopia. We observed an overall PDR rate of 14%, where all cases featured resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs): ~9% of participants harbored resistance solely to NNRTIs while ~5% harbored resistance to both NNRTIs and nucleoside reverse transcriptase inhibitors (NRTIs). No resistance to protease inhibitors was observed. No sociodemographic or clinical parameters were significantly associated with PDR, though limited statistical power is noted. The relatively high (14%) rate of NNRTI resistance in cART-naive children supports the use of non-NNRTI-based regimens in first-line pediatric treatment in Ethiopia and underscores the urgent need for access to additional antiretroviral classes in resource-limited settings.

There is limited data on virologic outcome and its correlates among HIV-infected children in resource-limited settings. We investigated rate and correlates of virologic outcome among treatment naïve HIV-infected Ethiopian children initiating cART, and were followed prospectively at baseline, 8, 12, 24 and 48 weeks using plasma viral load, clinical examination, laboratory tests and pretreatment HIV drug resistance (PDR) screening. Virologic outcome was assessed using two endpoints−virological suppression defined as having “undetectable” plasma viral load < 150 RNA copies/mL, and rebound defined as viral load ≥150 copies/mL after achieving suppression. Cox Proportional Hazards Regression was employed to assess correlates of outcome. At the end of follow up, virologic outcome was measured for 110 participants. Overall, 94(85.5%) achieved virological suppression, of which 36(38.3%) experienced virologic rebound. At 48 weeks, 9(8.2%) children developed WHO-defined virological treatment failure. Taking tenofovir-containing regimen (Hazard Ratio (HR) 3.1-[95% confidence interval (95%CI) 1.0−9.6], p = 0.049) and absence of pretreatment HIV drug resistance (HR 11.7-[95%CI 1.3−104.2], p = 0.028) were independently associated with earlier virologic suppression. In conclusion, PDR and cART regimen type correlate with rate of virologic suppression which was prominent during the first year of cART initiation. However, the impact of viral rebound in 38.3% of the children needs evaluation.

ABSTRACT HIV-1 Nef binds to the cytoplasmic region of HLA-A and HLA-B and downregulates these molecules from the surface of virus-infected cells, thus evading immune detection by CD8+ T cells. Polymorphic residues within the HLA cytoplasmic region may affect Nef’s downregulation activity. However, the effects of HLA polymorphisms on recognition by primary Nef isolates remain elusive, as do the specific Nef regions responsible for downregulation of HLA-A versus HLA-B. Here, we examined 46 Nef clones isolated from chronically HIV-1 subtype B-infected subjects for their ability to downregulate various HLA-A, HLA-B, and HLA-C molecules on the surface of virus-infected cells. Overall, HLA-B exhibited greater resistance to Nef-mediated downregulation than HLA-A, regardless of the cell type examined. As expected, no Nef clone downregulated HLA-C. Importantly, the differential abilities of patient-derived Nef clones to downregulate HLA-A and HLA-B correlated inversely with the sensitivities of HIV-infected target cells to recognition by effector cells expressing an HIV-1 Gag-specific T cell receptor. Nef codon function analysis implicated amino acid variation at position 202 (Nef-202) in differentially affecting the ability to downregulate HLA-A and HLA-B, an observation that was subsequently confirmed by experiments using Nef mutants constructed by site-directed mutagenesis. The in silico and mutagenesis analyses further suggested that Nef-202 may interact with the C-terminal Cys-Lys-Val residues of HLA-A, which are absent in HLA-B. Taken together, the results show that natural polymorphisms within Nef modulate its interaction with natural polymorphisms in the HLA cytoplasmic tails, thereby affecting the efficiency of HLA downregulation and consequent recognition by HIV-specific T cells. These results thus extend our understanding of this complex pathway of retroviral immune evasion. IMPORTANCE Recognition of genetically diverse pathogens by the adaptive immune system represents a primary strategy for host defense; however, pathogens such as HIV-1 can evade these responses to achieve persistent infection. The HIV-1 nef gene and the HLA class I locus rank among the most diverse genes of virus and host, respectively. The HIV-1 Nef protein interacts with the cytoplasmic region of HLA-A and HLA-B and downregulates these molecules to evade cellular immunity. By combining molecular, genetic, and in silico analyses, we demonstrate that patient-derived Nef clones downregulate HLA-A more effectively than HLA-B molecules. This in turn modulates the ability of HIV-specific T cells to recognize HIV-infected cells. We also identify a naturally polymorphic site at Nef codon 202 and HLA cytoplasmic motifs (GG314,315 and CKV339–341) that contribute to differential HLA downregulation by Nef. Our results highlight new interactions between HIV-1 and the human immune system that may contribute to pathogenesis.

Natural products originating from marine and plant materials are a rich source of chemical diversity and unique antimicrobials. Using an established in vitro model of HIV-1 latency, we screened 257 pure compounds from a marine natural product library and identified 4 (psammaplin A, aplysiatoxin, debromoaplysiatoxin, and previously-described alotaketal C) that induced expression of latent HIV-1 provirus in both cell line and primary cell models. Notably, aplysiatoxin induced similar levels of HIV-1 expression as prostratin but at up to 900-fold lower concentrations and without substantial effects on cell viability. Psammaplin A enhanced HIV-1 expression synergistically when treated in combination with the protein kinase C (PKC) activator prostratin, but not the histone deacetylase inhibitor (HDACi) panobinostat, suggesting that psammaplin A functions as a latency-reversing agent (LRA) of the HDACi class. Conversely, aplysiatoxin and debromoaplysiatoxin synergized with panobinostat but not prostratin, suggesting that they function as PKC activators. Our study identifies new compounds from previously untested marine natural products and adds to the repertoire of LRAs that can inform therapeutic “shock-and-kill”-based strategies to eliminate latent HIV-infected reservoirs.

Clinical monitoring of pediatric HIV treatment remains a major challenge in settings where drug resistance genotyping is not routinely available. As a result, our understanding of drug resistance, and its impact on subsequent therapeutic regimens available in these settings, remains limited. We investigate the prevalence and correlates of HIV-1 drug resistance among 94 participants of the Ethiopia Pediatric HIV Cohort failing first-line combination antiretroviral therapy (cART) using dried blood spot-based genotyping. Overall, 81% (73/90) of successfully genotyped participants harbored resistance mutations, including 69% (62/90) who harbored resistance to both Nucleoside Reverse Transcriptase Inhibitors (NRTIs) and Non-nucleoside Reverse Transcriptase Inhibitors (NNRTIs). Strikingly, 42% of resistant participants harbored resistance to all four NRTIs recommended for second-line use in this setting, meaning that there are effectively no remaining cART options for these children. Longer cART duration and prior regimen changes were significantly associated with detection of drug resistance mutations. Replicate genotyping increased the breadth of drug resistance detected in 34% of cases, and thus is recommended for consideration when typing from blood spots. Implementation of timely drug resistance testing and access to newer antiretrovirals and drug classes are urgently needed to guide clinical decision-making and improve outcomes for HIV-infected children on first-line cART in Ethiopia.