HIV-1 Evolution Research Articles

Efficient epistasis inference via higher-order covariance matrix factorization.

Epistasis can profoundly influence evolutionary dynamics. Temporal genetic data, consisting of sequences sampled repeatedly from a population over time, provides a unique resource to understand how epistasis shapes evolution. However, detecting epistatic interactions from sequence data is technically challenging. Existing methods for identifying epistasis are computationally demanding, limiting their applicability to real-world data. Here, we present a novel computational method for inferring epistasis that significantly reduces computational costs without sacrificing accuracy. We validated our approach in simulations and applied it to study HIV-1 evolution over multiple years in a data set of 16 individuals. There we observed a strong excess of negative epistatic interactions between beneficial mutations, especially mutations involved in immune escape. Our method is general and could be used to characterize epistasis in other large data sets.

A binary trait model reveals the fitness effects of HIV-1 escape from T cell responses.

Natural selection often acts on multiple traits simultaneously. For example, the virus HIV-1 faces pressure to evade host immunity while also preserving replicative fitness. While past work has studied selection during HIV-1 evolution, as in other examples where selection acts on multiple traits, it is challenging to quantitatively separate different contributions to fitness. This task is made more difficult because a single mutation can affect both immune escape and replication. Here, we develop an evolutionary model that disentangles the effects of escaping CD8+ T cell-mediated immunity, which we model as a binary trait, from other contributions to fitness. After validation in simulations, we applied this model to study within-host HIV-1 evolution in a clinical data set. We observed strong selection for immune escape, sometimes greatly exceeding past estimates, especially early in infection. Conservative estimates suggest that roughly half of HIV-1 fitness gains during the first months to years of infection can be attributed to T cell escape. Our approach is not limited to HIV-1 or viruses, and could be adapted to study the evolution of quantitative traits in other contexts.

Inverse uncertainty quantification for stochastic systems by resampling. Applications to modeling of alcohol consumption and infection by HIV

A random differential equation, or stochastic differential equation with parametric uncertainty, is a classical differential equation whose input values (coefficients, initial conditions, etc.) are random variables. Given data, the probability distributions of the input random parameters must be appropriately inferred, before proceeding to simulate the model’s output. This task is called inverse uncertainty quantification. In this paper, the goal is to study the applicability of the Bayesian bootstrap to draw inferences on the posterior distributions of the parameters, by resampling the residuals of the deterministic least-squares optimization with Dirichlet weights. The method is based on repeated deterministic calibrations. Thus, to alleviate the curse of dimensionality, the technique may be combined with the principle of maximum entropy for densities, when there are some parameters that are not optimized deterministically. For illustration of the methodology, two case studies on important health topics are conducted, with stochastic fitting to data. The first one, on past alcohol consumption in Spain, taking social contagion into account. The second one, on HIV evolution considering CD4+ T cells and viral load, with a patient in clinical follow-up. All these applied models are built from a compartmental viewpoint, with a randomized basic reproduction number that controls the long-term behavior of the system.

‘Not taking medications and taking medication, it was the same thing:’ perspectives of antiretroviral therapy among people hospitalised with advanced HIV disease

BackgroundDespite HIV's evolution to a chronic disease, the burden of advanced HIV disease (AHD, defined as a CD4 count of < 200 cells/uL or WHO clinical Stage 3 or 4 disease), remains high among People Living with HIV (PLHIV) who have previously been prescribed antiretroviral therapy (ART). As little is known about the experiences of patients hospitalised with AHD, this study sought to discern social forces driving hospitalisation with AHD. Understanding such forces could inform strategies to reduce HIV-related morbidity and mortality.MethodsWe conducted a qualitative study with patients hospitalised with AHD who had a history of poor adherence. Semi-structured interviews were conducted between October 1 and November 30, 2023. The Patient Health Engagement and socio-ecological theoretical models were used to guide a thematic analysis of interview transcripts.ResultsTwenty individuals participated in the research. Most reported repeated periods of disengagement with HIV services. The major themes identified as driving disengagement included: 1) feeling physically well; 2) life circumstances and relationships; and 3) health system factors, such as clinic staff attitudes and a perceived lack of flexible care. Re-engagement with care was often driven by new physical symptoms but was mediated through life circumstances/relationships and aspects of the health care system.ConclusionsCurrent practices fail to address the challenges to lifelong engagement in HIV care. A bold strategy for holistic care which involves people living with advanced HIV as active members of the health care team (i.e. ‘PLHIV as Partners’), could contribute to ensuring health care services are compatible with their lives, reducing periods of disengagement from care.

Identification of Two Novel HIV-1 Unique Recombinant Forms (CRF01_AE/CRF07_BC) and Genomic Characterization in Tongzhou District of Beijing, China.

Continuous recombination and variation during replication could lead to rapid evolution and genetic diversity of HIV-1. Some studies had identified that it was easy to develop new recombinant strains of HIV-1 among the populations of men who have sex with men (MSM). Surveillance of genetic variants of HIV-1 in key populations was crucial for comprehending the development of regional HIV-1 epidemics. The finding was reported the identification of two new unique recombinant forms (URF 20110561 and 21110743) from individuals infected with HIV-1 in Tongzhou, Beijing in 2020-2022. Sequences of near full-length genome (NFLG) were amplified, then identification of amplification products used phylogenetic analyses. The result showed that CRF01_AE was the main backbone of 20110561 and 21110743. In the gag region of the virus, 20110561 was inserted two fragments from CRF07_BC, while in the pol and tat regions of the virus, 21110743 was inserted four fragments from CRF07_BC. The CRF01_AE parental origin in the genomes of the two URFs was derived from the CRF01_AE Cluster 4. In the phylogenetic tree, the CRF07_BC parental origin of 20110561 clustered with 07BC_N and the CRF07_BC parental origin of 21110743 clustered with 07BC_O. In summary, the prevalence of novel second-generation URFs of HIV-1 was monitored in Tongzhou, Beijing. The emergence of the novel CRF01_AE/CRF07_BC recombination demonstrated that there was a great significance of continuous monitoring of new URFs in MSM populations to prevent and control the spreading of new HIV-1 URFs.

Higher HIV-1 evolutionary rate is associated with cytotoxic T lymphocyte escape mutations in infants.

Escape from cytotoxic T lymphocyte (CTL) responses toward HIV-1 Gag and Nef has been associated with reduced control of HIV-1 replication in adults. However, less is known about CTL-driven immune selection in infants as longitudinal studies of infants are limited. Here, 1,210 gag and 1,264 nef sequences longitudinally collected within 15 months after birth from 14 HIV-1 perinatally infected infants and their mothers were analyzed. The number of transmitted founder (T/F) viruses and associations between virus evolution, selection, CTL escape, and disease progression were determined. The analyses indicated that a paraphyletic-monophyletic relationship between the mother-infant sequences was common (80%), and that the HIV-1 infection was established by a single T/F virus in 10 of the 12 analyzed infants (83%). Furthermore, most HIV-1 CTL escape mutations among infants were transmitted from the mothers and did not revert during the first year of infection. Still, immune-driven selection was observed at approximately 3 months after HIV-1 infection in infants. Moreover, virus populations with CTL escape mutations in gag evolved faster than those without, independently of disease progression rate. These findings expand the current knowledge of HIV-1 transmission, evolution, and CTL escape in infant HIV-1 infection and are relevant for the development of immune-directed interventions in infants.IMPORTANCEDespite increased coverage in antiretroviral therapy for the prevention of perinatal transmission, paediatric HIV-1 infection remains a significant public health concern, especially in areas of high HIV-1 prevalence. Understanding HIV-1 transmission and the subsequent virus adaptation from the mother to the infant's host environment, as well as the viral factors that affect disease outcome, is important for the development of early immune-directed interventions for infants. This study advances our understanding of vertical HIV-1 transmission, and how infant immune selection pressure is shaping the intra-host evolutionary dynamics of HIV-1.

Parallel HIV-1 evolutionary dynamics in humans and rhesus macaques who develop broadly neutralizing antibodies.

Human immunodeficiency virus (HIV)-1 exhibits remarkable genetic diversity. For this reason, an effective HIV-1 vaccine must elicit antibodies that can neutralize many variants of the virus. While broadly neutralizing antibodies (bnAbs) have been isolated from HIV-1 infected individuals, a general understanding of the virus-antibody coevolutionary processes that lead to their development remains incomplete. We performed a quantitative study of HIV-1 evolution in two individuals who developed bnAbs. We observed strong selection early in infection for mutations affecting HIV-1 envelope glycosylation and escape from autologous strain-specific antibodies, followed by weaker selection for bnAb resistance later in infection. To confirm our findings, we analyzed data from rhesus macaques infected with viruses derived from the same two individuals. We inferred remarkably similar fitness effects of HIV-1 mutations in humans and macaques. Moreover, we observed a striking pattern of rapid HIV-1 evolution, consistent in both humans and macaques, that precedes the development of bnAbs. Our work highlights strong parallels between infection in rhesus macaques and humans, and it reveals a quantitative evolutionary signature of bnAb development.

The Intersection of HIV and Pulmonary Vascular Health: From HIV Evolution to Vascular Cell Types to Disease Mechanisms.

People living with HIV (PLWH) face a growing burden of chronic diseases, owing to the combinations of aging, environmental triggers, lifestyle choices, and virus-induced chronic inflammation. The rising incidence of pulmonary vascular diseases represents a major concern for PLWH. The study of HIV-associated pulmonary vascular complications ideally requires a strong understanding of pulmonary vascular cell biology and HIV pathogenesis at the molecular level for effective applications in infectious diseases and vascular medicine. Active HIV infection and/or HIV proteins disturb the delicate balance between vascular tone and constriction, which is pivotal for maintaining pulmonary vascular health. One of the defining features of HIV is its high genetic diversity owing to several factors including its high mutation rate, recombination between viral strains, immune selective pressures, or even geographical factors. The intrinsic HIV genetic diversity has several important implications for pathogenic outcomes of infection and the overall battle to combat HIV. Challenges in the field present themselves from two sides of the same coin: those imposed by the virus itself and those stemming from the host. The field may be advanced by further developing in vivo and in vitro models that are well described for both pulmonary vascular diseases and HIV for mechanistic studies. In essence, the study of HIV-associated pulmonary vascular complications requires a multidisciplinary approach, drawing upon insights from both infectious diseases and vascular medicine. In this review article, we discuss the fundamentals of HIV virology and their impact on pulmonary disease, aiming to enhance the understanding of either area or both simultaneously. Bridging the gap between preclinical research findings and clinical practice is essential for improving patient care. Addressing these knowledge gaps requires interdisciplinary collaborations, innovative research approaches, and dedicated efforts to prioritize HIV-related pulmonary complications on the global research agenda.

Sequence Analysis of Inducible, Replication-Competent Virus Reveals No Evidence of HIV-1 Evolution During Suppressive Antiviral Therapy, Indicating a Lack of Ongoing Viral Replication.

Persistence of HIV-1 in reservoirs necessitates life-long antiretroviral therapy (ART). There are conflicting data using genetic analysis on whether persistence includes an actively replicating reservoir with strong evidence arguing against replication. We investigated the possibility of ongoing viral evolution during suppressive therapy by comparing near full-length viral genomic sequences using phylogenetic analysis of viral RNA in plasma before therapy initiation early after infection and from virus induced to grow from the latent reservoir after a period of suppressive ART. We also focused our analysis on evidence of selective pressure by drugs in the treatment regimen and at sites of selective pressure by the adaptive immune response. Viral genomes induced to grow from the latent reservoir from 10 participants with up to 9 years on suppressive ART were highly similar to the nearly homogeneous sequences in plasma taken early after infection at ART initiation. This finding was consistent across the entire genome and when the analysis focused on sites targeted by the drug regimen and by host selective pressure of antibody and cytotoxic T cells. The lack of viral evolution away from pretherapy sequences in spite of demonstrated selective pressure is most consistent with a lack of viral replication during reservoir persistence. These results do not support ongoing viral replication as a mechanism of HIV-1 persistence during suppressive ART.

How to report and make sense of a new HIV-1 circulating recombinant form?

Co-circulation of multiple HIV-1 subtypes in the same high-risk groups leads to the on-going generation of various inter-subtype recombinants, including unique (URFs) and circulating (CRFs) recombinant forms, which brings a new challenge for the prevention and eradication of HIV/AIDS. Identification and prompt reporting of new CRFs will provide not only new insights into the understanding of genetic diversity and evolution of HIV-1, but also an early warning of potential prevalence of these variants. Currently, 140 HIV-1 CRFs have been described; however, their prevalence and clinical importance are less concerned. Apart from the mosaic genomic maps, less other valuable information, including the clinical and demographic data, genomic sequence characteristics, origin and evolutionary dynamics, as well as representative genomic fragments for determining the variants, are available for most of these CRFs. Accompanied with the growing increase of HIV-1 full-length genomic sequences, more and more CRFs will be identified in the near future due to the high recombination potential of HIV-1. Here, we discuss the prevalence and clinical importance of various HIV-1 CRFs and propose how to report and make sense of a new HIV-1 CRF.

HIV-1 Gag, Pol, and Env diversified with limited adaptation since the 1980s.

Global surveillance of HIV-1 sequences is critical for designing relevant prophylactic and therapeutic interventions to infection. We designed an open-source platform, Hervé, for analyzing and visualizing the diversification dynamics of HIV-1 protein sequences. We characterized the evolution of over 12,000 HIV-1 Env, Gag, and Pol protein sequences from 1980-2020 and found that, despite a steady increase in intra-subtype and circulating recombinant form diversity, the most frequent residue at each site, i.e., the consensus, has varied only moderately.

The replication-competent HIV reservoir is a genetically restricted, younger subset of the overall pool of HIV proviruses persisting during therapy, which is highly genetically stable over time.

Within-host HIV populations continually diversify during untreated infection, and this diversity persists within infected cell reservoirs during antiretroviral therapy (ART). Achieving a better understanding of on-ART proviral evolutionary dynamics, and a better appreciation of how the overall persisting pool of (largely genetically defective) proviruses differs from the much smaller replication-competent HIV reservoir, is critical to HIV cure efforts. We reconstructed within-host HIV evolutionary histories in blood from seven participants of the Women's Interagency HIV Study who experienced HIV seroconversion, and used these data to characterize the diversity, lineage origins, and ages of proviral env-gp120 sequences sampled longitudinally up to 12 years on ART. We also studied HIV sequences emerging from the reservoir in two participants. We observed that proviral clonality generally increased over time on ART, with clones frequently persisting long term. While on-ART proviral integration dates generally spanned the duration of untreated infection, HIV emerging in plasma was exclusively younger (i.e., dated to the years immediately pre-ART). The genetic and age distributions of distinct proviral sequences remained stable during ART in all but one participant, in whom there was evidence that younger proviruses had been preferentially eliminated after 12 years on ART. Analysis of the gag region in three participants corroborated our env-gp120-based observations, indicating that our observations are not influenced by the HIV region studied. Our results underscore the remarkable genetic stability of the distinct proviral sequences that persist in blood during ART. Our results also suggest that the replication-competent HIV reservoir is a genetically restricted, younger subset of this overall proviral pool.IMPORTANCECharacterizing the genetically diverse HIV sequences that persist in the reservoir despite antiretroviral therapy (ART) is critical to cure efforts. Our observations confirm that proviruses persisting in blood on ART, which are largely genetically defective, broadly reflect the extent of within-host HIV evolution pre-ART. Moreover, on-ART clonal expansion is not appreciably accompanied by the loss of distinct proviral lineages. In fact, on-ART proviral genetic composition remained stable in all but one participant, in whom, after 12 years on ART, proviruses dating to around near ART initiation had been preferentially eliminated. We also identified recombinant proviruses between parental sequence fragments of different ages. Though rare, such sequences suggest that reservoir cells can be superinfected with HIV from another infection era. Overall, our finding that the replication-competent reservoir in blood is a genetically restricted, younger subset of all persisting proviruses suggests that HIV cure strategies will need to eliminate a reservoir that differs in key respects from the overall proviral pool.

Evaluation of the ABL NGS assay for HIV-1 drug resistance testing

HIV evolution and variability around the world requires special monitoring of the viral strains in infected people. High-throughput HIV sequencing and drug resistance testing techniques have become routinely available over the last few years.We conducted a study to assess the new CE-marked ABL NGS HIV genotyping assay on an Illumina® platform, to compare the results (the detection of resistance associated mutations (RAMs) detected in the three main targets: reverse transcriptase, protease, and integrase) with those produced by three Sanger-based assays, and to compare the assays’ respective costs.For the 10 samples and a 20 % sensitivity threshold for the NGS technology, the percent agreement between the four assays ranged from 99.5 % to 100 %. We detected 4 more and 10 more RAMs of interest when we lowered the NGS assay's threshold to 10 % and 3 %, respectively. At a threshold of 3 %, the antiretroviral sensitivity interpretation algorithm (for protease inhibitors) was modified for only two patients. The NGS assay's unit cost fell rapidly as the number of samples per run increased.Compared with Sanger sequencing, the ABL NGS HIV genotyping assay is just as robust and somewhat more expensive but opens up interesting multiplexing perspectives for virology laboratories.

Accelerating Bayesian inference of dependency between mixed-type biological traits.

Inferring dependencies between mixed-type biological traits while accounting for evolutionary relationships between specimens is of great scientific interest yet remains infeasible when trait and specimen counts grow large. The state-of-the-art approach uses a phylogenetic multivariate probit model to accommodate binary and continuous traits via a latent variable framework, and utilizes an efficient bouncy particle sampler (BPS) to tackle the computational bottleneck-integrating many latent variables from a high-dimensional truncated normal distribution. This approach breaks down as the number of specimens grows and fails to reliably characterize conditional dependencies between traits. Here, we propose an inference pipeline for phylogenetic probit models that greatly outperforms BPS. The novelty lies in 1) a combination of the recent Zigzag Hamiltonian Monte Carlo (Zigzag-HMC) with linear-time gradient evaluations and 2) a joint sampling scheme for highly correlated latent variables and correlation matrix elements. In an application exploring HIV-1 evolution from 535 viruses, the inference requires joint sampling from an 11,235-dimensional truncated normal and a 24-dimensional covariance matrix. Our method yields a 5-fold speedup compared to BPS and makes it possible to learn partial correlations between candidate viral mutations and virulence. Computational speedup now enables us to tackle even larger problems: we study the evolution of influenza H1N1 glycosylations on around 900 viruses. For broader applicability, we extend the phylogenetic probit model to incorporate categorical traits, and demonstrate its use to study Aquilegia flower and pollinator co-evolution.

A snapshot on HIV-1 evolution through the identification of phylogenetic-specific properties of HIV-1 integrases M/O.

Transmissions of simian viruses to humans has originated the different groups of HIV-1. We recently identified a functional motif (CLA), in the C-terminal domain of the integrase, essential for integration in HIV-1 group M. Here, we found that the motif is instead dispensable in group O isolates, because of the presence, in the N-terminal domain of HIV-1 O of a specific sequence, Q7G27P41H44, that we define as the NOG motif. Alterations of reverse transcription and of 3' processing observed by mutating the CLA motif of IN M are fully rescued to wt levels by inserting the sequence of the NOG motif in the N-ter of the protein. These results indicate that the two motifs (CLA and NOG) functionally complement each other and a working model accounting for these observations is proposed. The establishment of these two alternative motifs seems to be due to the different phylogenetic origin and history of these two groups. Indeed, the NOG motif is already present in the ancestor of group O (SIVgor) while it is absent from SIVcpzPtt, the ancestor of group M. The CLA motif, instead, seems to have emerged after SIVcpzPtt has been transferred to humans, since no conservation is found at the same positions in these simian viruses. These results show the existence of two-group specific motifs in HIV-1 M and O integrases. In each group, only one of the motifs is functional, potentially leading the other motif to diverge from its original function and, in an evolutionary perspective, assist other functions of the protein, further increasing HIV genetic diversity.

Inferring the multiplicity of founder variants initiating HIV-1 infection: a systematic review and individual patient data meta-analysis

HIV-1 infections initiated by multiple founder variants are characterised by a higher viral load and a worse clinical prognosis than those initiated with single founder variants, yet little is known about the routes of exposure through which transmission of multiple founder variants is most probable. Here we used individual patient data to calculate the probability of multiple founders stratified by route of HIV exposure and study methodology. We conducted a systematic review and meta-analysis of studies that estimated founder variant multiplicity in HIV-1 infection, searching MEDLINE, Embase, and Global Health databases for papers published between Jan 1, 1990, and Sept 14, 2020. Eligible studies must have reported original estimates of founder variant multiplicity in people with acute or early HIV-1 infections, have clearly detailed the methods used, and reported the route of exposure. Studies were excluded if they reported data concerning people living with HIV-1 who had known or suspected superinfection, who were documented as having received pre-exposure prophylaxis, or if the transmitting partner was known to be receiving antiretroviral treatment. Individual patient data were collated from all studies, with authors contacted if these data were not publicly available. We applied logistic meta-regression to these data to estimate the probability that an HIV infection is initiated by multiple founder variants. We calculated a pooled estimate using a random effects model, subsequently stratifying this estimate across exposure routes in a univariable analysis. We then extended our model to adjust for different study methods in a multivariable analysis, recalculating estimates across the exposure routes. This study is registered with PROSPERO, CRD42020202672. We included 70 publications in our analysis, comprising 1657 individual patients. Our pooled estimate of the probability that an infection is initiated by multiple founder variants was 0·25 (95% CI 0·21-0·29), with moderate heterogeneity (Q=132·3, p<0·0001, I2=64·2%). Our multivariable analysis uncovered differences in the probability of multiple variant infection by exposure route. Relative to a baseline of male-to-female transmission, the predicted probability for female-to-male multiple variant transmission was significantly lower at 0·13 (95% CI 0·08-0·20), and the probabilities were significantly higher for transmissions in people who inject drugs (0·37 [0·24-0·53]) and men who have sex with men (0·30 [0·33-0·40]). There was no significant difference in the probability of multiple variant transmission between male-to-female transmission (0·21 [0·14-0·31]), post-partum transmission (0·18 [0·03-0·57]), pre-partum transmission (0·17 [0·08-0·33]), and intra-partum transmission (0·27 [0·14-0·45]). We identified that transmissions in people who inject drugs and men who have sex with men are significantly more likely to result in an infection initiated by multiple founder variants, and female-to-male infections are significantly less probable. Quantifying how the routes of HIV infection affect the transmission of multiple variants allows us to better understand how the evolution and epidemiology of HIV-1 determine clinical outcomes. Medical Research Council Precision Medicine Doctoral Training Programme and a European Research Council Starting Grant.

Antiretroviral APOBEC3 cytidine deaminases alter HIV-1 provirus integration site profiles

APOBEC3 (A3) proteins are host-encoded deoxycytidine deaminases that provide an innate immune barrier to retroviral infection, notably against HIV-1. Low levels of deamination are believed to contribute to the genetic evolution of HIV-1, while intense catalytic activity of these proteins can induce catastrophic hypermutation in proviral DNA leading to near-total HIV-1 restriction. So far, little is known about how A3 cytosine deaminases might impact HIV-1 proviral DNA integration sites in human chromosomal DNA. Using a deep sequencing approach, we analyze the influence of catalytic active and inactive APOBEC3F and APOBEC3G on HIV-1 integration site selections. Here we show that DNA editing is detected at the extremities of the long terminal repeat regions of the virus. Both catalytic active and non-catalytic A3 mutants decrease insertions into gene coding sequences and increase integration sites into SINE elements, oncogenes and transcription-silencing non-B DNA features. Our data implicates A3 as a host factor influencing HIV-1 integration site selection and also promotes what appears to be a more latent expression profile.

Ongoing HIV-1 evolution and reservoir reseeding in two elite controllers with genetically diverse peripheral proviral quasispecies.

Elite controllers (EC) are human immunodeficiency virus (HIV)-positive individuals who can maintain low viral loads for extended periods without antiretroviral therapy due to multifactorial and individual characteristics. Most have a small HIV-1 reservoir composed of identical proviral sequences maintained by clonal expansion of infected CD4+ T cells. However, some have a more diverse peripheral blood mononuclear cell (PBMC)-associated HIV-1 reservoir with unique sequences. To understand the turnover dynamics of the PBMC-associated viral quasispecies in ECs with relatively diverse circulating proviral reservoirs. We performed single genome amplification of the env gene at three time points during six years in two EC with high intra-host HIV DNA diversity. Both EC displayed quite diverse PBMCs-associated viral quasispecies (mean env diversity = 1.9-4.1%) across all time-points comprising both identical proviruses that are probably clonally expanded and unique proviruses with evidence of ongoing evolution. HIV-1 env glycosylation pattern suggests that ancestral and evolving proviruses may display different phenotypes of resistance to broadly neutralising antibodies consistent with persistent immune pressure. Evolving viruses may progressively replace the ancestral ones or may remain as minor variants in the circulating proviral population. These findings support that the high intra-host HIV-1 diversity of some EC resulted from long-term persistence of archival proviruses combined with the continuous reservoir's reseeding and low, but measurable, viral evolution despite undetectable viremia.

2073. Persistent HIV-1 Viremia Despite Intensive Antiviral Therapy Due to Non-responsive Clonal Viral Lineages

Abstract Background After initiation of antiviral therapy (ART), plasma HIV-1 RNA is usually undetectable after one month. In rare cases, viral suppression may not be achieved despite good adherence and with virus susceptible to ART. We used ultra-deep Primer ID next gen sequencing (NGS) to study the viral population and evolution of HIV-1 in two patients with persistent viremia on intensive ART. Methods We extracted viral RNA from plasma samples collected at multiple timepoints over the duration of the treatment from two patients (VEX1 and VEX2). We constructed Primer ID NGS libraries covering part of the pol gene and the env V1/V3 region and sequenced them on the Illumina MiSeq platform. We used the ‘tcs’ pipeline to construct template consensus sequences (TCS) for each region, and searched for drug resistance mutations (DRMs). The Geno2pheno pipeline was used to predict co-receptor tropisms. Results Patient VEX1 was followed for two years on ART. VEX2 restarted ART in the hospital and received directly observed therapy for nearly 6 months. Both had over 1 million viral copies/mL at the initiation of ART, and the CD4 cell counts were extremely low. Their viral loads slowly declined to approx. 10,000 copies/mL at the end of follow-up but complete viral suppression was not achieved for either patient despite appropriate ART. DRMs were not detected in either patient throughout the treatment with detection sensitivity as low as 0.1%. The sequencing results for VEX1 showed that there were both X4- and R5-tropic viruses at all timepoints and R5 viruses decayed much more slowly than X4 viruses, up to 35-fold more slowly in the initial 3 months of ART. Phylogenetic analysis revealed that the persistent R5 viruses were largely clonal while little clonality was found in the persistent X4 virus. In VEX2, all viruses were R5-tropic. There were three major distinct lineages in the viral population, and two of them completely disappeared after the initiation of ART while the other lineage persisted throughout therapy (Fig 1). The persistent lineage in VEX2 was highly clonal. Pooled maximum likelihood tree at env V1V3 region of 3 time points from patient VEX2. Sequences in red were from 2018 when we obtained the initial specimen from the patient. Sequences in blue were from Sept 2021 when the patient restarted ART. Sequences in green were from Oct 2021 when the patient were on ART for 3 weeks. The patient had 3 major viral lineages, L1, L2, and L3. From 2018 to Sept 2021, we could see the viral evolution of the three lineages (blue arrows). After 3 weeks of ART, L1 and L2 disappeared, while the L3 persisted. We could also see that L3 had several clonal populations which did not respond to ART nor evolve from 2018 to 2021. The additional sequences from Nov 2021 to Feb 2022 were identical to the sequences obtained on Oct 2021 (not shown on this figure). Conclusion Our study shows that persistent viremia on ART can come from clonal viral lineages that carry no DRMs. Clonally expanded and infected host cells might contribute to the phenomenon. Further study is needed to explore the origin of these clonal viral genomes. Disclosures Jonathan Parr, MD, Abbott Laboratories: Donation of laboratory testing and reagents|Gilead Sciences: Grant/Research Support|Virology Education: Honoraria|World Health Organization: Advisor/Consultant|World Health Organization: Grant/Research Support David A. Wohl, M.D., Gilead: Advisor/Consultant|Gilead: Grant/Research Support|Janssen: Advisor/Consultant|Lilly: Grant/Research Support|Merck: Grant/Research Support|ViiV: Advisor/Consultant|ViiV: Grant/Research Support Joseph J. Eron, MD, Adagio Therapeutics: data safety monitoring committee|Gilead Sciences: Advisor/Consultant|Gilead Sciences: Grant/Research Support|Glaxo Smith Kline: Advisor/Consultant|Merck: Advisor/Consultant|ViiV Healthcare: Advisor/Consultant|ViiV Healthcare: Grant/Research Support.

Contingency and Entrenchment of Drug-Resistance Mutations in HIV Viral Proteins.

The ability of HIV-1 to rapidly mutate leads to antiretroviral therapy (ART) failure among infected patients. Drug-resistance mutations (DRMs), which cause a fitness penalty to intrinsic viral fitness, are compensated by accessory mutations with favorable epistatic interactions which cause an evolutionary trapping effect, but the kinetics of this overall process has not been well characterized. Here, using a Potts Hamiltonian model describing epistasis combined with kinetic Monte Carlo simulations of evolutionary trajectories, we explore how epistasis modulates the evolutionary dynamics of HIV DRMs. We show how the occurrence of a drug-resistance mutation is contingent on favorable epistatic interactions with many other residues of the sequence background and that subsequent mutations entrench DRMs. We measure the time-autocorrelation of fluctuations in the likelihood of DRMs due to epistatic coupling with the sequence background, which reveals the presence of two evolutionary processes controlling DRM kinetics with two distinct time scales. Further analysis of waiting times for the evolutionary trapping effect to reverse reveals that the sequences which entrench (trap) a DRM are responsible for the slower time scale. We also quantify the overall strength of epistatic effects on the evolutionary kinetics for different mutations and show these are much larger for DRM positions than polymorphic positions, and we also show that trapping of a DRM is often caused by the collective effect of many accessory mutations, rather than a few strongly coupled ones, suggesting the importance of multiresidue sequence variations in HIV evolution. The analysis presented here provides a framework to explore the kinetic pathways through which viral proteins like HIV evolve under drug-selection pressure.