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Human endogenous retrovirus (HERV) transcripts are known to be highly expressed in cancers, yet their activity in non-diseased tissue is largely unknown. Using the GTEx RNA-seq dataset from normal tissue sampled at autopsy, we characterized individual expression of the recent HERV-K(HML-2) provirus group across 13,000 different samples of 54 different tissues from 948 individuals. HML-2 transcripts could be identified in every tissue sampled, and were elevated in the cerebellum, pituitary, testis, and thyroid. 37 different individual proviruses were expressed in one or more tissues, representing all three LTR5 subgroups. 9 proviruses were identified as having LTR-driven transcription, 7 of which belonged to the most recent LTR5HS subgroup. Proviruses of different subgroups displayed a bias in tissue expression which may be associated with differences in transcription factor binding sites in their LTRs. Provirus expression was greater in evolutionarily older proviruses with an earliest shared ancestor of gorilla or older. HML-2 expression was significantly affected by biological sex in one tissue, while age and timing of death (Hardy score) had little effect. Proviruses containing intact gag, pro and env ORFs were expressed in the dataset, with almost every tissue measured potentially expressing at least one intact ORF (gag).

The integration of human immunodeficiency virus type 1 (HIV-1) into chromosomal DNA establishes the long-term persistence of HIV-1 as proviruses despite effective antiretroviral therapy (ART). Characterizing proviruses is difficult because of their rarity in individuals on long-term suppressive ART, their highly polymorphic sequences and genetic structures, and the need for efficient amplification and sequencing of the provirus and its integration site.

HERV-K(HML-2), the youngest clade of human endogenous retroviruses (HERVs), includes many intact or nearly intact proviruses, but no replication competent HML-2 proviruses have been identified in humans. HML-2-related proviruses are present in other primates, including rhesus macaques, but the extent and timing of HML-2 activity in macaques remains unclear. We have identified 145 HML-2-like proviruses in rhesus macaques, including a clade of young, rhesus-specific insertions. Age estimates, intact ORFs, and insertional polymorphism of these insertions are consistent with recent or ongoing infectious activity in macaques. 108 of the proviruses form a clade characterized by an ~750 bp sequence between env and the 3’ LTR, derived from an ancient recombination with a HERV-K(HML-8)-related virus. This clade is found in Old World monkeys (OWM), but not great apes, suggesting it originated after the ape/OWM split. We identified similar proviruses in white-cheeked gibbons; the gibbon insertions cluster within the OWM recombinant clade, suggesting interspecies transmission from OWM to gibbons. The LTRs of the youngest proviruses have deletions in U3, which disrupt the Rec Response Element (RcRE), required for nuclear export of unspliced viral RNA. We show that the HML-8 derived region functions as a Rec-independent constitutive transport element (CTE), indicating the ancestral Rec-RcRE export system was replaced by a CTE mechanism.

The prevalence of HIV-1 drug resistance is increasing worldwide and monitoring its emergence is important for the successful management of populations receiving combination antiretroviral therapy. It is likely that pre-existing drug resistance mutations linked on the same viral genomes are predictive of treatment failure. Because of the large number of sequences generated by ultrasensitive single-genome sequencing (uSGS) and other similar next-generation sequencing methods, it is difficult to assess each sequence individually for linked drug resistance mutations. Several software/programs exist to report the frequencies of individual mutations in large data sets, but they provide no information on linkage of resistance mutations. In this study, we report the HIV-DRLink program, a research tool that provides resistance mutation frequencies as well as their genetic linkage by parsing and summarizing the Sierra output from the Stanford HIV Database. The HIV-DRLink program should only be used on data sets generated by methods that eliminate artifacts due to polymerase chain reaction recombination, for example, standard single-genome sequencing or uSGS. HIV-DRLink is exclusively a research tool and is not intended to inform clinical decisions.

BACKGROUNDHIV-1 viremia that is not suppressed by combination antiretroviral therapy (ART) is generally attributed to incomplete medication adherence and/or drug resistance. We evaluated individuals referred by clinicians for nonsuppressible viremia (plasma HIV-1 RNA above 40 copies/mL) despite reported adherence to ART and the absence of drug resistance to the current ART regimen.METHODSSamples were collected from at least 2 time points from 8 donors who had nonsuppressible viremia for more than 6 months. Single templates of HIV-1 RNA obtained from plasma and viral outgrowth of cultured cells and from proviral DNA were amplified by PCR and sequenced for evidence of clones of cells that produced infectious viruses. Clones were confirmed by host-proviral integration site analysis.RESULTSHIV-1 genomic RNA with identical sequences were identified in plasma samples from all 8 donors. The identical viral RNA sequences did not change over time and did not evolve resistance to the ART regimen. In 4 of the donors, viral RNA sequences obtained from plasma matched those sequences from viral outgrowth cultures, indicating that the viruses were replication competent. Integration sites for infectious proviruses from those 4 donors were mapped to the introns of the MATR3, ZNF268, ZNF721/ABCA11P, and ABCA11P genes. The sizes of the clones were estimated to be from 50 million to 350 million cells.CONCLUSIONThese findings show that clones of HIV-1-infected cells producing virus can cause failure of ART to suppress viremia. The mechanisms involved in clonal expansion and persistence need to be defined to effectively target viremia and the HIV-1 reservoir.FUNDINGNational Cancer Institute, NIH; Howard Hughes Medical Research Fellows Program, Howard Hughes Medical Institute; Bill and Melinda Gates Foundation; Office of AIDS Research; American Cancer Society; National Cancer Institute through a Leidos subcontract; National Institute for Allergy and Infectious Diseases, NIH, to the I4C Martin Delaney Collaboratory; University of Rochester Center for AIDS Research and University of Rochester HIV/AIDS Clinical Trials Unit.

COVID-19 vaccines were first administered on 15 December 2020, marking an important transition point for the spread of SARS-CoV-2 in the United States (U.S.). Prior to this point in time, the virus spread to an almost completely immunologically naïve population, whereas subsequently, vaccine-induced immune pressure and prior infections might be expected to influence viral evolution. Accordingly, we conducted a study to characterize the spread of SARS-CoV-2 in the U.S. pre-vaccination, investigate the depth and uniformity of genetic surveillance during this period, and measure and otherwise characterize changing viral genetic diversity, including by comparison with more recently emergent variants of concern (VOCs). In 2020, SARS-CoV-2 spread across the U.S. in three phases distinguishable by peaks in the numbers of infections and shifting geographical distributions. Virus was genetically sampled during this period at an overall rate of ~1.2%, though there was a substantial mismatch between case rates and genetic sampling nationwide. Viral genetic diversity tripled over this period but remained low in comparison to other widespread RNA virus pathogens, and although 54 amino acid changes were detected at frequencies exceeding 5%, linkage among them was not observed. Based on our collective observations, our analysis supports a targeted strategy for worldwide genetic surveillance as perhaps the most sensitive and efficient means of detecting new VOCs.

HIV infection is not curable with current antiretroviral therapy (ART) because a small fraction of CD4+ T cells infected prior to ART initiation persists. Understanding the nature of this latent reservoir and how it is created is essential to development of potentially curative strategies. The discovery that a large fraction of the persistently infected cells in individuals on suppressive ART are members of large clones greatly changed our view of the reservoir and how it arises. Rather than being the products of infection of resting cells, as was once thought, HIV persistence is largely or entirely a consequence of infection of cells that are either expanding or are destined to expand, primarily due to antigen-driven activation. Although most of the clones carry defective proviruses, some carry intact infectious proviruses; these clones comprise the majority of the reservoir. A large majority of both the defective and the intact infectious proviruses in clones of infected cells are transcriptionally silent; however, a small fraction expresses a few copies of unspliced HIV RNA. A much smaller fraction is responsible for production of low levels of infectious virus, which can rekindle infection when ART is stopped. Further understanding of the reservoir will be needed to clarify the mechanism(s) by which provirus expression is controlled in the clones of cells that constitute the reservoir.

In vivo clonal expansion of HIV-infected T cells is an important mechanism of viral persistence. In some cases, clonal expansion is driven by HIV proviral DNA integrated into one of a handful of genes. To investigate this phenomenon in vitro, we infected primary CD4+ T cells with an HIV construct expressing GFP and, after nearly 2 mo of culture and multiple rounds of activation, analyzed the resulting integration site distribution. In each of three replicates from each of two donors, we detected large clusters of integration sites with multiple breakpoints, implying clonal selection. These clusters all mapped to a narrow region within the STAT3 gene. The presence of hybrid transcripts splicing HIV to STAT3 sequences supports a model of LTR-driven STAT3 overexpression as a driver of preferential growth. Thus, HIV integration patterns linked to selective T cell outgrowth can be reproduced in cell culture. The single report of an HIV provirus in a case of AIDS-associated B-cell lymphoma with an HIV provirus in the same part of STAT3 also has implications for HIV-induced malignancy.

The characterisation of the HIV-1 reservoir, which consists of replication-competent integrated proviruses that persist on antiretroviral therapy (ART), is made difficult by the rarity of intact proviruses relative to those that are defective. While the only conclusive test for the replication-competence of HIV-1 proviruses is carried out in cell culture, genetic characterization of genomes by near full-length (NFL) PCR and sequencing can be used to determine whether particular proviruses have insertions, deletions, or substitutions that render them defective. Proviruses that are not excluded by having such defects can be classified as genetically intact and, possibly, replication competent. Identifying and quantifying proviruses that are potentially replication-competent is important for the development of strategies towards a functional cure. However, to date, there are no programs that can be incorporated into deep-sequencing pipelines for the automated characterization and annotation of HIV genomes. Existing programs that perform this work require manual intervention, cannot be widely installed, and do not have easily adjustable settings. Here, we present HIVIntact, a python-based software tool that characterises genomic defects in NFL HIV-1 sequences, allowing putative intact genomes to be identified in-silico. Unlike other applications that assess the genetic intactness of HIV genomes, this tool can be incorporated into existing sequence-analysis pipelines and applied to large next-generation sequencing datasets.

In 2020, SARS-CoV-2 spread across the United States (U.S.) in three phases distinguished by peaks in the numbers of infections and shifting geographical distribution. We investigated the viral genetic diversity in each phase using sequences publicly available prior to December 15th, 2020, when vaccination was initiated in the U.S. In Phase 1 (winter/spring), sequences were already dominated by the D614G Spike mutation and by Phase 3 (fall), genetic diversity of the viral population had tripled and at least 54 new amino acid changes had emerged at frequencies above 5%, several of which were within known antibody epitopes. These findings highlight the need to track the evolution of SARS-CoV-2 variants in the U.S. to ensure continued efficacy of vaccines and antiviral treatments., One Sentence Summary: SARS-CoV-2 genetic diversity in the U.S. increased 3-fold in 2020 and 54 emergent nonsynonymous mutations were detected.

Understanding HIV-1 persistence despite antiretroviral therapy (ART) is of paramount importance. Both single-genome sequencing (SGS) and integration site analysis (ISA) provide useful information regarding the structure of persistent HIV DNA populations; however, until recently, there was no way to link integration sites to their cognate proviral sequences. Here, we used multiple-displacement amplification (MDA) of cellular DNA diluted to a proviral endpoint to obtain full-length proviral sequences and their corresponding sites of integration. We applied this method to lymph node and peripheral blood mononuclear cells from 5 ART-treated donors to determine whether groups of identical subgenomic sequences in the 2 compartments are the result of clonal expansion of infected cells or a viral genetic bottleneck. We found that identical proviral sequences can result from both cellular expansion and viral genetic bottlenecks occurring prior to ART initiation and following ART failure. We identified an expanded T cell clone carrying an intact provirus that matched a variant previously detected by viral outgrowth assays and expanded clones with wild-type and drug-resistant defective proviruses. We also found 2 clones from 1 donor that carried identical proviruses except for nonoverlapping deletions, from which we could infer the sequence of the intact parental virus. Thus, MDA-SGS can be used for “viral reconstruction” to better understand intrapatient HIV-1 evolution and to determine the clonality and structure of proviruses within expanded clones, including those with drug-resistant mutations. Importantly, we demonstrate that identical sequences observed by standard SGS are not always sufficient to establish proviral clonality.

HIV-1 integrates its genome into the DNA of host cells. Consequently, HIV-1 genomes are copied with the host cell DNA during cellular division., Little is known about the emergence and persistence of human immunodeficiency virus (HIV)-infected T-cell clones in perinatally infected children. We analyzed peripheral blood mononuclear cells (PBMCs) for clonal expansion in 11 children who initiated antiretroviral therapy (ART) between 1.8 and 17.4 months of age and with viremia suppressed for 6 to 9 years. We obtained 8,662 HIV type 1 (HIV-1) integration sites from pre-ART samples and 1,861 sites from on-ART samples. Expanded clones of infected cells were detected pre-ART in 10/11 children. In 8 children, infected cell clones detected pre-ART persisted for 6 to 9 years on ART. A comparison of integration sites in the samples obtained on ART with healthy donor PBMCs infected ex vivo showed selection for cells with proviruses integrated in BACH2 and STAT5B. Our analyses indicate that, despite marked differences in T-cell composition and dynamics between children and adults, HIV-infected cell clones are established early in children, persist for up to 9 years on ART, and can be driven by proviral integration in proto-oncogenes.

The latent HIV-1 reservoir is comprised of stably integrated and intact proviruses with limited to no viral transcription. It has been proposed that latent infection may be maintained by methylation of pro-viral DNA. Here, for the first time, we investigate the cytosine methylation of a replication competent provirus (AMBI-1) found in a T cell clone in a donor on antiretroviral therapy (ART). Methylation profiles of the AMBI-1 provirus were compared to other proviruses in the same donor and in samples from three other individuals on ART, including proviruses isolated from lymph node mononuclear cells (LNMCs) and peripheral blood mononuclear cells (PBMCs). We also evaluated the apparent methylation of cytosines outside of CpG (i.e., CpH) motifs. We found no evidence for methylation in AMBI-1 or any other provirus tested within the 5′ LTR promoter. In contrast, CpG methylation was observed in the env-tat-rev overlapping reading frame. In addition, we found evidence for differential provirus methylation in cells isolated from LNMCs vs. PBMCs in some individuals, possibly from the expansion of infected cell clones. Finally, we determined that apparent low-level methylation of CpH cytosines is consistent with occasional bisulfite reaction failures. In conclusion, our data do not support the proposition that latent HIV infection is associated with methylation of the HIV 5′ LTR promoter.

HIV persists during antiretroviral therapy (ART) as integrated proviruses in cells descended from a small fraction of the CD4+ T cells infected prior to the initiation of ART. To better understand what controls HIV persistence and the distribution of integration sites (IS), we compared about 15,000 and 54,000 IS from individuals pre-ART and on ART, respectively, with approximately 395,000 IS from PBMC infected in vitro. The distribution of IS in vivo is quite similar to the distribution in PBMC, but modified by selection against proviruses in expressed genes, by selection for proviruses integrated into one of 7 specific genes, and by clonal expansion. Clones in which a provirus integrated in an oncogene contributed to cell survival comprised only a small fraction of the clones persisting in on ART. Mechanisms that do not involve the provirus, or its location in the host genome, are more important in determining which clones expand and persist., Author summary In HIV-infected individuals, a small fraction of the infected cells persist and divide. This reservoir persists during fully suppressive ART and can rekindle the infection if ART is discontinued. Because the number of possible sites of HIV DNA integration is very large, each infected cell, and all of its descendants, can be identified by the site where the provirus is integrated (IS). To understand the selective forces that determine the fates of infected cells in vivo, we compared the distribution of HIV IS in freshly-infected cells to cells from HIV-infected donors sampled both before and during ART. We found that, as previously reported, integration favors highly-expressed genes. However, over time, the fraction of cells with proviruses integrated in highly-expressed genes decreases, implying that they grow less well. There are exceptions to this broad negative selection. When a provirus is integrated in a specific region in one of seven genes, the proviruses affect the expression of the target gene, promoting growth and/or survival of the cell. Although this effect is striking, it is only a minor component of the forces that promote the growth and survival of the population of infected cells during ART.

The simultaneous discovery in 1970 of reverse transcriptase in virions of retroviruses by Howard Temin and David Baltimore was perhaps the most dramatic scientific moment of the second half of the 20th century. Ten years previously, Temin’s observation of cells transformed by Rous Sarcoma virus led him to the conclusion that retroviruses replicate through a DNA intermediate he called the provirus. This heretical hypothesis was greeted with derision by fellow scientists; Temin and Baltimore performed a simple experiment, rapidly reproduced, and convincing to all. Its result was a major paradigm shift—reversal of the central dogma of molecular biology. It immediately grabbed the attention of both the scientific and lay press. It also came at a key time for cancer research, at the start of the “War on Cancer.” As a theoretical base and fundamental molecular tool, it enabled a decade of (largely fruitless) search for human oncogenic retroviruses but laid the foundation for the discovery of HIV 13 years later, leading to the development of effective therapy. I had the good fortune, as a student in Temin’s lab, to witness these events. I am honored to be able to share my recollection on the occasion of their 50th anniversary.

HIV persists during antiretroviral therapy (ART) as integrated proviruses in cells descended from a small fraction of the CD4+ T cells infected prior to the initiation of ART. To better understand what controls HIV persistence and the distribution of integration sites (IS), we compared about 16,000 and 54,000 IS from individuals pre-ART and on ART, respectively, with approximately 385,000 IS from PBMC infected in vitro. The distribution of IS in vivo is quite similar to the distribution in PBMC, modified by selection against proviruses in expressed genes, by selection for proviruses integrated into one of 6 specific genes, and by clonal expansion. The clones in which a provirus integrated in an oncogene contributed to the survival of the clone comprise only a small fraction of the clones that persist in HIV-infected individuals on ART. Mechanisms that do not involve the provirus, or its location in the host genome, are more important in determining which clones expand and persist.Author SummaryIn HIV-infected individuals, a small fraction of the infected cells persist and divide. This reservoir persists on ART and can rekindle the infection if ART is discontinued. Because the number of possible sites of HIV DNA integration is very large, each infected cell, and all of its descendants, can be identified by the site where the provirus is integrated (IS). To understand the selective forces that determine the fates of infected cells in vivo, we compared the distribution of HIV IS in freshly-infected cells to cells from HIV-infected donors sampled both before and during ART. We found that, as has been previously reported, integration favors highly-expressed genes. However, over time the fraction of cells with proviruses integrated in highly-expressed genes decreases, implying that they grow less well. There are exceptions to this broad negative selection. When a provirus is integrated in a specific region in one of six genes, the proviruses affect the expression of the target gene, promoting growth and/or survival of the cell. Although this effect is striking, it is only a minor component of the forces that promote the growth and survival of the population of infected cells during ART.

Little is known about the emergence and persistence of HIV-infected T cell clones in perinatally-infected children. We analyzed peripheral blood mononuclear cells for clonal expansion in 11 children who initiated antiretroviral therapy (ART) between 1.8-17.4 months of age and with viremia suppressed for 6-9 years. We obtained 8,662 HIV-1 integration sites from pre-ART and 1,861 sites on ART. Expanded clones of infected cells were detected pre-ART in 10/11 children. In 8 children, infected cell clones detected pre-ART persisted for 6-9 years on ART. A comparison of integration sites in the samples obtained on ART with healthy donor PBMC infected ex-vivo showed selection for cells with proviruses integrated in BACH2 and STAT5B. Our analyses indicate that, despite marked differences in T cell composition and dynamics between children and adults, HIV-infected cell clones are established early in children, persist for up to 9 years on ART, and can be driven by proviral integration in proto-oncogenes.

An amendment to this paper has been published and can be accessed via the original article.

BACKGROUNDHIV-1 viremia that is not suppressed by combination antiretroviral therapy (ART) is generally attributed to incomplete medication adherence and/or drug resistance. We evaluated individuals referred for non-suppressible viremia (plasma HIV-1 RNA above 40 copies/ml) who reported adherence to ART and did not show drug resistance to their current regimen.METHODSSamples were collected from at least two time points from eight donors who had non-suppressible viremia for more than six months on ART. Single templates of HIV-1 RNA obtained from plasma and viral outgrowth of cultured cells and from proviral DNA were PCR-amplified and sequenced for evidence of clones of cells that produced infectious viruses. Clones were identified by host-proviral integration site analysis.RESULTSHIV-1 genomic RNAs with identical sequences were identified in plasma samples from all eight donors. The identical viral RNA sequences did not change over time and lacked resistance to the ART regimen. In four of the donors, viral RNA sequences obtained from plasma matched those sequences from viral outgrowth cultures, indicating that the viruses were replication-competent. Integration sites for infectious proviruses from those four donors were mapped to introns of theMATR3,ZNF268,ZNF721/ABCA11P, andABCA11Pgenes. The sizes of the clones were from 50 million to 350 million cells.CONCLUSIONClones of HIV-1-infected cells producing virus can cause failure of ART to suppress viremia despite medication adherence and absence of drug resistance. The mechanisms involved in clonal expansion and persistence need to be defined to eliminate viremia and the HIV-1 reservoir.

Combination antiretroviral therapy (cART) controls but does not eradicate HIV infection, HIV persistence is the principal obstacle to curing infections. The proportion of defective proviruses increases during cART, but the dynamics of this process are not well understood, and a quantitative analysis of how the proviral landscape is reshaped after cART is initiated is critical to understanding how HIV persists. Here, we studied longitudinal samples from HIV infected individuals undergoing long term cART using multiplexed Droplet Digital PCR (ddPCR) approaches to quantify the proportion of deleted proviruses in lymphocytes. In most individuals undergoing cART, HIV proviruses that contain gag are lost more quickly than those that lack gag. Increases in the fraction of gag-deleted proviruses occurred only after 1&ndash, 2 years of therapy, suggesting that the immune system, and/or toxicity of viral re-activation helps to gradually shape the proviral landscape. After 10&ndash, 15 years on therapy, there were as many as 3.5&ndash, 5 times more proviruses in which gag was deleted or highly defective than those containing intact gag. We developed a provirus-specific ddPCR approach to quantify individual clones. Investigation of a clone of cells containing a deleted HIV provirus integrated in the HORMAD2 gene revealed that the cells underwent a massive expansion shortly after cART was initiated until the clone, which was primarily in effector memory cells, dominated the population of proviruses for over 6 years. The expansion of this HIV-infected clone had substantial effects on the overall proviral population.

The prevalence of HIV-1 drug resistance is increasing worldwide and monitoring its emergence is important for the successful management of populations receiving combination antiretroviral therapy (cART). Using Ultrasensitive Single-Genome Sequencing (uSGS), a next-generation method that avoids PCR bias and PCR recombination, a recent report showed that pre-existing dual-class drug resistance mutations linked on the same viral genomes were predictive of treatment failure while unlinked mutations were not. Because of the large numbers of sequences generated by uSGS and other next-generation sequencing methods, it is difficult to assess each sequence individually for linked resistance mutations. Several software/programs exist to report the frequencies of individual mutations in large datasets but they provide no information on their linkage. Here, we report the HIV-DRLink program, a research tool that provides mutation frequencies in the total dataset as well as their linkage to other mutations conferring resistance to the same or different drug classes. The HIV-DRLink program should only be used on datasets generated by methods that eliminate artifacts due to PCR recombination, for example, standard Single-Genome Sequencing (SGS) or Ultrasensitive Single-Genome Sequencing (uSGS). HIV-DRLink is exclusively a research tool and is not intended to inform clinical decisions.

Background Increased transcription of the human endogenous retrovirus group HERV-K (HML-2) is often seen during disease. Although the mechanism of its tissue-specific activation is unclear, research shows that LTR CpG hypomethylation alone is not sufficient to induce its promoter activity and that the transcriptional milieu of a malignant cell contributes, at least partly, to differential HML-2 expression. Results We analyzed the relationship between LTR sequence variation and promoter expression patterns in human breast cancer cell lines, finding them to be positively correlated. In particular, two proviruses (3q12.3 and 11p15.4) displayed increased activity in almost all tumorigenic cell lines sampled. Using a transcription factor binding site prediction algorithm, we identified two unique binding sites in each 5′ LTR that appeared to be associated with inducing promoter activity during neoplasia. Genomic analysis of the homologous proviruses in several non-human primates indicated post-integration genetic drift in two transcription factor binding sites, away from the ancestral sequence and towards the active form. Based on the sequences of 2504 individuals from the 1000 Genomes Project, the active form of the 11p15.4 site was found to be polymorphic within the human population, with an allele frequency of 51%, whereas the activating mutation in the 3q12.3 provirus was fixed in humans but not present in the orthologous provirus in chimpanzees or gorillas. Conclusions These data suggest that stage-specific transcription factors at least partly contribute to LTR promoter activity during transformation and that, in some cases, transcription factor binding site polymorphisms may be responsible for the differential HML-2 expression often seen between individuals. Electronic supplementary material The online version of this article (10.1186/s12977-018-0441-2) contains supplementary material, which is available to authorized users.

AGS-004 consists of matured autologous dendritic cells co-electroporated with in vitro transcribed RNA encoding autologous HIV antigens. In an open-label, single arm sub-study of AGS-004-003, AGS-004 was administered monthly to suppressed participants who started antiretroviral therapy (ART) during acute HIV infection. HIV-1 specific T cell responses were measured by multicolor flow cytometry after 3–4 doses. The frequency of resting CD4+ T-cell infection (RCI) was measured by quantitative viral outgrowth assay. Participants demonstrating increased immune response postvaccination were eligible for analytic treatment interruption (ATI). AGS-004 induced a positive immune response defined as ≥2-fold increase from baseline in the number of multifunctional HIV-1 specific CD28+/CD45RA− CD8+ effector/memory cytoxic T-lymphocytes (CTLs) in all six participants. All participants underwent ATI with rebound viremia at a median of 29 days. Immune correlates between time to viral rebound and the induction of effector CTLs were determined. Baseline RCI was low in most participants (0.043–0.767 IUPM). One participant had a >2-fold decrease (0.179–0.067 infectious units per million [IUPM]) in RCI at week 10. One participant with the lowest RCI had the longest ATI. AGS-004 dendritic cell administration increased multifunctional HIV-specific CD28+/CD45RA− CD8+ memory T cell responses in all participants, but did not permit sustained ART interruption. However, greater expansion of CD28−/CCR7−/CD45RA− CD8+ effector T cell responses correlated with a longer time to viral rebound. AGS-004 may be a useful tool to augment immune responses in the setting of latency reversal and eradication strategies.

Efforts to cure HIV-1 infection require better quantification of the HIV-1 reservoir, particularly the clones of cells harboring replication-competent (intact) proviruses, termed repliclones. The digital droplet PCR assays commonly used to quantify intact proviruses do not differentiate among specific repliclones, thus the dynamics of repliclones are not well defined. The major challenge in tracking repliclones is the relative rarity of the cells carrying specific intact proviruses. To date, detection and accurate quantification of repliclones requires in-depth integration site sequencing. Here, we describe a simplified workflow using integration site-specific qPCR (IS-qPCR) to determine the frequencies of the proviruses integrated in individual repliclones. We designed IS-qPCR to determine the frequencies of repliclones and clones of cells that carry defective proviruses in samples from three donors. Comparing the results of IS-qPCR with deep integration site sequencing data showed that the two methods yielded concordant estimates of clone frequencies (r = 0.838). IS-qPCR is a potentially valuable tool that can be applied to multiple samples and cell types over time to measure the dynamics of individual repliclones and the efficacy of treatments designed to eliminate them.

We hypothesized that HIV-1 with dual-class but not single-class drug resistance mutations linked on the same viral genome, present in the virus population before initiation of antiretroviral therapy (ART), would be associated with failure of ART to suppress viremia. To test this hypothesis, we utilized an ultrasensitive single-genome sequencing assay that detects rare HIV-1 variants with linked drug resistance mutations (DRMs). A case (ART failure) control (nonfailure) study was designed to assess whether linkage of DRMs in pre-ART plasma samples was associated with treatment outcome in the nevirapine/tenofovir/emtricitabine arm of the AIDS Clinical Trials Group A5208/Optimal Combined Therapy After Nevirapine Exposure (OCTANE) Trial 1 among women who had received prior single-dose nevirapine. Ultrasensitive single-genome sequencing revealed a significant association between pre-ART HIV variants with DRMs to 2 drug classes linked on the same genome (dual class) and failure of combination ART with 3 drugs to suppress viremia. In contrast, linked, single-class DRMs were not associated with ART failure. We conclude that linked dual-class DRMs present before the initiation of ART are associated with ART failure, whereas linked single-class DRMs are not.

Background HIV-1 proviruses can persist during ART in clonally-expanded populations of CD4+ T cells. To date, few examples of an expanded clones containing replication-competent proviruses exist, although it is suspected to be common. One such clone, denoted AMBI-1 (Maldarelli et al., 2014), was also a source of persistent viremia on ART, begging the question of how the AMBI-1 clone can survive despite infection with a replication-competent, actively-expressing provirus. We hypothesized that only a small fraction of cells within the AMBI-1 clone are activated to produce virus particles during cell division while the majority remain latent despite division, ensuring their survival. To address this question, we determined the fraction of HIV-1 proviruses within the AMBI-1 clone that expresses unspliced cell-associated RNA during ART and compared this fraction to 33 other infected T cell clones within the same individual. Results In total, 34 different clones carrying either intact or defective proviruses in "Patient 1" from Maldarelli et al. (2014) were assessed. We found that 2.3% of cells within the AMBI-1 clone contained unspliced HIV-1 RNA. Highest levels of HIV-1 RNA were found in the effector memory (EM) T cell subset. The fraction of cells within clones that contained HIV-1 RNA was not different in clones with intact (median 2.3%) versus defective (median 3.5%) proviruses (p = 0.2). However, higher fractions and levels of RNA were found in cells with proviruses containing multiple drug resistance mutations, including those contributing to rebound viremia. Conclusion These findings show that the vast majority of HIV-1 proviruses within expanded T cell clones, including intact proviruses, may be transcriptionally silent at any given time, implying that infected T cells may be able to be activated to proliferate without inducing the expression of the integrated provirus or, alternatelively, may be able to proliferate without cellular activation. The results of this study suggest that the long, presumed correlation between the level of cellular and proviral activation may not be accurate and, therefore, requires further investigation.

To investigate the possibility that HIV-1 replication in lymph nodes sustains the reservoir during antiretroviral therapy (ART), we looked for evidence of viral replication in 5 donors after up to 13 years of viral suppression. We characterized proviral populations in lymph nodes and peripheral blood before and during ART, evaluated the levels of viral RNA expression in single lymph node and blood cells, and characterized the proviral integration sites in paired lymph node and blood samples. Proviruses with identical sequences, identical integration sites, and similar levels of RNA expression were found in lymph nodes and blood samples collected during ART, and no single sequence with significant divergence from the pretherapy population was detected in either blood or lymph nodes. These findings show that all detectable persistent HIV-1 infection is consistent with maintenance in lymph nodes by clonal proliferation of cells infected before ART and not by ongoing viral replication during ART.