Biology subjects

Kearney, M. F.

Publications and source records attributed to Kearney, M. F..

11 recordsLinked to original sources

bioRxiv2026

Ultrasensitive single-genome sequencing reveals strong purifying selection in acute HIV-1 infection

HIV transmission from one individual to another occurs by one or a small number of virions followed by spread and genetic diversification into a complex quasispecies. To understand the early events in this process, we investigated how HIV-1 genomes diversify within the first two to three weeks after transmission by use of ultra-deep single subgenomic sequencing of over 10,000 plasma RNA genomes in each of a cohort of 15 individuals in acute infection. This approach confirmed transmission of one or a few transmitted/founder (TF) viral lineages and very limited early divergence from the founder sequences. Most observed variants that differed from the TF included single nucleotide changes attributable to HIV-1 reverse transcriptase (RT) error or host APOBEC3G/F activity. Comparing the number of expected versus observed changes after transmission indicated that most de novo mutations do not persist in the virus population, consistent with strong purifying selection. We found little evidence that early diversification is driven by reversions to subtype consensus or by cytotoxic T lymphocyte pressure, although rare multi-mutation lineages suggest occasional influences. Together, these findings indicate that early HIV-1 evolution is influenced by stochastic and host-mediated mutational processes (e.g., APOBEC3G/F) filtered by strong purifying selection. The strong purifying selection observed in the early weeks of HIV-1 infection may provide an opportunity to investigate the potential of new interventions to induce viremic control, such as combinations of broadly neutralizing antibodies, cellular immunotherapy, or mRNA therapeutic vaccination.

microbiology↗

bioRxiv2025

HIV-1 envelopes from virions that persist in plasma on antiretroviral therapy show reduced susceptibility to autologous immunoglobulins and variable sensitivity to broadly neutralizing monoclonal antibodies

Despite adherence to antiretroviral therapy (ART), low levels of plasma virus persist in most individuals with HIV-1. In a small subset of these individuals, this non-suppressible viremia is detected by routine clinical testing (plasma HIV-1 RNA >20 copies/ml) and has been shown to be of clonal cellular origin. The mechanisms by which these virions escape immune clearance is not defined, but reduced binding and neutralization of their surface envelope protein (Env) by antibodies (Abs) may contribute. To assess this possibility, we measured the monoclonal antibody (mAb) sensitivity of HIV-1 Env in plasma virus from 4 well-characterized individuals on ART with non-suppressible viremia. Thirty-two env plasma sequences were used to produce pseudovirus for neutralization assays with both autologous plasma and a panel of 15 recombinant antibodies that targeted different regions of HIV-1 Env protein. We found that autologous plasma had no neutralizing activity against the pseudoviruses consistent with persistence of viremia. In general, the Envs from the non-suppressible plasma virus were also less sensitive to mAb neutralization compared controls: tier 1 (6535), tier 2 (TRO11) or tier 3 (PVO) subtype B envs, although variability across Envs was evident. Two Env protein variants from one donor, R-09_A8 and R-09_C2, were less sensitive to VRC01 likely due to an additional N-glycan site at a VRC01 contact site and longer V5 regions. Most of the donor Envs were sensitive to at least two of three V3-glycan mAbs except for variant C-03_A6, which showed reduced sensitivity to all three. The CD4 binding site mAb 3BNC117 and the Gp41-specific mAb 10E8 neutralized pseudoviruses from all donors, indicating the potential for clearance of persistent viremia in these individuals studied. Author summaryIn a fraction of individuals on suppressive antiretroviral therapy (ART), episodes of persistent low-level viremia can be observed that is not attributed to ineffective ART and/or non-adherence. Previously in three individuals, we have shown that this viremia results from large HIV-1 infected T cell clones harboring replication-competent proviruses. The factor(s) that allow these infectious virions to be detected and not cleared by the immune system is unknown, suggesting that these virions can evade humoral responses. Using single genome sequencing, we amplified the env from plasma derived virions from these individuals, cloned env -containing amplicons into an expression vector to produce pseudo viruses, which we tested against either their autologous contemporaneous autologous immunoglobulins (Ig) or a panel of 15 recombinant monoclonal antibodies. The results revealed that these pseudovirus were not neutralized by their autologous Igs and exhibited complex pseudovirus-specific susceptibility profiles for the monoclonal antibodies they were tested against. Collectively, our findings suggest that despite resistance to autologous Ig, likely combinations of monoclonal antibodies will be needed to clear this persistent viremia.

immunology↗

bioRxiv2025

Differential HIV-1 Proviral Defects in Children vs. Adults on Antiretroviral Therapy

HIV-1 proviral landscapes were investigated using near full-length HIV single-genome sequencing on blood samples from 5 children with vertically acquired infection and on ART for [~]7-9 years. Proviral structures were compared to published datasets in children prior to ART, children on short-term ART, and adults on ART. We found a strong selection for large internal proviral deletions in children, especially deletions of the env gene. Only 2.5% of the proviruses were sequence-intact, lower than in the comparative datasets from adults. Of the proviruses that retained the env gene, >80% contained two or more defects, most commonly stop codons and/or gag start mutations. Significantly fewer defects in the major splice donor site (MSD) and packaging signal were found in the children on short or long-term ART compared to the adults, and tat was more frequently defective in children. These results suggest that different selection pressures shape the proviral landscape in children compared to adults and reveal potentially different genetic regions to target for measuring the intact HIV reservoir and for achieving HIV remission in children.

microbiology↗

bioRxiv2024

In vivo detection of HIV-1 antisense transcripts in untreated and ART-treated individuals

Natural antisense transcripts are expressed in eukaryotes, prokaryotes, and viruses and can possess regulatory functions at the transcriptional and/or post-transcriptional levels. In vitro studies have shown that HIV-1 antisense transcripts (AST) promote viral latency through epigenetic silencing of the proviral 5' long terminal repeat (LTR). However, expression of HIV-1 AST in vivo have not been convincingly demonstrated. Here, we used single RNA template amplification, detection, and sequencing to demonstrate expression of AST in unstimulated PBMC collected from people with HIV-1 (PWH). We found that AST had high genetic diversity that matched proviruses in cells from blood and lymph nodes. We measured a median of 26 copies of AST per 100 infected cells in PWH on ART and a median of 2 copies per 100 infected cells in PWH not on ART. The expression of HIV-1 AST in vivo is consistent with a potential regulatory role in regulation of HIV-1 expression.

microbiology↗

bioRxiv2024

HIV-1 control in vivo is related to the number but not the fraction of infected cells with viral unspliced RNA

In the absence of antiretroviral therapy (ART), a subset of individuals, termed HIV controllers, have levels of plasma viremia that are orders of magnitude lower than non-controllers who are at higher risk for HIV disease progression. In addition to having fewer infected cells resulting in fewer cells with HIV RNA, it is possible that lower levels of plasma viremia in controllers is due to a lower fraction of the infected cells having HIV-1 unspliced RNA (HIV usRNA) compared with non-controllers. To directly test this possibility, we used sensitive and quantitative single cell sequencing methods to compare the fraction of infected cells that contain one or more copies of HIV usRNA in peripheral blood mononuclear cells (PBMC) obtained from controllers and non-controllers. The fraction of infected cells containing HIV usRNA did not differ between the two groups. Rather, the levels of viremia were strongly associated with the total number of infected cells that had HIV usRNA, as reported by others, with controllers having 34-fold fewer infected cells per million PBMC. These results reveal for the first time that viremic control is not associated with a lower fraction of proviruses expressing HIV usRNA, unlike what is reported for elite controllers, but is only related to having fewer infected cells overall, maybe reflecting greater immune clearance of infected cells. Our findings show that proviral silencing is not a key mechanism for viremic control and will help to refine strategies towards achieving HIV remission without ART.

microbiology↗

bioRxiv2021

Engineered Bacteriophage T4 Nanoparticle as a Potential Targeted Activator of HIV-1 Latency in CD4+ Human T cells

A major barrier for HIV-1 eradication is the latent virus reservoir containing stably integrated and silent proviruses in CD4+ T-cells. Targeted reactivation and removal of this latent reservoir is a potential strategy for HIV-1 cure but remains a major challenge. Here, we investigated whether CD4-targeted bacteriophage T4 capsid nanoparticles that mimic HIV envelope can reactivate HIV-1 latency. The nanoparticles were arrayed with CD4-binding CD4-DARPin, or HIV-1 gp140 envelope trimer. When exposed to J-Lat T-cell model of HIV-1 latency or primary T-lymphocytes from human PBMCs, these nanoparticles activated CD4+ T-cells without causing global T-cell activation, which led to activation of HIV-1 proviral transcription, viral protein production and release. Intriguingly, the observed T-cell activation and HIV-1 latency reversal do not involve the classic PKC or NFAT pathways and did not lead to cytokine storm. These studies indicate that engineered non-infectious bacteriophages can be exploited for HIV-1 cure and targeted T-cell therapies.

immunology↗

bioRxiv2020

Integration in or Near Oncogenes Plays Only a Minor Role in Determining the in Vivo Distribution of HIV Integration Sites Before or During Suppressive Antiretroviral Therapy

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.

microbiology↗

bioRxiv2020

Early Emergence and Long-Term Persistence of HIV-Infected T Cell Clones in Children

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.

microbiology↗

bioRxiv2020

Mechanistic Analysis of the Broad Antiretroviral Resistance Conferred by HIV-1 Envelope Glycoprotein Mutations

Despite the effectiveness of antiretroviral (ARV) therapy, virological failure can occur in some HIV-1 infected patients in the absence of mutations in the proteins targeted by these drugs. We previously reported that, in vitro, the lab-adapted NL4-3 strain of HIV-1 can acquire resistance to the integrase inhibitor dolutegravir (DTG) by acquiring mutations in the envelope glycoprotein (Env) that enhance the ability of HIV-1 to spread via cell-cell transmission. In this study, we investigated whether Env-mediated drug resistance extends to ARVs other than DTG and whether it occurs in other HIV-1 isolates. We demonstrate that Env mutations can broadly confer resistance to multiple classes of ARVs in the context of cell-cell but not cell-free infection and also increase resistance to ARVs when coupled with target-gene drug resistance mutations. To investigate the mechanism of Env-mediated drug resistance, we evaluated the impact of the Env mutations on Env stability and conformational dynamics. We observe that the NL4-3 Env mutants display a more stable and closed Env conformation compared to WT virus and reduced rates of gp120 shedding. We also selected for mutations in the gp41 ectodomain of clinically relevant, CCR5-tropic isolates in the presence of DTG. These Env mutants exhibit reduced susceptibility to DTG, with effects on replication kinetics and Env structure that are HIV-1 strain-dependent. Finally, to examine a possible in vivo relevance of Env-mediated drug resistance, we performed single-genome sequencing of plasma-derived virus from five patients failing an integrase inhibitor-containing regimen. This analysis revealed the presence of several mutations in the highly conserved gp120-gp41 interface despite low frequency of resistance mutations in integrase. These results suggest a "stepping-stone" model whereby mutations in Env that enhance the ability of HIV-1 to spread via a cell-cell route increase the opportunity for the virus to acquire high-level drug resistance mutations in ARV-target genes. Author summaryAlthough combination antiretroviral (ARV) therapy has proven highly effective in controlling the progression of HIV disease, drug resistance can be a major obstacle to long-term treatment, particularly in resource-limited settings. In most cases, resistance arises from the accumulation of mutations in the ARV-target genes; however, in some cases, resistance develops without ARV target-gene mutations. We previously reported that mutations in the HIV-1 envelope glycoprotein (Env) confer resistance to an integrase inhibitor. Here we investigated the mechanism of Env-mediated drug resistance and the possible contribution of Env to virological failure in vivo. We demonstrate that Env mutations can confer broad resistance to multiple classes of ARVs and define the effect of the Env mutations on Env subunit interactions and sensitivity to neutralizing antibodies. We also selected for drug resistance mutations in Env in clinically relevant HIV-1 isolates. We observed that many Env mutations accumulated in individuals failing integrase inhibitor therapy despite a low frequency of resistance mutations in integrase. Our findings suggest that broad-based, Env-mediated drug resistance may impact current and possibly future therapeutic strategies. Our findings also provide clues towards understanding how ARV-treated patients can experience virological failure without acquiring drug resistance mutations in ARV-target genes.

microbiology↗

bioRxiv2020

HIV-1 Viremia Not Suppressible By Antiretroviral Therapy Can Originate from Large T-Cell Clones Producing Infectious Virus

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 the MATR3, ZNF268, ZNF721/ABCA11P, and ABCA11P genes. 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.

genomics↗

bioRxiv2019

HIV-DRLink: A tool for detecting linked HIV-1 drug resistance mutations in next generation sequencing data

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.

bioinformatics↗