Publications by authors named "John W Mellors"

257 Publications

Can Broadly Neutralizing HIV-1 Antibodies Help Achieve an ART-Free Remission?

Front Immunol 2021 12;12:710044. Epub 2021 Jul 12.

U.S. Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, MD, United States.

Many broadly neutralizing antibodies (bnAbs) targeting the HIV-1 envelope glycoprotein are being assessed in clinical trials as strategies for HIV-1 prevention, treatment, and antiretroviral-free remission. BnAbs can neutralize HIV-1 and target infected cells for elimination. Concerns about HIV-1 resistance to single bnAbs have led to studies of bnAb combinations with non-overlapping resistance profiles. This review focuses on the potential for bnAbs to induce HIV-1 remission, either alone or in combination with latency reversing agents, therapeutic vaccines or other novel therapeutics. Key topics include preliminary activity of bnAbs in preclinical models and in human studies of HIV-1 remission, clinical trial designs, and antibody design strategies to optimize pharmacokinetics, coverage of rebound-competent virus, and enhancement of cellular immune functions.
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http://dx.doi.org/10.3389/fimmu.2021.710044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8311790PMC
July 2021

Tracking HIV-1-Infected Cell Clones Using Integration Site-Specific qPCR.

Viruses 2021 Jun 25;13(7). Epub 2021 Jun 25.

Department of Medicine, University of Pittsburgh, 3550 Terrace Street, Scaife Hall-818, Pittsburgh, PA 15261, USA.

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 . 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 ( = 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.
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http://dx.doi.org/10.3390/v13071235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8310066PMC
June 2021

Comparison of methods to quantify inducible HIV-1 outgrowth.

J Virus Erad 2021 Jun 17;7(2):100043. Epub 2021 May 17.

Division of Infectious Diseases, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.

The quantitative viral outgrowth assay (qVOA) is the gold standard for measuring inducible, replication-competent HIV-1. Using MOLT4-R5 and SupT1-R5 cell lines instead of allogeneic blasts and HIV-1 RNA detection rather than p24 enzyme-immunoassay (EIA) has been proposed to improve the sensitivity of the qVOA. It is unclear, however, how these alternative approaches affect qVOA performance. We compared three qVOAs methods across 15 persons with HIV-1 on suppressive antiretroviral therapy and found that the MOLT4-R5 method yielded a significantly higher proportion of p24-positive wells (42%) than both the allogeneic blast (29%) and SupT1-R5 (32%) assays. Additionally, 5 of 7 qVOAs that were negative by p24 EIA showed viral outgrowth by HIV-1 RNA quantification (>10-fold increase within 7 days). These findings reveal the potential for underestimation of the latent, inducible reservoir by qVOA depending on the target cells used and the measure of viral outgrowth. Use of MOLT4-R5 cells with both p24 EIA and HIV-1 RNA to detect viral outgrowth was the most sensitive method.
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http://dx.doi.org/10.1016/j.jve.2021.100043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8176359PMC
June 2021

Antibody Responses After mRNA-Based COVID-19 Vaccination in Residential Older Adults: Implications for Reopening.

J Am Med Dir Assoc 2021 Aug 12;22(8):1593-1598. Epub 2021 Jun 12.

Department of Pathology, University of Pittsburgh, Pittsburgh, PA, USA; Clinical Laboratory, University of Pittsburgh Medical Center, Pittsburgh, PA, USA.

Objective: COVID-19 disproportionately impacts residents in long-term care facilities. Our objective was to quantify the presence and magnitude of antibody response in vaccinated, older adult residents at assisted living, personal care, and independent living communities.

Design: A cross-sectional quality improvement study was conducted March 15 - April 1, 2021 in the greater Pittsburgh region.

Setting And Population: Participants were older adult residents at assisted living, personal care, and independent living communities, who received mRNA-based COVID-19 vaccine. Conditions that impair immune responses were exclusionary criteria.

Methods: Sera were collected to measure IgG anti-SARS-CoV-2 antibody level with reflex to total anti-SARS-CoV-2 immunoglobulin levels, and blinded evaluation of SARS-CoV-2 pseudovirus neutralization titers. Descriptive statistics, Pearson correlation coefficients, and multiple linear regression analysis evaluated relationships between factors potentially associated with antibody levels. Spearman correlations were calculated between antibody levels and neutralization titers.

Results: All participants (N = 70) had received two rounds of vaccination and were found to have antibodies with wide variation in relative levels. Antibody levels trended lower in males, advanced age, current use of steroids, and longer length of time from vaccination. Pseudovirus neutralization titer levels were strongly correlated (P < .001) with Beckman Coulter antibody levels [D614 G NT50, r = 0.91; B.1.1.7 (UK) NT50, r = 0.91].

Conclusions And Implications: Higher functioning, healthier, residential older adults mounted detectable antibody responses when vaccinated with mRNA-based COVID-19 vaccines. Data suggests some degree of immunity is present during the immediate period following vaccination. However, protective effects remain to be determined in larger studies as clinical protection is afforded by ongoing adaptive immunity, which is known to be decreased in older adults. This study provides important preliminary results on level of population risk in older adult residents at assisted living, personal care, and independent living communities to inform reopening strategies, but are not likely to be translatable for residents in nursing homes.
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http://dx.doi.org/10.1016/j.jamda.2021.06.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8196346PMC
August 2021

Frequency of Post Treatment Control Varies by ART Restart and Viral Load Criteria.

AIDS 2021 Jun 14. Epub 2021 Jun 14.

Brigham and Women's Hospital, Harvard Medical School Harvard T.H. Chan School of Public Health, Boston, Massachusetts University of California, San Francisco, San Francisco, California Case Western Reserve University, Cleveland, Ohio University of Arizona, Tucson, Arizona University of Massachusetts-Baystate, Springfield, Massachusetts University of North Carolina, Chapel Hill, North Carolina National Institute of Allergy and Infectious Diseases, Bethesda, Maryland University of Caliornia, San Diego, San Diego, California Weill Cornell Medicine, New York, New York University of Pittsburgh, Pittsburgh, Pennsylvania Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts University of California, Los Angeles, Los Angeles, California University of Minnesota, Minneapolis, Minnesota University of Pennsylvania, Philadelphia, Pennsylvania University of Washington, Seattle, Washington, USA.

Clinical trials including an analytical treatment interruption (ATI) are vital for evaluating the efficacy of novel strategies for HIV remissions. We briefly describe an interactive tool for predicting viral rebound timing in ATI trials and the impact of posttreatment controller (PTC) definitions on PTC frequency estimates. A 4-week viral load threshold of 1000 cps/ml provides both high specificity and sensitivity for PTC detection. PTC frequency varies greatly based on the definition of a PTC.
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http://dx.doi.org/10.1097/QAD.0000000000002978DOI Listing
June 2021

Improving the Outcomes of Immunocompromised Patients with COVID-19.

Clin Infect Dis 2021 May 5. Epub 2021 May 5.

Division of Infectious Diseases, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.

Recent case studies have highlighted that certain immunocompromised individuals are at risk for prolonged SARS-CoV-2 replication, intra-host viral evolution of multiply-mutated variants, and poor clinical outcomes. The immunologic determinants of this risk, the duration of infectiousness, and optimal treatment and prevention strategies in immunocompromised hosts are ill-defined. Of additional concern is the widespread use of immunosuppressive medications (corticosteroids, IL-6 antagonists) to treat COVID-19, which may enhance and prolong viral replication in the context of immunodeficiency. We outline the rationale for four inter-related approaches to usher in an era of evidence-based medicine for optimal management of immunocompromised patients with COVID-19: 1) multicenter pathogenesis and outcomes studies to relate the risk of severe disease to the type and degree of immunodeficiency; 2) studies evaluating immunologic responses to SARS-CoV-2 vaccines; 3) studies evaluating the efficacy of monoclonal antibodies for primary prophylaxis; and 4) clinical trials of novel antiviral agents for the treatment of COVID-19.
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http://dx.doi.org/10.1093/cid/ciab397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8135839PMC
May 2021

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

mBio 2021 04 8;12(2). Epub 2021 Apr 8.

HIV Dynamics and Replication Program, CCR, National Cancer Institute, Frederick, Maryland, USA.

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 showed selection for cells with proviruses integrated in and 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. 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. Pediatric immune systems differ significantly from adults, consisting primarily of naive T cells, which have low expression of the HIV-1 coreceptor CCR5. This difference may result in variances in the number or size of infected cell clones that persist in children on ART. Here, we provide the most extensive analysis of the integration landscape of HIV-1 in children. We found that, despite the largely naive cell populations in neonatal immune systems, patterns of HIV-1 integration and the size of infected cell clones are as large and widespread as those in adults. Furthermore, selection for integration events in proto-oncogenes were observed in children despite early ART. If such cell clones persist for the life span of these individuals, there may be long-term consequences that have yet to be realized.
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http://dx.doi.org/10.1128/mBio.00568-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8092253PMC
April 2021

Integration in oncogenes plays only a minor role in determining the in vivo distribution of HIV integration sites before or during suppressive antiretroviral therapy.

PLoS Pathog 2021 04 7;17(4):e1009141. Epub 2021 Apr 7.

National Cancer Institute, Frederick, Maryland, United States of America.

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.
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http://dx.doi.org/10.1371/journal.ppat.1009141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8055010PMC
April 2021

Scaling Up Covid-19 Vaccination in Africa - Lessons from the HIV Pandemic.

N Engl J Med 2021 Jul 31;385(3):196-198. Epub 2021 Mar 31.

From the Departments of Epidemiology and of Infectious Diseases and Microbiology and the Center for Global Health, University of Pittsburgh Graduate School of Public Health (J.B.N.); and the Division of Infectious Diseases, Department of Medicine, University of Pittsburgh School of Medicine (J.W.M.) - both in Pittsburgh; the Department of Medicine and the Center for Infectious Diseases, Stellenbosch University, Faculty of Medicine and Health Sciences, Cape Town, South Africa (J.B.N.); the Departments of Epidemiology and International Health, Johns Hopkins Bloomberg School of Public Health (J.B.N.), and the Department of Pediatrics and the Institute of Human Virology, University of Maryland School of Medicine (N.A.S.-A.) - both in Baltimore; the Pediatric/Adolescent HIV Unit and International Research Center of Excellence, Institute of Human Virology Nigeria, Abuja, Nigeria (N.A.S.-A.); the Department of Pediatrics and Child Health, School of Medical Sciences, University of Cape Coast, Cape Coast, Ghana (N.A.S.-A.); the Division of Infection and Immunity, University College London, and the NIHR Biomedical Research Centre, University College London Hospitals - both in London (A.Z.); the African Forum for Research and Education in Health, Kumasi, Ghana (J.B.N., N.A.S.-A., A.Z.); and the Elizabeth Glaser Pediatric AIDS Foundation, Washington, DC (L.M.M.).

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http://dx.doi.org/10.1056/NEJMp2103313DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8291168PMC
July 2021

The emerging plasticity of SARS-CoV-2.

Science 2021 03;371(6536):1306-1308

Division of Infectious Diseases, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA.

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http://dx.doi.org/10.1126/science.abg4493DOI Listing
March 2021

Preprocedural SARS-CoV-2 Testing to Sustain Medically Needed Health Care Delivery During the COVID-19 Pandemic: A Prospective Observational Study.

Open Forum Infect Dis 2021 Feb 18;8(2):ofab022. Epub 2021 Jan 18.

Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.

Background: We implemented a preprocedural severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) screening initiative designed to sustain health care during a time when the extent of SARS-CoV-2 infection was unknown.

Methods: This was a prospective study of patients undergoing procedures at 3 academic hospitals in Pittsburgh, Pennsylvania (April 21-June 11), and 19 community hospitals across Middle/Western Pennsylvania and Southwestern New York (May 1-June 11). Patients at academic hospitals underwent symptom screening ≤7 days preprocedure, then SARS-CoV-2 nasopharyngeal polymerase chain reaction (PCR) testing 1-4 days preprocedure. A subset also underwent day-of-procedure testing. Community hospital patients underwent testing per local protocols. We report SARS-CoV-2 PCR positivity rates, impact, and barriers to testing encountered through June 11. PCR positivity rates of optional preprocedural SARS-CoV-2 testing for 2 consecutive periods following the screening initiative are also reported.

Results: Of 5881 eligible academic hospital patients, 2415 (41.1%) were tested (April 21-June 11). Lack of interest, distance, self-isolation, and nursing home/incarceration status were barriers. There were 11 PCR-positive patients (10 asymptomatic) among 10 539 patients tested (0.10%; 95% CI, 0.05%-0.19%): 3/2415 (0.12%; 95% CI, 0.02%-0.36%) and 8/8124 (0.10%; 95% CI, 0.04%-0.19%) at academic and community hospitals, respectively. Procedures were performed as scheduled in 40% (4/10) of asymptomatic PCR-positive patients. Positivity increased during subsequent coronavirus disease 2019 (COVID-19) surges: 54/34948 (0.15%; 95% CI, 0.12%-0.20%) and 101/24741 (0.41%; 95% CI, 0.33%-0.50%) PCR-positive patients from June 12-September 10 and September 11-December 15, respectively ( < .0001).

Conclusions: Implementing preprocedural PCR testing was complex and revealed low infection rates (0.24% overall), which increased during COVID-19 surges. Additional studies are needed to define the COVID-19 prevalence threshold at which universal preprocedural screening is warranted.
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http://dx.doi.org/10.1093/ofid/ofab022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7880268PMC
February 2021

Safety, uptake, and use of a dapivirine vaginal ring for HIV-1 prevention in African women (HOPE): an open-label, extension study.

Lancet HIV 2021 02;8(2):e87-e95

Magee-Womens Research Institute, Pittsburgh, PA, USA.

Background: Two phase 3 clinical trials showed that use of a monthly vaginal ring containing 25 mg dapivirine was well tolerated and reduced HIV-1 incidence in women by approximately 30% compared with placebo. We aimed to evaluate use and safety of the dapivirine vaginal ring (DVR) in open-label settings with high background rates of HIV-1 infection, an important step for future implementation.

Methods: We did a phase 3B open-label extension trial of the DVR (MTN-025/HIV Open-label Prevention Extension [HOPE]). Women who were HIV-1-negative and had participated in the MTN-020/ASPIRE phase 3 trial were offered 12 months of access to the DVR at 14 clinical research centres in Malawi, South Africa, Uganda, and Zimbabwe. At each visit (monthly for 3 months, then once every 3 months), women chose whether or not to accept the offer of the ring. Used, returned rings were tested for residual amounts of dapivirine as a surrogate marker for adherence. HIV-1 serological testing was done at each visit. Dapivirine amounts in returned rings and HIV-1 incidence were compared with data from the ASPIRE trial, and safety was assessed. This study is registered with ClinicalTrials.gov, NCT02858037.

Findings: Between July 16, 2016, and Oct 10, 2018, of 1756 women assessed for eligibility, 1456 were enrolled and participated in the study. Median age was 31 years (IQR 27-37). At baseline, 1342 (92·2%) women chose to take the DVR; ring acceptance was more than 79% at each visit up until 12 months and 936 (73·2%) of 1279 chose to take the ring at all visits. 12 530 (89·3%) of 14 034 returned rings had residual dapivirine amounts consistent with some use during the previous month (>0·9 mg released) and the mean dapivirine amount released was greater than in the ASPIRE trial (by 0·21 mg; p<0·0001). HIV-1 incidence was 2·7 per 100 person-years (95% CI 1·9-3·8, 35 infections), compared with an expected incidence of 4·4 per 100 person-years (3·2-5·8) among a population matched on age, site, and presence of a sexually transmitted infection from the placebo group of ASPIRE. No serious adverse events or grade 3 or higher adverse events observed were assessed as related to the DVR.

Interpretation: High uptake and persistent use in this open-label extension study support the DVR as an HIV-1 prevention option for women. With an increasing number of HIV-1 prophylaxis choices on the horizon, these results suggest that the DVR will be an acceptable and practical option for women in Africa.

Funding: The Microbicide Trials Network and the National Institute of Allergy and Infectious Diseases, The Eunice Kennedy Shriver National Institute of Child Health and Human Development, and the National Institute of Mental Health, all components of the US National Institutes of Health.
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http://dx.doi.org/10.1016/S2352-3018(20)30304-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8038210PMC
February 2021

Intractable COVID-19 and Prolonged SARS-CoV-2 Replication in a CAR-T-cell Therapy Recipient: A Case Study.

Clin Infect Dis 2021 Jan 28. Epub 2021 Jan 28.

Division of Infectious Diseases, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.

A CAR-T-cell recipient developed severe COVID-19, intractable RNAemia, and viral replication lasting >2 months. Pre-mortem endotracheal aspirate contained 2x10 10 SARS-CoV-2 RNA copies/mL and infectious virus. Deep sequencing revealed multiple sequence variants consistent with intra-host virus evolution. SARS-CoV-2 humoral and cell-mediated immunity were minimal. Prolonged transmission from immunosuppressed patients is possible.
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http://dx.doi.org/10.1093/cid/ciab072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7929077PMC
January 2021

Dendritic cells focus CTL responses toward highly conserved and topologically important HIV-1 epitopes.

EBioMedicine 2021 Jan 12;63:103175. Epub 2021 Jan 12.

Department of Infectious Diseases and Microbiology, University of Pittsburgh, Graduate School of Public Health, Pittsburgh, PA, United States. Electronic address:

Background: During early HIV-1 infection, immunodominant T cell responses to highly variable epitopes lead to the establishment of immune escape virus variants. Here we assessed a type 1-polarized monocyte-derived dendritic cell (MDC1)-based approach to selectively elicit cytotoxic T lymphocyte (CTL) responses against highly conserved and topologically important HIV-1 epitopes in HIV-1-infected individuals from the Thailand RV254/SEARCH 010 cohort who initiated antiretroviral therapy (ART) during early infection (Fiebig stages I-IV).

Methods: Autologous MDC1 were used as antigen presenting cells to induce in vitro CTL responses against HIV-1 Gag, Pol, Env, and Nef as determined by flow cytometry and ELISpot assay. Ultra-conserved or topologically important antigens were respectively identified using the Epigraph tool and a structure-based network analysis approach and compared to overlapping peptides spanning the Gag proteome.

Findings: MDC1 presenting either the overlapping Gag, Epigraph, or Network 14-21mer peptide pools consistently activated and expanded HIV-1-specific T cells to epitopes identified at the 9-13mer peptide level. Interestingly, some CTL responses occurred outside known or expected HLA associations, providing evidence of new HLA-associated CTL epitopes. Comparative analyses demonstrated more sequence conservation among Epigraph antigens but a higher magnitude of CTL responses to Network and Gag peptide groups. Importantly, CTL responses against topologically constrained Gag epitopes contained in both the Network and Gag peptide pools were selectively enhanced in the Network pool-initiated cultures.

Interpretation: Our study supports the use of MDC1 as a therapeutic strategy to induce and focus CTL responses toward putative fitness-constrained regions of HIV-1 to prevent immune escape and control HIV-1 infection.

Funding: A full list of the funding sources is detailed in the Acknowledgment section of the manuscript.
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http://dx.doi.org/10.1016/j.ebiom.2020.103175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7811131PMC
January 2021

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

mBio 2021 01 12;12(1). Epub 2021 Jan 12.

Virus-Cell Interaction Section, HIV Dynamics and Replication Program, Center for Cancer Research, National Cancer Institute, Frederick, Maryland, USA

Despite the effectiveness of antiretroviral (ARV) therapy, virological failure can occur in some HIV-1-infected patients in the absence of mutations in drug target genes. We previously reported that, , the lab-adapted HIV-1 NL4-3 strain can acquire resistance to the integrase inhibitor dolutegravir (DTG) by acquiring mutations in the envelope glycoprotein (Env) that enhance viral 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 reduce susceptibility to multiple classes of ARVs and also increase resistance to ARVs when coupled with target-gene mutations. We observe that the NL4-3 Env mutants display a more stable and closed Env conformation and lower rates of gp120 shedding than the WT virus. We also selected for Env mutations in clinically relevant HIV-1 isolates in the presence of ARVs. These Env mutants exhibit reduced susceptibility to DTG, with effects on replication and Env structure that are HIV-1 strain dependent. Finally, to examine a possible 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 that mutations in Env that enhance the ability of HIV-1 to spread via a cell-cell route may increase the opportunity for the virus to acquire high-level drug resistance mutations in ARV target genes. Although combination antiretroviral (ARV) therapy is highly effective in controlling the progression of HIV disease, drug resistance can be a major obstacle. Recent findings suggest that resistance can develop 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 We demonstrate that Env mutations can reduce sensitivity to major classes of ARVs in multiple viral isolates and define the effect of the Env mutations on Env subunit interactions. 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 therapeutic strategies and provide clues toward understanding how ARV-treated individuals fail therapy without acquiring mutations in drug target genes.
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http://dx.doi.org/10.1128/mBio.03134-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7844542PMC
January 2021

HIV-specific T cell responses reflect substantive in vivo interactions with antigen despite long-term therapy.

JCI Insight 2021 02 8;6(3). Epub 2021 Feb 8.

Division of Infectious Diseases, Department of Medicine, Weill Cornell Medicine, New York, New York, USA.

Antiretroviral therapies (ARTs) abrogate HIV replication; however, infection persists as long-lived reservoirs of infected cells with integrated proviruses, which reseed replication if ART is interrupted. A central tenet of our current understanding of this persistence is that infected cells are shielded from immune recognition and elimination through a lack of antigen expression from proviruses. Efforts to cure HIV infection have therefore focused on reactivating latent proviruses to enable immune-mediated clearance, but these have yet to succeed in reducing viral reservoirs. Here, we revisited the question of whether HIV reservoirs are predominately immunologically silent from a new angle: by querying the dynamics of HIV-specific T cell responses over long-term ART for evidence of ongoing recognition of HIV-infected cells. In longitudinal assessments, we show that the rates of change in persisting HIV Nef-specific responses, but not responses to other HIV gene products, were associated with residual frequencies of infected cells. These Nef-specific responses were highly stable over time and disproportionately exhibited a cytotoxic, effector functional profile, indicative of recent in vivo recognition of HIV antigens. These results indicate substantial visibility of the HIV-infected cells to T cells on stable ART, presenting both opportunities and challenges for the development of therapeutic approaches to curing infection.
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http://dx.doi.org/10.1172/jci.insight.142640DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7934865PMC
February 2021

Novel Criteria for Diagnosing Acute and Early HIV Infection in a Multi-National Study of Early Antiretroviral Therapy Initiation.

Clin Infect Dis 2020 Dec 31. Epub 2020 Dec 31.

Lundquist Institute at Harbor-UCLA Medical Center, Torrance, CA, USA.

Background: Antiretroviral therapy (ART) initiation during acute and early HIV infection (AEHI) limits HIV reservoir formation and may facilitate post-ART control but is logistically challenging. We evaluated the performance of new AEHI diagnostic criteria from a multi-national prospective study of ART initiation during AEHI.

Methods: ACTG 5354 enrolled adults at 30 sites in the Americas, Africa, and Asia who met any one of six criteria based on combinations of results of HIV RNA, HIV antibody, Western blot or Geenius assay, and/or the signal-to-cutoff (S/CO) ratio of the ARCHITECT HIV Combo Ag/Ab CMIA or GS HIV COMBO Ag/Ab EIA. HIV infection and Fiebig stage were subsequently confirmed by centralized testing.

Results: From 2017-2019, 195 participants were enrolled with median age 27 (interquartile range 23-39) years. Thirty (15.4%) were female. ART was started by 171 (87.7%) on the day of enrollment and 24 (12.3%) the next day. AEHI was confirmed in 188 (96.4%) participants after centralized testing, four (2.0%) participants were retrospectively found to have chronic infection, and three (1.5%) found not to have HIV discontinued ART and were withdrawn. Retrospectively, a nonreactive or indeterminate HIV antibody on the Geenius assay combined with ARCHITECT S/CO ≥10 correctly identified 99 of 122 (81.2%) Fiebig II-IV AEHI cases with no false-positive results.

Conclusions: Novel AEHI criteria incorporating ARCHITECT S/CO into diagnostic algorithms facilitated rapid and efficient ART initiation without waiting for an HIV RNA result. These new criteria may facilitate AEHI diagnosis, staging, and immediate ART initiation in future research studies and clinical practice.
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http://dx.doi.org/10.1093/cid/ciaa1893DOI Listing
December 2020

A phase I/II randomized, placebo-controlled trial of romidepsin in persons with HIV-1 on suppressive antiretroviral therapy to assess safety and activation of HIV-1 expression (A5315).

J Infect Dis 2020 Dec 22. Epub 2020 Dec 22.

University of Pittsburgh, Pittsburgh, PA.

Background: Romidepsin (RMD) is a histone deacetylase inhibitor reported to reverse HIV-1 latency. We sought to identify doses of RMD that were safe and induced HIV-1 expression.

Methods: Enrollees had HIV-1 RNA <40 copies/ml on ART. Measurements included RMD levels, plasma viremia by single copy HIV-1 RNA assay, HIV-1 DNA, cell-associated unspliced HIV-1 RNA (CA-RNA), acetylation of histone H3-lysine-9 (H3K9ac+) and phosphorylation of transcription factor P-TEFb. Wilcoxon tests were used for comparison.

Results: 43 participants enrolled in the single dose Cohorts 1-3 of 0.5, 2, and 5 mg/m 2 (36 RMD; 7 placebo) and 16 enrolled in the multi-dose Cohort 4 of 5 mg/m 2 (13 RMD; 3 placebo). One grade 3 event (neutropenia) was possibly treatment-related. No significant changes in viremia were observed in Cohorts 1-4 compared to placebo. In Cohort 4, observed pharmacodynamic effects of RMD were reduced proportions of CD4+ T cells 24 hours after infusions 2, 3, and 4 (median -3.5% to -4.5%) vs. placebo (+0.5% to 1%; p≤0.022) and increases in H3K9ac+ and phosphorylated P-TEFb in CD4 + T cells compared to placebo (p≤0.02).

Conclusions: RMD infusions were safe but did not increase plasma viremia or unspliced CA-RNA despite pharmacodynamic effects on CD4 + T cells. The trial is registered with ClinicalTrials.gov, number NCT01933594.
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http://dx.doi.org/10.1093/infdis/jiaa777DOI Listing
December 2020

Adaptation of advanced clinical virology assays from HIV-1 to SARS-CoV-2.

Curr Opin HIV AIDS 2021 01;16(1):3-10

Division of Infectious Diseases, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

Purpose Of Review: In response to the HIV-AIDS pandemic, great strides have been made in developing molecular methods that accurately quantify nucleic acid products of HIV-1 at different stages of viral replication and to assess HIV-1 sequence diversity and its effect on susceptibility to small molecule inhibitors and neutralizing antibodies. Here, we review how knowledge gained from these approaches, including viral RNA quantification and sequence analyses, have been rapidly applied to study SARS-CoV-2 and the COVID-19 pandemic.

Recent Findings: Recent studies have shown detection of SARS-CoV-2 RNA in blood of infected individuals by reverse transcriptase PCR (RT-PCR); and, as in HIV-1 infection, there is growing evidence that the level of viral RNA in plasma may be related to COVID disease severity. Unlike HIV-1, SARS-CoV-2 sequences are highly conserved limiting SARS-CoV-2 sequencing applications to investigating interpatient genetic diversity for phylogenetic analysis. Sensitive sequencing technologies, originally developed for HIV-1, will be needed to investigate intrapatient SARS-CoV-2 genetic variation in response to antiviral therapeutics and vaccines.

Summary: Methods used for HIV-1 have been rapidly applied to SARS-CoV-2/COVID-19 to understand pathogenesis and prognosis. Further application of such methods should improve precision of therapy and outcome.
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http://dx.doi.org/10.1097/COH.0000000000000656DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7752249PMC
January 2021

Brief Report: Dipyridamole Decreases Gut Mucosal Regulatory T-Cell Frequencies Among People With HIV on Antiretroviral Therapy.

J Acquir Immune Defic Syndr 2020 12;85(5):665-669

Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA.

Background: We had previously conducted a double-blind, randomized placebo-controlled, partial cross-over trial showing that 12 weeks of dipyridamole decreased CD8 T-cell activation among treated HIV(+) individuals by increasing extracellular adenosine levels.

Methods: In this substudy, rectosigmoid biopsies were obtained from 18 participants (9 per arm), to determine whether 12 weeks of dipyridamole affects mucosal immune cells. Participants randomized to placebo were then switched to dipyridamole for 12 weeks while the treatment arm continued dipyridamole for another 12 weeks. We evaluated T-cell frequencies and plasma markers of microbial translocation and intestinal epithelial integrity. Linear regression models on log-transformed outcomes were used for the primary 12-week analysis.

Results: Participants receiving dipyridamole had a median 70.2% decrease from baseline in regulatory T cells (P = 0.007) and an 11.3% increase in CD8 T cells (P = 0.05). There was a nonsignificant 10.80% decrease in plasma intestinal fatty acid binding protein levels in the dipyridamole arm compared with a 9.51% increase in the placebo arm. There were no significant differences in plasma levels of β-D-glucan. In pooled analyses, there continued to be a significant decrease in regulatory T cells (-44%; P = 0.004). There was also a trend for decreased CD4 and CD8 T-cell activation.

Conclusion: Increasing extracellular adenosine levels using dipyridamole in virally suppressed HIV (+) individuals on antiretroviral therapy can affect regulation of gut mucosal immunity.
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http://dx.doi.org/10.1097/QAI.0000000000002488DOI Listing
December 2020

Rapid identification of a human antibody with high prophylactic and therapeutic efficacy in three animal models of SARS-CoV-2 infection.

Proc Natl Acad Sci U S A 2020 11 2;117(47):29832-29838. Epub 2020 Nov 2.

Department of Medicine, Division of Infectious Diseases, Center for Antibody Therapeutics, University of Pittsburgh Medical School, Pittsburgh, PA 15261;

Effective therapies are urgently needed for the SARS-CoV-2/COVID-19 pandemic. We identified panels of fully human monoclonal antibodies (mAbs) from large phage-displayed Fab, scFv, and VH libraries by panning against the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) glycoprotein. A high-affinity Fab was selected from one of the libraries and converted to a full-size antibody, IgG1 ab1, which competed with human ACE2 for binding to RBD. It potently neutralized replication-competent SARS-CoV-2 but not SARS-CoV, as measured by two different tissue culture assays, as well as a replication-competent mouse ACE2-adapted SARS-CoV-2 in BALB/c mice and native virus in hACE2-expressing transgenic mice showing activity at the lowest tested dose of 2 mg/kg. IgG1 ab1 also exhibited high prophylactic and therapeutic efficacy in a hamster model of SARS-CoV-2 infection. The mechanism of neutralization is by competition with ACE2 but could involve antibody-dependent cellular cytotoxicity (ADCC) as IgG1 ab1 had ADCC activity in vitro. The ab1 sequence has a relatively low number of somatic mutations, indicating that ab1-like antibodies could be quickly elicited during natural SARS-CoV-2 infection or by RBD-based vaccines. IgG1 ab1 did not aggregate, did not exhibit other developability liabilities, and did not bind to any of the 5,300 human membrane-associated proteins tested. These results suggest that IgG1 ab1 has potential for therapy and prophylaxis of SARS-CoV-2 infections. The rapid identification (within 6 d of availability of antigen for panning) of potent mAbs shows the value of large antibody libraries for response to public health threats from emerging microbes.
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http://dx.doi.org/10.1073/pnas.2010197117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7703590PMC
November 2020

Vesatolimod, a Toll-like Receptor 7 Agonist, Induces Immune Activation in Virally Suppressed Adults Living With Human Immunodeficiency Virus-1.

Clin Infect Dis 2021 06;72(11):e815-e824

Gilead Sciences Inc., Foster City, California, USA.

Background: Treatment with vesatolimod, an investigational, oral, toll-like receptor 7 (TLR7) agonist, leads to sustained viral remission in some non-human primates when combined with anti-envelope antibodies or therapeutic vaccines. We report results of a Phase Ib study evaluating safety, pharmacokinetics, and pharmacodynamics of vesatolimod in adults living with human immunodeficiency virus (HIV)-1.

Methods: In this double-blind, multicenter, placebo-controlled trial, participants on antiretroviral therapy with screening plasma HIV-1 RNA levels <50 copies/mL were randomized (6:2) to receive 6-10 doses of vesatolimod (1-12 mg) or matching placebo orally every other week in sequential dose-escalation cohorts. The primary study objectives included establishing the safety and virologic effects of vesatolimod (change from baseline in plasma HIV-1 RNA). Pharmacokinetics and pharmacodynamic/immunologic activity were assessed as secondary objectives.

Results: A total of 48 individuals were randomly assigned to vesatolimod (n = 36) or placebo (n = 12). Vesatolimod was generally well tolerated, with no study drug-related serious adverse events or adverse events leading to study drug discontinuation. There were no statistically significant changes from baseline in plasma HIV-1 RNA in the vesatolimod groups, compared to placebo.Vesatolimod plasma exposures increased dose proportionally; consistent responses in cytokines, interferon-stimulated gene expression, and lymphocyte activation were observed with increasing dose levels above 4 mg. Peak elevations 24 hours after receipt of a 6 mg dose were >3.9-fold higher for interferon gamma-induced protein 10 (IP-10), interleukin-1 receptor antagonist (IL-1RA), interferon-inducible T-cell alpha chemoattractant (ITAC) when compared to baseline values.

Conclusions: Vesatolimod was well tolerated at doses ranging from 1 to 12 mg. Immune stimulation was observed at doses above 4 mg, providing rationale for future combination trials in people living with HIV.

Clinical Trials Registration: NCT02858401.
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http://dx.doi.org/10.1093/cid/ciaa1534DOI Listing
June 2021

HIV-1 viremia not suppressible by antiretroviral therapy can originate from large T cell clones producing infectious virus.

J Clin Invest 2020 11;130(11):5847-5857

Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

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.
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http://dx.doi.org/10.1172/JCI138099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7598056PMC
November 2020

Enhanced elicitation of potent neutralizing antibodies by the SARS-CoV-2 spike receptor binding domain Fc fusion protein in mice.

Vaccine 2020 10 22;38(46):7205-7212. Epub 2020 Sep 22.

Center for Antibody Therapeutics, Division of Infectious Diseases, Department of Medicine, University of Pittsburgh Medical School, 3550 Terrace St, Pittsburgh, PA 15261, USA; Abound Bio, 1401 Forbes Ave, Pittsburgh, PA 15219, USA. Electronic address:

The development of an effective vaccine against SARS-CoV-2 is urgently needed. We generated SARS-CoV-2 RBD-Fc fusion protein and evaluated its potency to elicit neutralizing antibody response in mice. RBD-Fc elicited a higher neutralizing antibodies titer than RBD as evaluated by a pseudovirus neutralization assay and a live virus based microneutralization assay. Furthermore, RBD-Fc immunized sera better inhibited cell-cell fusion, as evaluated by a quantitative cell-cell fusion assay. The cell-cell fusion assay results correlated well with the virus neutralization potency and could be used for high-throughput screening of large panels of anti-SARS-CoV-2 antibodies and vaccines without the requirement of live virus infection in BSL3 containment. Moreover, the anti-RBD sera did not enhance the pseudotyped SARS-CoV-2 infection of K562 cells. These results demonstrate that Fc fusion can significantly improve the humoral immune response to recombinant RBD immunogen, and suggest that RBD-Fc could serve as a useful component of effective vaccines against SARS-CoV-2.
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http://dx.doi.org/10.1016/j.vaccine.2020.09.058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7508516PMC
October 2020

Clinical Characteristics and Outcomes of Patients Hospitalized for COVID-19 in Africa: Early Insights from the Democratic Republic of the Congo.

Am J Trop Med Hyg 2020 12 2;103(6):2419-2428. Epub 2020 Oct 2.

Department of Internal Medicine, School of Medicine, University of Kinshasa, Kinshasa, Democratic Republic of Congo.

Little is known about the clinical features and outcomes of SARS-CoV-2 infection in Africa. We conducted a retrospective cohort study of patients hospitalized for COVID-19 between March 10, 2020 and July 31, 2020 at seven hospitals in Kinshasa, Democratic Republic of the Congo (DRC). Outcomes included clinical improvement within 30 days (primary) and in-hospital mortality (secondary). Of 766 confirmed COVID-19 cases, 500 (65.6%) were male, with a median (interquartile range [IQR]) age of 46 (34-58) years. One hundred ninety-one (25%) patients had severe/critical disease requiring admission in the intensive care unit (ICU). Six hundred twenty patients (80.9%) improved and were discharged within 30 days of admission. Overall in-hospital mortality was 13.2% (95% CI: 10.9-15.8), and almost 50% among those in the ICU. Independent risk factors for death were age < 20 years (adjusted hazard ratio [aHR] = 6.62, 95% CI: 1.85-23.64), 40-59 years (aHR = 4.45, 95% CI: 1.83-10.79), and ≥ 60 years (aHR = 13.63, 95% CI: 5.70-32.60) compared with those aged 20-39 years, with obesity (aHR = 2.30, 95% CI: 1.24-4.27), and with chronic kidney disease (aHR = 5.33, 95% CI: 1.85-15.35). In marginal structural model analysis, there was no statistically significant difference in odds of clinical improvement (adjusted odds ratio [aOR] = 1.53, 95% CI: 0.88-2.67, = 0.132) nor risk of death (aOR = 0.65, 95% CI: 0.35-1.20) when comparing the use of chloroquine/azithromycin versus other treatments. In this DRC study, the high mortality among patients aged < 20 years and with severe/critical disease is of great concern, and requires further research for confirmation and targeted interventions.
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http://dx.doi.org/10.4269/ajtmh.20-1240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7695108PMC
December 2020

Automated Multireplicate Quantification of Persistent HIV-1 Viremia in Individuals on Antiretroviral Therapy.

J Clin Microbiol 2020 11 18;58(12). Epub 2020 Nov 18.

Division of Infectious Diseases, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.

Clearance of low-level viremia that persists in most HIV-1-positive individuals on antiretroviral therapy (ART) is an important milestone for efforts to cure HIV-1 infection. The level of persistent viremia on ART is generally below the lower limit of quantification (LOQ) of current FDA-cleared plasma HIV-1 RNA assays (20 to 40 copies/ml) but can be quantified by reverse transcriptase PCR (RT-PCR) assays with single-copy sensitivity. Such assays require multistep manual methods, and their low throughput limits the capacity to monitor the effects of interventions on persistent viremia. Recently, S. Bakkour, X. Deng, P. Bacchetti, E. Grebe, et al. (J Clin Microbiol 58:e01400-20, 2020, https://doi.org/10.1128/JCM.01400-20), reported the use of multiple replicates and Poisson statistics to infer HIV-1 RNA concentrations below the commercial LOQ of an automated platform (Hologic Panther Aptima). Here, we evaluate the detection and quantitation of low-level viremia using the following two adaptions of the automated platform: a multireplicate strategy (9×) and a concentrated single-replicate strategy in which 5 ml of plasma is concentrated by centrifugation (1×, concentrated). We compare these new methods to a recently reported manual -targeting single-copy assay version 2 (iSCA v2). Using laboratory-generated HIV-1 RNA plasma samples at known concentrations, all three methods had similar sensitivity for HIV-1 RNA detection, although iSCA v2 was most sensitive (95% LOD, 2.3 copies/ml), 9× was marginally less sensitive (95% LOD, 3.0 copies/ml), and 1×, concentrated was least sensitive (95% LOD, 3.9 copies/ml). In contrast, for clinical plasma samples, 9× had greater sensitivity than iSCA v2 (82% of samples were quantifiable compared with 62% of samples by iSCA v2). These results support 9× as an acceptable high-throughput alternative to iSCA v2 for quantifying low-level viremia in individuals on ART.
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http://dx.doi.org/10.1128/JCM.01442-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7685899PMC
November 2020

High Potency of a Bivalent Human V Domain in SARS-CoV-2 Animal Models.

Cell 2020 10 4;183(2):429-441.e16. Epub 2020 Sep 4.

Center for Antibody Therapeutics, Division of Infectious Diseases, Department of Medicine, University of Pittsburgh Medical School, 3550 Terrace St., Pittsburgh, PA 15261, USA; Abound Bio, 1401 Forbes Ave., Pittsburgh, PA 15219, USA. Electronic address:

Novel COVID-19 therapeutics are urgently needed. We generated a phage-displayed human antibody V domain library from which we identified a high-affinity V binder ab8. Bivalent V, V-Fc ab8, bound with high avidity to membrane-associated S glycoprotein and to mutants found in patients. It potently neutralized mouse-adapted SARS-CoV-2 in wild-type mice at a dose as low as 2 mg/kg and exhibited high prophylactic and therapeutic efficacy in a hamster model of SARS-CoV-2 infection, possibly enhanced by its relatively small size. Electron microscopy combined with scanning mutagenesis identified ab8 interactions with all three S protomers and showed how ab8 neutralized the virus by directly interfering with ACE2 binding. V-Fc ab8 did not aggregate and did not bind to 5,300 human membrane-associated proteins. The potent neutralization activity of V-Fc ab8 combined with good developability properties and cross-reactivity to SARS-CoV-2 mutants provide a strong rationale for its evaluation as a COVID-19 therapeutic.
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http://dx.doi.org/10.1016/j.cell.2020.09.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7473018PMC
October 2020

Detection of SARS-CoV-2 RNA in Blood of Patients with COVID-19: What Does It Mean?

Clin Infect Dis 2020 Sep 8. Epub 2020 Sep 8.

Division of Infectious Diseases, Department of Medicine, University of Pittsburgh School of Medicine.

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http://dx.doi.org/10.1093/cid/ciaa1316DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7499541PMC
September 2020

Selective Decay of Intact HIV-1 Proviral DNA on Antiretroviral Therapy.

J Infect Dis 2021 Feb;223(2):225-233

Division of Infectious Diseases, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

Background: HIV-1 proviruses persist in people on antiretroviral therapy (ART) but most are defective and do not constitute a replication-competent reservoir. The decay of infected cells carrying intact compared with defective HIV-1 proviruses has not been well defined in people on ART.

Methods: We separately quantified intact and defective proviruses, residual plasma viremia, and markers of inflammation and activation in people on long-term ART.

Results: Among 40 participants tested longitudinally from a median of 7.1 years to 12 years after ART initiation, intact provirus levels declined significantly over time (median half-life, 7.1 years; 95% confidence interval [CI], 3.9-18), whereas defective provirus levels did not decrease. The median half-life of total HIV-1 DNA was 41.6 years (95% CI, 13.6-75). The proportion of all proviruses that were intact diminished over time on ART, from about 10% at the first on-ART time point to about 5% at the last. Intact provirus levels on ART correlated with total HIV-1 DNA and residual plasma viremia, but there was no evidence for associations between intact provirus levels and inflammation or immune activation.

Conclusions: Cells containing intact, replication-competent proviruses are selectively lost during suppressive ART. Defining the mechanisms involved should inform strategies to accelerate HIV-1 reservoir depletion.
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http://dx.doi.org/10.1093/infdis/jiaa532DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7857155PMC
February 2021

Intact proviral DNA assay analysis of large cohorts of people with HIV provides a benchmark for the frequency and composition of persistent proviral DNA.

Proc Natl Acad Sci U S A 2020 08 20;117(31):18692-18700. Epub 2020 Jul 20.

Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205.

A scalable approach for quantifying intact HIV-1 proviruses is critical for basic research and clinical trials directed at HIV-1 cure. The intact proviral DNA assay (IPDA) is a novel approach to characterizing the HIV-1 reservoir, focusing on the genetic integrity of individual proviruses independent of transcriptional status. It uses multiplex digital droplet PCR to distinguish and separately quantify intact proviruses, defined by a lack of overt fatal defects such as large deletions and APOBEC3G-mediated hypermutation, from the majority of proviruses that have such defects. This distinction is important because only intact proviruses cause viral rebound on ART interruption. To evaluate IPDA performance and provide benchmark data to support its implementation, we analyzed peripheral blood samples from 400 HIV-1 adults on ART from several diverse cohorts, representing a robust sample of treated HIV-1 infection in the United States. We provide direct quantitative evidence that defective proviruses greatly outnumber intact proviruses (by >12.5 fold). However, intact proviruses are present at substantially higher frequencies (median, 54/10 CD4 T cells) than proviruses detected by the quantitative viral outgrowth assay, which requires induction and in vitro growth (∼1/10 CD4 T cells). IPDA amplicon signal issues resulting from sequence polymorphisms were observed in only 6.3% of individuals and were readily apparent and easily distinguished from low proviral frequency, an advantage of the IPDA over standard PCR assays which generate false-negative results in such situations. The large IPDA dataset provided here gives the clearest quantitative picture to date of HIV-1 proviral persistence on ART.
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http://dx.doi.org/10.1073/pnas.2006816117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7414172PMC
August 2020
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