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Single-cell immunology of SARS-CoV-2 infection

Nat Biotechnol

2021 Michael Zager; Yuan Tian; Raphael Gottardo; Lindsay Carpp; Helen Rodgers Miller; Evan Newell

Gaining a better understanding of the immune cell subsets and molecular factors associated with protective or pathological immunity against severe acute respiratory syndrome coronavirus (SARS-CoV)-2 could aid the development of vaccines and therapeutics for coronavirus disease 2019 (COVID-19). Single-cell technologies, such as flow cytometry, mass cytometry, single-cell transcriptomics and single-cell multi-omic profiling, offer considerable promise in dissecting the heterogeneity of immune responses among individual cells and uncovering the molecular mechanisms of COVID-19 pathogenesis. Single-cell immune-profiling studies reported to date have identified innate and adaptive immune cell subsets that correlate with COVID-19 disease severity, as well as immunological factors and pathways of potential relevance to the development of vaccines and treatments for COVID-19. For facilitation of integrative studies and meta-analyses into the immunology of SARS-CoV-2 infection, we provide standardized, download-ready versions of 21 published single-cell sequencing datasets (over 3.2 million cells in total) as well as an interactive visualization portal for data exploration.

HIV reservoir quantification by five-target multiplex droplet digital PCR

STAR Protoc

2021 Florian Hladik; Dara Lehman; Chelsea Amstuz; Keith Jerome

Most latent human immunodeficiency virus (HIV) proviruses are defective and cannot produce infectious virions. Thus, the number of HIV proviruses with intact genomes is a relevant clinical parameter to assess therapies for HIV cure. We describe high-molecular-weight DNA isolation, followed by restriction enzyme fragmentation that limits cutting within the HIV genome. Multiplexed droplet digital PCR quantifies five targets spanning the HIV genome to estimate potentially intact proviral copies. A reference assay counts the number of T lymphocytes and assesses the level of DNA shearing. For complete details on the use and execution of this protocol, please refer to Levy et al. (2021).

Toward a better understanding of T cells in cancer

Cancer Cell

2021 Evan Newell

T cells mediate anti-tumor immune responses and are the key target of immune checkpoint therapy, but they can also promote immune tolerance. A clear understanding of the specific contributions and biology of different T cell subsets is required to fully harness the curative potential of immunotherapies. Experts discuss the state of the field and key challenges for moving forward.

Viral Suppression Levels in Newly Diagnosed Men Who Have Sex with Men and Transgender Women with Newly Diagnosed HIV and Alcohol Use Disorder in Peru: Results from a Randomized, Double-Blind, Placebo-Controlled Trial Using Oral Naltrexone

J Acquir Immune Defic Syndr

2021 Angela Ulrich; Arielle Grieco; Ann Duerr

BACKGROUND: Alcohol use disorders (AUD) are common in men who have sex with men (MSM) and transgender women (TGW) in Peru and undermine antiretroviral therapy (ART) adherence. Oral naltrexone (NTX) is an evidenced-based treatment for AUD that has not been assessed in co-treating AUD in MSM/TGW with HIV. SETTING: and Design: A two-site, randomized, double-blind, placebo-controlled trial among MSM/TGW with AUD and newly diagnosed with HIV in Lima, Peru. METHODS: Newly diagnosed MSM/TGW with AUD were prescribed a single-treatment regimen of EFV/TDF/FTC from 2014-2015 and randomized 2:1 to oral NTX (N=103) or placebo (N=53) for 24 weeks. Primary and secondary outcomes were proportion achieving viral suppression (VS: HIV-1 RNA<400 copies/mL) or maximal viral suppression (MVS: HIV-1 RNA<40 copies/mL) at 24 weeks. RESULTS: The were no significant differences between arms in VS (81.6% NTX vs 75.5% placebo; p=0.37) or MVS (61.2% NTX vs 66.0% placebo; p=0.48). Adherence to study medication was low (mean=34.6%) overall with only 21.4% of participants meeting recommended levels (80% daily doses/month). Participants allocated to NTX had significantly lower adherence compared to placebo for both 12-week study periods (44.0% vs 35.2%, p=0.04; 31.4% vs 35.2%, p=0.03). CONCLUSIONS: Findings are inconclusive regarding use of NTX for treatment of AUD in MSM/TGW newly diagnosed with HIV. VS and MVS levels were high irrespective of allocation. Adherence to study medication was low, requiring further exploration of strategies to optimize adherence to NTX as AUD treatment.

Integration features of intact latent HIV-1 in CD4(+) T cell clones contribute to viral persistence

J Exp Med

2021 Lillian Cohn

Latent intact HIV-1 proviruses persist in a small subset of long-lived CD4+ T cells that can undergo clonal expansion in vivo. Expanded clones of CD4+ T cells dominate latent reservoirs in individuals on long-term antiretroviral therapy (ART) and represent a major barrier to HIV-1 cure. To determine how integration landscape might contribute to latency, we analyzed integration sites of near full length HIV-1 genomes from individuals on long-term ART, focusing on individuals whose reservoirs are highly clonal. We find that intact proviruses in expanded CD4+ T cell clones are preferentially integrated within Krüppel-associated box (KRAB) domain-containing zinc finger (ZNF) genes. ZNF genes are associated with heterochromatin in memory CD4+ T cells; nevertheless, they are expressed in these cells under steady-state conditions. In contrast to genes carrying unique integrations, ZNF genes carrying clonal intact integrations are down-regulated upon cellular activation. Together, the data suggest selected genomic sites, including ZNF genes, can be especially permissive for maintaining HIV-1 latency during memory CD4+ T cell expansion.

Preexisting memory CD4(+) T cells contribute to the primary response in an HIV-1 vaccine trial

J Clin Invest

2021 Kristen Cohen; Nicole Frahm; Janine Maenza; Julie McElrath

Naive and memory CD4+ T cells reactive with human immunodeficiency virus type 1 (HIV-1) are detectable in unexposed, unimmunized individuals. The contribution of preexisting CD4+ T cells to a primary immune response was investigated in 20 HIV-1-seronegative volunteers vaccinated with an HIV-1 envelope (Env) plasmid DNA prime and recombinant modified vaccinia virus Ankara (MVA) boost in the HVTN 106 vaccine trial (clinicaltrials.gov NCT02296541). Prevaccination naive or memory CD4+ T cell responses directed against peptide epitopes in Env were identified in 14 individuals. After priming with DNA, 40% (8/20) of the elicited responses matched epitopes detected in the corresponding preimmunization memory repertoires, and clonotypes were shared before and after vaccination in 2 representative volunteers. In contrast, there were no shared epitope specificities between the preimmunization memory compartment and responses detected after boosting with recombinant MVA expressing a heterologous Env. Preexisting memory CD4+ T cells therefore shape the early immune response to vaccination with a previously unencountered HIV-1 antigen.

Calibration of two validated SARS-CoV-2 pseudovirus neutralization assays for COVID-19 vaccine evaluation

Sci Rep

2021 Michelle Karuna; Christine Posavad; Julie McElrath; Lindsay Carpp; Yunda Huang; Larry Corey; Jia Jin Kee; Peter Gilbert; John Hural

Vaccine-induced neutralizing antibodies (nAbs) are key biomarkers considered to be associated with vaccine efficacy. In United States government-sponsored phase 3 efficacy trials of COVID-19 vaccines, nAbs are measured by two different validated pseudovirus-based SARS-CoV-2 neutralization assays, with each trial using one of the two assays. Here we describe and compare the nAb titers obtained in the two assays. We observe that one assay consistently yielded higher nAb titers than the other when both assays were performed on the World Health Organization's anti-SARS-CoV-2 immunoglobulin International Standard, COVID-19 convalescent sera, and mRNA-1273 vaccinee sera. To overcome the challenge this difference in readout poses in comparing/combining data from the two assays, we evaluate three calibration approaches and show that readouts from the two assays can be calibrated to a common scale. These results may aid decision-making based on data from these assays for the evaluation and licensure of new or adapted COVID-19 vaccines.

Mucosal tissue regulatory T cells are integral in balancing immunity and tolerance at portals of antigen entry

Mucosal Immunol

2021 Laura Richert Spuhler; Jennifer Lund; Brianna Traxinger

Foxp3+ regulatory T cells (Tregs) are a subset of CD4+ T cells that exert suppressive control over other immune cells. Tregs are critical for preventing systemic autoimmunity and maintaining peripheral tolerance, and yet they also assist in orchestration of immunity to pathogenic insult, wherein they limit collateral immunopathology and assist in facilitating a fine balance between immune tolerance and effector activity. Tregs have been extensively studied in lymphoid tissues, and a growing body of work has characterized phenotypically distinct Tregs localized in various nonlymphoid tissue compartments. These tissue Tregs can perform location-specific, alternative functions, highlighting their dynamic, context-dependent roles. Tregs have also been identified in mucosal tissues where specialized physiological functions are paramount, including helping the host to respond appropriately to pathogenic versus innocuous antigens that are abundant at mucosal portals of antigen entry. As in other tissue Treg compartments, mucosal Tregs in the respiratory, gastrointestinal, and genitourinary tracts are distinct from circulating counterparts and can carry out mucosa-specific functions as well as classic suppressive functions that are the hallmark of Tregs. In this review, we summarize current knowledge regarding mucosal Tregs in both health and disease.

Sequence and vector shapes vaccine induced antibody effector functions in HIV vaccine trials

PLoS Pathog

2021 Julie McElrath; Nicole Frahm; Shannon Grant; Felicity Moodie

Despite the advent of long-acting anti-retroviral therapy able to control and prevent infection, a preventative vaccine remains a global priority for the elimination of HIV. The moderately protective RV144 vaccine trial suggested functional IgG1 and IgG3 antibodies were a potential correlate of protection, but the RV144-inspired HVTN702 validation trial failed to demonstrate efficacy despite inducing targeted levels of IgG1/IgG3. Alterations in inserts, and antigens, adjuvant, and regimen also resulted in vaccine induced target quantitative levels of the immune correlates, but drove qualitative changes to the humoral immune response, pointing to the urgent need to define the influence of vaccine strategies on shaping antibody quality, not just quantity. Thus, defining how distinct prime/boost approaches tune long-lived functional antibodies represents an important goal in vaccine development. Here, we compared vaccine responses in Phase I and II studies in humans utilizing various combinations of DNA/vector, vector/vector and DNA/protein HIV vaccines. We found that adenoviral vector immunization, compared to pox-viral vectors, resulted in the most potent IgG1 and IgG3 responses, linked to highly functional antibody activity, including assisting NK cell related functions. Minimal differences were observed in the durability of the functional humoral immune response across vaccine regimens, except for antibody dependent phagocytic function, which persisted for longer periods in the DNA/rAd5 and rAd35/rAd5 regimen, likely driven by higher IgG1 levels. Collectively, these findings suggest adenoviral vectors drive superior antibody quality and durability that could inform future clinical vaccine studies. Trial registration: ClinicalTrials.gov NCT00801697, NCT00961883, NCT02207920, NCT00125970, NCT02852005).

A review of ex vivo placental perfusion models: an underutilized but promising method to study maternal-fetal interactions

J Matern Fetal Neonatal Med

2021 Florian Hladik

Studying the placenta can provide information about the mechanistic pathways of pregnancy disease. However, analyzing placental tissues and manipulating placental function in real-time during pregnancy is not feasible. The ex vivo placental perfusion model allows observing important aspects of the physiology and pathology of the placenta, while maintaining its viability and functional integrity, and without causing harm to mother or fetus. In this review, we describe and compare setups for this technically complex model and summarize outcomes from various published studies. We hope that our review will encourage wider use of ex vivo placental perfusion, which in turn would generate more knowledge to improve pregnancy outcomes.