A Randomized Placebo-Controlled Trial of Leronlimab in Mild-To-Moderate COVID-19.

PURPOSE: Early in the course of the SARS-CoV-2 pandemic it was hypothesised that host genetics played a role in the pathophysiology of COVID-19 including a suggestion that the CCR5-Δ32 mutation may be protective in SARS-CoV-2 infection. Leronlimab is an investigational CCR5-specific humanized IgG4 monoclonal antibody currently in development for HIV-1 infection. We aimed to explore the impact of leronlimab on the severity of disease symptoms among participants with mild-to-moderate COVID-19. METHODS: The TEMPEST trial was a randomized, double-blind, placebo-controlled study in participants with mild-to-moderate COVID-19. Participants were randomly assigned in a 2:1 ratio to receive subcutaneous leronlimab (700 mg) or placebo on days 0 and 7. The primary efficacy endpoint was assessed by change in total symptom score based on fever, myalgia, dyspnea, and cough, at end of treatment (day 14). FINDINGS: Overall, 84 participants were randomized and treated with leronlimab (n = 56) or placebo (n = 28). No difference was observed in change in total symptom score (P = 0.8184) or other pre-specified secondary endpoints between treatments. However, in a post hoc analysis, 50.0% of participants treated with leronlimab demonstrated improvements from baseline in National Early Warning Score 2 (NEWS2) at day 14, compared with 20·8% of participants in the placebo group (post hoc; p = 0.0223). Among participants in this trial with mild-to-moderate COVID-19 adverse events rates were numerically but not statistically significantly lower in leronlimab participants (33.9%) compared with placebo participants (50.0%). IMPLICATIONS: At the time the TEMPEST trial was designed although CCR5 was known to be implicated in COVID-19 disease severity the exact pathophysiology of SARS-CoV-2 infection was poorly understood. Today it is well accepted that SARS-CoV-2 infection in asymptomatic-to-mild cases is primarily characterized by viral replication, with a heightened immune response, accompanied by diminished viral replication in moderate-to-severe disease and a peak in inflammatory responses with excessive production of pro-inflammatory cytokines in critical disease. It is therefore perhaps not surprising that no differences between treatments were observed in the primary endpoint or in pre-specified secondary endpoints among participants with mild-to-moderate COVID-19. However, the results of the exploratory post hoc analysis showing that participants in the leronlimab group had greater improvement in NEWS2 assessment compared to placebo provided a suggestion that leronlimab may be associated with a lower likelihood of people with mild-to-moderate COVID-19 progressing to more severe disease and needs to be confirmed in other appropriately designed clinical trials. CLINICALTRIALS: gov number, NCT04343651 https://classic. CLINICALTRIALS: gov/ct2/show/NCT04343651.

Human cytomegalovirus UL18 prevents priming of MHC-E- and MHC-II-restricted CD8+ T cells.

Rhesus cytomegalovirus (RhCMV) vectors elicit major histocompatibility complex (MHC)-E-restricted CD8+ T cells that stringently control simian immunodeficiency virus (SIV) in rhesus macaques. These responses require deletion of eight RhCMV chemokine-like open reading frames (ORFs) that are conserved in human cytomegalovirus (HCMV). To determine whether HCMV encodes additional, nonconserved inhibitors of unconventional T cell priming, we inserted 41 HCMV-specific ORFs into a chemokine-deficient strain (68-1 RhCMV). Monitoring of epitope recognition revealed that HCMV UL18 prevented unconventional T cell priming, resulting in MHC-Ia-targeted responses. UL18 is homologous to MHC-I but does not engage T cell receptors and, instead, binds with high affinity to inhibitory leukocyte immunoglobulin-like receptor-1 (LIR-1). UL18 lacking LIR-1 binding no longer interfered with MHC-E-restricted T cell stimulation by RhCMV-infected cells or the induction of unconventionally restricted T cells. Thus, LIR-1 binding needs to be deleted from UL18 of HCMV/HIV vaccines to allow for the induction of protective MHC-E-restricted T cells.

Functional genomic analysis of the 68-1 RhCMV-Mycobacteria tuberculosis vaccine reveals an IL-15 response signature that is conserved with vector attenuation.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) is a deadly infectious disease having a major impact on global health. Using the CMV vector for development of novel vaccines is a promising new strategy that elicits strong and durable, high frequency memory T cell responses against heterologous immunogens. We conducted functional transcriptomic analysis of whole blood samples collected from cohorts of rhesus (Rh) macaques that were administered RhCMV/TB vector using a prime-boost strategy. Two modified CMV vectors were used in this study, including 68-1 RhCMV/TB-6Ag (encoding 6 Mtb protein immunogens, including Ag85A, ESAT-6, Rv3407, Rv2626, Rpf A, and Rpf D) and its attenuated variant, 68-1 RhCMV/Δpp71-TB-6Ag (a cell-to-cell spread-deficient vaccine vector lacking the Rh110 gene encoding the pp71 tegument protein). Bulk mRNA sequencing, differential gene expression, and functional enrichment analyses showed that these RhCMV/TB vaccines induce the innate and adaptive immune responses with specific transcriptomic signatures, including the IL-15-induced protective gene signature previously defined to be linked with protection against simian immunodeficiency virus (SIV) by the 68-1 RhCMV/SIV vaccine. While both vectors exhibited a transcriptomic response of the IL-15 protective signature in whole blood, we show that lack of pp71 does not maintain induction of the protective signature for the full duration of the study compared to the parental non-attenuated vector. Our observations indicate that RhCMV vector vaccines induce a transcriptomic response in whole blood that include a conserved IL-15 signature of which vector-encoded pp71 is an important component of response durability that upon future Mtb challenge may define specific vaccine protection outcomes against Mtb infection.

Pre-challenge gut microbial signature predicts RhCMV/SIV vaccine efficacy in rhesus macaques.

Rhesus cytomegalovirus expressing simian immunodeficiency virus (RhCMV/SIV) vaccines protect ~59% of vaccinated rhesus macaques against repeated limiting-dose intra-rectal exposure with highly pathogenic SIVmac239M, but the exact mechanism responsible for the vaccine efficacy is unknown. It is becoming evident that complex interactions exist between gut microbiota and the host immune system. Here, we aimed to investigate if the rhesus gut microbiome impacts RhCMV/SIV vaccine-induced protection. Three groups of 15 rhesus macaques naturally pre-exposed to RhCMV were vaccinated with RhCMV/SIV vaccines. Rectal swabs were collected longitudinally both before SIV challenge (after vaccination) and post-challenge and were profiled using 16S rRNA based microbiome analysis. We identified ~2,400 16S rRNA amplicon sequence variants (ASVs), representing potential bacterial species/strains. Global gut microbial profiles were strongly associated with each of the three vaccination groups, and all animals tended to maintain consistent profiles throughout the pre-challenge phase. Despite vaccination group differences, by using newly developed compositional data analysis techniques, we identified a common gut microbial signature predictive of vaccine protection outcome across the three vaccination groups. Part of this microbial signature persisted even after SIV challenge. We also observed a strong correlation between this microbial signature and an early signature derived from whole blood transcriptomes in the same animals. Our findings indicate that changes in gut microbiomes are associated with RhCMV/SIV vaccine-induced protection and early host response to vaccination in rhesus macaques.IMPORTANCEThe human immunodeficiency virus (HIV) has infected millions of people worldwide. Unfortunately, still there is no vaccine that can prevent or treat HIV infection. A promising pre-clinical HIV vaccine based on rhesus cytomegalovirus (RhCMV) expressing simian immunodeficiency virus (SIV) antigens (RhCMV/SIV) provides sustained, durable protection against SIV challenge in ~59% of vaccinated rhesus macaques. There is an urgent need to understand the cause of this protection vs non-protection outcome. In this study, we profiled the gut microbiomes of 45 RhCMV/SIV vaccinated rhesus macaques and identified gut microbial signatures that were predictive of RhCMV/SIV vaccination groups and vaccine protection outcomes. These vaccine protection-associated microbial features were significantly correlated with early vaccine-induced host immune signatures in whole blood from the same animals. These findings show that the gut microbiome may be involved in RhCMV/SIV vaccine-induced protection, warranting further research into the impact of the gut microbiome in human vaccine trials.

Viral escape mutations do not account for non-protection from SIVmac239 challenge in RhCMV/SIV vaccinated rhesus macaques.

Simian immunodeficiency virus (SIV) vaccines based upon 68-1 Rhesus Cytomegalovirus (RhCMV) vectors show remarkable protection against pathogenic SIVmac239 challenge. Across multiple independent rhesus macaque (RM) challenge studies, nearly 60% of vaccinated RM show early, complete arrest of SIVmac239 replication after effective challenge, whereas the remainder show progressive infection similar to controls. Here, we performed viral sequencing to determine whether the failure to control viral replication in non-protected RMs is associated with the acquisition of viral escape mutations. While low level viral mutations accumulated in all animals by 28 days-post-challenge, which is after the establishment of viral control in protected animals, the dominant circulating virus in virtually all unprotected RMs was nearly identical to the challenge stock, and there was no difference in mutation patterns between this cohort and unvaccinated controls. These data definitively demonstrate that viral mutation does not explain lack of viral control in RMs not protected by RhCMV/SIV vaccination. We further demonstrate that during chronic infection RhCMV/SIV vaccinated RMs do not acquire escape mutation in epitopes targeted by RhCMV/SIV, but instead display mutation in canonical MHC-Ia epitopes similar to unvaccinated RMs. This suggests that after the initial failure of viral control, unconventional T cell responses induced by 68-1 RhCMV/SIV vaccination do not exert strong selective pressure on systemically replicating SIV.

Cytomegalovirus vaccine vector-induced effector memory CD4 + T cells protect cynomolgus macaques from lethal aerosolized heterologous avian influenza challenge.

An influenza vaccine approach that overcomes the problem of viral sequence diversity and provides long-lived heterosubtypic protection is urgently needed to protect against pandemic influenza viruses. Here, to determine if lung-resident effector memory T cells induced by cytomegalovirus (CMV)-vectored vaccines expressing conserved internal influenza antigens could protect against lethal influenza challenge, we immunize Mauritian cynomolgus macaques (MCM) with cynomolgus CMV (CyCMV) vaccines expressing H1N1 1918 influenza M1, NP, and PB1 antigens (CyCMV/Flu), and challenge with heterologous, aerosolized avian H5N1 influenza. All six unvaccinated MCM died by seven days post infection with acute respiratory distress, while 54.5% (6/11) CyCMV/Flu-vaccinated MCM survived. Survival correlates with the magnitude of lung-resident influenza-specific CD4 + T cells prior to challenge. These data demonstrate that CD4 + T cells targeting conserved internal influenza proteins can protect against highly pathogenic heterologous influenza challenge and support further exploration of effector memory T cell-based vaccines for universal influenza vaccine development.

Pre-challenge gut microbial signature predicts RhCMV/SIV vaccine efficacy in rhesus macaques.

Background: RhCMV/SIV vaccines protect ∼59% of vaccinated rhesus macaques against repeated limiting-dose intra-rectal exposure with highly pathogenic SIVmac239M, but the exact mechanism responsible for the vaccine efficacy is not known. It is becoming evident that complex interactions exist between gut microbiota and the host immune system. Here we aimed to investigate if the rhesus gut microbiome impacts RhCMV/SIV vaccine-induced protection. Methods: Three groups of 15 rhesus macaques naturally pre-exposed to RhCMV were vaccinated with RhCMV/SIV vaccines. Rectal swabs were collected longitudinally both before SIV challenge (after vaccination) and post challenge and were profiled using 16S rRNA based microbiome analysis. Results: We identified ∼2,400 16S rRNA amplicon sequence variants (ASVs), representing potential bacterial species/strains. Global gut microbial profiles were strongly associated with each of the three vaccination groups, and all animals tended to maintain consistent profiles throughout the pre-challenge phase. Despite vaccination group differences, using newly developed compositional data analysis techniques we identified a common gut microbial signature predictive of vaccine protection outcome across the three vaccination groups. Part of this microbial signature persisted even after SIV challenge. We also observed a strong correlation between this microbial signature and an early signature derived from whole blood transcriptomes in the same animals. Conclusions: Our findings indicate that changes in gut microbiomes are associated with RhCMV/SIV vaccine-induced protection and early host response to vaccination in rhesus macaques.

Rhesus Cytomegalovirus-encoded Fcγ-binding glycoproteins facilitate viral evasion from IgG-mediated humoral immunity.

Human cytomegalovirus (HCMV) encodes four viral Fc-gamma receptors (vFcγRs) that counteract antibody-mediated activation in vitro , but their role in infection and pathogenesis is unknown. To examine the in vivo function of vFcγRs in animal hosts closely related to humans, we identified and characterized vFcγRs encoded by rhesus CMV (RhCMV). We demonstrate that Rh05, Rh152/151 and Rh173 represent the complete set of RhCMV vFcγRs, each displaying functional similarities to their respective HCMV orthologs with respect to antagonizing host FcγR activation in vitro . When RhCMV-naïve rhesus macaques were infected with vFcγR-deleted RhCMV, peak plasma viremia levels and anti-RhCMV antibody responses were comparable to wildtype infections. However, the duration of plasma viremia was significantly shortened in immunocompetent, but not in CD4+ T cell-depleted animals. Since vFcγRs were not required for superinfection, we conclude that vFcγRs delay control by virus-specific adaptive immune responses, particularly antibodies, during primary infection.

The pentameric complex is not required for vertical transmission of cytomegalovirus in seronegative pregnant rhesus macaques.

Congenital cytomegalovirus (cCMV) infection is the leading infectious cause of neonatal neurological impairment but essential virological determinants of transplacental CMV transmission remain unclear. The pentameric complex (PC), composed of five subunits, glycoproteins H (gH), gL, UL128, UL130, and UL131A, is essential for efficient entry into non-fibroblast cells in vitro . Based on this role in cell tropism, the PC is considered a possible target for CMV vaccines and immunotherapies to prevent cCMV. To determine the role of the PC in transplacental CMV transmission in a non-human primate model of cCMV, we constructed a PC-deficient rhesus CMV (RhCMV) by deleting the homologues of the HCMV PC subunits UL128 and UL130 and compared congenital transmission to PC-intact RhCMV in CD4+ T cell-depleted or immunocompetent RhCMV-seronegative, pregnant rhesus macaques (RM). Surprisingly, we found that the transplacental transmission rate was similar for PC-intact and PC-deleted RhCMV based on viral genomic DNA detection in amniotic fluid. Moreover, PC-deleted and PC-intact RhCMV acute infection led to similar peak maternal plasma viremia. However, there was less viral shedding in maternal urine and saliva and less viral dissemination in fetal tissues in the PC-deleted group. As expected, dams inoculated with PC-deleted RhCMV demonstrated lower plasma IgG binding to PC-intact RhCMV virions and soluble PC, as well as reduced neutralization of PC-dependent entry of the PC-intact RhCMV isolate UCD52 into epithelial cells. In contrast, binding to gH expressed on the cell surface and neutralization of entry into fibroblasts by the PC-intact RhCMV was higher for dams infected with PC-deleted RhCMV compared to those infected with PC-intact RhCMV. Our data demonstrates that the PC is dispensable for transplacental CMV infection in our non-human primate model. One Sentence Summary: Congenital CMV transmission frequency in seronegative rhesus macaques is not affected by the deletion of the viral pentameric complex.

Correction: Mitigation of endemic GI-tract pathogen-mediated inflammation through development of multimodal treatment regimen and its impact on SIV acquisition in rhesus macaques.

[This corrects the article DOI: 10.1371/journal.ppat.1009565.].

Programming cytomegalovirus as an HIV vaccine.

The initial development of cytomegalovirus (CMV) as a vaccine vector for HIV/simian immunodeficiency virus (SIV) was predicated on its potential to pre-position high-frequency, effector-differentiated, CD8+ T cells in tissues for immediate immune interception of nascent primary infection. This goal was achieved and also led to the unexpected discoveries that non-human primate (NHP) CMVs can be programmed to differentially elicit CD8+ T cell responses that recognize viral peptides via classical MHC-Ia, and/or MHC-II, and/or MHC-E, and that MHC-E-restricted CD8+ T cell responses can uniquely mediate stringent arrest and subsequent clearance of highly pathogenic SIV, an unprecedented type of vaccine-mediated protection. These discoveries delineate CMV vector-elicited MHC-E-restricted CD8+ T cells as a functionally distinct T cell response with the potential for superior efficacy against HIV-1, and possibly other infectious agents or cancers.

Late gene expression-deficient cytomegalovirus vectors elicit conventional T cells that do not protect against SIV.

Rhesus cytomegalovirus-based (RhCMV-based) vaccine vectors induce immune responses that protect ~60% of rhesus macaques (RMs) from SIVmac239 challenge. This efficacy depends on induction of effector memory-based (EM-biased) CD8+ T cells recognizing SIV peptides presented by major histocompatibility complex-E (MHC-E) instead of MHC-Ia. The phenotype, durability, and efficacy of RhCMV/SIV-elicited cellular immune responses were maintained when vector spread was severely reduced by deleting the antihost intrinsic immunity factor phosphoprotein 71 (pp71). Here, we examined the impact of an even more stringent attenuation strategy on vector-induced immune protection against SIV. Fusion of the FK506-binding protein (FKBP) degradation domain to Rh108, the orthologue of the essential human CMV (HCMV) late gene transcription factor UL79, generated RhCMV/SIV vectors that conditionally replicate only when the FK506 analog Shield-1 is present. Despite lacking in vivo dissemination and reduced innate and B cell responses to vaccination, Rh108-deficient 68-1 RhCMV/SIV vectors elicited high-frequency, durable, EM-biased, SIV-specific T cell responses in RhCMV-seropositive RMs at doses of ≥ 1 × 106 PFU. Strikingly, elicited CD8+ T cells exclusively targeted MHC-Ia-restricted epitopes and failed to protect against SIVmac239 challenge. Thus, Rh108-dependent late gene expression is required for both induction of MHC-E-restricted T cells and protection against SIV.

Cytomegalovirus-vaccine-induced unconventional T cell priming and control of SIV replication is conserved between primate species.

Strain 68-1 rhesus cytomegalovirus expressing simian immunodeficiency virus (SIV) antigens (RhCMV/SIV) primes MHC-E-restricted CD8+ T cells that control SIV replication in 50%-60% of the vaccinated rhesus macaques. Whether this unconventional SIV-specific immunity and protection is unique to rhesus macaques or RhCMV or is intrinsic to CMV remains unknown. Here, using cynomolgus CMV vectors expressing SIV antigens (CyCMV/SIV) and Mauritian cynomolgus macaques, we demonstrate that the induction of MHC-E-restricted CD8+ T cells requires matching CMV to its host species. RhCMV does not elicit MHC-E-restricted CD8+ T cells in cynomolgus macaques. However, cynomolgus macaques vaccinated with species-matched 68-1-like CyCMV/SIV mounted MHC-E-restricted CD8+ T cells, and half of the vaccinees stringently controlled SIV post-challenge. Protected animals manifested a vaccine-induced IL-15 transcriptomic signature that is associated with efficacy in rhesus macaques. These findings demonstrate that the ability of species-matched CMV vectors to elicit MHC-E-restricted CD8+ T cells that are required for anti-SIV efficacy is conserved in nonhuman primates, and these data support the development of HCMV/HIV for a prophylactic HIV vaccine.

Myeloid cell tropism enables MHC-E-restricted CD8+ T cell priming and vaccine efficacy by the RhCMV/SIV vaccine.

The strain 68-1 rhesus cytomegalovirus (RhCMV)-based vaccine for simian immunodeficiency virus (SIV) can stringently protect rhesus macaques (RMs) from SIV challenge by arresting viral replication early in primary infection. This vaccine elicits unconventional SIV-specific CD8+ T cells that recognize epitopes presented by major histocompatibility complex (MHC)-II and MHC-E instead of MHC-Ia. Although RhCMV/SIV vaccines based on strains that only elicit MHC-II- and/or MHC-Ia-restricted CD8+ T cells do not protect against SIV, it remains unclear whether MHC-E-restricted T cells are directly responsible for protection and whether these responses can be separated from the MHC-II-restricted component. Using host microRNA (miR)-mediated vector tropism restriction, we show that the priming of MHC-II and MHC-E epitope-targeted responses depended on vector infection of different nonoverlapping cell types in RMs. Selective inhibition of RhCMV infection in myeloid cells with miR-142-mediated tropism restriction eliminated MHC-E epitope-targeted CD8+ T cell priming, yielding an exclusively MHC-II epitope-targeted response. Inhibition with the endothelial cell-selective miR-126 eliminated MHC-II epitope-targeted CD8+ T cell priming, yielding an exclusively MHC-E epitope-targeted response. Dual miR-142 + miR-126-mediated tropism restriction reverted CD8+ T cell responses back to conventional MHC-Ia epitope targeting. Although the magnitude and differentiation state of these CD8+ T cell responses were generally similar, only the vectors programmed to elicit MHC-E-restricted CD8+ T cell responses provided protection against SIV challenge, directly demonstrating the essential role of these responses in RhCMV/SIV vaccine efficacy.

BFF and cellhashR: analysis tools for accurate demultiplexing of cell hashing data.

MOTIVATION: Single-cell sequencing methods provide previously impossible resolution into the transcriptome of individual cells. Cell hashing reduces single-cell sequencing costs by increasing capacity on droplet-based platforms. Cell hashing methods rely on demultiplexing algorithms to accurately classify droplets; however, assumptions underlying these algorithms limit accuracy of demultiplexing, ultimately impacting the quality of single-cell sequencing analyses. RESULTS: We present Bimodal Flexible Fitting (BFF) demultiplexing algorithms BFFcluster and BFFraw, a novel class of algorithms that rely on the single inviolable assumption that barcode count distributions are bimodal. We integrated these and other algorithms into cellhashR, a new R package that provides integrated QC and a single command to execute and compare multiple demultiplexing algorithms. We demonstrate that BFFcluster demultiplexing is both tunable and insensitive to issues with poorly behaved data that can confound other algorithms. Using two well-characterized reference datasets, we demonstrate that demultiplexing with BFF algorithms is accurate and consistent for both well-behaved and poorly behaved input data. AVAILABILITY AND IMPLEMENTATION: cellhashR is available as an R package at https://github.com/BimberLab/cellhashR. cellhashR version 1.0.3 was used for the analyses in this manuscript and is archived on Zenodo at https://www.doi.org/10.5281/zenodo.6402477. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Reduced Cell Surface Levels of C-C Chemokine Receptor 5 and Immunosuppression in Long Coronavirus Disease 2019 Syndrome.

In an exploratory trial treating "long COVID" with the CCR5-binding antibody leronlimab, we observed significantly increased blood cell surface CCR5 in treated symptomatic responders but not in nonresponders or placebo-treated participants. These findings suggest an unexpected mechanism of abnormal immune downmodulation in some persons that is normalized by leronlimab. Clinical Trials Registration. NCT04678830.

Suppression of human and simian immunodeficiency virus replication with the CCR5-specific antibody Leronlimab in two species.

The CCR5-specific antibody Leronlimab is being investigated as a novel immunotherapy that can suppress HIV replication with minimal side effects. Here we studied the virological and immunological consequences of Leronlimab in chronically CCR5-tropic HIV-1 infected humans (n = 5) on suppressive antiretroviral therapy (ART) and in ART-naïve acutely CCR5-tropic SHIV infected rhesus macaques (n = 4). All five human participants transitioned from daily combination ART to self-administered weekly subcutaneous (SC) injections of 350 mg or 700 mg Leronlimab and to date all participants have sustained virologic suppression for over seven years. In all participants, Leronlimab fully occupied CCR5 receptors on peripheral blood CD4+ T cells and monocytes. In ART-naïve rhesus macaques acutely infected with CCR5-tropic SHIV, weekly SC injections of 50 mg/kg Leronlimab fully suppressed plasma viremia in half of the macaques. CCR5 receptor occupancy by Leronlimab occurred concomitant with rebound of CD4+ CCR5+ T-cells in peripheral blood, and full CCR5 receptor occupancy was found in multiple anatomical compartments. Our results demonstrate that weekly, self-administered Leronlimab was safe, well-tolerated, and efficacious for long-term virologic suppression and should be included in the arsenal of safe, easily administered, longer-acting antiretroviral treatments for people living with HIV-1. Trial Registration: ClinicalTrials.gov Identifiers: NCT02175680 and NCT02355184.

CCR5 Receptor Occupancy Analysis Reveals Increased Peripheral Blood CCR5+CD4+ T Cells Following Treatment With the Anti-CCR5 Antibody Leronlimab.

CCR5 plays a central role in infectious disease, host defense, and cancer progression, thereby making it an ideal target for therapeutic development. Notably, CCR5 is the major HIV entry co-receptor, where its surface density correlates with HIV plasma viremia. The level of CCR5 receptor occupancy (RO) achieved by a CCR5-targeting therapeutic is therefore a critical predictor of its efficacy. However, current methods to measure CCR5 RO lack sensitivity, resulting in high background and overcalculation. Here, we report on two independent, flow cytometric methods of calculating CCR5 RO using the anti-CCR5 antibody, Leronlimab. We show that both methods led to comparable CCR5 RO values, with low background on untreated CCR5+CD4+ T cells and sensitive measurements of occupancy on both blood and tissue-resident CD4+ T cells that correlated longitudinally with plasma concentrations in Leronlimab-treated macaques. Using these assays, we found that Leronlimab stabilized cell surface CCR5, leading to an increase in the levels of circulating and tissue-resident CCR5+CD4+ T cells in vivo in Leronlimab-treated macaques. Weekly Leronlimab treatment in a chronically SIV-infected macaque led to increased CCR5+CD4+ T cells levels and fully suppressed plasma viremia, both concomitant with full CCR5 RO on peripheral blood CD4+ T cells, demonstrating that CCR5+CD4+ T cells were protected from viral replication by Leronlimab binding. Finally, we extended these results to Leronlimab-treated humans and found that weekly 700 mg Leronlimab led to complete CCR5 RO on peripheral blood CD4+ T cells and a statistically significant increase in CCR5+CD4+ T cells in peripheral blood. Collectively, these results establish two RO calculation methods for longitudinal monitoring of anti-CCR5 therapeutic antibody blockade efficacy in both macaques and humans, demonstrate that CCR5+CD4+ T cell levels temporarily increase with Leronlimab treatment, and facilitate future detailed investigations into the immunological impacts of CCR5 inhibition in multiple pathophysiological processes.

Identification and Characterization of Antigen-Specific CD8+ T Cells Using Surface-Trapped TNF-α and Single-Cell Sequencing.

CD8+ T cells are key mediators of antiviral and antitumor immunity. The isolation and study of Ag-specific CD8+ T cells, as well as mapping of their MHC restriction, has practical importance to the study of disease and the development of therapeutics. Unfortunately, most experimental approaches are cumbersome, owing to the highly variable and donor-specific nature of MHC-bound peptide/TCR interactions. Here we present a novel system for rapid identification and characterization of Ag-specific CD8+ T cells, particularly well suited for samples with limited primary cells. Cells are stimulated ex vivo with Ag of interest, followed by live cell sorting based on surface-trapped TNF-α. We take advantage of major advances in single-cell sequencing to generate full-length sequence data from the paired TCR α- and ß-chains from these Ag-specific cells. The paired TCR chains are cloned into retroviral vectors and used to transduce donor CD8+ T cells. These TCR transductants provide a virtually unlimited experimental reagent, which can be used for further characterization, such as minimal epitope mapping or identification of MHC restriction, without depleting primary cells. We validated this system using CMV-specific CD8+ T cells from rhesus macaques, characterizing an immunodominant Mamu-A1*002:01-restricted epitope. We further demonstrated the utility of this system by mapping a novel HLA-A*68:02-restricted HIV Gag epitope from an HIV-infected donor. Collectively, these data validate a new strategy to rapidly identify novel Ags and characterize Ag-specific CD8+ T cells, with applications ranging from the study of infectious disease to immunotherapeutics and precision medicine.