Chimeric antigen receptor T (CAR-T) cell therapy has demonstrated curative potential in B cell malignancies, yet translating this success to chronic infections like human immunodeficiency virus (HIV) remains a major challenge. In people living with HIV (PLWH) on suppressive antiretroviral therapy (ART), low antigen levels limit CAR-T cell expansion and persistence. We previously reported data from a pilot study which suggested that HIV-targeted CD4CAR-T cells could overcome this barrier through exogenous antigen supplementation, leading to robust in vivo expansion. Here, we sought to comprehensively confirm and expand on those findings. We tested a broad array of strategies to enhance CD4CAR-T cell efficacy, including CRISPR-Cas9-mediated gene editing of immune checkpoint and HIV-associated genes, single and pooled competitive infusions of engineered CAR-T cells, distinct CAR constructs incorporating either CD28 or 4-1BB costimulatory domains, and exogenous antigen boosting. We also developed highly sensitive droplet digital PCR (ddPCR) assays both to quantify CAR-T cell frequency and corroborate flow cytometry-based quantification of CD4CAR T-cell expansion. We evaluated these new approaches across multiple NHP models of HIV, including both simian immunodeficiency virus (SIV)- and simian-human immunodeficiency virus (SHIV)-infected, ART-suppressed NHPs. Although CD4CAR-T cell products exhibited antigen-specific proliferation and cytotoxicity ex vivo, they failed to expand, persist, or control viremia in vivo. We were also unable to confirm previously observed CD4CAR T cell expansions from our earlier studies, which will be retracted. Together these data highlight the need for alternative strategies to potentiate anti-HIV CD4CAR-T cells in the immunocompetent setting.

BACKGROUND:We recently reported that cytomegalovirus (CMV) viremia was associated with worse clinical outcomes in hospitalized children starting antiretroviral therapy (ART). Here, we interrogate associations between CMV and Epstein-Barr (EBV) viremia and HIV viral suppression, CD4 recovery, and growth after ART initiation. SETTING:In-patient setting, 4 hospitals in Kenya. METHODS:We used regression models to assess associations between CMV and EBV viremia and 6-month growth and immunologic and virologic responses to ART in children aged 2 months to 12 years. RESULTS:Overall, 142 (78%) of 181 children survived 6 months, of whom 94 (66%) had CMV and EBV DNA assessments pre-ART. Adjusting for baseline HIV RNA, children with baseline plasma CMV DNA ≥1000 IU//mL (coefficient=-5.65; 95% CI: -10.9 to -0.41, P = 0.035) and children with EBV DNA ≥1000 copies/mL had slower rates of CD4% increase (coefficient=-4.66; 95% CI: -7.06 to -2.27, P =<0.001). Age, WHO clinical stage, and stunting predicted growth recovery. CONCLUSIONS:High CMV or EBV viremia at admission predicted slower CD4 gain over 6 months in infants starting ART. These data suggest that herpesvirus viremia at baseline may identify a subset of children who will have a slower immunologic response to ART. Further research is needed to determine whether herpesvirus viremia delays ART responses and recovery in children.

BackgroundHuman herpesvirus 6B (HHV-6B) is associated with various central nervous system (CNS) disorders, particularly in immunocompromised patients. We present a rare case of inherited chromosomally integrated HHV-6B (iciHHV-6B), also referred to as endogenous HHV-6B (eHHV-6B) discovered during the workup for encephalitis in a child with relapsed non-WNT/non-SHH medulloblastoma.Case presentationA preschool aged female was treated in infancy for non-WNT, non-SHH medulloblastoma with high dose chemotherapy and autologous stem cell transplant. She had leptomeningeal recurrence 6 months after transplant and received salvage therapy with high dose craniospinal proton therapy. Six months following radiation, she developed high fever, acute encephalopathy and seizures. Neuroimaging revealed left posterior temporal gyral edema, while extensive infectious/paraneoplastic/autoimmune workup demonstrated markedly elevated HHV-6B viral loads, consistent with inherited chromosomally integrated HHV-6B (iciHHV-6B) which was confirmed through blood digital droplet PCR analysis and PCR-based nail clipping analysis demonstrating paternal inheritance. Retrospective immunohistochemical analysis of the original medulloblastoma revealed HHV-6 late structural glycoprotein H antigens in peritumoral lymphocytes. Serum HHV-6 U100 glycoprotein mRNA detection was consistent with iciHHV-6B. Treatment included intravenous immunoglobulin (IVIG) and ganciclovir followed by a 12-month course of IVIG monotherapy with clinical improvement.ConclusionOur case highlights the diagnostic challenges, controversies and clinical implications of ici-HHV-6/eHHV-6 in immunocompromised patients with encephalitis, where qualitative CSF viral detection alone may be insufficient for accurate diagnosis and cannot identify ici-HHV-6 status which complicates treatment decisions. This case demonstrates the importance of multimodal testing including chromosomal integration analysis in patients with high HHV-6 viral loads.

Background Limited understanding of the immunopathogenesis of human herpesvirus 6B (HHV-6B) has prevented its recognition as a pulmonary pathogen after allogeneic hematopoietic cell transplant (HCT). Methods We conducted a prospective study of allogeneic HCT recipients undergoing bronchoalveolar lavage (BAL) for lower respiratory tract disease (LRTD) within 120 days of HCT at three cancer centers from 2015-2019. We tested blood and BAL fluid (BALF) for HHV-6B DNA and mRNA transcripts associated with lytic infection and performed RNA-seq on paired blood. We distinguished between viral shedding and lytic infection based on a receiver operating characteristic (ROC) curve analysis to determine a BALF HHV-6B DNA viral load threshold predictive of HHV-6B mRNA transcript detection. We used genome-wide host expression profiling to examine whether HHV-6B detection was associated with unique transcriptional signatures. Results Among 116 participants, HHV-6B DNA was detected in 37% of BALs, 49% of which had HHV-6B mRNA detection. We established an HHV-6B DNA threshold (≥2.3 log10 copies/ml in BALF) that was highly predictive of HHV-6B mRNA detection and associated with increased risk for death from respiratory failure (adjusted HR, 2.35; 95% CI, 1.08-5.11; Figure 1). We identified a subgroup of 54 participants with a well-documented single cause of LRTD based on clinical testing (bacterial, n=7; viral, n=17; fungal, n=17; IPS, n=13) and who had a whole blood sample with sufficient quality of extracted RNA for RNA-seq. Principal components analysis (PCA) of the entire transcriptome suggested segregation between patients with viral LRTD who also had HHV-6B detection in BALF or plasma comparted to those without HHV-6B detection; similar segregation by HHV-6B detection was evident in the idiopathic pneumonia syndrome (IPS) subgroup but not in the fungal subgroup (Figure 2A-2C). Pathway analysis to define distinct groups of genes that share common biological function demonstrated enrichment of distinct transcriptional programs between participants with HHV-6B detection in BALF versus those without (Figure 2D-2F). Notably, HHV-6B detection in participants with clinically diagnosed IPS and fungal LRTD was associated with a strong host interferon response and enrichment of a variety of pathways consistent with active viral infection. Conclusions These data implicate HHV-6B as a pulmonary pathogen after allogeneic HCT.

Limited understanding of the immunopathogenesis of human herpesvirus 6B (HHV-6B) has prevented its acceptance as a pulmonary pathogen after hematopoietic cell transplant (HCT). In this prospective multicenter study of patients undergoing bronchoalveolar lavage (BAL) for pneumonia after allogeneic HCT, we test blood and BAL fluid (BALF) for HHV-6B DNA and mRNA transcripts associated with lytic infection and perform RNA-seq on paired blood. Among 116 participants, HHV-6B DNA is detected in 37% of BALs, 49% of which also have HHV-6B mRNA detection. We establish HHV-6B DNA viral load thresholds in BALF that are highly predictive of HHV-6B mRNA detection and associated with increased risk for overall mortality and death from respiratory failure. Participants with HHV-6B DNA in BALF exhibit distinct host gene expression signatures, notable for enriched interferon signaling pathways in participants clinically diagnosed with idiopathic pneumonia. These data implicate HHV-6B as a pulmonary pathogen after allogeneic HCT.

BACKGROUND:Although antivirals remain important for the treatment COVID-19, methods to assess treatment efficacy are lacking. Here, we investigated the impact of remdesivir on viral dynamics and their contribution to understanding antiviral efficacy in the multicenter Adaptive COVID-19 Treatment Trial 1, which randomized patients to remdesivir or placebo. METHODS:Longitudinal specimens collected during hospitalization from a substudy of 642 patients with COVID-19 were measured for viral RNA (upper respiratory tract and plasma), viral nucleocapsid antigen (serum), and host immunologic markers. Associations with clinical outcomes and response to therapy were assessed. RESULTS:Higher baseline plasma viral loads were associated with poorer clinical outcomes, and decreases in viral RNA and antigen in blood but not the upper respiratory tract correlated with enhanced benefit from remdesivir. The treatment effect of remdesivir was most pronounced in patients with elevated baseline nucleocapsid antigen levels: the recovery rate ratio was 1.95 (95% CI, 1.40-2.71) for levels >245 pg/mL vs 1.04 (95% CI, .76-1.42) for levels <245 pg/mL. Remdesivir also accelerated the rate of viral RNA and antigen clearance in blood, and patients whose blood levels decreased were more likely to recover and survive. CONCLUSIONS:Reductions in SARS-CoV-2 RNA and antigen levels in blood correlated with clinical benefit from antiviral therapy. CLINICAL TRIAL REGISTRATION:NCT04280705 (ClinicalTrials.gov).

The skin at the site of HSV-2 reactivation is enriched for HSV-2-specific T cells. To evaluate whether an immunotherapeutic vaccine could elicit skin-based memory T cells, we studied skin biopsies and HSV-2-reactive CD4+ T cells from PBMCs by T cell receptor (TCR) β chain (TRB) sequencing before and after vaccination with a replication-incompetent whole-virus HSV-2 vaccine candidate (HSV529). The representation of HSV-2-reactive CD4+ TRB sequences from PBMCs in the skin TRB repertoire increased after the first vaccine dose. We found sustained expansion after vaccination of unique, skin-based T cell clonotypes that were not detected in HSV-2-reactive CD4+ T cells isolated from PBMCs. In one participant, a switch in immunodominance occurred with the emergence of a TCR αβ pair after vaccination that was not detected in blood. This TCRαβ was shown to be HSV-2 reactive by expression of a synthetic TCR in a Jurkat-based NR4A1 reporter system. The skin in areas of HSV-2 reactivation possessed an oligoclonal TRB repertoire that was distinct from the circulation. Defining the influence of therapeutic vaccination on the HSV-2-specific TRB repertoire requires tissue-based evaluation.

s / Transplant Cell Ther 28 3S (2021) S1–S509 S213 cell recipients, 1 had evidence of prior SARS-CoV-2 infection (positive anti-N); this individual had a post-vaccine anti-S titer >2500 AU/mL and was excluded from analyses. 50% (n=5) developed positive anti-S IgG (median, 340 AU/mL), but only 10% (n=1) had a positive neutralization assay. 50% (n=5) had detectable S-specific T cells, of whom 20% (n=2) also had positive anti-S IgG (median, 844 AU/mL). Among the 22 post CAR-T cell patients, none had evidence of prior SARS-CoV-2 infection. 50% (n=11) developed positive anti-S IgG (median, 2100 AU/ mL), most of whom also had neutralizing antibodies (91%; n=10). 59% (n=13) developed S-specific T cells, of whom 54% (n=7) had positive anti-S IgG (median, 2942 AU/mL). Anti-S IgG titers correlated with neutralization and T cell levels (Fig. 1). Antibody responses appeared more frequent later after CAR-T cell therapy (Fig. 2). Interpretation: SARS-CoV-2 vaccination elicited humoral and/ or T cell responses in most individuals preand post-CAR-T cell therapy. Among individuals with cellular immunity, half did not have detectable humoral immune responses. Humoral immune responses in the pre-CAR-T cell cohort were of low level, and most did not generate neutralizing antibodies. Booster vaccination will likely play an important role in this patient population. 265 Brexucabtagene Autoleucel for Relapsed/Refractory Mantle Cell Lymphoma: Real World Experience from the US Lymphoma CAR T Consortium Javier Muñoz, MD, MS, MBA, FACP1; Yucai Wang, MD, PhD2; Preetesh Jain3; Frederick L. Locke, MD4; Matthew J Maurer5; Amer Beitinjaneh, MD6; Matthew J. Frank, MD, PhD7; Saurabh Dahiya, MD8; Joseph P. McGuirk, DO9; Miriam T Jacobs, MD10; Andre H. Goy, MD, MS11; Julie M. Vose, MD, MBA12; Brian T. Hill, MD, PhD13; Olalekan O. Oluwole, MBBS, MPH14; Abhinav Deol, MD15; Bijal Shah, MD16; Jonas Paludo, MD2; Trent P Wang, DO, MPH17; Lazaros J. Lekakis, MD17; David B. Miklos, MD, PhD7; Aaron P. Rapoport, MD18; Armin Ghobadi, MD19; Sattva S. Neelapu, MD20; Yi Lin, MD, PhD2; Michael Wang21 and Michael D Jain, MD, PhD4. 1 Division of Hematology and Medical Oncology, Mayo Clinic, Phoenix, AZ; 2 Division of Hematology, Mayo Clinic, Rochester, MN; 3 The University of Texas MD Anderson Cancer Center, Houston, TX; 4 Blood and Marrow Transplantation and Cellular Immunotherapy, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL; 5 Department of Health Sciences Research, Mayo Clinic, Rochester, MN; 6 University of Miami/Jackson Memorial Hospital, Miami, FL; 7 Division of BMT and Cellular Therapy, Stanford University School of Medicine, Stanford, CA; 8 University of Maryland Medical School of Medicine, Baltimore, MD; 9 Division of Hematologic Malignancies & Cellular Therapeutics, University of Kansas Medical Center, Kansas city, KS; 10 3Siteman Cancer Center, Washington University School of Medicine, St. Louis, MO, St. Louis, MO; 11 John Theurer Cancer Center, Hackensack, NJ; 12 Hematology/Oncology, University of Nebraska Medical Center, Omaha, NE; 13 Blood and Marrow Transplant Program, Department of Hematology and Medical Oncology, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH; 14 Vanderbilt-Ingram Cancer Center, Nashville, TN; 15 Karmanos Cancer Institute, Wayne State University, Detroit, MI; 16 Malignant Hematology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL; 17 Division of Transplantation & Cellular Therapy, University of Miami Hospital and Clinics, Sylvester Comprehensive Cancer Center, Miami, FL; 18 The Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD; 19 Washington University School of Medicine, Saint Louis, MO; 20 Department of Lymphoma and Myeloma, The University of Texas MD Anderson Cancer Center, Houston, TX; 21 Department of Lymphoma and Myeloma, University of Texas, MD Anderson Cancer Center, Houston, TX Introduction: Brexucabtagene autoleucel (brexu-cel) was approved by US FDA for relapsed/refractory (R/R) mantle cell lymphoma (MCL) on July 24, 2020 based on results from the pivotal ZUMA-2 (NCT02601313) study, in which an objective response rate (ORR) of 93% and a complete response (CR) rate of 67% were achieved. Methods: Fourteen centers participated in this retrospective study. Patients who underwent leukapheresis by 6/15/2021 with an intent to manufacture brexu-cel were included. Duration of response, progression-free survival (PFS) and overall survival (OS) were analyzed using the Kaplan-Meier method. Results: At the data cut-off date of 7/9/2021, 107 patients underwent leukapheresis, among whom 93 (87%) completed brexu-cel infusion, 2 (2%) were waiting for infusion, and 12 (11%) did not receive infusion (manufacture failure n=6, organ dysfunction n=1, death n=5). Baseline clinical characteristics are shown in Table 1. The median age was 67 years and 81% were male. 32% had high risk simplified MIPI, 77% had Ki-67≥30%, 45% had blastoid or pleomorphic variant, 46% had TP53 alteration, 29% had complex karyotype, and 7% had CNS

BACKGROUND:Antiviral resistance in human herpes simplex viruses (HSV) remains a significant clinical challenge in immunocompromised populations. Although molecular tests have largely replaced viral culture for HSV diagnosis and molecular antiviral resistance testing is available for many viruses, HSV resistance testing continues to rely on phenotypic, viral culture-based methods, requiring weeks for results. Consequently, treatment of suspected HSV resistance remains largely empiric.METHODS:We used HSV whole genome sequencing and a database of previously characterized HSV acyclovir and foscarnet resistance mutations to evaluate the performance of genotypic antiviral resistance testing among 19 control strains compared to in-house plaque reduction assay (PRA) and 25 clinical isolates sent for reference lab PRA antiviral resistance testing.RESULTS:Among control strains, 23/29 (79.3%) results were concordant, 5 (17.2%) were indeterminate, and 1 (3.4%) was discordant. Indeterminate results were caused by variants of uncertain significance (VUS), including mutations without published phenotypes and mutations with contradictory results. Among clinical isolates, 14/40 (35%) results were concordant, 17 (42.5%) were indeterminate, and 9 (22.5%) were discordant. All discordant results were in reportedly phenotypically-susceptible HSV-1 strains yet possessed resistance mutations. Three contained resistant subpopulations. 6/8 (75%) discordant phenotypes were concordant with resistant genotypes upon repeat PRA.CONCLUSIONS:These data support the combination of genotypic and phenotypic testing to diagnose HSV resistance more accurately and likely more rapidly than phenotypic testing alone. Genotypic context of resistance mutations and the ability of viral strains to form plaques in culture may affect phenotypic resistance results, highlighting the limitations of PRA alone as a gold standard method.

HIV-specific T cells are necessary for control of HIV-1 replication but are largely insufficient for viral clearance. This is due in part to these cells' recognition of immunodominant but variable regions of the virus, which facilitates viral escape via mutations that do not incur viral fitness costs. HIV-specific T cells targeting conserved viral elements are associated with viral control but are relatively infrequent in people living with HIV (PLWH). The goal of this study was to increase the number of these cells via an ex vivo cell manufacturing approach derived from our clinically-validated HIV-specific expanded T-cell (HXTC) process. Using a nonhuman primate (NHP) model of HIV infection, we sought to determine i) the feasibility of manufacturing ex vivo-expanded virus-specific T cells targeting viral conserved elements (CE, CE-XTCs), ii) the in vivo safety of these products, and iii) the impact of simian/human immunodeficiency virus (SHIV) challenge on their expansion, activity, and function. NHP CE-XTCs expanded up to 10-fold following co-culture with the combination of primary dendritic cells (DCs), PHA blasts pulsed with CE peptides, irradiated GM-K562 feeder cells, and autologous T cells from CE-vaccinated NHP. The resulting CE-XTC products contained high frequencies of CE-specific, polyfunctional T cells. However, consistent with prior studies with human HXTC and these cells' predominant CD8(+) effector phenotype, we did not observe significant differences in CE-XTC persistence or SHIV acquisition in two CE-XTC-infused NHP compared to two control NHP. These data support the safety and feasibility of our approach and underscore the need for continued development of CE-XTC and similar cell-based strategies to redirect and increase the potency of cellular virus-specific adaptive immune responses.

Background and Aims:. Adeno-associated virus (AAV) vectors are widely used to deliver therapeutic transgenes to distinct tissues, including the liver. Vectors based on naturally occurring AAV serotypes as well as vectors using engineered capsids have shown variations in tissue tropism and level of transduction between different mouse models. Moreover, results obtained in rodents frequently lack translatability into large animal studies. In light of the increasing interest in AAV vectors for human gene therapy, an increasing number of studies are being performed in nonhuman primates. To keep animal numbers to a minimum and thus optimize the process of AAV capsid selection, we developed a multiplex barcoding approach to simultaneously evaluate the in vivo vector performance for a set of serotypes and capsid-engineered AAV vectors across multiple organs. Approach and Results:. Vector biodistribution and transgene expression were assessed by quantitative PCR, quantitative reverse transcription PCR, vector DNA amplicon Illumina sequencing and vRNAseq in male and female rhesus macaques simultaneously dosed with a mixture of barcoded naturally occurring or engineered AAV vectors encoding the same transgene. As expected, our findings show animal-to-animal variation in both the biodistribution and tissue transduction pattern, which was partly influenced by each animal’s distinctive serological status. Conclusions:. This method offers a robust approach to AAV vector optimization that can be used to identify and validate AAV vectors for gene delivery to potentially any anatomical site or cell type.

Kidney disease in allogeneic hematopoietic cell transplantation (HCT) recipients is associated with increased mortality rates. BK virus (BKV) viremia has been associated with kidney dysfunction in pediatric HCT recipients; however, few studies have investigated longer-term kidney outcomes in association with BKV in this population. Here we assessed the relationship between BK viremia and changes in estimated glomerular filtration rate (eGFR) in children in the first year post-HCT. We selected 136 patients age <26 years who underwent HCT in 2007 to 2018 at a single center and had plasma BK viral load data available at 2 time points, weeks 4 to 7 post-HCT and weeks 10 to 13 post-HCT from prospectively collected stored plasma samples. A total of 272 samples were analyzed for BKV using quantitative PCR. We used multivariate linear models to determine the association of BK viremia and change in eGFR by 1 year post-HCT. Forty percent of the patients (54 of 136) had BKV detection in weeks 4 to 7, 13% of whom (7 of 54) had a BK viral load of >10,000 copies/mL, and 46% (62 of 136) had BKV detected in weeks 10 to 13, 34% (21 of 62) of whom had a BK viral load of >10,000 copies/mL. The mean decline in eGFR was 25.73 mL/min/1.73 m2 by 1 year post-HCT. In multivariate models, a BK viral load of >10,000 copies/mL during weeks 4 to 7 was associated with a mean decline in eGFR of 30.6 mL/min/1.73 m2 (95% confidence interval, -55.94 to -5.17; P = .019) compared with a BK viral load <10,000 copies/mL. In adjusted analyses, a high BK viral load in the blood (>10,000 copies/mL) was associated with a significant decline in eGFR by 1 year post-HCT. (c) 2022 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.

The major barrier to an HIV cure is the persistence of infected cells that evade host immune surveillance despite effective antiretroviral therapy (ART). Most prior host genetic HIV studies have focused on identifying DNA polymorphisms (e.g., CCR5Δ32 , MHC class I alleles) associated with viral load among untreated "elite controllers" (~1% of HIV+ individuals who are able to control virus without ART). However, there have been few studies evaluating host genetic predictors of viral control for the majority of people living with HIV (PLWH) on ART. We performed host RNA sequencing and HIV reservoir quantification (total DNA, unspliced RNA, intact DNA) from peripheral CD4+ T cells from 191 HIV+ ART-suppressed non-controllers. Multivariate models included covariates for timing of ART initiation, nadir CD4+ count, age, sex, and ancestry. Lower HIV total DNA (an estimate of the total reservoir) was associated with upregulation of tumor suppressor genes NBL1 (q=0.012) and P3H3 (q=0.012). Higher HIV unspliced RNA (an estimate of residual HIV transcription) was associated with downregulation of several host genes involving inflammasome ( IL1A, CSF3, TNFAIP5, TNFAIP6, TNFAIP9 , CXCL3, CXCL10 ) and innate immune ( TLR7 ) signaling, as well as novel associations with potassium ( KCNJ2 ) and gap junction ( GJB2 ) channels, all q<0.05. Gene set enrichment analyses identified significant associations with TLR4/microbial translocation (q=0.006), IL-1β/NRLP3 inflammasome (q=0.008), and IL-10 (q=0.037) signaling. HIV intact DNA (an estimate of the "replication-competent" reservoir) demonstrated trends with thrombin degradation ( PLGLB1 ) and glucose metabolism ( AGL ) genes, but data were (HIV intact DNA detected in only 42% of participants). Our findings demonstrate that among treated PLWH, that inflammation, innate immune responses, bacterial translocation, and tumor suppression/cell proliferation host signaling play a key role in the maintenance of the HIV reservoir during ART. Further data are needed to validate these findings, including functional genomic studies, and expanded epidemiologic studies in female, non-European cohorts.Author Summary:Although lifelong HIV antiretroviral therapy (ART) suppresses virus, the major barrier to an HIV cure is the persistence of infected cells that evade host immune surveillance despite effective ART, "the HIV reservoir." HIV eradication strategies have focused on eliminating residual virus to allow for HIV remission, but HIV cure trials to date have thus far failed to show a clinically meaningful reduction in the HIV reservoir. There is an urgent need for a better understanding of the host-viral dynamics during ART suppression to identify potential novel therapeutic targets for HIV cure. This is the first epidemiologic host gene expression study to demonstrate a significant link between HIV reservoir size and several well-known immunologic pathways (e.g., IL-1β, TLR7, TNF-α signaling pathways), as well as novel associations with potassium and gap junction channels (Kir2.1, connexin 26). Further data are needed to validate these findings, including functional genomic studies and expanded epidemiologic studies in female, non-European cohorts.

The major barrier to an HIV cure is the HIV reservoir: latently-infected cells that persist despite effective antiretroviral therapy (ART). There have been few cohort-based studies evaluating host genomic or transcriptomic predictors of the HIV reservoir. We performed host RNA sequencing and HIV reservoir quantification (total DNA [tDNA], unspliced RNA [usRNA], intact DNA) from peripheral CD4+ T cells from 191 ART-suppressed people with HIV (PWH). After adjusting for nadir CD4+ count, timing of ART initiation, and genetic ancestry, we identified two host genes for which higher expression was significantly associated with smaller total DNA viral reservoir size, P3H3 and NBL1, both known tumor suppressor genes. We then identified 17 host genes for which lower expression was associated with higher residual transcription (HIV usRNA). These included novel associations with membrane channel (KCNJ2, GJB2), inflammasome (IL1A, CSF3, TNFAIP5, TNFAIP6, TNFAIP9, CXCL3, CXCL10), and innate immunity (TLR7) genes (FDR-adjusted q<0.05). Gene set enrichment analyses further identified significant associations of HIV usRNA with TLR4/microbial translocation (q = 0.006), IL-1/NRLP3 inflammasome (q = 0.008), and IL-10 (q = 0.037) signaling. Protein validation assays using ELISA and multiplex cytokine assays supported these observed inverse host gene correlations, with P3H3, IL-10, and TNF-α protein associations achieving statistical significance (p<0.05). Plasma IL-10 was also significantly inversely associated with HIV DNA (p = 0.016). HIV intact DNA was not associated with differential host gene expression, although this may have been due to a large number of undetectable values in our study. To our knowledge, this is the largest host transcriptomic study of the HIV reservoir. Our findings suggest that host gene expression may vary in response to the transcriptionally active reservoir and that changes in cellular proliferation genes may influence the size of the HIV reservoir. These findings add important data to the limited host genetic HIV reservoir studies to date.

Objectives: Letermovir for cytomegalovirus (CMV) prophylaxis in allogeneic haematopoietic cell trans-plant (HCT) recipients has decreased anti-CMV therapy use. Contrary to letermovir, anti-CMV antivirals are also active against human herpesvirus-6 (HHV-6). We assessed changes in HHV-6 epidemiology in the post-letermovir era. Methods: We conducted a retrospective cohort study of CMV-seropositive allogeneic HCT recipients comparing time periods before and after routine use of prophylactic letermovir. HHV-6 testing was at the discretion of clinicians. We computed the cumulative incidence of broad-spectrum antiviral initiation (foscarnet, (val)ganciclovir, and/or cidofovir), HHV-6 testing, and HHV-6 detection in blood and cere-brospinal fluid within 100 days after HCT. We used Cox proportional-hazards models with stabilized inverse probability of treatment weights to compare outcomes between cohorts balanced for baseline factors. Results: We analysed 738 patients, 376 in the pre-letermovir and 362 in the post-letermovir cohort. Broad-spectrum antiviral initiation incidence decreased from 65% (95% CI, 60-70%) pre-letermovir to 21% (95% CI, 17-25%) post-letermovir. The cumulative incidence of HHV-6 testing (17% [95% CI, 13-21%] pre-letermovir versus 13% [95% CI, 10-16%] post-letermovir), detection (3% [95% CI, 1-5%] in both co-horts), and HHV-6 encephalitis (0.5% [95% CI, 0.1-1.8%] pre-letermovir and 0.6% [95% CI, 0.1-1.9%] post-letermovir) were similar between cohorts. First HHV-6 detection occurred at a median of 37 days (interquartile range, 18-58) in the pre-letermovir cohort and 27 (interquartile range, 25-34) in the post-letermovir cohort. In a weighted model, there was no association between the pre-versus post-leter-movir cohort and HHV-6 detection (adjusted hazard ratio, 1.08; 95% CI, 0.44-2.62). Discussion: : Despite a large decrease in broad-spectrum antivirals after the introduction of letermovir prophylaxis in CMV-seropositive allogeneic HCT recipients, there was no evidence for increased clinically detected HHV-6 reactivation and disease. Eleftheria Kampouri, Clin Microbiol Infect 2023;29:1450.e1 -1450.e7 (c) 2023 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Liver-humanized mouse models have become the gold standard for the in vivo study of hepatitis B virus (HBV), yet their complexity and cost have prohibited widespread use of existing models in research. Here, we show that the NSG-PiZ liver-humanized mouse model, which is relatively inexpensive and simple to establish, can support chronic HBV infection.

Background Co-infection with HIV can result in impaired control of cytomegalovirus (CMV) replication, increasing the likelihood of disease and onward transmission. The objective of this analysis was to measure the impact of HIV on CMV replication in an intensively-sampled cohort in Kampala, Uganda. Methods CMV seropositive men and women aged 18–65, with or without HIV co-infection, were followed for one month. Daily oral swabs and weekly anogenital swabs and plasma were collected. Quantitative CMV PCR was performed on all samples. Results Eighty-five participants were enrolled and provided ≥1 oral swab; 43 (51%) were HIV-seropositive. People living with HIV (PLWH; median CD4 count 439 cells/mm 3 ; none on antiretrovirals) had 2–4 times greater risk of CMV detection at each anatomical site assessed. At the oral site, 773 of 1272 (61%) of samples from PLWH had CMV detected, compared to 214 of 1349 (16%) among people without HIV. Similarly, the mean CMV quantity was higher among PLWH at all anatomical sites, with the largest difference seen for oral swabs (mean difference 1.63 log/mL; 95% CI 1.13–2.13). Among PLWH, absolute quantity of CD4+ T-cells was not associated with risk of CMV detection. HIV plasma RNA quantity was positively correlated with oral CMV shedding frequency, but not detection at other sites. Conclusions Mucosal and systemic CMV replication occurs at higher levels in PLWH than people without HIV, particularly oral shedding, which is a major mode of CMV transmission. Increased CMV replication despite relatively preserved CD4+ T-cell counts suggests that additional interventions are required to improve CMV control in PLWH.

Abstract Background Remdesivir is approved for treatment of coronavirus disease 2019 (COVID-19) in nonhospitalized and hospitalized adult and pediatric patients. Here we present severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) resistance analyses from the phase 3 ACTT-1 randomized placebo-controlled trial conducted in adult participants hospitalized with COVID-19. Methods Swab samples were collected at baseline and longitudinally through day 29. SARS-CoV-2 genomes were sequenced using next-generation sequencing. Phenotypic analysis was conducted directly on participant virus isolates and/or using SARS-CoV-2 subgenomic replicons expressing mutations identified in the Nsp12 target gene. Results Among participants with both baseline and postbaseline sequencing data, emergent Nsp12 substitutions were observed in 12 of 31 (38.7%) and 12 of 30 (40.0%) participants in the remdesivir and placebo arms, respectively. No emergent Nsp12 substitutions in the remdesivir arm were observed in more than 1 participant. Phenotyping showed low to no change in susceptibility to remdesivir relative to wild-type Nsp12 reference for the substitutions tested: A16V (0.8-fold change in EC50), P323L + V792I (2.2-fold), C799F (2.5-fold), K59N (1.0-fold), and K59N + V792I (3.4-fold). Conclusions The similar rate of emerging Nsp12 substitutions in the remdesivir and placebo arms and the minimal change in remdesivir susceptibility among tested substitutions support a high barrier to remdesivir resistance development in COVID-19 patients. Clinical Trials Registration. NCT04280705.