CMV reactivation during pretransplantation evaluation: a novel risk factor for posttransplantation CMV reactivation.

ABSTRACT: Cytomegalovirus (CMV) disease occurs occasionally before allogeneic hematopoietic cell transplantation (HCT) and is associated with poor post-HCT outcomes; however, the impact of pre-HCT CMV reactivation is unknown. Pre-HCT CMV reactivation was assessed in HCT candidates from the preemptive antiviral therapy (2007-2017) and letermovir prophylaxis (2018-2021) eras. CMV DNA polymerase chain reaction (PCR) surveillance was routinely performed during the pre-HCT workup period, and antiviral therapy was recommended according to risk of progression to CMV disease. Risk factors for pre-HCT CMV reactivation were characterized, and the associations of pre-HCT CMV reactivation with post-HCT outcomes were examined using logistic regression and Cox proportional hazard models, respectively. A total of 1694 patients were identified, and 11% had pre-HCT CMV reactivation 14 days (median; interquartile range [IQR], 6-23) before HCT. Lymphopenia (≤0.3 × 103/µL) was the strongest risk factor for pre-HCT CMV reactivation at multiple PCR levels. In the preemptive therapy era, patients with pre-HCT CMV reactivation had a significantly increased risk of CMV reactivation by day 100 as well as CMV disease and death by 1 year after HCT. Clearance of pre-HCT CMV reactivation was associated with a lower risk of post-HCT CMV reactivation. Similar associations with post-HCT CMV end points were observed in a cohort of patients receiving letermovir prophylaxis. Pre-HCT CMV reactivation can be routinely detected in high-risk HCT candidates and is a significant risk factor for post-HCT CMV reactivation and disease. Pre-HCT CMV DNA PCR surveillance is recommended in high-risk HCT candidates, and antiviral therapy may be indicated to prevent post-HCT CMV reactivation.

Gene editing for latent herpes simplex virus infection reduces viral load and shedding in vivo.

Anti-HSV therapies are only suppressive because they do not eliminate latent HSV present in ganglionic neurons, the source of recurrent disease. We have developed a potentially curative approach against HSV infection, based on gene editing using HSV-specific meganucleases delivered by adeno-associated virus (AAV) vectors. Gene editing performed with two anti-HSV-1 meganucleases delivered by a combination of AAV9, AAV-Dj/8, and AAV-Rh10 can eliminate 90% or more of latent HSV DNA in mouse models of orofacial infection, and up to 97% of latent HSV DNA in mouse models of genital infection. Using a pharmacological approach to reactivate latent HSV-1, we demonstrate that ganglionic viral load reduction leads to a significant decrease of viral shedding in treated female mice. While therapy is well tolerated, in some instances, we observe hepatotoxicity at high doses and subtle histological evidence of neuronal injury without observable neurological signs or deficits. Simplification of the regimen through use of a single serotype (AAV9) delivering single meganuclease targeting a duplicated region of the HSV genome, dose reduction, and use of a neuron-specific promoter each results in improved tolerability while retaining efficacy. These results reinforce the curative potential of gene editing for HSV disease.

Variant mutation in SARS-CoV-2 nucleocapsid enhances viral infection via altered genomic encapsidation.

The evolution of SARS-CoV-2 variants and their respective phenotypes represents an important set of tools to understand basic coronavirus biology as well as the public health implications of individual mutations in variants of concern. While mutations outside of Spike are not well studied, the entire viral genome is undergoing evolutionary selection, particularly the central disordered linker region of the nucleocapsid (N) protein. Here, we identify a mutation (G215C), characteristic of the Delta variant, that introduces a novel cysteine into this linker domain, which results in the formation of a disulfide bond and a stable N-N dimer. Using reverse genetics, we determined that this cysteine residue is necessary and sufficient for stable dimer formation in a WA1 SARS-CoV-2 background, where it results in significantly increased viral growth both in vitro and in vivo. Finally, we demonstrate that the N:G215C virus packages more nucleocapsid per virion and that individual virions are larger, with elongated morphologies.

SARS-CoV-2 RNA and Nucleocapsid Antigen Are Blood Biomarkers Associated With Severe Disease Outcomes That Improve in Response to Remdesivir.

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).

Human herpesvirus 6 reactivation and disease are infrequent in chimeric antigen receptor T-cell therapy recipients.

ABSTRACT: Human herpesvirus 6B (HHV-6B) reactivation and disease are increasingly reported after chimeric antigen receptor (CAR) T-cell therapy (CARTx). HHV-6 reactivation in the CAR T-cell product was recently reported, raising questions about product and patient management. Because of overlapping manifestations with immune effector cell-associated neurotoxicity syndrome, diagnosing HHV-6B encephalitis is challenging. We provide 2 lines of evidence assessing the incidence and outcomes of HHV-6B after CARTx. First, in a prospective study with weekly HHV-6B testing for up to 12 weeks after infusion, HHV-6B reactivation occurred in 8 of 89 participants; 3 had chromosomally integrated HHV-6 and were excluded, resulting in a cumulative incidence of HHV-6B reactivation of 6% (95% confidence interval [CI], 2.2-12.5). HHV-6B detection was low level (median peak, 435 copies per mL; interquartile range, 164-979) and did not require therapy. Second, we retrospectively analyzed HHV-6B detection in the blood and/or cerebrospinal fluid (CSF) within 12 weeks after infusion in CARTx recipients. Of 626 patients, 24 had symptom-driven plasma testing, with detection in 1. Among 34 patients with CSF HHV-6 testing, 1 patient had possible HHV-6 encephalitis for a cumulative incidence of 0.17% (95% CI, 0.02-0.94), although symptoms improved without treatment. Our data demonstrate that HHV-6B reactivation and disease are infrequent after CARTx. Routine HHV-6 monitoring is not warranted.

Late cytomegalovirus disease after hematopoietic cell transplantation: significance of novel transplantation techniques.

ABSTRACT: Preemptive therapy (PET) and letermovir prophylaxis are effective in preventing cytomegalovirus (CMV) disease within the first 100 days after allogeneic hematopoietic cell transplantation (HCT) but are associated with late-onset CMV disease. We retrospectively examined the clinical manifestations, risk factors, prevention algorithm, and outcome of late CMV disease in CMV seropositive day 100 survivors transplanted between 2001-2017 (PET cohort) and 2018-2021 (letermovir cohort). There were 203 episodes of late CMV disease among 2469 day 100 survivors, and the estimated cumulative incidence of first late CMV disease was 7.2% (95% confidence interval [CI], 6.2-8.3) with no difference between the PET (7.4%; 95% CI, 6.4-8.6) and the letermovir group (5.4%; 95% CI, 3.2-8.3). Thirty-seven patients (1.5%) had a second episode of CMV disease. In multivariable Cox regression models, posttransplant cyclophosphamide was associated with an increased risk of gastrointestinal CMV disease. CMV viremia or disease detected before day 100, corticosteroid treatment after day 100 at dose ≥1 mg/kg, acute and chronic graft-versus-host disease, lymphopenia, HLA-mismatched related donor status, were also associated with late CMV disease. HLA-mismatched donor status and late use of corticosteroids (≥1 mg/kg) were risk factors for late CMV disease recurrence. Late CMV disease occurred most frequently in a setting of prolonged low-level untreated viremia and was independently associated with death by 2 years after HCT. In summary, late CMV disease continues to occur in the present era. Improved prevention strategies for late CMV disease are needed.

CD20 CAR T cells safely and reversibly ablate B cell follicles in a non-human primate model of HIV persistence.

Chimeric antigen receptor (CAR) T cell therapies have demonstrated immense clinical success for B cell and plasma cell malignancies. We tested their impact on the viral reservoir in a macaque model of HIV persistence, comparing the functions of CD20 CAR T cells between animals infected with simian/human immunodeficiency virus (SHIV) and uninfected controls. We focused on the potential of this approach to disrupt B cell follicles (BCFs), exposing infected cells for immune clearance. In SHIV-infected animals, CAR T cells were highly functional, with rapid expansion and trafficking to tissue-associated viral sanctuaries, including BCFs and gut-associated lymphoid tissue (GALT). CD20 CAR T cells potently ablated BCFs and depleted lymph-node-associated follicular helper T (TFH) cells, with complete restoration of BCF architecture and TFH cells following CAR T cell contraction. BCF ablation decreased the splenic SHIV reservoir but was insufficient for effective reductions in systemic viral reservoirs. Although associated with moderate hematologic toxicity, CD20 CAR T cells were well tolerated in SHIV-infected and control animals, supporting the feasibility of this therapy in people living with HIV with underlying B cell malignancies. Our findings highlight the unique ability of CD20 CAR T cells to safely and reversibly unmask TFH cells within BCF sanctuaries, informing future combinatorial HIV cure strategies designed to augment antiviral efficacy.

HHV-6B detection and host gene expression implicate HHV-6B as pulmonary pathogen after hematopoietic cell transplant.

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.

Cytomegalovirus (CMV) Reactivation and CMV-Specific Cell-Mediated Immunity After Chimeric Antigen Receptor T-Cell Therapy.

BACKGROUND: The epidemiology of cytomegalovirus (CMV) after chimeric antigen receptor-modified T-cell immunotherapy (CARTx) is poorly understood owing to a lack of routine surveillance. METHODS: We prospectively enrolled 72 adult CMV-seropositive CD19-, CD20-, or BCMA-targeted CARTx recipients and tested plasma samples for CMV before and weekly up to 12 weeks after CARTx. We assessed CMV-specific cell-mediated immunity (CMV-CMI) before and 2 and 4 weeks after CARTx, using an interferon γ release assay to quantify T-cell responses to IE-1 and pp65. We tested pre-CARTx samples to calculate a risk score for cytopenias and infection (CAR-HEMATOTOX). We used Cox regression to evaluate CMV risk factors and evaluated the predictive performance of CMV-CMI for CMV reactivation in receiver operator characteristic curves. RESULTS: CMV was detected in 1 patient (1.4%) before and in 18 (25%) after CARTx, for a cumulative incidence of 27% (95% confidence interval, 16.8-38.2). The median CMV viral load (interquartile range) was 127 (interquartile range, 61-276) IU/mL, with no end-organ disease observed; 5 patients received preemptive therapy based on clinical results. CMV-CMI values reached a nadir 2 weeks after infusion and recovered to baseline levels by week 4. In adjusted models, BCMA-CARTx (vs CD19/CD20) and corticosteroid use for >3 days were significantly associated with CMV reactivation, and possible associations were detected for lower week 2 CMV-CMI and more prior antitumor regimens. The cumulative incidence of CMV reactivation almost doubled when stratified by BCMA-CARTx target and use of corticosteroids for >3 days (46% and 49%, respectively). CONCLUSIONS: CMV testing could be considered between 2 and 6 weeks in high-risk CARTx recipients.

Within-Host Rhinovirus Evolution in Upper and Lower Respiratory Tract Highlights Capsid Variability and Mutation-Independent Compartmentalization.

BACKGROUND: Rhinovirus (RV) infections can progress from the upper (URT) to lower (LRT) respiratory tract in immunocompromised individuals, causing high rates of fatal pneumonia. Little is known about how RV evolves within hosts during infection. METHODS: We sequenced RV complete genomes from 12 hematopoietic cell transplant patients with infection for up to 190 days from both URT (nasal wash, NW) and LRT (bronchoalveolar lavage, BAL). Metagenomic and amplicon next-generation sequencing were used to track the emergence and evolution of intrahost single nucleotide variants (iSNVs). RESULTS: Identical RV intrahost populations in matched NW and BAL specimens indicated no genetic adaptation is required for RV to progress from URT to LRT. Coding iSNVs were 2.3-fold more prevalent in capsid over nonstructural genes. iSNVs modeled were significantly more likely to be found in capsid surface residues, but were not preferentially located in known RV-neutralizing antibody epitopes. Newly emergent, genotype-matched iSNV haplotypes from immunocompromised individuals in 2008-2010 could be detected in Seattle-area community RV sequences in 2020-2021. CONCLUSIONS: RV infections in immunocompromised hosts can progress from URT to LRT with no specific evolutionary requirement. Capsid proteins carry the highest variability and emergent mutations can be detected in other, including future, RV sequences.

A cohort-based study of host gene expression: tumor suppressor and innate immune/inflammatory pathways associated with the HIV reservoir size.

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.

Efficient long-term multilineage engraftment of CD33-edited hematopoietic stem/progenitor cells in nonhuman primates.

Current immunotherapeutic targets are often shared between neoplastic and normal hematopoietic stem and progenitor cells (HSPCs), leading to unwanted on-target, off-tumor toxicities. Deletion or modification of such targets to protect normal HSPCs is, therefore, of great interest. Although HSPC modifications commonly aim to mimic naturally occurring phenotypes, the long-term persistence and safety of gene-edited cells need to be evaluated. Here, we deleted the V-set domain of CD33, the immune-dominant domain targeted by most anti-CD33 antibodies used to treat CD33-positive malignancies, including acute myeloid leukemia, in the HSPCs of two rhesus macaques, performed autologous transplantation after myeloablative conditioning, and followed the animals for up to 3 years. CD33-edited HSPCs engrafted without any delay in recovery of neutrophils, the primary cell type expressing CD33. No impact on the blood composition, reconstitution of the bone marrow stem cell compartment, or myeloid differentiation potential was observed. Up to 20% long-term gene editing in HSPCs and blood cell lineages was seen with robust loss of CD33 detection on myeloid lineages. In conclusion, deletion of the V-set domain of CD33 on HSPCs, progenitors, and myeloid lineages did not show any adverse effects on their homing and engraftment potential or the differentiation and functionality of myeloid progenitors and lineages.

Viral Resistance Analyses From the Remdesivir Phase 3 Adaptive COVID-19 Treatment Trial-1 (ACTT-1).

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.

Within-host rhinovirus evolution in upper and lower respiratory tract highlights capsid variability and mutation-independent compartmentalization.

Background: Human rhinovirus (HRV) infections can progress from the upper (URT) to lower (LRT) respiratory tract in immunocompromised individuals, causing high rates of fatal pneumonia. Little is known about how HRV evolves within hosts during infection. Methods: We sequenced HRV complete genomes from 12 hematopoietic cell transplant patients with prolonged infection for up to 190 days from both URT (nasal wash, NW) and LRT (bronchoalveolar lavage, BAL) specimens. Metagenomic (mNGS) and amplicon-based NGS were used to study the emergence and evolution of intra-host single nucleotide variants (iSNVs). Results: Identical HRV intra-host populations in matched NW and BAL specimens indicated no genetic adaptation is required for HRV to progress from URT to LRT. Microbial composition between matched NW and BAL confirmed no cross-contamination during sampling procedure. Coding iSNVs were 2.3-fold more prevalent in capsid over non-structural genes, adjusted for length. iSNVs modeled onto HRV capsid structures were significantly more likely to be found in surface residues, but were not preferentially located in known HRV neutralizing antibody epitopes. Newly emergent, serotype-matched iSNV haplotypes from immunocompromised individuals from 2008-2010 could be detected in Seattle-area community HRV sequences from 2020-2021. Conclusion: HRV infections in immunocompromised hosts can progress from URT to LRT with no specific evolutionary requirement. Capsid proteins carry the highest variability and emergent mutations can be detected in other, including future, HRV sequences.

Liver-Humanized NSG-PiZ Mice Support the Study of Chronic Hepatitis B Virus Infection and Antiviral Therapies.

Hepatitis B virus (HBV) is a pathogen of major public health importance that is largely incurable once a chronic infection is established. Only humans and great apes are fully permissive to HBV infection, and this species restriction has impacted HBV research by limiting the utility of small animal models. To combat HBV species restrictions and enable more in vivo studies, liver-humanized mouse models have been developed that are permissive to HBV infection and replication. Unfortunately, these models can be difficult to establish and are expensive commercially, which has limited their academic use. As an alternative mouse model to study HBV, we evaluated liver-humanized NSG-PiZ mice and showed that they are fully permissive to HBV. HBV selectively replicates in human hepatocytes within chimeric livers, and HBV-positive (HBV+) mice secrete infectious virions and hepatitis B surface antigen (HBsAg) into blood while also harboring covalently closed circular DNA (cccDNA). HBV+ mice develop chronic infections lasting at least 169 days, which should enable the study of new curative therapies targeting chronic HBV, and respond to entecavir therapy. Furthermore, HBV+ human hepatocytes in NSG-PiZ mice can be transduced by AAV3b and AAV.LK03 vectors, which should enable the study of gene therapies that target HBV. In summary, our data demonstrate that liver-humanized NSG-PiZ mice can be used as a robust and cost-effective alternative to existing chronic hepatitis B (CHB) models and may enable more academic research labs to study HBV disease pathogenesis and antiviral therapy. IMPORTANCE 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. Infected mice are fully permissive to hepatitis B, supporting both active replication and spread, and can be used to study novel antiviral therapies. This model is a viable and cost-effective alternative to other liver-humanized mouse models that are used to study HBV.

Adeno-associated virus vectors and neurotoxicity-lessons from preclinical and human studies.

Over 15 years after hepatotoxicity was first observed following administration of an adeno-associated virus (AAV) vector during a hemophilia B clinical trial, recent reports of treatment-associated neurotoxicity in animals and humans have brought the potential impact of AAV-associated toxicity back to prominence. In both pre-clinical studies and clinical trials, systemic AAV administration has been associated with neurotoxicity in peripheral nerve ganglia and spinal cord. Neurological signs have also been seen following direct AAV injection into the brain, both in non-human primates and in a clinical trial for late infantile Batten disease. Neurotoxic events appear variable across species, and preclinical animal studies do not fully predict clinical observations. Accumulating data suggest that AAV-associated neurotoxicity may be underdiagnosed and may differ between species in terms of frequency and/or severity. In this review, we discuss the different animal models that have been used to demonstrate AAV-associated neurotoxicity, its potential causes and consequences, and potential approaches to blunt AAV-associated neurotoxicity.

A multiplexed barcode approach to simultaneously evaluate gene delivery by adeno-associated virus capsid variants in nonhuman primates.

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.

Cytomegalovirus breakthrough and resistance during letermovir prophylaxis.

Letermovir is a relatively new antiviral for prophylaxis against cytomegalovirus (CMV) after allogeneic hematopoietic cell transplantation (HCT). CMV-seropositive HCT recipients who received letermovir prophylaxis from 2018 to 2020 at our center were evaluated for letermovir resistance and breakthrough CMV reactivation. Two-hundred twenty-six letermovir recipients were identified and 7/15 (47%) with CMV DNAemia ≥200 IU/mL were successfully genotyped for UL56 resistance. A single C325Y resistance mutation was identified in an umbilical cord blood recipient. Ninety-five (42%), 43 (19%), and 15 (7%) patients had breakthrough CMV at any level, ≥150 IU/mL, and ≥500 IU/mL, respectively. Risk factors for breakthrough CMV reactivation at each viral threshold were examined. Cumulative steroid exposure was the strongest risk factor for CMV at all evaluated viral thresholds. Graft-versus-host disease prophylaxis with post-transplantation cyclophosphamide (aHR 2.34, 95% CI 1.28-4.28, p = 0.001) or calcineurin inhibitors plus mycophenolate (aHR 2.24, 95% CI 1.30-3.86, p = 0.004) were also associated with an increased risk of CMV reactivation at any level. De novo letermovir resistance is rare and can be successfully treated using other antivirals. Letermovir effectively prevents clinically significant CMV, however, subclinical CMV reactivation occurs frequently at our center.

Differences in expression of tumor suppressor, innate immune, inflammasome, and potassium/gap junction channel host genes significantly predict viral reservoir size during treated HIV infection.

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-1ß/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-1ß, 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.