BACKGROUND:Respiratory virus infections (RVIs) are common after hematopoietic cell transplantation (HCT), but their effect on pulmonary outcomes, including bronchiolitis obliterans syndrome (BOS), and mortality is poorly defined. METHODS:Prospective cohort study of 471 allogeneic HCT recipients transplanted in the pre-COVID-19 pandemic era in an academic cancer center. Participants were prospectively followed for 1 year with serial handheld spirometry, symptom questionnaires, and multiplex 11-virus PCR. Pulmonary function testing occurred at recommended intervals. Cox proportional hazard and generalized estimating equation models were used to estimate associations between RVI and weekly spirometry with late airflow obstruction (AFO), BOS, and overall mortality. RESULTS:The 1-year cumulative incidence of at least one RVI was 62%; lower respiratory tract disease (LRTD) occurred in 7.6% of patients. Late AFO developed in 15.6% of patients and BOS in 3.9% of patients. Any symptomatic viral upper respiratory tract infections (URTI) were associated with AFO (adjusted HR [aHR] 1.87, 95% CI 1.11-3.16) and BOS (aHR 2.65, 95% CI 1.02-6.91). Individually, PIV-3 URTIs were associated with AFO (aHR 2.83, 95% CI 1.01-7.97) and RSV URTIs were associated with BOS (aHR 6.32, 95% CI 2.04-19.6). Short-term airflow decline was associated with AFO. Any LRTD (aHR 3.49, 95% CI 2.18-5.57), as well as symptomatic influenza URTI (aHR 2.68, 95% CI 1.52-4.72), were associated with mortality. CONCLUSIONS:RVI after HCT, particularly those caused by RSV, PIV-3, and influenza, increase the risk of pulmonary impairment and mortality. These infections should be targeted for specific anti-viral approaches and intensified monitoring for late onset pulmonary disease.

Herpes simplex virus 1 (HSV-1) causes lifelong recurrent infections. Following primary infection of the oral or genital mucosa, HSV-1 travels retrogradely through axons and establishes latency in the cell body of ganglionic neurons of the peripheral nervous system. Periodic reactivation in neurons and anterograde transport of virions back to peripheral regions cause oral or genital ulcerations. Many host and viral factors implicated in retrograde and anterograde transport of HSV-1 have been identified. In particular, studies reported that introducing five amino acid substitutions in the R2 region of the viral tegument protein UL37 was sufficient to completely eliminate retrograde transport of HSV-1 strain F. Here, we introduced the same R2 mutations in the highly neurovirulent HSV-1 strain 17+. We show that this R217 virus is highly attenuated in mice and acts as a potent vaccine that protects mice against acute HSV-1 infection. However, we report that the R217 virus has residual retrograde transport. We show that R217 can establish latency in mouse models of ocular and vaginal infection and reactivate. These results contradict published evidence and show that the R2 mutation is not sufficient to fully prevent retrograde transport of HSV-1.IMPORTANCEHerpes simplex virus 1 (HSV-1) is a ubiquitous pathogen without a cure or vaccine. HSV-1 travels through nerves between the oral and genital mucosa and the peripheral nervous system, where it establishes lifelong latency. Studies reported that introducing five amino acid substitutions in the R2 region of the viral tegument protein UL37 was sufficient to completely eliminate the retrograde transport of HSV-1 strain F from the mucosa to the nervous system. Here, we present contradictory findings. We report that an HSV-1 virus from strain 17+ with the same R2 mutation has residual retrograde transport. This shows that the R2 mutation is not sufficient to fully prevent the retrograde transport of HSV-1 in all settings. This finding may be particularly relevant for assessing the safety of prospective live-attenuated vaccines that include the R2 mutation.

Herpes simplex virus 1 (HSV-1) infection of epithelial cells is lytic, while infection of neurons typically results in long-term latency. However, the rates at which HSV-1 replicates and spreads in epithelial cells versus neurons under low and high multiplicity of infection (MOI) conditions remain undefined. Identifying these rates requires the application of mathematical models to carefully designed viral kinetic experiments. It is also critical to differentiate the dynamics of infectious viral particles versus viral DNA, as both quantities are routinely measured in in vitro experiments and human studies using plaque assays and polymerase chain reactions, respectively. Here, we developed mechanistic mathematical models to describe HSV-1 dynamics after infection of epithelial Vero cells and neuronal N2A cells, at high (3) and low (0.01) MOI. Our model recapitulates the dynamics of cell-free and cell-associated viral DNA and plaque-forming units (PFU). In epithelial cells, the model describes a pre-productive eclipse phase with a mean duration of 10.9 and 12.8 hours prior to HSV DNA replication and PFU production, respectively. Cells exited the eclipse phase as early and late as 2.5 and 32 hours, respectively. Infected cells produced a single PFU for every 224 HSV DNA genomes. PFU egressed at a constant rate, whereas the HSV DNA egress rate increased over time, before saturating at a 15 times higher rate. Under low relative to high MOI conditions, Vero cells spent 7 hours longer in the eclipse phase, had a 12-hour delay prior to egress, and had a longer mean duration of productive infection (14 versus 3.5-hour half-life). Secondary epithelial cell infection in low MOI experiments was overwhelmingly due to cell-to-cell viral spread and originated from a small number of early-producer cells. Neuronal cells produced viruses at a 5-fold lower rate and had a longer (mean: 42 hours) and more variable eclipse phase, with some neurons remaining in eclipse for more than a week. Our results highlighted large differences in HSV egress rates, as well as infected cell eclipse phase duration and death rates, in epithelial cells versus neurons during low and high MOI infection. The observed viral dynamics in neurons reflect a balance between active replication and latency.

ABSTRACT Human herpesviruses 6A and 6B (HHV-6A/B) can integrate into the germline, resulting in inherited viral DNA—now proposed to be called “endogenous HHV-6A/B (eHHV-6A/B).” Present in 0.2–3% of humans, this integrated DNA is passed to offspring and may reactivate, posing health risks such as angina or lupus. To reduce confusion caused by varied terminology, researchers advocate using “eHHV-6A/B” for inherited forms and reserving “chromosomally integrated” for somatic integrations only.

Lipid nanoparticles (LNPs) can efficiently deliver nucleic acid therapeutics to a range of tissues, particularly hepatocytes to treat diseases of the liver. We initially investigated whether three LNPs with different ionizable lipids, previously validated in non-human primates (NHPs), could deliver functional GFP mRNA to human hepatocytes in chimeric NSG-PiZ and FRG mice. After intravenous delivery, GFP expression was observed throughout the livers but was restricted to mouse hepatocytes because the payload mRNA was not internalized by human hepatocytes. LNP transfection was also restricted to mouse hepatocytes in NSG-PiZ mice administered a different LNP containing the ionizable lipid SM-102. In vitro, primary human hepatocytes (PHHs) were transfected by LNPs containing lipids SM-102, LP01, or ALC0315 in the presence of normal mouse serum, but not chimeric NSG-PiZ serum. SM-102 LNP transfection of PHH was also inhibited by naive untransplanted NSG-PiZ serum. However, serum from NSG mice supported PHH transfection by SM-102 LNP. These results suggest that inhibitory factors in NSG-PiZ mouse serum are responsible for the lack of human hepatocyte transduction in chimeric mice. Finally, we found that LNPs displaying trivalent N-acetylgalactosamine (TriGalNAc), which targets them to the asialoglycoprotein receptor, can overcome species restriction, transfecting both mouse and human hepatocytes in chimeric NSG-PiZ mice.

ABSTRACT:Chimeric antigen receptor T-cell (CAR-T) therapy has revolutionized the treatment of B-cell malignancies; however, >60% of patients relapse within 1 year, often due to insufficient CAR-T persistence. Although mouse and primary cell models have been instrumental in advancing CAR-T therapy, they frequently fail to predict clinical outcomes, underscoring the need for more translationally relevant models. To address this limitation, we conducted, to our knowledge, the first systematic evaluation of CAR structure-function relationships in an immunocompetent nonhuman primate (NHP) model. We engineered an array of 20 CD20-targeted CARs with distinct combinations of hinge, transmembrane, and costimulatory domains. After ex vivo characterization, we administered pooled autologous CAR-T arrays to 3 NHPs and tracked CAR abundance longitudinally using a novel digital droplet polymerase chain reaction assay. Ex vivo, CAR-T cells incorporating the MyD88-CD40 costimulatory domain exhibited markedly distinct functional profiles, including increased activation, unique cytokine secretion, tonic signaling, and resistance to exhaustion. In vivo, MyD88-CD40 CARs expanded dramatically, comprising up to 100% of peripheral CAR-T cells and significantly outperforming canonical CD28- and 4-1BB-based CARs. This expansion was associated with robust B-cell depletion across all animals. MyD88-CD40 CARs, particularly those with a CD28 hinge and transmembrane domain, demonstrated superior trafficking to secondary lymphoid tissues and persistence through study end point, unlike other CARs, which waned by day 28. Our findings highlight the value of NHP models for screening CAR designs and identify MyD88-CD40 CARs as candidates with unmatched potency. The unique functional attributes conferred by this domain may provide key insights into features that drive enhanced CAR-T activity.

Despite antiretroviral therapy (ART), people with HIV (PWH) on ART experience higher rates of morbidity and mortality vs. age-matched HIV negative controls, which may be driven by chronic inflammation due to persistent virus. We performed bulk RNA sequencing (RNA-seq) on peripheral CD4+ T cells, as well as quantified plasma immune marker levels from 154 PWH on ART to identify host immune signatures associated with immune recovery (CD4:CD8) and HIV persistence (cell-associated HIV DNA and RNA). Using a novel dimension reduction tool - Pairwise Controlled Manifold Approximation (PaCMAP), we defined three distinct participant transcriptomic clusters. We found that these three clusters were largely defined by differential expression of genes regulated by the transcription factor NF-κB. While clustering was not associated with HIV reservoir size, we observed an association with CD4:CD8 ratio, a marker of immune recovery and prognostic factor for mortality in PWH on ART. Furthermore, distinct patterns of plasma IL-1β, TNF-α and GCSF were also strongly associated with the clusters, suggesting that these immune markers play a key role in CD4+ T cell transcriptomic diversity and immune recovery in PWH on ART. These findings reveal novel subgroups of PWH on ART with distinct immunological characteristics, and define a transcriptional signature associated with clinically significant immune parameters for PWH. A deeper understanding of these subgroups could advance clinical strategies to treat HIV-associated immune dysfunction.

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, with several variants containing mutations in 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 more 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. Mechanistically, we show that the N:G215C mutant has more encapsidation as measured by increased RNA binding to N, N incorporation into virions, and electron microscopy showing that individual virions are larger, with elongated morphologies.

Chimeric antigen receptor T (CAR-T) cell therapy has revolutionized treatment for B-cell malignancies, yet over 60% of patients relapse within one year, often due to insufficient CAR-T persistence. While mouse and primary cell models have been instrumental in advancing CAR-T therapy, they frequently fail to predict clinical outcomes, underscoring the need for more translationally relevant models. To address this limitation, we conducted the first systematic evaluation of CAR structure-function relationships in an immunocompetent nonhuman primate (NHP) model. We engineered an array of 20 CD20-targeted CARs with distinct combinations of hinge, transmembrane, and costimulatory domains. Following ex vivo characterization, we administered pooled autologous CAR-T arrays to three NHPs and tracked CAR abundance longitudinally using a novel digital droplet PCR assay. Ex vivo, CAR-T cells incorporating the MyD88-CD40 costimulatory domain exhibited markedly distinct functional profiles, including increased activation, unique cytokine secretion, tonic signaling, and resistance to exhaustion. In vivo, MyD88 CD40 CARs expanded dramatically, comprising up to 100% of peripheral T cells and significantly outperforming canonical CD28- and 4-1BB-based CARs. This expansion was associated with robust B-cell depletion across all animals. MyD88-CD40 CARs, particularly those with a CD28 hinge and transmembrane domain, demonstrated superior trafficking to secondary lymphoid tissues and persistence through study endpoint, unlike other CARs which waned by day 28. Our findings highlight the value of NHP models for screening CAR designs and identify MyD88-CD40 CARs as candidates with unmatched potency. The unique functional attributes conferred by this domain may provide key insights into features that drive enhanced CAR-T cell activity. ### Competing Interest Statement H.-P.K. is or was a consultant to and has or had ownership interests in Rocket Pharmaceuticals, Homology Medicines, Vor Biopharma, and Ensoma, Inc. H.-P.K. is a member of the scientific advisory board at Umoja Biopharma. The remaining authors declare no competing financial interests.

Abstract Background Limited data exist on the association between specific symptoms and viral loads of common cold coronavirus (ccCOV). We investigated this potential association in allogeneic hematopoietic cell transplant (HCT) recipients.Figure 1.Proportion of sampling time points with each symptom Methods Four hundred seventy-one children and adults were prospectively followed with weekly symptom questionnaires through one year after allogeneic HCT at the Fred Hutchinson Cancer Center (12/2005-2/2010). Multiplex PCR for 11 respiratory viruses was performed on combined nasal wash and throat swab samples collected weekly through day 100 post-HCT, and then every three months or if symptoms were present through one-year post-HCT. Positive samples were evaluated for viral loads with quantitative real-time PCR (copies/ml). We included time points with the detection of ccCOV only and corresponding 15-point symptom survey data available post-HCT. To assess the association between specific symptoms and viral loads, the Mann-Whitney or Jonckheere-Terpstra tests were used to compare 2 or >2 groups for continuous variables, respectively.Figure 2.Symptoms associated with higher viral loads Results Among 6,276 samples tested, 251 samples (79 patients) were positive for ccCoV. One hundred thirty-six sampling time points (52 patients) met the inclusion criteria. The proportion of sample time points with each symptom present is shown in Figure 1. When we compared viral loads with the presence of each of the 15 symptoms, higher viral loads were associated with the presence of four respiratory tract symptoms (rhinorrhea, sneezing, sore throat, and coughing) and one systemic symptom (diarrhea) (p< 0.01 for each) (Figure 2). Viral loads appeared to increase with increasing number of respiratory tract symptoms (p< 0.001) (Figure 3).Figure 3.Viral loads compared with the number of respiratory tract symptoms Conclusion The presence of specific respiratory tract symptoms is associated with higher viral loads of ccCOV in the upper respiratory tract in allogeneic HCT recipients. The association with diarrhea suggests the possibility of fecal shedding, but this was not evaluated in our study. Patients with specific symptoms may be more likely to transmit virus given their higher viral loads. Studies of these associations for other respiratory viruses are warranted. Disclosures Chikara Ogimi, MD, PhD, AstraZeneca: Honoraria|bioMerieux Japan Ltd.: Honoraria|ELSEVIER: Honoraria|KYORIN: Honoraria|Miyarisan: Honoraria|MSD: Honoraria|NOVARTIS: Honoraria|Pfizer: Honoraria Alpana waghmare, MD, Allovir: Grant/Research Support|Ansun Biopharma: Grant/Research Support|GlaxoKlineSmith: Advisor/Consultant|GlaxoKlineSmith: Grant/Research Support|Pfizer: Grant/Research Support|Vir: Advisor/Consultant Janet A. Englund, MD, Abbvie: Advisor/Consultant|AstraZeneca: Advisor/Consultant|AstraZeneca: Grant/Research Support|GlaxoSmithKline: Advisor/Consultant|GlaxoSmithKline: Grant/Research Support|Meissa Vaccines: Advisor/Consultant|Merck: Advisor/Consultant|Pfizer: Board Member|Pfizer: Grant/Research Support|Pfizer: Speaker at meeting|SanofiPasteur: Advisor/Consultant|Shinogi: Advisor/Consultant Michael J. Boeckh, MD PhD, Allovir: Advisor/Consultant|Allovir: Grant/Research Support|AstraZeneca: Advisor/Consultant|AstraZeneca: Grant/Research Support|Merck: Advisor/Consultant|Merck: Grant/Research Support|Moderna: Advisor/Consultant|Moderna: Grant/Research Support|Symbio: Advisor/Consultant

Herpes Simplex Virus 2 (HSV-2) infection results in variable rates of local viral shedding in anogenital skin. The effect of episodic viral exposures on immune cells in adjacent mucosal tissues, including the genital tract, is unknown. However, any immune responses at this site could affect protective mucosal immunity, tissue homeostasis, and adverse health outcomes. To investigate the effect of HSV-2 on cervicovaginal tract immunity, we applied flow cytometry, immunofluorescence imaging, analysis of soluble immune factors, and spatial transcriptomics to cervicovaginal tissue and blood samples provided by a total of 232 HSV-2-seropositive and seronegative participants, with genital HSV-2 shedding evaluated at the time of biopsy. This unique dataset was used to define and spatially map immune cell subsets and localized gene expression via spatial transcriptomics. HSV-2 seropositivity alone was associated with minimal differences in cervicovaginal and circulating T cell phenotypes. However, the vaginal mucosa during active HSV-2 shedding was associated with alterations in T cell, macrophage, and DC localization and gene expression, consistent with increased immune surveillance, with immune activating and suppressing signals potentially reinforcing mucosal tissue homeostasis.

Abstract: The treatment of monogenetic disorders, such as hemoglobinopathies and lysosomal storage diseases, has markedly improved with the advent of cell and gene therapies, particularly allogeneic or gene-modified autologous stem cell transplantations. However, therapeutic efficacy is reliant on maintaining engraftment above a critical threshold. To maintain such engraftment levels, we and others have pursued approaches to shield edited cells from antibody or chimeric antigen receptor (CAR) T-cell–mediated selection. Here, we focused on CD33, which is expressed early on hematopoietic stem and progenitor cells (HSPCs) as well as on myeloid progenitors. Rhesus macaques were engrafted with HSPCs edited to ablate CD33 using either CRISPR/CRISPR-associated protein 9 or adenine base editor. Both editing strategies showed similar post-transplant recovery kinetics and yielded equivalent levels of engraftment. We then created a V-set domain–specific CAR construct (CAR33), validated its functionality in vitro, and treated both animals with autologous CAR33 T cells. CAR33 T cells expanded after infusion and caused specific depletion of CD33WT but not CD33null progeny, leading to a transient enrichment for gene-edited cells in the blood. No depletion was seen in the bone marrow stem cell compartment with CD34+CD90+ HSCs expressing lower levels of CD33 in comparison to monocytes. Thus, we show proof of concept and safety of an epitope editing–based enrichment/protection strategy in macaques.

Abstract Background We previously identified novel risk factors for progression to lower respiratory tract disease (LRTD) among allogeneic hematopoietic cell transplant (HCT) recipients presenting with upper respiratory tract infection (URTI) with 12 viruses in the PCR era and proposed a simple prediction model by a number of risk factors (BMT Ogimi C, et al. Bone Marrow Transplant 2022; 57(4):649-657). We aimed to investigate whether the presence of these risk factors at the time of URTI diagnosis also predicts overall mortality and pulmonary death.Figure 1.Kaplan-Meier plot of overall death and cumulative incidence plot of pulmonary death by day 90 among patients with any viral URTI (N=947) Methods We retrospectively analyzed patients with the first respiratory virus detected by multiplex PCR after allogeneic HCT (4/2008-9/2018). We created Kaplan-Meier plots for overall mortality and cumulative incidence plots for pulmonary death within 90 days among patients presenting with URTI by a single virus. Candidate risk factors were age >=40 years, a history of multiple HCT, early timing post-HCT (< =30 days), albumin < =3 g/dL, monocytopenia (< =100 cells/µL), highest glucose value >200 mg/dl, and systemic steroid use within 14 days.Figure 2.Kaplan-Meier plot of overall death by day 90 among patients with viral URTI Results A total of 947 patients (199 children and 748 adults) presented with URTI only as follows: 407 human rhinoviruses (HRV), 174 parainfluenza viruses (PIV) 1–4, 140 common cold coronaviruses, 84 respiratory syncytial virus (RSV), 58 influenza A/B, 43 human metapneumovirus (MPV), and 41 adenoviruses. Among these, 81 (18%) died with 44 pulmonary deaths within 90 days from URTI. No patients with any viral URTI without risk factors died (Figure 1). Patients with 3 or more risk factors appear to have high overall mortality, and pulmonary death accounted for more than half of cases as a cause of death (Figure 1). These trends were seen across viruses evaluated (Figure 2). Conclusion Established risk factors for progression from viral URTI to LRTD also predict overall mortality and pulmonary death. Patients without any risk factors appeared to be completely protected from death, while patients with >3 risk factors were at the highest risk for death, suggesting an intervention opportunity and close monitoring. These trends were observed across several viruses and future studies are needed to validate our findings in different cohorts. Disclosures Chikara Ogimi, MD, PhD, AstraZeneca: Honoraria|bioMerieux Japan Ltd.: Honoraria|ELSEVIER: Honoraria|KYORIN: Honoraria|Miyarisan: Honoraria|MSD: Honoraria|NOVARTIS: Honoraria|Pfizer: Honoraria Alpana waghmare, MD, Allovir: Grant/Research Support|Ansun Biopharma: Grant/Research Support|GlaxoKlineSmith: Advisor/Consultant|GlaxoKlineSmith: Grant/Research Support|Pfizer: Grant/Research Support|Vir: Advisor/Consultant Janet A. Englund, MD, Abbvie: Advisor/Consultant|AstraZeneca: Advisor/Consultant|AstraZeneca: Grant/Research Support|GlaxoSmithKline: Advisor/Consultant|GlaxoSmithKline: Grant/Research Support|Meissa Vaccines: Advisor/Consultant|Merck: Advisor/Consultant|Pfizer: Board Member|Pfizer: Grant/Research Support|Pfizer: Speaker at meeting|SanofiPasteur: Advisor/Consultant|Shinogi: Advisor/Consultant Michael J. Boeckh, MD PhD, Allovir: Advisor/Consultant|Allovir: Grant/Research Support|AstraZeneca: Advisor/Consultant|AstraZeneca: Grant/Research Support|Merck: Advisor/Consultant|Merck: Grant/Research Support|Moderna: Advisor/Consultant|Moderna: Grant/Research Support|Symbio: Advisor/Consultant

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.