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Background and Purpose: To improve radiotherapy (RT) planning efficiency and plan quality, knowledge-based planning (KBP) and deep learning (DL) solutions have been developed. We aimed to make a direct comparison of these models for breast cancer planning using the same training, validation, and testing sets. Materials and Methods: Two KBP models were trained and validated with 90 RT plans for left-sided breast cancer with 15 fractions of 2.6 Gy. The versions either used the full dataset (non-clean model) or a cleaned dataset (clean model), thus eliminating geometric and dosimetric outliers. Results were compared with a DL U-net model (previously trained and validated with the same 90 RT plans) and manually produced RT plans, for the same independent dataset of 15 patients. Clinically relevant dose volume histogram parameters were evaluated according to established consensus criteria. Results: Both KBP models underestimated the mean heart and lung dose equally 0.4 Gy (0.3–1.1 Gy) and 1.4 Gy (1.1–2.8 Gy) compared to the clinical plans 0.8 Gy (0.5–1.8 Gy) and 1.7 Gy (1.3–3.2 Gy) while in the final calculations the mean lung dose was higher 1.9–2.0 Gy (1.5–3.5 Gy) for both KPB models. The U-Net model resulted in a mean planning target volume dose of 40.7 Gy (40.4–41.3 Gy), slightly higher than the clinical plans 40.5 Gy (40.1–41.0 Gy). Conclusions: Only small differences were observed between the estimated and final dose calculation and the clinical results for both KPB models and the DL model. With a good set of breast plans, the data cleaning module is not needed and both KPB and DL models lead to clinically acceptable results.

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)., Competing Interests: Potential conflicts of interest . A. L. G. reports contract testing to his institution from Abbott, Cepheid, Novavax, Pfizer, Janssen, and Hologic and research support to his institution from Gilead Sciences, Inc, and Merck outside of the described work. D. P. is an employee and shareholder of Gilead Sciences, Inc. K. J. is a shareholder of and was employed by Gilead Sciences, Inc, at the time of manuscript development. C. A. B. has received grants/contracts to her institution from Gilead Sciences, Inc, and consultant fees from NDA Partners, LLC, related to drug development. C. A. B. served as the president of the CROI Foundation Board of Directors, vice president of the Zimbabwe AIDS Treatment Assistance Project Board of Directors, and secretary/treasurer of the IAS-USA Board of Directors (all nonprofit organizations). C. A. B. recently served as deputy editor for Clinical Infectious Diseases for the Infectious Diseases Society of America. N. J. reports salary support by Sagent Pharmaceuticals to his institution. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest., (Published by Oxford University Press on behalf of Infectious Diseases Society of America 2024.)

Gene drives are genetic modifications designed to propagate efficiently through a population. Most applications rely on homologous recombination during sexual reproduction in diploid organisms such as insects, but we recently developed a gene drive in herpesviruses that relies on co-infection of cells by wild-type and engineered viruses. Here, we report on a viral gene drive against human herpes simplex virus 1 (HSV-1) and show that it propagates efficiently in cell culture and during HSV-1 infection in mice. We describe high levels of co-infection and gene drive-mediated recombination in neuronal tissues during herpes encephalitis as the infection progresses from the site of inoculation to the peripheral and central nervous systems. In addition, we show evidence that a superinfecting gene drive virus could recombine with wild-type viruses during latent infection. These findings indicate that HSV-1 achieves high rates of co-infection and recombination during viral infection, a phenomenon that is currently underappreciated. Overall, this study shows that a viral gene drive could spread in vivo during HSV-1 infection, paving the way toward therapeutic applications., (© 2024. The Author(s).)

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., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

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., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

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., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

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., (© 2024. The Author(s).)

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 (T FH ) cells, with complete restoration of BCF architecture and T FH 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 T FH cells within BCF sanctuaries, informing future combinatorial HIV cure strategies designed to augment antiviral efficacy., Competing Interests: Declaration of interests The authors declare the following competing interests: 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., (Copyright © 2024 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Most individuals are latently infected with herpes simplex virus type 1 (HSV-1), and it is well-established that HSV-1 establishes latency in sensory neurons of peripheral ganglia. However, it was recently proposed that latent HSV-1 is also present in immune cells recovered from the ganglia of experimentally infected mice. Here, we reanalyzed the single-cell RNA sequencing (scRNA-Seq) data that formed the basis for that conclusion. Unexpectedly, off-target priming in 3' scRNA-Seq experiments enabled the detection of non-polyadenylated HSV-1 latency-associated transcript ( LAT ) intronic RNAs. However, LAT reads were near-exclusively detected in mixed populations of cells undergoing cell death. Specific loss of HSV-1 LAT and neuronal transcripts during quality control filtering indicated widespread destruction of neurons, supporting the presence of contaminating cell-free RNA in other cells following tissue processing. In conclusion, the reported detection of latent HSV-1 in non-neuronal cells is best explained using compromised scRNA-Seq datasets.IMPORTANCEMost people are infected with herpes simplex virus type 1 (HSV-1) during their life. Once infected, the virus generally remains in a latent (silent) state, hiding within the neurons of peripheral ganglia. Periodic reactivation (reawakening) of the virus may cause fresh diseases such as cold sores. A recent study using single-cell RNA sequencing (scRNA-Seq) proposed that HSV-1 can also establish latency in the immune cells of mice, challenging existing dogma. We reanalyzed the data from that study and identified several flaws in the methodologies and analyses performed that invalidate the published conclusions. Specifically, we showed that the methodologies used resulted in widespread destruction of neurons which resulted in the presence of contaminants that confound the data analysis. We thus conclude that there remains little to no evidence for HSV-1 latency in immune cells., Competing Interests: The authors declare no conflict of interest.

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., Competing Interests: Potential conflicts of interest. E. K. reports travel support from Merck to attend the International Immunocompromised Host Society (ICHS) symposium 2022. D. J. G. has served as an advisor and has received research funding and royalties from Juno Therapeutics, a Bristol-Myers Squibb company; has served as an advisor and received research funding from Seattle Genetics; has served as an advisor for GlaxoSmithKline, Celgene, Ensoma, Janssen Biotech, and Legend Biotech; and has received research funding from SpringWorks Therapeutics, Sanofi, and Cellectar Biosciences. J. G. has served as ad hoc consultant and has received honoraria from Sobi, Legend Biotech, Janssen, Kite Pharma, and MorphoSys; has received research funding from Sobi, Juno Therapeutics (a Bristol-Myers Squibb company), Celgene (a Bristol-Myers Squibb company), and Angiocrine Bioscience; and has participated in the independent data review committee for Century Therapeutics. D. G. M. has served as ad hoc consultant and has received honoraria from BMS, Celgene, Genentech, Juno Therapeutics, and Kite. D. G. M.'s institution, Fred Hutchinson Cancer Center, has received research funding, including salary support, from the following companies for clinical trials on which D. G. M. is a principal investigator or subinvestigator: Kite Pharma, Juno Therapeutics, Celgene, Legend Biotech, and BMS. D. G. M. has the rights to royalties from the Fred Hutchinson Cancer Center for patents licensed to Juno Therapeutics/BMS; has stock options from A2 Biotherapeutics and Navan Technologies; has served as member with compensation for A2 Biotherapeutics, Navan, and Chimeric Therapeutics (member of the Scientific Advisory Board), Genentech (member and chair of the Lymphoma Steering Committee), BMS (member of the JCAR017 EAP-001 Safety Review Committee), BMS (member of the CLL Strategic Council), ImmPACT Bio (member of the Clinical Advisory Board and the CD19/CD20 bispecific CAR-T Cell Therapy Program), Gilead Sciences (member of the Scientific Review Committee and Research Scholars Program in Hematologic Malignancies), and Interius (member of Clinical Advisory Board); has served as member without compensation for BMS (member of the JCAR017-BCM-03 Scientific Steering Committee). M. J. B has served as consultant and has received research funding from Merck; has served as consultant for Symbio, Helocyte, Moderna, and Allovir; and has served as a consultant and had option to acquire stock for EvrysBio. J. A. H. has served as a consultant for Moderna, Allovir, Gilead, SentiBio, Modulus, and Allogene and received research funding from Allovir, Gilead, and Merck. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

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., Competing Interests: Potential conflicts of interest . A. L. G. reports contract testing from Abbott, Cepheid, Novavax, Pfizer, Janssen, and Hologic; and research support from Gilead and Merck, outside of the described work. A. W. reports research support from Pfizer, Amazon, GlaxoSmithKline/Vir, Ansun Biopharma, and Allovir; receiving personal fees from Kyorin Pharmaceutical and Vir Biotechnology; and receiving grants from Amazon and VB Tech, all outside the submitted work. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

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., (© 2024. The Author(s).)