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Human cytomegalovirus (HCMV) encodes four viral Fc-gamma receptors (vFcγRs) that counteract antibody-mediated activation in vitro, but their role in infection and pathogenesis is unknown. To examine their in vivo function in an animal model evolutionarily closely related to humans, we identified and characterized Rh05, Rh152/151 and Rh173 as the complete set of vFcγRs encoded by rhesus CMV (RhCMV). Each one of these proteins displays functional similarities to their prospective HCMV orthologs with respect to antagonizing host FcγR activation in vitro. When RhCMV-naïve male rhesus macaques were infected with vFcγR-deleted RhCMV, peak plasma DNAemia levels and anti-RhCMV antibody responses were comparable to wildtype infections of both male and female animals. However, the duration of plasma DNAemia was significantly shortened in immunocompetent, but not in CD4 + T cell-depleted animals. Since vFcγRs were not required for superinfection of rhesus macaques, we conclude that these proteins can prolong lytic replication during primary infection by evading virus-specific adaptive immune responses, particularly antibodies., Competing Interests: Competing interests: S.R.P. has served as a consultant to Merck, Moderna, Pfizer, GSK and Dynavax and has led sponsored programs with Moderna and Merck. P.K. has received funding from Biotest AG. None of these activities have impacted this work. The remaining authors declare no competing interests., (© 2025. The Author(s).)

The emergence of bloodstream infections (BSI) caused by vancomycin-resistant Enterococci (VRE) has caused concern. Nonetheless, it remains unclear whether these types are associated with an excess risk of severe outcomes when compared with infections caused by vancomycin-susceptible Enterococci (VSE). This cohort study included hospitalized patients in Denmark with Enterococcus faecium -positive blood cultures collected between 2010 and 2019 identified in the Danish Microbiology Database. We estimated 30-day hazard ratio (HR) of death or discharge among VRE compared to VSE patients adjusted for age, sex, and comorbidity. The cohort included 6071 patients with E. faecium BSI (335 VRE, 5736 VSE) among whom VRE increased (2010-13, 2.6%; 2014-16, 6.3%; 2017-19; 9.4%). Mortality (HR 1.08, 95%CI 0.90-1.29; 126 VRE, 37.6%; 2223 VSE, 37.0%) or discharge (HR 0.89, 95%CI 0.75-1.06; 126 VRE, 37.6%; 2386 VSE, 41.6%) was not different between VRE and VSE except in 2014 (HR 1.87, 95% CI 1.18-2.96). There was no interaction between time from admission to BSI (1-2, 3-14, and >14 days) and HR of death ( P  = 0.14) or discharge ( P  = 0.45) after VRE compared to VSE, despite longer time for VRE patients (17 vs. 10 days for VSE, P  < 0.0001). In conclusion, VRE BSI was not associated with excess morbidity and mortality. The excess mortality in 2014 only may be attributed to improved diagnostic- and patient-management practices after 2014, reducing time to appropriate antibiotic therapy. The high level of mortality after E. faecium BSI warrants further study.

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

Purpose: Early in the course of the SARS-CoV-2 pandemic it was hypothesised that host genetics played a role in the pathophysiology of COVID-19 including a suggestion that the CCR5-Δ32 mutation may be protective in SARS-CoV-2 infection. Leronlimab is an investigational CCR5-specific humanized IgG4 monoclonal antibody currently in development for HIV-1 infection. We aimed to explore the impact of leronlimab on the severity of disease symptoms among participants with mild-to-moderate COVID-19., Methods: The TEMPEST trial was a randomized, double-blind, placebo-controlled study in participants with mild-to-moderate COVID-19. Participants were randomly assigned in a 2:1 ratio to receive subcutaneous leronlimab (700 mg) or placebo on days 0 and 7. The primary efficacy endpoint was assessed by change in total symptom score based on fever, myalgia, dyspnea, and cough, at end of treatment (day 14)., Findings: Overall, 84 participants were randomized and treated with leronlimab (n = 56) or placebo (n = 28). No difference was observed in change in total symptom score (P = 0.8184) or other pre-specified secondary endpoints between treatments. However, in a post hoc analysis, 50.0% of participants treated with leronlimab demonstrated improvements from baseline in National Early Warning Score 2 (NEWS2) at day 14, compared with 20·8% of participants in the placebo group (post hoc; p = 0.0223). Among participants in this trial with mild-to-moderate COVID-19 adverse events rates were numerically but not statistically significantly lower in leronlimab participants (33.9%) compared with placebo participants (50.0%)., Implications: At the time the TEMPEST trial was designed although CCR5 was known to be implicated in COVID-19 disease severity the exact pathophysiology of SARS-CoV-2 infection was poorly understood. Today it is well accepted that SARS-CoV-2 infection in asymptomatic-to-mild cases is primarily characterized by viral replication, with a heightened immune response, accompanied by diminished viral replication in moderate-to-severe disease and a peak in inflammatory responses with excessive production of pro-inflammatory cytokines in critical disease. It is therefore perhaps not surprising that no differences between treatments were observed in the primary endpoint or in pre-specified secondary endpoints among participants with mild-to-moderate COVID-19. However, the results of the exploratory post hoc analysis showing that participants in the leronlimab group had greater improvement in NEWS2 assessment compared to placebo provided a suggestion that leronlimab may be associated with a lower likelihood of people with mild-to-moderate COVID-19 progressing to more severe disease and needs to be confirmed in other appropriately designed clinical trials., Clinicaltrials: gov number, NCT04343651 https://classic., Clinicaltrials: gov/ct2/show/NCT04343651., Competing Interests: Declaration of competing interest O.O.Y. declares being on the scientific board and being a scientific consultant (compensated in stock options and cash) for CytoDyn and reports being co-founder and board member for CDR3 Therapeutics Corp (stock) as well as being on the Board of Directors for Applied Medical Inc (stock and cash). O.O. declares receiving honoraria from Gilead Sciences for advisory board membership. J.B.S. declares having a significant financial interest in and serves on the scientific advisory board of CytoDyn, a company that may have a financial interest in the results of this research and technology. This potential individual conflict of interest has been reviewed and managed by the Oregon Health & Science University. S.G.H. declares recieving compensation for consulting for CytoDyn. This potential conflict of interest has been reviewed and managed by the Oregon Health & Science University. C.A. and J.P.L. declares they are paid employees of CytoDyn and own stock. H.S., D.S., R.L., A.M., and S.O. declare that they have no known financial interests or personal relationships that could have appreared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Tuberculosis (TB), caused by Mycobacterium tuberculosis ( Mtb ) is a deadly infectious disease having a major impact on global health. Using the CMV vector for development of novel vaccines is a promising new strategy that elicits strong and durable, high frequency memory T cell responses against heterologous immunogens. We conducted functional transcriptomic analysis of whole blood samples collected from cohorts of rhesus (Rh) macaques that were administered RhCMV/TB vector using a prime-boost strategy. Two modified CMV vectors were used in this study, including 68-1 RhCMV/TB-6Ag (encoding 6 Mtb protein immunogens, including Ag85A, ESAT-6, Rv3407, Rv2626, Rpf A, and Rpf D) and its attenuated variant, 68-1 RhCMV/Δpp71-TB-6Ag (a cell-to-cell spread-deficient vaccine vector lacking the Rh110 gene encoding the pp71 tegument protein). Bulk mRNA sequencing, differential gene expression, and functional enrichment analyses showed that these RhCMV/TB vaccines induce the innate and adaptive immune responses with specific transcriptomic signatures, including the IL-15-induced protective gene signature previously defined to be linked with protection against simian immunodeficiency virus (SIV) by the 68-1 RhCMV/SIV vaccine. While both vectors exhibited a transcriptomic response of the IL-15 protective signature in whole blood, we show that lack of pp71 does not maintain induction of the protective signature for the full duration of the study compared to the parental non-attenuated vector. Our observations indicate that RhCMV vector vaccines induce a transcriptomic response in whole blood that include a conserved IL-15 signature of which vector-encoded pp71 is an important component of response durability that upon future Mtb challenge may define specific vaccine protection outcomes against Mtb infection., Competing Interests: OHSU and LP and SH have a significant financial interest in Vir Biotechnology, Inc., a company that may have a financial interest in the results of CMV-based vector research and technology. This potential individual and institutional conflict of interest has been reviewed and managed by OHSU. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Sung, Whitmore, Smith, Chang, Tisoncik-Go, Barber-Axthelm, Selseth, Feltham, Ojha, Hansen, Picker and Gale.)

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

Simian immunodeficiency virus (SIV) vaccines based upon 68-1 Rhesus Cytomegalovirus (RhCMV) vectors show remarkable protection against pathogenic SIVmac239 challenge. Across multiple independent rhesus macaque (RM) challenge studies, nearly 60% of vaccinated RM show early, complete arrest of SIVmac239 replication after effective challenge, whereas the remainder show progressive infection similar to controls. Here, we performed viral sequencing to determine whether the failure to control viral replication in non-protected RMs is associated with the acquisition of viral escape mutations. While low level viral mutations accumulated in all animals by 28 days-post-challenge, which is after the establishment of viral control in protected animals, the dominant circulating virus in virtually all unprotected RMs was nearly identical to the challenge stock, and there was no difference in mutation patterns between this cohort and unvaccinated controls. These data definitively demonstrate that viral mutation does not explain lack of viral control in RMs not protected by RhCMV/SIV vaccination. We further demonstrate that during chronic infection RhCMV/SIV vaccinated RMs do not acquire escape mutation in epitopes targeted by RhCMV/SIV, but instead display mutation in canonical MHC-Ia epitopes similar to unvaccinated RMs. This suggests that after the initial failure of viral control, unconventional T cell responses induced by 68-1 RhCMV/SIV vaccination do not exert strong selective pressure on systemically replicating SIV., Competing Interests: Oregon Health & Science University (OHSU), LP, JS, BB, and SH have a substantial financial interest in Vir Biotechnology Inc., a company that may have a commercial interest in the results of this research and technology. LP, and SH have received compensation for consulting for Vir. The potential individual and institutional conflicts of interest have been reviewed and managed by OHSU. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Bimber, Sunshine, McElfresh, Reed, Pathak, Bateman, Hughes, Gilbride, Ford, Morrow, Lifson, Sacha, Hansen and Picker.)

An influenza vaccine approach that overcomes the problem of viral sequence diversity and provides long-lived heterosubtypic protection is urgently needed to protect against pandemic influenza viruses. Here, to determine if lung-resident effector memory T cells induced by cytomegalovirus (CMV)-vectored vaccines expressing conserved internal influenza antigens could protect against lethal influenza challenge, we immunize Mauritian cynomolgus macaques (MCM) with cynomolgus CMV (CyCMV) vaccines expressing H1N1 1918 influenza M1, NP, and PB1 antigens (CyCMV/Flu), and challenge with heterologous, aerosolized avian H5N1 influenza. All six unvaccinated MCM died by seven days post infection with acute respiratory distress, while 54.5% (6/11) CyCMV/Flu-vaccinated MCM survived. Survival correlates with the magnitude of lung-resident influenza-specific CD4 + T cells prior to challenge. These data demonstrate that CD4 + T cells targeting conserved internal influenza proteins can protect against highly pathogenic heterologous influenza challenge and support further exploration of effector memory T cell-based vaccines for universal influenza vaccine development., (© 2024. The Author(s).)

Background: Previous studies have indicated that glucagon-like peptide-1 (GLP-1) receptor agonists (GLP-1RAs) may enhance bone formation and have neutral or beneficial effects on fracture risk. We evaluated the effect of the GLP-1RA semaglutide on the bone formation marker Procollagen type I N-terminal propeptide (PINP) in adults with increased fracture risk., Methods: This randomised, placebo-controlled, double-blinded, phase 2 clinical trial was conducted at two public hospitals in Denmark. We enrolled 64 men and women with increased fracture risk based on a T-score < -1.0 at the total hip or lumbar spine and/or low-energy fracture within three years of recruitment. Participants were randomised (1:1) to receive once-weekly subcutaneous semaglutide 1.0 mg or placebo. The primary outcome was changes in plasma (P)-PINP from baseline to week 52. Primary and safety outcomes were assessed and evaluated for all participants. This trial is complete and registered with ClinicalTrials.gov, NCT04702516., Findings: Between March 24 and December 8, 2021, 55 (86%) postmenopausal women and nine men with a mean age of 63 years (SD 5.5) and BMI of 27.5 kg/m 2 (SD 4.5) were enrolled. There was no effect on changes in P-PINP from baseline to week 52 between the two groups (estimated treatment difference (ETD) semaglutide versus placebo 3.8 μg/L [95% CI -5.6 to 13.3]; p = 0.418), and no difference in P-PINP levels between groups at week 52 (semaglutide 64.3 μg/L versus placebo 62.3 μg/L [95% CI -10.8 to 15.0]; p = 0.749). The secondary outcomes showed higher plasma levels of bone resorption marker Collagen type I cross-linked C-terminal telopeptide (P-CTX) in the semaglutide group than in the placebo group (ETD 166.4 ng/L [95% CI 25.5-307.3]; p = 0.021). Compared to placebo, lumbar spine and total hip areal bone mineral densities (aBMD) were lower in the semaglutide group after 52 weeks ((ETD lumbar spine -0.018 g/cm 3 [95% CI -0.031 to -0.005]; p = 0.007); ETD total hip -0.020 g/cm 2 ([95% CI -0.032 to -0.008]; p = 0.001). Treatment differences in femoral neck aBMD were not observed ([95% CI [-0.017 to 0.006]; p = 0.328). Further, body weight was lower in the semaglutide group than in the placebo group after 52 weeks (ETD -6.8 kg [95% CI -8.8 to -4.7]; p < 0.001). Thirty-one [97%] in the semaglutide group and 18 [56%] in the placebo group experienced at least one adverse event, including four serious events (two in each group). No episodes of hypoglycaemia or deaths were reported., Interpretation: In adults with increased fracture risk, semaglutide once weekly did not increase bone formation based on the bone formation marker P-PINP. The observed increase in bone resorption in the semaglutide group may be explained by the accompanying weight loss., Funding: Region of Southern Denmark, Novo Nordisk Foundation, and Gangsted Foundation. Novo Nordisk provided the investigational drug and placebo., Competing Interests: EMW, SJ, SGH, JJM, and CE declare no conflicts of interest. MSH and MF have received funding from the Novo Nordisk Foundation. RE receives consultancy funding from Immunodiagnostic Systems, Sandoz, Samsung, CL Bio, Biocon, Takeda, UCB, meeting presentations for Pharmacosmos, Alexion, UCB and Amgen, and grant funding from Alexion. NRJ has received assays and reagents from IDS and Roche for clinical studies. MF has received consultancy funding from Novo Nordisk and is shareholder at Novo Nordisk and Eli Lily. Novo Nordisk, Denmark, provided the investigational drug and placebo., (© 2024 The Author(s).)

Angelman syndrome (AS) is a rare genetic developmental disability that presents with high rates of co-occurring sleep difficulties. Most existing research has focused on the pathophysiology of sleep problems in people with AS, and suggests that sleep problems are the result of genetic and neurobiological factors. However, little is known about the role of the social environment and learning in sleep problems in children with AS. This descriptive study used survey data from 139 parents of children with AS to investigate: 1) the type, topography and severity of children's sleep problems; 2) the collateral child, parent and family impacts of the sleep problems; 3) treatment selection practices and the perceived effectiveness of these treatments; and 4) sources of support and treatment advice received. Parents reported that the majority of children experienced sleep problems, resulting in numerous deleterious effects on child and family functioning. They also reported high levels of concern about these sleep problems, but low levels of perceived support. Study findings highlight the need to establish a disability-specific profile of the type and impact of sleep problems experienced by children with AS, and have further implications for the delivery of clinical services and support provided to parents of children with AS.

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

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

Purpose: We investigated whether selenium supplementation improves quality-of-life (QoL) in patients with autoimmune thyroiditis (ID:NCT02013479)., Methods: We included 412 patients ≥18 years with serum thyroid peroxidase antibody (TPOAb) level ≥100 IU/mL in a multicentre double-blinded randomised clinical trial. The patients were allocated 1:1 to daily supplementation with either 200 μg selenium as selenium-enriched yeast or matching placebo tablets for 12 months, as add-on to levothyroxine (LT4) treatment. QoL, assessed by the Thyroid-related Patient-Reported-Outcome questionnaire (ThyPRO-39), was measured at baseline, after six weeks, three, six, 12, and 18 months., Results: In total, 332 patients (81%) completed the intervention period, of whom 82% were women. Although QoL improved during the trial, no difference in any of the ThyPRO-39 scales was found between the selenium group and the placebo group after 12 months of intervention. In addition, employing linear mixed model regression no difference between the two groups was observed in the ThyPRO-39 composite score (28.8 [95%CI:24.5-33.6] and 28.0 [24.5-33.1], respectively; P=0.602). Stratifying the patients according to duration of the disease at inclusion, ThyPRO-39 composite score, TPOAb level, or selenium status at baseline did not significantly change the results. TPOAb levels after 12 months of intervention were lower in the selenium group than in the placebo group (1995 [95%CI:1512-2512] vs. 2344 kIU/L [1862-2951]; P=0.016) but did not influence LT4 dosage or free triiodothyronine/free thyroxin ratio., Conclusion: In hypothyroid patients on LT4 therapy due to autoimmune thyroiditis, daily supplementation with 200 μg selenium or placebo for 12 months improved QoL to the same extent.

Inclusive postsecondary education (IPSE) programs support adults with intellectual disabilities' participation in higher education. Students in IPSE programs may have limited knowledge of sexuality, relationships, and social skills, which can be addressed through sexuality and relationship education (SRE). This project evaluated the effect of the Positive Choices© curriculum on the SRE knowledge of 7 students attending an IPSE program in the southeastern United States. Students attended 15 weekly classes taught by two graduate assistants and faculty supervisor during spring 2020; half of course meetings occurred in person and half via Zoom due to COVID-19 restrictions. The authors analyzed results of pre-post one sample t-test of student scores on five instructor-created assessments. All students showed statistically significant increases in knowledge for each assessment and overall. Future research should assess the effect of other curricula in use in IPSE programs and evaluate the need for and use of supplemental materials for instruction.

Competing Interests: Declaration of competing interest All authors state that they have no conflict of interest.

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

The initial development of cytomegalovirus (CMV) as a vaccine vector for HIV/simian immunodeficiency virus (SIV) was predicated on its potential to pre-position high-frequency, effector-differentiated, CD8 + T cells in tissues for immediate immune interception of nascent primary infection. This goal was achieved and also led to the unexpected discoveries that non-human primate (NHP) CMVs can be programmed to differentially elicit CD8 + T cell responses that recognize viral peptides via classical MHC-Ia, and/or MHC-II, and/or MHC-E, and that MHC-E-restricted CD8 + T cell responses can uniquely mediate stringent arrest and subsequent clearance of highly pathogenic SIV, an unprecedented type of vaccine-mediated protection. These discoveries delineate CMV vector-elicited MHC-E-restricted CD8 + T cells as a functionally distinct T cell response with the potential for superior efficacy against HIV-1, and possibly other infectious agents or cancers., Competing Interests: Declaration of interests L.J.P., S.G.H., and K.F. have a substantial financial interest in Vir Biotechnology, Inc., a company that may have a commercial interest in the results of this research and technology. L.J.P., S.G.H., and K.F. are also consultants to Vir Biotechnology, Inc. These potential individual and institutional conflicts of interest have been reviewed and managed by Oregon Health and Science University (OHSU)., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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

Objectives: The purpose of this review is to describe the variety and effectiveness of instructional technologies used in the early childhood setting., Methods: A systematic review of three databases was completed, and studies were reviewed by two independent coders to determine if they met inclusion criteria. Studies were excluded from this review if (a) the technology was used to train teachers and was not directly used with early childhood students, (b) participants were all enrolled in 2nd grade or higher, (c) the setting was not an early childhood education setting, or (d) studies were descriptive in nature or utilized a survey methodology. Data were extracted from each article related to participant characteristics, setting characteristics, research design, technology type, and dependent variables., Results: Thirty-five studies met criteria were included in this review. A wide range of technologies were used to provide or facilitate instruction on (a) academics, (b) social and communication skills, and (c) cognitive skills. Academic outcomes targeted in Head Start preschools were the most common across studies. The results ranged from no effect to highly effective., Conclusions: The findings from the included studies varied widely in their outcomes from reporting no difference between traditional instruction and technology-aided instruction to reporting significant difference between groups or reporting a functional relation between the technology-based intervention and the target behavior or skill. Studies that included students identified with neurodevelopmental disorders demonstrated a positive impact in the outcomes of students who experience an intervention that included technology-aided instruction. Future research is needed to identify critical components of effective technology-based interventions in early childhood educational settings., Competing Interests: Competing InterestsThe authors declare no competing interests., (© The Author(s), under exclusive licence to Springer Nature Switzerland AG 2023, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

Pelvic organ prolapse (POP) affects many women, with an estimated lifetime risk of surgical intervention of 18.7%. There is a need for alternative approaches as the use of synthetic nondegradable mesh was stopped due to severe adverse events, and as current methods for pelvic floor repair have high POP recurrence rates. Thus, we hypothesized that electrospun degradable meshes with stem cells and growth factor were safe and durable for the long term in elderly rats. In an abdominal repair model, electrospun polycaprolactone (PCL) meshes coated with connective tissue growth factor (CTGF)/PEG-fibrinogen (PF) and rat mesenchymal stem cells were implanted in elderly female rats and removed after in average 53 weeks (53-week group). Collagen amount and production were quantified by qPCR and Western blotting. Moreover, histological appearance and biomechanical properties were evaluated. Results were compared with previous results of young rats with identical mesh implanted for 24 weeks (24-week group). The 53-week group differed from the 24-week group in terms of (1) reduced collagen III, (2) strong reduction in foreign body response, and (3) altered histological appearance. We found comparable biomechanical properties, aside from higher, not significant, mean tissue stiffness in the 53-week group. Lastly, we identified mesh components 53 weeks after implantation. This study provides new insights into future POP repair in postmenopausal women by showing how CTGF/PF-coated electrospun PCL meshes with stem cells exhibit sufficient support, biocompatibility, and no mesh-related complications long term in an abdominal repair model in elderly rats., (© 2022 The Authors. Journal of Biomedical Materials Research Part B: Applied Biomaterials published by Wiley Periodicals LLC.)

There is controversy regarding the association between nonalcoholic fatty liver disease (NAFLD) and osteoporosis. Our study aim was to assess bone mineral density (BMD) in patients with biopsy-proven NAFLD and examine if the severity of NAFLD affects BMD. A total of 147 adult women ( n  = 108) and men ( n  = 39) aged 18-76 years (mean ± standard deviation [SD] age 45.3 ± 12.5) were recruited in this cross-sectional study and underwent a liver biopsy and dual-energy X-ray absorptiometry (DXA). NAFLD activity score (NAS) based on the degree of steatosis, lobular inflammation and hepatocellular ballooning was used to assess NAFLD severity. The majority of subjects, 53%, had steatosis, 25% had nonalcoholic steatohepatitis (NASH) whereas 23% served as control subjects with no evidence of NAFLD. There were no significant differences in the lumbar spine (1.09 ± 0.12, 1.11 ± 0.18, and 1.12 ± 0.15 g/cm 2 , p  = 0.69, in controls, steatosis, and NASH, respectively) or hip BMD (1.10 ± 0.15, 1.12 ± 0.13, and 1.09 ± 0.13 g/cm 2 , p  = 0.48, in controls, steatosis, and NASH, respectively) between the groups. Adjusting for age, gender, body mass index, and diabetes in multiple regression models did not alter the results. There was no correlation between NAS and neither lumbar spine BMD ( r  = 0.06, p  = 0.471), nor hip BMD ( r  = -0.03, p  = 0.716). In conclusion, BMD was similar across the spectrum of NAFLD in both genders and not related to the severity of the underlying histological lesions, suggesting that neither steatosis nor NASH exerts a detrimental effect on BMD in these relatively young patients. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research., (© 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.)

X-linked hypophosphatemic rickets (XLH) and m.3243A&gt;G mitochondrial disease share several clinical findings, including short stature, hearing impairment (HI), nephropathy, and hypertension. Here, we report on a case with the rare coincidence of these two genetic conditions. In early childhood, the patient presented with hypophosphatemia and bone deformities and was clinically diagnosed with XLH. This was genetically verified in adulthood with the identification of a de novo pathogenic deletion in phosphate-regulating endopeptidase homolog X-linked (PHEX). In addition, the patient developed HI and hypertension and when his mother was diagnosed with m.3243A&gt;G, subsequent genetic testing confirmed the patient to carry the same variant. Over the next two decades, the patient developed progressive renal impairment however without nephrocalcinosis known to associate with XLH which could indicate an m.3243A&gt;G-related kidney disease. Parallel with the progression of renal impairment, the patient developed hyperphosphatemia and secondary hyperparathyroidism. In conclusion, this case represents a complex clinical phenotype with the reversal of hypo- to hyperphosphatemia in XLH potentially mediated by the development of an m.3243A&gt;G-associated nephropathy., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Exercise addiction describes a pattern of excessive and obsessive exercise and is associated with hypoleptinemia and low testosterone that may have adverse skeletal effects. We used a validated questionnaire to identify males with high and low risk of exercise addiction. In a cross-sectional design, males (aged 21-49 years) with high ( n = 20, exercise addictive) and low risk ( n = 20, exercise controls) of exercise addiction had examinations of bone mass, bone microarchitecture, and estimated bone strength performed using dual-energy x-ray absorptiometry of the hip and spine and high-resolution peripheral quantitative computed tomography of the distal radius and tibia. Findings were compared between the groups and to a population-based sample of healthy men aged 20-80 years ( n = 236). We found similar hip and spine bone mineral density in exercise addictive and controls. Cortical and trabecular bone microarchitecture and estimated bone strength in radius and tibia did not differ significantly between the groups. Multiple regression analyses adjusting for age, body weight, free testosterone, and hours of weekly training did not alter findings. Also, bone indices from both groups were within 95% prediction bands derived from the population-based sample for the vast majority of indices. Neither group had no associations between circulating leptin or free testosterone and bone outcomes. In conclusion, in a study on younger males, we found no associations between high risk of exercise addiction and various indices of bone mass and bone quality indicative of altered skeletal health., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hansen, Lichtenstein, Johansen and Støving.)

Background: Bariatric surgery has adverse effects on the muscular-skeletal system with loss of bone mass and muscle mass and an increase in the risk of fracture. Zoledronic acid is widely used in osteoporosis and prevents bone loss and fracture. Bisphosphonates may also have positive effects on skeletal muscle. The aim of this study is to investigate the effects of zoledronic acid for the prevention of bone and muscle loss after bariatric surgery.  METHODS/DESIGN: This is a randomized double-blind placebo-controlled study. Sixty women and men with obesity aged 35 years or older will complete baseline assessments before randomization to either zoledronic acid (5 mg in 100 ml isotonic saline) or placebo (100 ml isotonic saline only) 3 weeks before surgery with Roux-en-Y-gastric bypass (RYGB) or sleeve gastrectomy (SG). Follow-up assessments are performed 12 and 24 months after surgery. The primary outcome is changes in lumbar spine volumetric bone mineral density (vBMD) assessed by quantitative computed tomography (QCT). Secondary bone outcomes are changes in proximal femur vBMD assessed by QCT. Changes in cortical and trabecular bone microarchitecture and estimated bone strength will be assessed by high-resolution peripheral quantitative computed tomography (HR-pQCT). Cortical material bone strength at the mid-tibia diaphysis will be assessed using microindentation and fasting blood samples will be obtained to assess biochemical markers of bone turnover and calcium metabolism.  Secondary muscle outcomes include whole body lean mass assessed using dual-energy X-ray absorptiometry. Dynamometers will be used to assess handgrip, shoulder, ankle, and knee muscle strength. Short Physical Performance Battery, 7.6-m walking tests, 2-min walking test, and a stair climb test will be assessed as biomarkers of physical function. Self-reported physical activity level is assessed using International Physical Activity Questionnaire (IPAQ)., Discussion: Results from this study will be instrumental for the evidence-based care of patients undergoing bariatric surgery., Trial Registration: ClinicalTrials.gov NCT04742010. Registered on 5 February 2021., (© 2022. The Author(s).)

In an exploratory trial treating "long COVID" with the CCR5-binding antibody leronlimab, we observed significantly increased blood cell surface CCR5 in treated symptomatic responders but not in nonresponders or placebo-treated participants. These findings suggest an unexpected mechanism of abnormal immune downmodulation in some persons that is normalized by leronlimab. Clinical Trials Registration. NCT04678830., Competing Interests: Potential conflicts of interest. S. A. K., N. Z. P., and C. R. are officers of CytoDyn Inc and report stock options and consulting fees from CytoDyn, assistance with manuscript preparation (Medical Expressions, paid by CytoDyn), and other financial or nonfinancial interests as employees of CytoDyn. J. B. S., S. G. H., and O. O. Y. are paid consultants for CytoDyn Inc. J. B. S. reports stock options available for purchase from CytoDyn and assistance with manuscript preparation (Medical Expressions, paid by CytoDyn). S. G. H. reports stock options and receipt of leronlimab for CCR5 receptor occupancy assay from CytoDyn and assistance with manuscript preparation (Medical Expression, paid by CytoDyn). O. O. Y. reports assistance in writing the manuscript (paid by CytoDyn). M. T. reports assistance with manuscript preparation (Medical Expressions, paid by CytoDyn). N. B. G. was a principal investigator for this trial, serves on the CytoDyn Inc Scientific Advisory Board with stock options, and reports assistance with manuscript preparation (Medical Expressions, paid by CytoDyn). A. R. was a principal investigator for this trial and worked as an employee at the Center for Advanced Research & Education, Gainesville, Georgia, which is performing a clinical research trial for CytoDyn. A. R. reports assistance with manuscript preparation (Medical Expressions, paid by CytoDyn). 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) 2022. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Strain 68-1 rhesus cytomegalovirus expressing simian immunodeficiency virus (SIV) antigens (RhCMV/SIV) primes MHC-E-restricted CD8+ T cells that control SIV replication in 50%-60% of the vaccinated rhesus macaques. Whether this unconventional SIV-specific immunity and protection is unique to rhesus macaques or RhCMV or is intrinsic to CMV remains unknown. Here, using cynomolgus CMV vectors expressing SIV antigens (CyCMV/SIV) and Mauritian cynomolgus macaques, we demonstrate that the induction of MHC-E-restricted CD8+ T cells requires matching CMV to its host species. RhCMV does not elicit MHC-E-restricted CD8+ T cells in cynomolgus macaques. However, cynomolgus macaques vaccinated with species-matched 68-1-like CyCMV/SIV mounted MHC-E-restricted CD8+ T cells, and half of the vaccinees stringently controlled SIV post-challenge. Protected animals manifested a vaccine-induced IL-15 transcriptomic signature that is associated with efficacy in rhesus macaques. These findings demonstrate that the ability of species-matched CMV vectors to elicit MHC-E-restricted CD8+ T cells that are required for anti-SIV efficacy is conserved in nonhuman primates, and these data support the development of HCMV/HIV for a prophylactic HIV vaccine., Competing Interests: Declaration of interests OHSU and Drs. Malouli, Früh, Picker, Hansen, and Sacha have a significant financial interest in Vir Biotechnology, Inc., a company that may have a financial interest in the results of this research and technology. This potential individual and institutional conflict of interest has been reviewed and managed by OHSU., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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

Motivation: Single-cell sequencing methods provide previously impossible resolution into the transcriptome of individual cells. Cell hashing reduces single-cell sequencing costs by increasing capacity on droplet-based platforms. Cell hashing methods rely on demultiplexing algorithms to accurately classify droplets; however, assumptions underlying these algorithms limit accuracy of demultiplexing, ultimately impacting the quality of single-cell sequencing analyses., Results: We present Bimodal Flexible Fitting (BFF) demultiplexing algorithms BFFcluster and BFFraw, a novel class of algorithms that rely on the single inviolable assumption that barcode count distributions are bimodal. We integrated these and other algorithms into cellhashR, a new R package that provides integrated QC and a single command to execute and compare multiple demultiplexing algorithms. We demonstrate that BFFcluster demultiplexing is both tunable and insensitive to issues with poorly behaved data that can confound other algorithms. Using two well-characterized reference datasets, we demonstrate that demultiplexing with BFF algorithms is accurate and consistent for both well-behaved and poorly behaved input data., Availability and Implementation: cellhashR is available as an R package at https://github.com/BimberLab/cellhashR. cellhashR version 1.0.3 was used for the analyses in this manuscript and is archived on Zenodo at https://www.doi.org/10.5281/zenodo.6402477., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The CCR5-specific antibody Leronlimab is being investigated as a novel immunotherapy that can suppress HIV replication with minimal side effects. Here we studied the virological and immunological consequences of Leronlimab in chronically CCR5-tropic HIV-1 infected humans (n = 5) on suppressive antiretroviral therapy (ART) and in ART-naïve acutely CCR5-tropic SHIV infected rhesus macaques (n = 4). All five human participants transitioned from daily combination ART to self-administered weekly subcutaneous (SC) injections of 350 mg or 700 mg Leronlimab and to date all participants have sustained virologic suppression for over seven years. In all participants, Leronlimab fully occupied CCR5 receptors on peripheral blood CD4+ T cells and monocytes. In ART-naïve rhesus macaques acutely infected with CCR5-tropic SHIV, weekly SC injections of 50 mg/kg Leronlimab fully suppressed plasma viremia in half of the macaques. CCR5 receptor occupancy by Leronlimab occurred concomitant with rebound of CD4+ CCR5+ T-cells in peripheral blood, and full CCR5 receptor occupancy was found in multiple anatomical compartments. Our results demonstrate that weekly, self-administered Leronlimab was safe, well-tolerated, and efficacious for long-term virologic suppression and should be included in the arsenal of safe, easily administered, longer-acting antiretroviral treatments for people living with HIV-1. Trial Registration: ClinicalTrials.gov Identifiers: NCT02175680 and NCT02355184., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: JBS and LCN have received compensation for serving on the scientific advisory board of CytoDyn, a company that may have commercial interests in the results of this research. JBS, HLW, and GMW have also received compensation for consulting for CytoDyn. The potential conflict of interest has been reviewed and managed by the Oregon Health & Science University. LCN has served as an advisor for Abbvie and ViiV Healthcare unrelated to this study. NP and SK are employees of CytoDyn, owner and developer of Leronlimab. KD is an employee of Amarex Clinical Research, a company that manages clinical trials and regulatory matters for CytoDyn. All other authors declare no competing interests. Authors Kush Dhody, Nader Pourhassan, and Cassandra Moats were unavailable to confirm their authorship contributions. On their behalf, the corresponding author has reported their contributions to the best of their knowledge.

CD8 + T cells are key mediators of antiviral and antitumor immunity. The isolation and study of Ag-specific CD8 + T cells, as well as mapping of their MHC restriction, has practical importance to the study of disease and the development of therapeutics. Unfortunately, most experimental approaches are cumbersome, owing to the highly variable and donor-specific nature of MHC-bound peptide/TCR interactions. Here we present a novel system for rapid identification and characterization of Ag-specific CD8 + T cells, particularly well suited for samples with limited primary cells. Cells are stimulated ex vivo with Ag of interest, followed by live cell sorting based on surface-trapped TNF-α. We take advantage of major advances in single-cell sequencing to generate full-length sequence data from the paired TCR α- and β-chains from these Ag-specific cells. The paired TCR chains are cloned into retroviral vectors and used to transduce donor CD8 + T cells. These TCR transductants provide a virtually unlimited experimental reagent, which can be used for further characterization, such as minimal epitope mapping or identification of MHC restriction, without depleting primary cells. We validated this system using CMV-specific CD8 + T cells from rhesus macaques, characterizing an immunodominant Mamu-A1*002:01-restricted epitope. We further demonstrated the utility of this system by mapping a novel HLA-A*68:02-restricted HIV Gag epitope from an HIV-infected donor. Collectively, these data validate a new strategy to rapidly identify novel Ags and characterize Ag-specific CD8 + T cells, with applications ranging from the study of infectious disease to immunotherapeutics and precision medicine., (Copyright © 2021 by The American Association of Immunologists, Inc.)

CCR5 plays a central role in infectious disease, host defense, and cancer progression, thereby making it an ideal target for therapeutic development. Notably, CCR5 is the major HIV entry co-receptor, where its surface density correlates with HIV plasma viremia. The level of CCR5 receptor occupancy (RO) achieved by a CCR5-targeting therapeutic is therefore a critical predictor of its efficacy. However, current methods to measure CCR5 RO lack sensitivity, resulting in high background and overcalculation. Here, we report on two independent, flow cytometric methods of calculating CCR5 RO using the anti-CCR5 antibody, Leronlimab. We show that both methods led to comparable CCR5 RO values, with low background on untreated CCR5+CD4+ T cells and sensitive measurements of occupancy on both blood and tissue-resident CD4+ T cells that correlated longitudinally with plasma concentrations in Leronlimab-treated macaques. Using these assays, we found that Leronlimab stabilized cell surface CCR5, leading to an increase in the levels of circulating and tissue-resident CCR5+CD4+ T cells in vivo in Leronlimab-treated macaques. Weekly Leronlimab treatment in a chronically SIV-infected macaque led to increased CCR5+CD4+ T cells levels and fully suppressed plasma viremia, both concomitant with full CCR5 RO on peripheral blood CD4+ T cells, demonstrating that CCR5+CD4+ T cells were protected from viral replication by Leronlimab binding. Finally, we extended these results to Leronlimab-treated humans and found that weekly 700 mg Leronlimab led to complete CCR5 RO on peripheral blood CD4+ T cells and a statistically significant increase in CCR5+CD4+ T cells in peripheral blood. Collectively, these results establish two RO calculation methods for longitudinal monitoring of anti-CCR5 therapeutic antibody blockade efficacy in both macaques and humans, demonstrate that CCR5+CD4+ T cell levels temporarily increase with Leronlimab treatment, and facilitate future detailed investigations into the immunological impacts of CCR5 inhibition in multiple pathophysiological processes., Competing Interests: Authors NP, SK and CPR were employees of company CytoDyn, owner and developer of Leronlimab. JBS, SH, HLW, and GMW have received compensation for consulting for CytoDyn, a company that may have commercial interests in the results of this research. JBS has also received compensation for serving on the scientific advisory board of CytoDyn. This potential conflict of interest has been reviewed and managed by the Oregon Health & Science University. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Chang, Wu, Webb, Tiwary, Hughes, Reed, Hwang, Waytashek, Boyle, Pessoa, Sylwester, Morrow, Belica, Fischer, Kelly, Pourhassan, Bochart, Smedley, Recknor, Hansen and Sacha.)

Simian immunodeficiency virus (SIV) challenge of rhesus macaques (RMs) vaccinated with strain 68-1 Rhesus Cytomegalovirus (RhCMV) vectors expressing SIV proteins (RhCMV/SIV) results in a binary outcome: stringent control and subsequent clearance of highly pathogenic SIV in ~55% of vaccinated RMs with no protection in the remaining 45%. Although previous work indicates that unconventionally restricted, SIV-specific, effector-memory (EM)-biased CD8+ T cell responses are necessary for efficacy, the magnitude of these responses does not predict efficacy, and the basis of protection vs. non-protection in 68-1 RhCMV/SIV vector-vaccinated RMs has not been elucidated. Here, we report that 68-1 RhCMV/SIV vector administration strikingly alters the whole blood transcriptome of vaccinated RMs, with the sustained induction of specific immune-related pathways, including immune cell, toll-like receptor (TLR), inflammasome/cell death, and interleukin-15 (IL-15) signaling, significantly correlating with subsequent vaccine efficacy. Treatment of a separate RM cohort with IL-15 confirmed the central involvement of this cytokine in the protection signature, linking the major innate and adaptive immune gene expression networks that correlate with RhCMV/SIV vaccine efficacy. This change-from-baseline IL-15 response signature was also demonstrated to significantly correlate with vaccine efficacy in an independent validation cohort of vaccinated and challenged RMs. The differential IL-15 gene set response to vaccination strongly correlated with the pre-vaccination activity of this pathway, with reduced baseline expression of IL-15 response genes significantly correlating with higher vaccine-induced induction of IL-15 signaling and subsequent vaccine protection, suggesting that a robust de novo vaccine-induced IL-15 signaling response is needed to program vaccine efficacy. Thus, the RhCMV/SIV vaccine imparts a coordinated and persistent induction of innate and adaptive immune pathways featuring IL-15, a known regulator of CD8+ T cell function, that support the ability of vaccine-elicited unconventionally restricted CD8+ T cells to mediate protection against SIV challenge., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Louis J. Picker and Scott G. Hansen declare their role as consultants for, and their substantial financial interest in Vir Biotechnology, Inc. as a Conflict of Interest that is managed by OHSU. No other authors have any conflicts to disclose.

In the absence of a prophylactic vaccine, the use of antiretroviral therapy (ART) as pre-exposure prophylaxis (PrEP) to prevent HIV acquisition by uninfected individuals is a promising approach to slowing the epidemic, but its efficacy is hampered by incomplete patient adherence and ART-resistant variants. Here, we report that competitive inhibition of HIV Env-CCR5 binding via the CCR5-specific antibody Leronlimab protects rhesus macaques against infection following repeated intrarectal challenges of CCR5-tropic SHIV SF162P3 . Injection of Leronlimab weekly at 10 mg/kg provides significant but partial protection, while biweekly 50 mg/kg provides complete protection from SHIV acquisition. Tissue biopsies from protected macaques post challenge show complete CCR5 receptor occupancy and an absence of viral nucleic acids. After Leronlimab washout, protected macaques remain aviremic, and adoptive transfer of hematologic cells into naïve macaques does not transmit viral infection. These data identify CCR5 blockade with Leronlimab as a promising approach to HIV prophylaxis and support initiation of clinical trials.

Here, we assessed the efficacy of a short-course multimodal therapy (enrofloxacin, azithromycin, fenbendazole, and paromomycin) to eliminate common macaque endemic pathogens (EPs) and evaluated its impact on gastrointestinal (GI) microbiota, mucosal integrity, and local and systemic inflammation in sixteen clinically healthy macaques. Treatment combined with expanded practices resulted in successful maintenance of rhesus macaques (RM) free of common EPs, with no evidence of overt microbiota diversity loss or dysbiosis and instead resulted in a more defined luminal microbiota across study subjects. Creation of a GI pathogen free (GPF) status resulted in improved colonic mucosal barrier function (histologically, reduced colonic MPO+, and reduced pan-bacterial 16s rRNA in the MLN), reduced local and systemic innate and adaptive inflammation with reduction of colonic Mx1 and pSTAT1, decreased intermediate (CD14+CD16+) and non-classical monocytes (CD14-CD16+), reduced populations of peripheral dendritic cells, Ki-67+ and CD38+ CD4+ T cells, Ki-67+IgG+, and Ki-67+IgD+ B cells indicating lower levels of background inflammation in the distal descending colon, draining mesenteric lymph nodes, and systemically in peripheral blood, spleen, and axillary lymph nodes. A more controlled rate of viral acquisition resulted when untreated and treated macaques were challenged by low dose intrarectal SIVmac239x, with an ~100 fold increase in dose required to infect 50% (AID50) of the animals receiving treatment compared to untreated controls. Reduction in and increased consistency of number of transmitted founder variants resulting from challenge seen in the proof of concept study directly correlated with post-treatment GPF animal's improved barrier function and reduction of key target cell populations (Ki-67+ CD4+T cells) at the site of viral acquisition in the follow up study. These data demonstrate that a therapeutic and operational strategy can successfully eliminate varying background levels of EPs and their associated aberrant immunomodulatory effects within a captive macaque cohort, leading to a more consistent, better defined and reproducible research model., Competing Interests: The authors have declared that no competing interests exist.

Strain 68-1 rhesus cytomegalovirus (RhCMV) vectors expressing simian immunodeficiency virus (SIV) antigens elicit CD8 + T cells recognizing epitopes presented by major histocompatibility complex II (MHC-II) and MHC-E but not MHC-Ia. These immune responses mediate replication arrest of SIV in 50 to 60% of monkeys. We show that the peptide VMAPRTLLL (VL9) embedded within the RhCMV protein Rh67 promotes intracellular MHC-E transport and recognition of RhCMV-infected fibroblasts by MHC-E-restricted CD8 + T cells. Deletion or mutation of viral VL9 abrogated MHC-E-restricted CD8 + T cell priming, resulting in CD8 + T cell responses exclusively targeting MHC-II-restricted epitopes. These responses were comparable in magnitude and differentiation to responses elicited by 68-1 vectors but did not protect against SIV. Thus, Rh67-enabled direct priming of MHC-E-restricted T cells is crucial for RhCMV/SIV vaccine efficacy., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

To define the contribution of CD8+ T cell responses to control of SIV reactivation during and following antiretroviral therapy (ART), we determined the effect of long-term CD8+ T cell depletion using a rhesusized anti-CD8β monoclonal antibody on barcoded SIVmac239 dynamics on stable ART and after ART cessation in rhesus macaques (RMs). Among the RMs with full CD8+ T cell depletion in both blood and tissue, there were no significant differences in the frequency of viral blips in plasma, the number of SIV RNA+ cells and the average number of RNA copies/infected cell in tissue, and levels of cell-associated SIV RNA and DNA in blood and tissue relative to control-treated RMs during ART. Upon ART cessation, both CD8+ T cell-depleted and control RMs rebounded in fewer than 12 days, with no difference in the time to viral rebound or in either the number or growth rate of rebounding SIVmac239M barcode clonotypes. However, effectively CD8+ T cell-depleted RMs showed a stable, approximately 2-log increase in post-ART plasma viremia relative to controls. These results indicate that while potent antiviral CD8+ T cell responses can develop during ART-suppressed SIV infection, these responses effectively intercept post-ART SIV rebound only after systemic viral replication, too late to limit reactivation frequency or the early spread of reactivating SIV reservoirs.

CD4 T cell effector function is required for optimal containment of Mycobacterium tuberculosis (Mtb) infection. IFNɣ produced by CD4 T cells is a key cytokine that contributes to protection. However, lung-infiltrating CD4 T cells have a limited ability to produce IFNɣ, and IFNɣ plays a lesser protective role within the lung than at sites of Mtb dissemination. In a murine infection model, we observed that IFNɣ production by Mtb-specific CD4 T cells is rapidly extinguished within the granuloma but not within unaffected lung regions, suggesting localized immunosuppression. We identified a signature of TGFβ signaling within granuloma-infiltrating T cells in both mice and rhesus macaques. Selective blockade of TGFβ signaling in T cells resulted in an accumulation of terminally differentiated effector CD4 T cells, improved IFNɣ production within granulomas, and reduced bacterial burdens. These findings uncover a spatially localized immunosuppressive mechanism associated with Mtb infection and provide potential targets for host-directed therapy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021. Published by Elsevier Inc.)

Simian immunodeficiency virus (SIV) insert-expressing, 68-1 rhesus cytomegalovirus (RhCMV/SIV) vectors elicit major histocompatibility complex E (MHC-E)- and MHC-II-restricted, SIV-specific CD8 + T cell responses, but the basis of these unconventional responses and their contribution to demonstrated vaccine efficacy against SIV challenge in the rhesus monkeys (RMs) have not been characterized. We show that these unconventional responses resulted from a chance genetic rearrangement in 68-1 RhCMV that abrogated the function of eight distinct immunomodulatory gene products encoded in two RhCMV genomic regions ( Rh157.5/Rh157.4 and Rh158-161 ), revealing three patterns of unconventional response inhibition. Differential repair of these genes with either RhCMV-derived or orthologous human CMV (HCMV)-derived sequences ( UL128/UL130 ; UL146/UL147 ) leads to either of two distinct CD8 + T cell response types-MHC-Ia-restricted only or a mix of MHC-II- and MHC-Ia-restricted CD8 + T cells. Response magnitude and functional differentiation are similar to RhCMV 68-1, but neither alternative response type mediated protection against SIV challenge. These findings implicate MHC-E-restricted CD8 + T cell responses as mediators of anti-SIV efficacy and indicate that translation of RhCMV/SIV vector efficacy to humans will likely require deletion of all genes that inhibit these responses from the HCMV/HIV vector., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Tuberculosis (TB) is a heterogeneous disease manifesting in a subset of individuals infected with aerosolized Mycobacterium tuberculosis (Mtb). Unlike human TB, murine infection results in uniformly high lung bacterial burdens and poorly organized granulomas. To develop a TB model that more closely resembles human disease, we infected mice with an ultra-low dose (ULD) of between 1-3 founding bacteria, reflecting a physiologic inoculum. ULD-infected mice exhibited highly heterogeneous bacterial burdens, well-circumscribed granulomas that shared features with human granulomas, and prolonged Mtb containment with unilateral pulmonary infection in some mice. We identified blood RNA signatures in mice infected with an ULD or a conventional Mtb dose (50-100 CFU) that correlated with lung bacterial burdens and predicted Mtb infection outcomes across species, including risk of progression to active TB in humans. Overall, these findings highlight the potential of the murine TB model and show that ULD infection recapitulates key features of human TB., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Antimicrobial agents are used extensively off-label in mink, as almost no agents are registered for this animal species. Pharmacokinetic (PK) and pharmacodynamic (PD) data are required to determine antimicrobial dosages specifically targeting mink bacterial pathogens. The aims of this study were to assess, in a PKPD framework, the empirical dosage regimen for a combination of trimethoprim (TMP) and sulfadiazine (SDZ) in mink, and secondarily to produce data for future setting of clinical breakpoints. TMP and SDZ PK parameters were obtained experimentally in 22 minks following IV or oral administration of TMP/SDZ (30 mg/kg, i.e. 5 mg/kg TMP and 25 mg/kg SDZ). fAUC/MIC with a target value of 24 hr was selected as the PKPD index predictive of TMP/SDZ efficacy. Using a modeling approach, PKPD cutoffs for TMP and SDZ were determined as 0.062 and 16 mg/L, respectively. By incorporating an anticipated potentiation effect of SDZ on TMP against Escherichia coli and Staphylococcus delphini, the PKPD cutoff of TMP was revised to 0.312 mg/L, which is above the tentative epidemiological cutoffs (TECOFF) for these species. The current empirical TMP/SDZ dosage regimen (30 mg/kg, PO, once daily) therefore appears adequate for treatment of wild-type E. coli and S. delphini infections in mink., (© 2020 John Wiley & Sons Ltd.)

Cytomegaloviruses (CMVs) are highly adapted to their host species resulting in strict species specificity. Hence, in vivo examination of all aspects of CMV biology employs animal models using host-specific CMVs. Infection of rhesus macaques (RM) with rhesus CMV (RhCMV) has been established as a representative model for infection of humans with HCMV due to the close evolutionary relationships of both host and virus. However, the only available RhCMV clone that permits genetic modifications is based on the 68-1 strain which has been passaged in fibroblasts for decades resulting in multiple genomic changes due to tissue culture adaptations. As a result, 68-1 displays reduced viremia in RhCMV-naïve animals and limited shedding compared to non-clonal, low passage isolates. To overcome this limitation, we used sequence information from primary RhCMV isolates to construct a full-length (FL) RhCMV by repairing all mutations affecting open reading frames (ORFs) in the 68-1 bacterial artificial chromosome (BAC). Inoculation of adult, immunocompetent, RhCMV-naïve RM with the reconstituted virus resulted in significant viremia in the blood similar to primary isolates of RhCMV and furthermore led to high viral genome copy numbers in many tissues at day 14 post infection. In contrast, viral dissemination was greatly reduced upon deletion of genes also lacking in 68-1. Transcriptome analysis of infected tissues further revealed that chemokine-like genes deleted in 68-1 are among the most highly expressed viral transcripts both in vitro and in vivo consistent with an important immunomodulatory function of the respective proteins. We conclude that FL-RhCMV displays in vitro and in vivo characteristics of a wildtype virus while being amenable to genetic modifications through BAC recombineering techniques., Competing Interests: OHSU and Drs. S.G.H., L.J.P., K.F. and D.M. have a significant financial interest in VirBiotechnology, Inc., a company that may have a commercial interest in the results of this research and technology. The potential individual and institutional conflicts of interest have been reviewed and managed by OHSU.

Lay Abstract: Sleep problems are commonly reported among parents of children with autism spectrum disorder (ASD). Without effective treatment, such problems are unlikely to resolve. To date, we know very little about how and why parents of children with ASD seek help for sleep disturbance. Via an online survey, we gathered information about how parents make sense of their children's sleep problems, beliefs about their causes, sources of information, and help-seeking behavior. The analysis of responses from 244 parents revealed that parents commonly view sleep problems (a) as a consequence of their child's ASD, and unlikely to change over time (stable), and (b) as located within the child (intrinsic), stable over time, and difficult to treat. Despite this, parents also rated sleep problems as being important to treat. Eighty-two percent of parents surveyed reported seeking some kind of help for their child's sleep disturbance, and the average parent had tried six different treatment strategies, most commonly medical approaches (e.g. melatonin). The alignment between parents' treatment choices and those strategies that are supported by research was poor, but belief in the effectiveness of treatments was closely related to how often the treatment was used. These findings have important implications for parental education and clinical practice in the treatment of sleep problems in children with ASD.

The diversity of Ig and TCR repertoires is a focal point of immunological studies. Rhesus macaques ( Macaca mulatta ) are key for modeling human immune responses, placing critical importance on the accurate annotation and quantification of their Ig and TCR repertoires. However, because of incomplete reference resources, the coverage and accuracy of the traditional targeted amplification strategies for profiling rhesus Ig and TCR repertoires are largely unknown. In this study, using long read sequencing, we sequenced four Indian-origin rhesus macaque tissues and obtained high-quality, full-length sequences for over 6000 unique Ig and TCR transcripts, without the need for sequence assembly. We constructed, to our knowledge, the first complete reference set for the constant regions of all known isotypes and chain types of rhesus Ig and TCR repertoires. We show that sequence diversity exists across the entire variable regions of rhesus Ig and TCR transcripts. Consequently, existing strategies using targeted amplification of rearranged variable regions comprised of V(D)J gene segments miss a significant fraction (27-53% and 42-49%) of rhesus Ig/TCR diversity. To overcome these limitations, we designed new rhesus-specific assays that remove the need for primers conventionally targeting variable regions and allow single cell level Ig and TCR repertoire analysis. Our improved approach will enable future studies to fully capture rhesus Ig and TCR repertoire diversity and is applicable for improving annotations in any model organism., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Although a sizable minority of students who are deaf or hard of hearing (DHH) are also diagnosed with autism spectrum disorder (ASD), there is little research examining teachers' feelings of aptitude for working with these students, nor the instructional strategies used with this population. This study reports results from a researcher-designed survey of teachers working with children who are both DHH and have ASD. Our results suggest that teachers working with this population felt under-resourced, under-prepared, and under-supported in their work with dually diagnosed students. Perhaps as a result, participants tended to use instructional strategies common to their certification area. We identify a need for cross-training teachers across disability areas. In addition, we call for research that tests the applicability of practices in either Deaf Education or ASD Education for dually diagnosed children who may have needs that are unique from children either group.

Currently 247 million people are living with chronic hepatitis B virus infection (CHB), and the development of novel curative treatments is urgently needed. Immunotherapy is an attractive approach to treat CHB, yet therapeutic approaches to augment the endogenous hepatitis B virus (HBV)-specific T cell response in CHB patients have demonstrated little success. In this study, we show that strain 68-1 rhesus macaque (RM) CMV vaccine vectors expressing HBV Ags engender HBV-specific CD8 + T cells unconventionally restricted by MHC class II and the nonclassical MHC-E molecule in RM. Surface staining of human donor and RM primary hepatocytes (PH) ex vivo revealed the majority of PH expressed MHC-E but not MHC class II. HBV-specific, MHC-E-restricted CD8 + T cells from RM vaccinated with RM CMV vaccine vectors expressing HBV Ags recognized HBV-infected PH from both human donor and RM. These results provide proof-of-concept that MHC-E-restricted CD8 + T cells could be harnessed for the treatment of CHB, either through therapeutic vaccination or adoptive immunotherapy., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Surgical outcome following pelvic organ prolapse (POP) repair needs improvement. We suggest a new approach based on a tissue-engineering strategy. In vivo, the regenerative potential of an electrospun biodegradable polycaprolactone (PCL) mesh was studied. Six different biodegradable PCL meshes were evaluated in a full-thickness abdominal wall defect model in 84 rats. The rats were assigned into three groups: (1) hollow fiber PCL meshes delivering two dosages of basic fibroblast growth factor (bFGF), (2) solid fiber PCL meshes with and without bFGF, and (3) solid fiber PCL meshes delivering connective tissue growth factor (CTGF) and rat mesenchymal stem cells (rMSC). After 8 and 24 weeks, we performed a histological evaluation, quantitative analysis of protein content, and the gene expression of collagen-I and collagen-III, and an assessment of the biomechanical properties of the explanted meshes. Multiple complications were observed except from the solid PCL-CTGF mesh delivering rMSC. Hollow PCL meshes were completely degraded after 24 weeks resulting in herniation of the mesh area, whereas the solid fiber meshes were intact and provided biomechanical reinforcement to the weakened abdominal wall. The solid PCL-CTGF mesh delivering rMSC demonstrated improved biomechanical properties after 8 and 24 weeks compared to muscle fascia. These meshes enhanced biomechanical and biochemical properties, demonstrating a great potential of combining tissue engineering with stem cells as a new therapeutic strategy for POP repair. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 108B:48-55, 2020., (© 2019 Wiley Periodicals, Inc.)

Vaccines based on cytomegalovirus (CMV) demonstrate protection in animal models of infectious disease and cancer. Vaccine efficacy is associated with the ability of CMV to elicit and indefinitely maintain high frequencies of circulating effector memory T cells (T EM ) providing continuous, life-long anti-pathogen immune activity. To allow for the clinical testing of human CMV (HCMV)-based vaccines we constructed and characterized as a vector backbone the recombinant molecular clone TR3 representing a wildtype genome. We demonstrate that TR3 can be stably propagated in vitro and that, despite species incompatibility, recombinant TR3 vectors elicit high frequencies of T EM to inserted antigens in rhesus macaques (RM). Live-attenuated versions of TR3 were generated by deleting viral genes required to counteract intrinsic and innate immune responses. In addition, we eliminated subunits of a viral pentameric glycoprotein complex thus limiting cell tropism. We show in a humanized mouse model that such modified vectors were able to establish persistent infection but lost their ability to reactivate from latency. Nevertheless, attenuated TR3 vectors preserved the ability to elicit and maintain T EM to inserted antigens in RM. We further demonstrate that attenuated TR3 can be grown in approved cell lines upon elimination of an anti-viral host factor using small interfering RNA, thus obviating the need for a complementing cell line. In sum, we have established a versatile platform for the clinical development of live attenuated HCMV-vectored vaccines and immunotherapies.

Paraeducators are ideal candidates for delivering communication interventions to children with developmental disabilities and delays (DD) because they spend a significant amount of time with these children. However, professional development is often inadequate and limited research supports best practices. Additionally, paraeducators work with multiple children with varying skill levels. Little research has been conducted on the use of existing strategies with multiple children. This single-case study examines the effect of a training package on paraeducators' fidelity of intervention implementation with a child dyad and subsequent child outcomes. Results suggest that formal coaching contributed to improved fidelity of intervention implementation. Furthermore, paraeducators were able to use intervention strategies with children with varying communication skills and goals. Variable increases in child communication were also detected.