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Purification of Human CD34+CD90+ HSCs Reduces Target Cell Population and Improves Lentiviral Transduction for Gene Therapy

Mol Ther Methods Clin Dev

2020 Yan-Yi Chan; Andrew Berger; Mark Enstrom; Thomas Eunson; Hans-Peter Kiem; Stefan Radtke; Anai Perez; Stefanie Schmuck; Margaret Cui; Jennifer Adair; Dnyanada Pande

Hematopoietic stem cell (HSC) gene therapy has the potential to cure many genetic, malignant, and infectious diseases. We have shown in a nonhuman primate gene therapy and transplantation model that the CD34+CD90+ cell fraction was exclusively responsible for multilineage engraftment and hematopoietic reconstitution. In this study, we show the translational potential of this HSC-enriched CD34 subset for lentivirus-mediated gene therapy. Alternative HSC enrichment strategies include the purification of CD133+ cells or CD38low/- subsets of CD34+ cells from human blood products. We directly compared these strategies to the isolation of CD90+ cells using a good manufacturing practice (GMP) grade flow-sorting protocol with clinical applicability. We show that CD90+ cell selection results in about 30-fold fewer target cells in comparison to CD133+ or CD38low/- CD34+ hematopoietic stem and progenitor cell (HSPC) subsets without compromising the engraftment potential in vivo. Single-cell RNA sequencing confirmed nearly complete depletion of lineage-committed progenitor cells in CD90+ fractions compared to alternative selections. Importantly, lentiviral transduction efficiency in purified CD90+ cells resulted in up to 3-fold higher levels of engrafted gene-modified blood cells. These studies should have important implications for the manufacturing of patient-specific HSC gene therapy and gene-engineered cell products.

Protection at First Sexual Intercourse Among Adolescent Girls and Young Women in Kenya (Jul, 10.1007/s10508-020-01785-w, 2020)

Arch Sex Behav

2020 Nelly Mugo; Anna Wald

There was an error in this article as originally published.

Humoral response to HPV16 proteins in persons with anal high-grade squamous intraepithelial lesion or anal cancer

Cancer Epidemiol Biomarkers Prev

2020 Anna Wald; Margaret Madeleine; Denise Galloway; David Doody

BACKGROUND: This study was launched to evalute the association of early and late antibodies to human papillomavirus (HPV)16 detection and risk of anal high-grade intraepithelial lesions (HSIL) or cancer. METHODS: We analyzed data from persons with anal HSIL or cancer and controls from a case-control study in Seattle, Washington. Sera were evaluated for HPV16 early (E1, E2, E4, E6, E7) and late (L1) antibodies by multiplex serology. Logistic regression models were used to assess serologic associations with risk of anal HSIL or cancer. RESULTS: The study included 67 participants with anal HSIL, 116 with anal cancer, and 830 population-based controls. HPV16 seropositivity to L1 (aOR 13.8, 95% CI 7.4-25.8), E4 (aOR 2.3, 95% CI 1.1-4.5) and E6 (aOR 4.9, 95% CI 1.1-21.2) was associated with HSIL; and detection of all antibodies to HPV16 late and early proteins was associated with increased risk of anal cancer ranging from aOR 1.7 to 32.5 (L1 aOR 12.5 [95% CI 7.3-21.7], E1 aOR 24.9 [95% CI 10.3-59.9], E2 aOR 6.3 [95% CI 3.4-11.7], E4 aOR 2.8 [95% CI 1.6-4.8], E6 aOR 32.5 [95% CI 14.2-74.4] and E7 aOR 1.7 [95% CI 1.0-3.0]). CONCLUSIONS: HPV serologic markers proved to be specific for identifying anal cancer. HPV16 E6 seropositivity is relatively uncommon in persons without anal cancer. IMPACT: This large study comprehensively describes the distinct antibody responses to the HPV16 proteins in persons with anal HSIL or anal cancer. Antibodies to HPV16 E6 should be further evaluated as a potential biomarker for anal cancer prevention.

Integrating genotypes and phenotypes improves long-term forecasts of seasonal influenza A/H3N2 evolution


2020 Trevor Bedford

Seasonal influenza virus A/H3N2 is a major cause of death globally. Vaccination remains the most effective preventative. Rapid mutation of hemagglutinin allows viruses to escape adaptive immunity. This antigenic drift necessitates regular vaccine updates. Effective vaccine strains need to represent H3N2 populations circulating one year after strain selection. Experts select strains based on experimental measurements of antigenic drift and predictions made by models from hemagglutinin sequences. We developed a novel influenza forecasting framework that integrates phenotypic measures of antigenic drift and functional constraint with previously published sequence-only fitness estimates. Forecasts informed by phenotypic measures of antigenic drift consistently outperformed previous sequence- only estimates, while sequence-only estimates of functional constraint surpassed more comprehensive experimentally-informed estimates. Importantly, the best models integrated estimates of both functional constraint and either antigenic drift phenotypes or recent population growth.

Cross-Sectional Prevalence of SARS-CoV-2 Among Skilled Nursing Facility Employees and Residents Across Facilities in Seattle

J Gen Intern Med

2020 Lea Starita; Trevor Bedford

BACKGROUND: Skilled nursing facilities (SNFs) are high-risk settings for SARS-CoV-2 transmission. Infection rates among employees are infrequently described. OBJECTIVE: To describe SARS-CoV-2 rates among SNF employees and residents during a non-outbreak time period, we measured cross-sectional SARS-CoV-2 prevalence across multiple sites in the Seattle area. DESIGN: SARS-CoV-2 testing was performed for SNF employees and residents using quantitative real-time reverse transcription polymerase chain reaction. A subset of employees completed a sociodemographic and symptom questionnaire. PARTICIPANTS: Between March 29 and May 13, 2020, we tested 1583 employees and 1208 residents at 16 SNFs for SARS-CoV-2. MAIN MEASURE: SARS-CoV-2 testing results and symptom report among employees and residents. KEY RESULTS: Eleven of the 16 SNFs had one or more resident or employee test positive. Overall, 46 (2.9%) employees had positive or inconclusive testing for SARS-CoV-2, and among those who completed surveys, most were asymptomatic and involved in direct patient care. The majority of employees tested were female (934, 73%), and most employees were Asian (392, 30%), Black (360, 28%), or white (360, 28%). Among the 1208 residents tested, 110 (9.1%) had positive or inconclusive results. There was no association between the presence of positive residents and positive employees within a SNF (p=0.62, McNemar's test). CONCLUSIONS: In the largest study of SNFs to date, SARS-CoV-2 infections were detected among both employees and residents. Employees testing positive were often asymptomatic and involved in direct patient care. Surveillance testing is needed for SNF employees and residents during the pandemic response.

In vivo antiviral host transcriptional response to SARS-CoV-2 by viral load, sex, and age

PLoS Biol

2020 Pavitra Roychoudhury; Alex Greninger; Keith Jerome

Despite limited genomic diversity, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has shown a wide range of clinical manifestations in different patient populations. The mechanisms behind these host differences are still unclear. Here, we examined host response gene expression across infection status, viral load, age, and sex among shotgun RNA sequencing profiles of nasopharyngeal (NP) swabs from 430 individuals with PCR-confirmed SARS-CoV-2 and 54 negative controls. SARS-CoV-2 induced a strong antiviral response with up-regulation of antiviral factors such as OAS1-3 and IFIT1-3 and T helper type 1 (Th1) chemokines CXCL9/10/11, as well as a reduction in transcription of ribosomal proteins. SARS-CoV-2 culture in human airway epithelial (HAE) cultures replicated the in vivo antiviral host response 7 days post infection, with no induction of interferon-stimulated genes after 3 days. Patient-matched longitudinal specimens (mean elapsed time = 6.3 days) demonstrated reduction in interferon-induced transcription, recovery of transcription of ribosomal proteins, and initiation of wound healing and humoral immune responses. Expression of interferon-responsive genes, including ACE2, increased as a function of viral load, while transcripts for B cell-specific proteins and neutrophil chemokines were elevated in patients with lower viral load. Older individuals had reduced expression of the Th1 chemokines CXCL9/10/11 and their cognate receptor CXCR3, as well as CD8A and granzyme B, suggesting deficiencies in trafficking and/or function of cytotoxic T cells and natural killer (NK) cells. Relative to females, males had reduced B cell-specific and NK cell-specific transcripts and an increase in inhibitors of nuclear factor kappa-B (NF-κB) signaling, possibly inappropriately throttling antiviral responses. Collectively, our data demonstrate that host responses to SARS-CoV-2 are dependent on viral load and infection time course, with observed differences due to age and sex that may contribute to disease severity.

Post-Prescription Review with Threat of Infectious Disease Consultation and Sustained Reduction in Meropenem Use Over Four Years

Clin Infect Dis

2020 Elizabeth Krantz; Catherine Liu

BACKGROUND: Following a meropenem shortage, we implemented a post-prescription review with feedback (PPRF) in November 2015 with mandatory infectious disease (ID) consultation for all meropenem and imipenem courses > 72 hours. Providers were made aware of the policy via an electronic alert at the time of ordering. METHODS: A retrospective study was conducted at the University of Washington Medical Center (UWMC) and Harborview Medical Center (HMC) to evaluate the impact of the policy on antimicrobial consumption and clinical outcomes pre- and post-intervention during a 6-year period. Antimicrobial use was tracked using days of therapy (DOT) per 1,000 patient-days, and data were analyzed by an interrupted time series. RESULTS: There were 4,066 and 2,552 patients in the pre- and post-intervention periods, respectively. Meropenem and imipenem use remained steady until the intervention, when a marked reduction in DOT/1,000 patient-days occurred at both hospitals (UWMC: percentage change -72.1%, (95% CI -76.6, -66.9), P & 0.001; HMC: percentage change -43.6%, (95% CI -59.9, -20.7), P = 0.001). Notably, although the intervention did not address antibiotic use until 72 hours after initiation, there was a significant decline in meropenem and imipenem initiation ("first starts") in the post-intervention period, with a 64.9% reduction (95% CI 58.7, 70.2; P &0.001) at UWMC and 44.7% reduction (95% CI 28.1, 57.4; P & 0.001) at HMC. CONCLUSIONS: Mandatory ID consultation and PPRF for meropenem and imipenem beyond 72 hours resulted in a significant and sustained reduction in the use of these antibiotics and notably impacted their up-front usage.

Model-based predictions of HIV incidence among African women using HIV risk behaviors and community-level data on male HIV prevalence and viral suppression

J Acquir Immune Defic Syndr

2020 Dobromir Dimitrov; Deborah Donnell; James Moore

BACKGROUND: Pre-exposure prophylaxis (PrEP) with Tenofovir Disoproxil Fumarate and Emtricitabine (TDF-FTC) has proven highly effective in preventing HIV acquisition and is therefore offered to all participants in the control group as part of the standard of care package in many new HIV prevention studies. We propose a methodology for predicting HIV incidence in a hypothetical "placebo arm" for open-label studies or clinical trials with active control among African women. We apply the method to an open-label PrEP study, HIV Prevention Trials Network (HPTN) 082, which tested strategies to improve PrEP adherence in young African women all of whom were offered PrEP. METHODS: Our model predicted HIV infection risk for female study cohorts in sub-Saharan Africa using baseline behavioral risk factors and contemporary HIV prevalence and viral suppression in the local male population. The model was calibrated to HIV incidence in the Vaginal and Oral Interventions to Control the Epidemic study (VOICE). RESULTS: Our model reproduced the annual HIV incidence of 3.2%-4.8% observed over one year of follow-up in the placebo groups of four completed clinical studies. We predicted an annual HIV incidence of 3.7% (95% CI 3.2-4.2) among HPTN 082 participants in the absence of PrEP and other risk reduction interventions. CONCLUSIONS: We demonstrated the potential of the proposed methodology to provide HIV incidence predictions based on assessment of individual risk behaviors and community and time-specific HIV exposure risk using HIV treatment and viral suppression data. These estimates may serve as comparators in HIV prevention trials without a placebo group.

Anti-PD-1 and Anti-PD-L1 Monoclonal Antibodies in People Living with HIV and Cancer


2020 Thomas Uldrick

PURPOSE OF REVIEW: Immune checkpoint inhibitors targeting the programmed cell death-1 (PD-1) pathway are a class of anti-cancer immunotherapy agents changing treatment paradigms of many cancers that occur at higher rates in people living with HIV (PLWH) than in the general population. However, PLWH have been excluded from most of the initial clinical trials with these agents. RECENT FINDINGS: Two recent prospective studies of anti-PD-1 agents, along with observational studies and a meta-analysis, have demonstrated acceptable safety in PLWH. Preliminary evidence indicates activity in a range of tumors and across CD4+ T cell counts. Safety and preliminary activity data suggest monoclonal antibodies targeting PD-1 or its ligand, PD-L1, are generally appropriate for PLWH and cancers for which there are FDA-approved indications. Ongoing and future trials of anti-PD-1 and anti-PD-L1 therapy alone or in combination for HIV-associated cancers may further improve outcomes for this underserved population.

Neutralizing antibodies correlate with protection from SARS-CoV-2 in humans during a fishery vessel outbreak with high attack rate

J Clin Microbiol

2020 Katharine Dusenbury; Alex Greninger; Meei-Li Huang; Pavitra Roychoudhury; Adam Dingens; Jesse Bloom; Keith Jerome

The development of vaccines against SARS-CoV-2 would be greatly facilitated by the identification of immunological correlates of protection in humans. However, to date, studies on protective immunity have only been performed in animal models and correlates of protection have not been established in humans. Here, we describe an outbreak of SARS-CoV-2 on a fishing vessel associated with a high attack rate. Predeparture serological and viral RT-PCR testing along with repeat testing after return to shore was available for 120 of the 122 persons on board over a median follow-up of 32.5 days (range 18.8 to 50.5 days). A total of 104 individuals had an RT-PCR positive viral test with Ct <35 or seroconverted during the follow-up period, yielding an attack rate on board of 85.2% (104/122 individuals). Metagenomic sequencing of 39 viral genomes suggested the outbreak originated largely from a single viral clade. Only three crewmembers tested seropositive prior to the boat's departure in initial serological screening and also had neutralizing and spike-reactive antibodies in follow-up assays. None of these crewmembers with neutralizing antibody titers showed evidence of bona fide viral infection or experienced any symptoms during the viral outbreak. Therefore, the presence of neutralizing antibodies from prior infection was significantly associated with protection against re-infection (Fisher's exact test, p=0.002).