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CRISPR-Cas9 gene editing of hepatitis B virus in chronically infected humanized mice

Mol Ther Methods Clin Dev

2021 Michelle Loprieno; Nicholas Weber; Rossana Colon-Thillet; Larry Corey; Lindsay Klouser; Martine Aubert; Karsten Eichholz; Keith Jerome; Stephan Polyak; Harshana De Silva Feelixge; Zhenhua Li; Daniel Stone

Chronic hepatitis B virus (HBV) infection is a major public health problem. New treatment approaches are needed because current treatments do not target covalently closed circular DNA (cccDNA), the template for HBV replication, and rarely clear the virus. We harnessed adeno-associated virus (AAV) vectors and CRISPR-Staphylococcus aureus (Sa)Cas9 to edit the HBV genome in liver-humanized FRG mice chronically infected with HBV and receiving entecavir. Gene editing was detected in livers of five of eight HBV-specific AAV-SaCas9-treated mice, but not control mice, and mice with detectable HBV gene editing showed higher levels of SaCas9 delivery to HBV+ human hepatocytes than those without gene editing. HBV-specific AAV-SaCas9 therapy significantly improved survival of human hepatocytes, showed a trend toward decreasing total liver HBV DNA and cccDNA, and was well tolerated. This work provides evidence for the feasibility and safety of in vivo gene editing for chronic HBV infections, and it suggests that with further optimization, this approach may offer a plausible way to treat or even cure chronic HBV infections.

Viral load and contact heterogeneity predict SARS-CoV-2 transmission and super-spreading events


2021 Bryan Mayer; Joshua Schiffer; Erwing Cardozo Ojeda; Ashish Goyal; Daniel Reeves

SARS-CoV-2 is difficult to contain because many transmissions occur during pre-symptomatic infection. Unlike influenza, most SARS-CoV-2 infected people do not transmit while a small percentage infect large numbers of people. We designed mathematical models which link observed viral loads with epidemiologic features of each virus, including distribution of transmissions attributed to each infected person and duration between symptom onset in the transmitter and secondarily infected person. We identify that people infected with SARS-CoV-2 or influenza can be highly contagious for less than one day, congruent with peak viral load. SARS-CoV-2 super-spreader events occur when an infected person is shedding at a very high viral load and has a high number of exposed contacts. The higher predisposition of SARS-CoV-2 towards super-spreading events cannot be attributed to additional weeks of shedding relative to influenza. Rather, a person infected with SARS-CoV-2 exposes more people within equivalent physical contact networks, likely due to aerosolization.

Megasphaera lornae sp. nov., Megasphaera hutchinsoni sp. nov., and Megasphaera vaginalis sp. nov.: novel bacteria isolated from the female genital tract

Int J Syst Evol Microbiol

2021 Susan Strenk; Tina Fiedler; David Fredricks; Daniel Raftery; Sujatha Srinivasan

Six strictly anaerobic Gram-negative bacteria representing three novel species were isolated from the female reproductive tract. The proposed type strains for each species were designated UPII 199-6T, KA00182T and BV3C16-1T. Phylogenetic analyses based on 16S rRNA gene sequencing indicated that the bacterial isolates were members of the genus Megasphaera. UPII 199-6T and KA00182T had 16S rRNA gene sequence identities of 99.9 % with 16S rRNA clone sequences previously amplified from the human vagina designated as Megasphaera type 1 and Megasphaera type 2, members of the human vaginal microbiota associated with bacterial vaginosis, preterm birth and HIV acquisition. UPII 199-6T exhibited sequence identities ranging from 92.9 to 93.6 % with validly named Megasphaera isolates and KA00182T had 16S rRNA gene sequence identities ranging from 92.6-94.2 %. BV3C16-1T was most closely related to Megasphaera cerevisiae with a 16S rRNA gene sequence identity of 95.4 %. Cells were coccoid or diplococcoid, non-motile and did not form spores. Genital tract isolates metabolized organic acids but were asaccharolytic. The isolates also metabolized amino acids. The DNA G+C content for the genome sequences of UPII 199-6T, KA00182T and BV3C16-1T were 46.4, 38.9 and 49.8 mol%, respectively. Digital DNA-DNA hybridization and average nucleotide identity between the genital tract isolates and other validly named Megasphaera species suggest that each isolate type represents a new species. The major fatty acid methyl esters include the following: C12 : 0, C16 : 0, C16 : 0 dimethyl acetal (DMA) and summed feature 5 (C15 : 0 DMA and/or C14 : 0 3-OH) in UPII 199-6T; C16 : 0 and C16 : 1cis 9 in KA00182T; C12 : 0; C14 : 0 3-OH; and summed feature 5 in BV3C16-1T. The isolates produced butyrate, isobutyrate, and isovalerate but there were specific differences including production of formate and propionate. Together, these data indicate that UPII 199-6T, KA00182T and BV3C16-1T represent novel species within the genus Megasphaera. We propose the following names: Megasphaera lornae sp. nov. for UPII 199-6T representing the type strain of this species (=DSM 111201T=ATCC TSD-205T), Megasphaera hutchinsoni sp. nov. for KA00182T representing the type strain of this species (=DSM 111202T=ATCC TSD-206T) and Megasphaera vaginalis sp. nov. for BV3C16-1T representing the type strain of this species (=DSM 111203T=ATCC TSD-207T).

Targeting the membrane-proximal C2-set domain of CD33 for improved CD33-directed immunotherapy


2021 Colin Correnti; Salvatore Fiorenza; Cameron Turtle; Roland Walter; George Laszlo; Eliotte Garling; Margaret Lunn; Olivia Bates; Colin Godwin; Olivier Humbert; Hans-Peter Kiem; Benjamin Hoffstrom; Tinhdoan Phi

There is increasing interest in targeting CD33 in malignant and non-malignant disorders. In acute myeloid leukemia, longer survival with the CD33 antibody-drug conjugate gemtuzumab ozogamicin (GO) validates this strategy. Still, GO benefits only some patients, prompting efforts to develop more potent CD33-directed therapeutics. As one limitation, CD33 antibodies typically recognize the membrane-distal V-set domain. Using various artificial CD33 proteins, in which this domain was differentially positioned within the extracellular portion of the molecule, we tested whether targeting membrane-proximal epitopes enhances the effector functions of CD33 antibody-based therapeutics. Consistent with this idea, a CD33V-set/CD3 bispecific antibody (BsAb) and CD33V-set-directed chimeric antigen receptor (CAR)-modified T cells elicited substantially greater cytotoxicity against cells expressing a CD33 variant lacking the entire C2-set domain than cells expressing full-length CD33, whereas cytotoxic effects induced by GO were independent of the position of the V-set domain. We therefore raised murine and human antibodies against the C2-set domain of human CD33 and identified antibodies that bound CD33 regardless of the presence/absence of the V-set domain ("CD33PAN antibodies"). These antibodies internalized when bound to CD33 and, as CD33PAN/CD3 BsAb, had potent cytolytic effects against CD33+ cells. Together, our data provide the rationale for further development of CD33PAN antibody-based therapeutics.

DAS181 Treatment of Severe Lower Respiratory Tract Parainfluenza Virus Infection in Immunocompromised Patients: A Phase 2 Randomized, Placebo-Controlled Study

Clin Infect Dis

2021 Michael Boeckh

BACKGROUND: There are no antiviral therapies for Parainfluenza virus (PIV) infections. DAS181, a sialidase fusion protein, has demonstrated activity in in vitro and in animal models of PIV. METHODS: Adult immunocompromised patients diagnosed with PIV lower respiratory tract infection (LRTI) who required oxygen supplementation were randomized 2:1 to nebulized DAS181 (4.5mg/day) or matching placebo for up to 10 days. Randomization was stratified by need for mechanical ventilation (MV) or supplemental oxygen (SO). The primary endpoint was the proportion of patients reaching clinical stability survival (CSS) defined as returning to breathing room air (RTRA), normalization of vital signs for at least 24 hours, and survival up to day 45 from enrollment. RESULTS: A total of 111 patients were randomized to DAS181 (n=74) or placebo (n=37). CSS was achieved by 45.0% DAS181 treated patients in the SO stratum compared with 31.0% for placebo (p=0.15), while patients on MV had no benefit from DAS181. The proportion of patients achieving RTRA was numerically higher for SO stratum DAS181 patients (51.7%) compared with Placebo (34.5%) at Day 28 (p=0.17). In a post-hoc analysis of solid organ transplant, hematopoietic cell transplantation within one year, or chemotherapy within one year, more SO stratum patients achieved RTRA on DAS181 (51.8%) when compared to placebo (15.8%) by day 28 (p=0.012). CONCLUSION: The primary endpoint was not met, but post-hoc analysis of the RTRA component suggests DAS181 may have clinical activity in improving oxygenation in select severely immunocompromised patients with PIV LRTI who are not on mechanical ventilation.

Evaluating Specimen Quality and Results from a Community-Wide, Home-Based Respiratory Surveillance Study

J Clin Microbiol

2021 Jover Lee; Trevor Bedford; Michael Boeckh; Janet Englund; Debbie Nickerson; Mikaela Ilcisin; Thomas Sibley; Matthew Thompson; Kairsten Fay; Lea Starita

Introduction. While influenza and other respiratory pathogens cause significant morbidity and mortality, the community-based burden of these infections remains incompletely understood. The development of novel methods to detect respiratory infections is essential for mitigating epidemics and developing pandemic-preparedness infrastructure.Methods. From October 2019 to March 2020, we conducted a home-based cross-sectional study in the greater Seattle area, utilizing electronic consent and data collection instruments. Participants received nasal swab collection kits via rapid delivery within 24 hours of self-reporting respiratory symptoms. Samples were returned to the laboratory and were screened for 26 respiratory pathogens and a housekeeping gene. Participant data were recorded via online survey at the time of sample collection and one week later.Results. Of the 4,572 consented participants, 4,359 (95.3%) received a home swab kit, and 3,648 (83.7%) returned a nasal specimen for respiratory pathogen screening. The 3,638 testable samples had a mean RNase P CRT value of 19.0 (SD: 3.4) and 1,232 (33.9%) samples had positive results for one or more pathogens, including 645 (17.7%) influenza-positive specimens. Among the testable samples, the median time between shipment of the home swab kit and completion of laboratory testing was 8 days [IQR: 7.0-14.0]. A single adverse event occurred and did not cause long-term effects or require medical attention.Discussion. Home-based surveillance using online participant enrollment and specimen self-collection is a safe and feasible method for community-level monitoring of influenza and other respiratory pathogens, which can readily be adapted for use during pandemics.

Challenges and Opportunities for COVID-19 Vaccines in Patients with Cancer

Cancer Invest

2021 Paul Carpenter; Gary Lyman; Joshua Hill

Given the rapidly expanding global spread of the SARS-Co-V-2 virus and the expanding number of individuals with the serious and potentially fatal illness, COVID-19, there is an urgent need for safe and effective vaccines. Based on compelling evidence that patients with cancer are at increased risk for greater morbidity and mortality with COVID-19, several professional organizations have provided early guidance on the role of COVID-19 vaccines in patients with malignant disease. In this commentary we review the available data on the efficacy and safety of the approved and forthcoming vaccines in patients with cancer. Based on a review of the totality of available evidence, we recommend that most patients with cancer should receive the recommended dose and schedule of one of the COVID-19 vaccines when available. We encourage industry, regulators and professional research organizations to carefully track the efficacy and safety of COVID-19 vaccination in patients with cancer in the real world setting and routinely report unanticipated adverse events and signs of loss of efficacy. Particular attention is needed for patients on active cancer therapy to carefully evaluate efficacy and safety in relationship to the timing of vaccination relative to that of active cancer treatment and immunosuppression.

Assessment of the Inclusion of Racial/Ethnic Minority, Female, and Older Individuals in Vaccine Clinical Trials

JAMA Netw Open

2021 Elizabeth Krantz; Michele Andrasik; Steven Pergam

Importance: Medical research has not equitably included members of racial/ethnic minority groups or female and older individuals. There are limited data on participant demographic characteristics in vaccine trials despite the importance of these data to current trials aimed at preventing coronavirus disease 2019. Objective: To investigate whether racial/ethnic minority groups and female and older adults are underrepresented among participants in vaccine clinical trials. Design, Setting, and Participants: This cross-sectional study examined data from completed US-based vaccine trials registered on from July 1, 2011, through June 30, 2020. The terms vaccine, vaccination, immunization, and inoculation were used to identify trials. Only those addressing vaccine immunogenicity or efficacy of preventative vaccines were included. Main Outcomes and Measures: The numbers and percentages of racial/ethnic minority, female, and older individuals compared with US census data from 2011 and 2018. Secondary outcome measures were inclusion by trial phase and year of completion. Results: A total of 230 US-based trials with 219555 participants were included in the study. Most trials were randomized (180 [78.3%]), included viral vaccinations (159 [69.1%]), and represented all trial phases. Every trial reported age and sex; 134 (58.3%) reported race and 79 (34.3%) reported ethnicity. Overall, among adult study participants, White individuals were overrepresented (77.9%; 95% CI, 77.4%-78.4%), and Black or African American individuals (10.6%; 95% CI, 10.2%-11.0%) and American Indian or Alaska Native individuals (0.4%; 95% CI, 0.3%-0.5%) were underrepresented compared with US census data; enrollment of Asian individuals was similar (5.7%; 95% CI, 5.5%-6.0%). Enrollment of Hispanic or Latino individuals (11.6%; 95% CI, 11.1%-12.0%) was also low even among the limited number of adult trials reporting ethnicity. Adult trials were composed of more female participants (75325 [56.0%]), but among those reporting age as a percentage, enrollment of participants who were aged 65 years or older was low (12.1%; 95% CI, 12.0%-12.3%). Black or African American participants (10.1%; 95% CI, 9.7%-10.6%) and Hispanic or Latino participants (22.5%; 95% CI, 21.6%-23.4%) were also underrepresented in pediatric trials. Among trials reporting race/ethnicity, 65 (48.5%) did not include American Indian or Alaska Native participants and 81 (60.4%) did not include Hawaiian or Pacific Islander participants. Conclusions and Relevance: This cross-sectional study found that among US-based vaccine clinical trials, members of racial/ethnic minority groups and older adults were underrepresented, whereas female adults were overrepresented. These findings suggest that diversity enrollment targets should be included for all vaccine trials targeting epidemiologically important infections.

Immune response to the hepatitis B vaccine among HIV-infected adults in Uganda


2021 Warren Phipps; Scott Adams; Emmanuel Seremba; Corey Casper; Jackson Orem

BACKGROUND: Co-infection with hepatitis B virus (HBV) and human immunodeficiency virus (HIV) is common in sub-Saharan Africa (SSA) and can rapidly progress to cirrhosis and hepatocellular carcinoma. Recent data demonstrate ongoing HBV transmission among HIV-infected adults in SSA, suggesting that complications of HIV/HBV co-infection could be prevented with HBV vaccination. Because HBV vaccine efficacy is poorly understood among HIV-infected persons in SSA, we sought to characterize the humoral response to the HBV vaccine in HIV-seropositive Ugandan adults. METHODS: We enrolled HIV-infected adults in Kampala, Uganda without serologic evidence of prior HBV infection. Three HBV vaccine doses were administered at 0, 1 and 6months. Anti-HBs levels were measured 4weeks after the third vaccine dose. "Response" to vaccination was defined as anti-HBs levels10IU/L and "high response" as100IU/L. Regression analysis was used to determine predictors of response. RESULTS: Of 251 HIV-positive adults screened, 132 (53%) had no prior HBV infection or immunity and were enrolled. Most participants were women [89 (67%)]; median (IQR) age was 32years (27-41), and 68 (52%) had received antiretroviral therapy (ART) for>3months. Median (IQR) CD4 count was 426 (261-583), and 64 (94%) of the 68 receiving ART had undetectable plasma HIV RNA. Overall, 117 (92%) participants seroconverted to the vaccine (anti-HBs10IU/L), with 109 (86%) participants having high-level response (anti-HBs100IU/L). In multivariate analysis, only baseline CD4>200 cells/mm3 was associated with response [OR=6.97 (1.34-34.71), p=0.02] and high-level response [OR=4.25 (1.15-15.69)], p=0.03]. CONCLUSION: HBV vaccination was effective in eliciting a protective humoral response, particularly among those with higher CD4 counts. Half of the screened patients did not have immunity to HBV infection, suggesting a large at-risk population for HBV infection among HIV-positive adults in Uganda. Our findings support including HBV vaccination as part of routine care among HIV-positive adults.

Beyond the storm - subacute toxicities and late effects in children receiving CAR T cells

Nat Rev Clin Oncol

2021 Colleen Annesley; Rebecca Gardner; Joshua Hill

As clinical advances with chimeric antigen receptor (CAR) T cells are increasingly described and the potential for extending their therapeutic benefit grows, optimizing the implementation of this therapeutic modality is imperative. The recognition and management of cytokine release syndrome (CRS) marked a milestone in this field; however, beyond the understanding gained in treating CRS, a host of additional toxicities and/or potential late effects of CAR T cell therapy warrant further investigation. A multicentre initiative involving experts in paediatric cell therapy, supportive care and/or study of late effects from cancer and haematopoietic stem cell transplantation was convened to facilitate the comprehensive study of extended CAR T cell-mediated toxicities and establish a framework for new systematic investigations of CAR T cell-related adverse events. Together, this group identified six key focus areas: extended monitoring of neurotoxicity and neurocognitive function, psychosocial considerations, infection and immune reconstitution, other end organ toxicities, evaluation of subsequent neoplasms, and strategies to optimize remission durability. Herein, we present the current understanding, gaps in knowledge and future directions of research addressing these CAR T cell-related outcomes. This systematic framework to study extended toxicities and optimization strategies will facilitate the translation of acquired experience and knowledge for optimal application of CAR T cell therapies.