Evaluation of Pro-Inflammatory Biomarkers as Potential Early Indicators of Acute Respiratory Distress Syndrome (ARDS)

Infections with the 2009 pandemic influenza A(HlNl) virus often leads to acute respiratory distress syndrome (ARDS) in high-risk patients. In these patients, ARDS has a mortality rate of 40-50 % [1]. Patients may die before any intervention is possible since there are few if any early biomarkers that are indicative of ARDS and can be used for diagnosis. In a search for biomarkers that signal early disease progression in mice, multiple acute phase proteins involved with inflammatory responses to infectious stimuli were proposed. Three biomarkers were evaluated for this project. These included C-reactive protein (CRP), Serum Amyloid A (SAA) and Transferrin. To validate these biomarkers as predictors of ARDS, mice were infected with the influenza A Califomia/04/2009 (H 1 N 1 )pdm09 virus and serum was collected from infected mice at different time points (0 - 72 hours post infection). Serum samples were tested for amounts of CRP, SAA and Transferrin using an enzyme-linked immunosorbent assay (ELISA). Control mice were mock-infected and tested alongside for comparison. Our results indicate that of the three biomarkers tested, SAA shows strong potential as an early indicator of ARDS with a peak at 72 hours post infection in infected mice serum. Further investigation is in progress to observe the levels of SAA past day 3 (72 hours post infection)

Optimization of non-coding regions for a non-modified mRNA COVID-19 vaccine

The CVnCoV (CureVac) mRNA vaccine for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was recently evaluated in a phase 2b/3 efficacy trial in human

Deletion of the SARS-CoV-2 Spike Cytoplasmic Tail Increases Infectivity in Pseudovirus Neutralization Assays

Pseudotyped viruses are valuable tools for studying virulent or lethal viral pathogens that need to be handled in biosafety level 3 (BSL-3) or higher facilities. With the explosive spread of the coronavirus disease 2019 (COVID-19) pandemic, the establishment of a BSL-2 adapted SARS-CoV-2 pseudovirus neutralization assay is needed to facilitate the development of countermeasures. Here we describe an approach to generate a single-round lentiviral vector-based SARS-CoV-2 pseudovirus, which produced a signal more than 2 logs above background. Specifically, a SARS-CoV-2 spike variant with a cytoplasmic tail deletion of 13 amino acids, termed SΔCT13, conferred enhanced spike incorporation into pseudovirions and increased viral entry into cells as compared with full-length spike (S). We further compared S and SΔCT13 in terms of their sensitivity to vaccine sera, purified convalescent IgG, hACE2-mIgG, and the virus entry inhibitor BafA1. We developed a SΔCT13-based pseudovirus neutralization assay and defined key assay characteristics, including linearity, limit of detection, and intra- and intermediate-assay precision. Our data demonstrate that the SΔCT13-based pseudovirus shows enhanced infectivity in target cells, which will facilitate the assessment of humoral immunity to SARS-CoV-2 infection, antibody therapeutics, and vaccination. This pseudovirus neutralization assay can also be readily adapted to SARS-CoV-2 variants that emerge. IMPORTANCE SARS-CoV-2 is the etiologic agent of the COVID-19 pandemic. The development of a high throughput pseudovirus neutralization assay is critical for the development of vaccines and immune-based therapeutics. In this study, we show that deletion of the cytoplasmic tail of the SARS-CoV-2 spike leads to pseudoviruses with enhanced infectivity. This SΔCT13-based pseudovirus neutralization assay should be broadly useful for the field

Epidemiological and Immunological Features of Obesity and SARS-CoV-2

Obesity is a key correlate of severe SARS-CoV-2 outcomes while the role of obesity on risk of SARS-CoV-2 infection, symptom phenotype, and immune response remain poorly defined. We examined data from a prospective SARS-CoV-2 cohort study to address these questions. Serostatus, body mass index, demographics, comorbidities, and prior COVID-19 compatible symptoms were assessed at baseline and serostatus and symptoms monthly thereafter. SARS-CoV-2 immunoassays included an IgG ELISA targeting the spike RBD, multiarray Luminex targeting 20 viral antigens, pseudovirus neutralization, and T cell ELISPOT assays. Our results from a large prospective SARS-CoV-2 cohort study indicate symptom phenotype is strongly influenced by obesity among younger but not older age groups; we did not identify evidence to suggest obese individuals are at higher risk of SARS-CoV-2 infection; and remarkably homogenous immune activity across BMI categories suggests immune protection across these groups may be similar

Comparison of Chikungunya Virus Strains in Disease Severity and Susceptibility to T-705 (Favipiravir), In vitro and In vivo

Chikungunya is a mosquito-transmitted disease caused by Chikungunya virus (CHIKV). Symptoms of Chikungunya include debilitating joint pain and swelling, fever and rash. CHIKV was first discovered in 1953 in Tanzania, and has since caused periodic outbreaks of disease. The virus reemerged recently in 2004 and has since spread around the world affecting more than 3 million people. The different strains of CHIKV have been grouped into three phylogenetic clades: West African, Asian and East/Central/South African (ECSA). There are no FDA approved medicines or vaccines used to treat or prevent CHIKV infection. The antiviral drug, T-705 (commercially known as Favipiravir), has recently been shown to have activity against CHIKV. T-705 has already been approved in Japan for the treatment of influenza and is currently going through clinical trials in the US. Since there may be phenotypic differences between the clades of CHIKV, it is important to first characterize distinctions between the strains and determine the susceptibility of these strains to treatment. To do this, we obtained two different CHIKV strains from each of the three phylogenetic groups. These CHIKV strains displayed differences in their ability to replicate in cell culture and exhibited only slight differences in susceptibility to T-705 treatment. However, more profound differences were observed in mouse models where differences in disease severity and response to T-705 treatment were observed

CRISPR–Cas9 Genetic Analysis of Virus–Host Interactions

Clustered regularly interspaced short palindromic repeats (CRISPR) has greatly expanded the ability to genetically probe virus–host interactions. CRISPR systems enable focused or systematic, genomewide studies of nearly all aspects of a virus lifecycle. Combined with its relative ease of use and high reproducibility, CRISPR is becoming an essential tool in studies of the host factors important for viral pathogenesis. Here, we review the use of CRISPR–Cas9 for the loss-of-function analysis of host dependency factors. We focus on the use of CRISPR-pooled screens for the systematic identification of host dependency factors, particularly in Epstein–Barr virus-transformed B cells. We also discuss the use of CRISPR interference (CRISPRi) and gain-of-function CRISPR activation (CRISPRa) approaches to probe virus–host interactions. Finally, we comment on the future directions enabled by combinatorial CRISPR screens

Alpha-defensin 5 differentially modulates adenovirus vaccine vectors from different serotypes in vivo.

Adenoviral vectors have shown significant promise as vaccine delivery vectors due to their ability to elicit both innate and adaptive immune responses. α-defensins are effector molecules of the innate immune response and have been shown to modulate natural infection with adenoviruses, but the majority of α-defensin-adenovirus interactions studied to date have only been analyzed in vitro. In this study, we evaluated the role of α-defensin 5 (HD5) in modulating adenovirus vaccine immunogenicity using various serotype adenovirus vectors in mice. We screened a panel of human adenoviruses including Ad5 (species C), Ad26 (species D), Ad35 (species B), Ad48 (species D) and a chimeric Ad5HVR48 for HD5 sensitivity. HD5 inhibited transgene expression from Ad5 and Ad35 but augmented transgene expression from Ad26, Ad48, and Ad5HVR48. HD5 similarly suppressed antigen-specific IgG and CD8+ T cell responses elicited by Ad5 vectors in mice, but augmented IgG and CD8+ T cell responses and innate cytokine responses elicited by Ad26 vectors in mice. Moreover, HD5 suppressed the protective efficacy of Ad5 vectors but enhanced the protective efficacy of Ad26 vectors expressing SIINFEKL against a surrogate Listeria-OVA challenge in mice. These data demonstrate that HD5 differentially modulates adenovirus vaccine delivery vectors in a species-specific manner in vivo