COHORT PROFILE: IMMUNE RESPONSES TO SARS-COV-2 VACCINATION AND INFECTION IN A LONGITUDINAL SAMPLING AMIDST THE COVID-19 PANDEMIC (LONGTONG-SARS2) IN MALAYSIA (preprint)

Purpose: This prospective, longitudinal study aims to evaluate the durability and functionality of SARS-CoV-2 Ancestral strain (Wuhan-Hu-1)-specific immune responses induced by COVID-19 vaccination and natural infection over a 12-month period. This article reviews the study protocol, design, methodology, ongoing data collection, analysis procedures, and demographic characteristics of the cohort enrolled. Participants: Between March 2021 and May 2022, 400 participants were enrolled with a 12-month follow-up, concluding in May 2023. Two main groups of participants: (1) serologically SARS-CoV-2-naive individuals receiving the BNT162b2 primary series vaccination (referred to as VAC) and (2) those who recently recovered from COVID-19 infection within 30 days, regardless of vaccination history (referred to as COV). Additionally, a subset of 45 participants with selected COVID-19 exposure histories provided peripheral blood mononuclear cells (PBMCs) for cross-sectional analysis six months after enrollment. Findings to date: Out of 400 participants, 66.8% (n=267) completed the follow-up. Among them, 52.8% (n=141) were in VAC, and 47.2% (n=126) were in COV. As the study progressed, we acknowledged cross-over between initial groups, leading to restructuring into five revised groups based on sequential exposure events. Sociodemographic factors revealed statistically significant age distribution differences (p=0.001) in both initial and revised groups, with no significant differences observed for sex. Future plans: LONGTONG-SARS2 assesses the host-pathogen interactions central to the development of COVID-19 immunity. With enrollment spanning two years of the pandemic, most participants exhibited mixed SARS-CoV-2 exposures-via vaccination and infection-resulting in diverse subgroups of interest. Notably, the inclusion of SARS-CoV-2-naive, pre-exposure serum samples allowed for robust comparator and reduced potential biases. Ongoing analyses will include serology kinetics, memory cells ELISpots, B cells repertoire analysis, cytokine/chemokine profiling, and proteomic pathway to comprehensively examine the immune response against the SARS-CoV-2, thus informing and potentially predicting dynamic longitudinal responses against new more transmissible, immune-evasive SARS-CoV-2 variants.

Mapping disease regulatory circuits at cell-type resolution from single-cell multiomics data (preprint)

Resolving chromatin remodeling-linked gene expression changes at cell type resolution is important for understanding disease states. We describe MAGICAL, a hierarchical Bayesian approach that leverages paired scRNA-seq and scATAC-seq data from different conditions to map disease-associated transcription factors, chromatin sites, and genes as regulatory circuits. By simultaneously modeling signal variation across cells and conditions in both omics data types, MAGICAL achieved high accuracy on circuit inference. We applied MAGICAL to study Staphylococcus aureus sepsis from peripheral blood mononuclear single-cell data that we generated from infected subjects with bloodstream infection and from uninfected controls. MAGICAL identified sepsis-associated regulatory circuits predominantly in CD14 monocytes, known to be activated by bacterial sepsis. We addressed the challenging problem of distinguishing host regulatory circuit responses to methicillin-resistant- (MRSA) and methicillin-susceptible Staphylococcus aureus (MSSA) infections. While differential expression analysis failed to show predictive value, MAGICAL identified epigenetic circuit biomarkers that distinguished MRSA from MSSA.

Multi-omics Characterization of Neutrophil Extracellular Trap Formation in Severe and Mild COVID-19 Infections (preprint)

The detailed mechanisms of COVID-19 infection pathology remain poorly understood. To improve our understanding of SARS-CoV-2 pathology, we performed a multi-omics analysis of an immunologically naive SARS-CoV-2 clinical cohort from the plasma of uninfected controls, mild, and severe infections. A comparison of healthy controls and patient samples showed activation of neutrophil degranulation pathways and formation of neutrophil extracellular trap (NET) complexes that were activated in a subset of the mild infections and more prevalent in severe infections (containing multiple NET proteins in individual patient samples). As a potential mechanism to suppress NET formation, multiple redox enzymes were elevated in the mild and severe symptom population. Analysis of metabolites from the same cohort showed a 24- and 60-fold elevation in plasma L-cystine, the oxidized form of cysteine, which is a substrate of the powerful antioxidant glutathione, in mild and severe patients, respectively. Unique to patients with mild infections, the carnosine dipeptidase modifying enzyme (CNDP1) was up-regulated. The strong protein and metabolite oxidation signatures suggest multiple compensatory pathways working to suppress oxidation and NET formation in SARS-CoV-2 infections.

Adverse effects and antibody titers in response to the BNT162b2 mRNA COVID-19 vaccine in a prospective study of healthcare workers (preprint)

Background: mRNA COVID-19 vaccines are playing a key role in controlling the COVID-19 pandemic. The relationship between post-vaccination symptoms and strength of antibody responses is unclear. Objective: To determine whether adverse effects caused by vaccination with the Pfizer/BioNTech BNT162b2 vaccine are associated with the magnitude of vaccine-induced antibody levels. Design: Single center, prospective, observational cohort study. Setting: Participants worked at Walter Reed National Military Medical Center and were seen monthly at the Naval Medical Research Center Clinical Trials Center. Participants: Generally healthy adults that were not severely immunocompromised, had no history of COVID-19, and were seronegative for SARS-CoV-2 spike protein prior to vaccination. Measures: Severity of vaccine-associated symptoms was obtained through participant completed questionnaires. Testing for IgG antibodies against SARS-CoV-2 spike protein and receptor binding domain was conducted using microsphere-based multiplex immunoassays. Results: 206 participants were evaluated (69.4% female, median age 41.5 years old). We found no correlation between vaccine-associated symptom severity scores and vaccine-induced antibody titers one month after vaccination. We also observed that 1) post-vaccination symptoms were inversely correlated with age and weight and more common in women, 2) systemic symptoms were more frequent after the second vaccination, 3) high symptom scores after first vaccination were predictive of high symptom scores after second vaccination, and 4) older age was associated with lower titers. Limitations: Study only observes antibody responses and consists of healthy participants. Conclusions: Lack of post-vaccination symptoms following receipt of the BNT162b2 vaccine does not equate to lack of vaccine-induced antibodies one month after vaccination. This study also suggests that it may be possible to design future mRNA vaccines that confer robust antibody responses with lower frequencies of vaccine-associated symptoms.

SARS-CoV-2 seropositivity and subsequent infection risk in healthy young adults: a prospective cohort study (preprint)

Background: The risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) subsequent infection among seropositive young adults was studied prospectively. Methods: The study population comprised 3,249 predominantly male, 18-20-year-old Marine recruits. Upon arrival at a Marine-supervised two-week quarantine, participants were assessed for baseline SARS-CoV-2 IgG seropositivity, defined as a 1:150 dilution or greater on receptor binding domain and full-length spike protein enzyme-linked immunosorbent (ELISA) assays. SARS-CoV-2 infection was assessed by PCR at initiation, middle and end of the quarantine. After appropriate exclusions, including participants with a positive PCR during quarantine, we performed three biweekly PCR tests in both seropositive and in seronegative groups once recruits left quarantine and entered basic training and baseline neutralizing antibody titers on all subsequently infected seropositive and selected seropositive uninfected participants. Findings: Among 189 seropositive participants, 19 (10.1%) had at least one positive PCR test for SARS-CoV-2 during the six-week follow-up (1.1 cases per person-year). In contrast, 1,079 (48.0%) of the 2,247 seronegative participants tested positive (6.2 cases per person-year). The incidence rate ratio was 0.18 (95% CI 0.11-0.28, p<0.00001). Among seropositive recruits, infection was associated with lower baseline full-length spike protein IgG titers (p<0.0001). Compared with seronegative recruits, seropositive recruits had about 10-fold lower viral loads (ORF1ab gene, p<0.005), and trended towards shorter duration of PCR positivity (p=0.18) and more frequent asymptomatic infections (p=0.13). Among seropositive participants, baseline neutralizing titers were detected in 45 of 54 (83.3%) uninfected and in 6 of 19 (31.6%) infected participants during the 6 weeks of observation (ID50 difference p