LETHAL COVID-19 ASSOCIATES WITH RAAS-INDUCED INFLAMMATION FOR MULTIPLE ORGAN DAMAGE INCLUDING MEDIASTINAL LYMPH NODES (preprint)

Lethal COVID-19 causation most often invokes classic cytokine storm and attendant excessive immune signaling. We re-visit this question using RNA sequencing in nasopharyngeal and 40 autopsy samples from both COVID-19-positive and negative individuals. In nasal swabs, the top 100 genes expressed, and significantly correlated with COVID-19 viral load, indeed include many canonical innate immune genes. However, 22 much less studied \"non-canonical\" genes are found and despite the absence of viral transcripts, subsets of these are upregulated in heart, lung, kidney, and liver, but not mediastinal lymph nodes. An important regulatory potential emerges for the non-canonical genes for over-activating the renin-angiotensin-activation-system (RAAS) pathway, resembling this phenomenon in hereditary angioedema (HAE) and its overlapping multiple features with lethal COVID-19 infections. Specifically, RAAS overactivation links increased fibrin deposition, leaky vessels, thrombotic tendency, and initiating the PANoptosis death pathway, as suggested in heart, lung, and especially mediastinal lymph nodes, and a tight association mitochondrial dysfunction linked to immune responses. For mediastinal lymph nodes, immunohistochemistry studies correlate showing abnormal architecture, excess fibrin and collagen deposition, and pathogenic fibroblasts. Further, our findings overlap these for COVID-19 infected hamsters, C57BL/6 and BALB/c mouse models, and importantly peripheral blood mononuclear cell (PBMC) and whole blood samples from COVID-19 patients infected with early alpha but also later COVID-19 omicron strains. We thus present cytokine storm in lethal COVID-19 disease as an interplay between upstream immune gene signaling producing downstream RAAS overactivation with resultant severe organ damage, especially compromising mediastinal lymph node function.

Post-vaccination Omicron infections induce broader immunity across antigenic space than prototype mRNA COVID-19 booster vaccination or primary infection (preprint)

The rapid emergence of new SARS-CoV-2 variants challenges vaccination strategies. Here, we measured antigenic diversity among variants and interpreted neutralizing antibody responses following single and multiple exposures in longitudinal infection and vaccine cohorts. Antigenic cartography using primary infection antisera showed that BA.2, BA.4/BA.5, and BA.2.12.1 are distinct from BA.1 and closer to the Beta cluster. Three doses of an mRNA COVID-19 vaccine increased breadth to BA.1 more than to BA.4/BA.5 or BA.2.12.1. Omicron BA.1 post-vaccination infection elicited antibody landscapes characterized by broader immunity across antigenic space than three doses alone, although with less breadth than expected to BA.2.12.1 and BA.4/BA.5. Those with Omicron BA.1 infection after two or three vaccinations had similar neutralizing titer magnitude and antigenic breadth. Accounting for antigenic differences among variants of concern when interpreting neutralizing antibody titers aids understanding of complex patterns in humoral immunity and informs selection of future COVID-19 vaccine strains.

Topological data analysis identifies distinct biomarker phenotypes during the inflammatory phase of COVID-19 (preprint)

OBJECTIVES: The relationships between baseline clinical phenotypes and the cytokine milieu of the peak inflammatory phase of coronavirus 2019 (COVID-19) are not yet well understood. We used Topological Data Analysis (TDA), a dimensionality reduction technique to identify patterns of inflammation associated with COVID-19 severity and clinical characteristics. DESIGN: Exploratory analysis from a multi-center prospective cohort study. SETTING: Eight military hospitals across the United States between April 2020 and January 2021. PATIENTS: Adult ([≥]18 years of age) SARS-CoV-2 positive inpatient and outpatient participants were enrolled with plasma samples selected from the putative inflammatory phase of COVID-19, defined as 15-28 days post symptom onset. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Concentrations of 12 inflammatory protein biomarkers were measured using a broad dynamic range immunoassay. TDA identified 3 distinct inflammatory protein expression clusters. Peak severity (outpatient, hospitalized, ICU admission or death), Charlson Comorbidity Index (CCI), and body mass index (BMI) were evaluated with logistic regression for associations with each cluster. The study population (n=129, 33.3% female, median 41.3 years of age) included 77 outpatient, 31 inpatient, 16 ICU-level, and 5 fatal cases. Three distinct clusters were found that differed by peak disease severity (p <0.001), age (p <0.001), BMI (p<0.001), and CCI (p=0.001). CONCLUSIONS: Exploratory clustering methods can stratify heterogeneous patient populations and identify distinct inflammation patterns associated with comorbid disease, obesity, and severe illness due to COVID-19.

SARS-CoV-2 Omicron neutralization by therapeutic antibodies, convalescent sera, and post-mRNA vaccine booster (preprint)

The rapid spread of the highly contagious Omicron variant of SARS-CoV-2 along with its high number of mutations in the spike gene has raised alarm about the effectiveness of current medical countermeasures. To address this concern, we measured neutralizing antibodies against Omicron in three important settings: (1) post-vaccination sera after two and three immunizations with the Pfizer/BNT162b2 vaccine, (2) convalescent sera from unvaccinated individuals infected by different variants, and (3) clinical-stage therapeutic antibodies. Using a pseudovirus neutralization assay, we found that titers against Omicron were low or undetectable after two immunizations and in most convalescent sera. A booster vaccination significantly increased titers against Omicron to levels comparable to those seen against the ancestral (D614G) variant after two immunizations. Neither age nor sex were associated with differences in post-vaccination antibody responses. Only three of 24 therapeutic antibodies tested retained their full potency against Omicron and high-level resistance was seen against fifteen. These findings underscore the potential benefit of booster mRNA vaccines for protection against Omicron and the need for additional therapeutic antibodies that are more robust to highly mutated variants. One Sentence Summary Third dose of Pfizer/BioNTech COVID-19 vaccine significantly boosts neutralizing antibodies to the Omicron variant compared to a second dose, while neutralization of Omicron by convalescent sera, two-dose vaccine-elicited sera, or therapeutic antibodies is variable and often low.

Durability of SARS-CoV-2-specific T cell responses at 12-months post-infection (preprint)

BackgroundCharacterizing the longevity and quality of cellular immune responses to SARS-CoV-2 is critical to understanding immunologic approaches to protection against COVID-19. Prior studies suggest SARS-CoV-2-specific T cells are present in peripheral blood 10 months after infection. Further analysis of the function, durability, and diversity of the cellular response long after natural infection, over a wider range of ages and disease phenotypes, is needed to further identify preventative and therapeutic interventions. MethodsWe identified participants in our multi-site longitudinal, prospective cohort study 12-months post SARS-CoV-2 infection representing a range of disease severity. We investigated the function, phenotypes, and frequency of T cells specific for SARS-CoV-2 using intracellular cytokine staining and spectral flow cytometry. In parallel, the magnitude of SARS-CoV-2-specific antibodies was compared. ResultsSARS-CoV-2-specific antibodies and T cells were detected at 12-months post-infection. Severity of acute illness was associated with higher frequencies of SARS-CoV-2-specific CD4 T cells and antibodies at 12-months. In contrast, polyfunctional and cytotoxic T cells responsive to SARS-CoV-2 were identified in participants over a wide spectrum of disease severity. ConclusionsOur data show that SARS-CoV-2 infection induces polyfunctional memory T cells detectable at 12-months post-infection, with higher frequency noted in those who originally experienced severe disease.

A betacoronavirus multiplex microsphere immunoassay detects early SARS-CoV-2 seroconversion and antibody cross reactions (preprint)

Sensitive and specific SARS-CoV-2 antibody assays remain critical for community and hospital-based SARS-CoV-2 surveillance. Here, we developed and applied a multiplex microsphere-based immunoassay (MMIA) for COVD-19 antibody studies that incorporates spike protein trimers of SARS-CoV-2, SARS-CoV-1, MERS-CoV, and the seasonal human betacoronaviruses, HCoV-HKU1 and HCoV-OC43, that enables measurement of off-target pre-existing cross-reactive antibodies. The MMIA performances characteristics are: 98% sensitive and 100% specific for human subject samples collected as early as 10 days from symptom onset. The MMIA permitted the simultaneous identification of SARS-CoV-2 seroconversion and the induction of SARS-CoV-2 IgG antibody cross reactions to SARS-CoV-1 and MERS-CoV. Further, synchronous increases of HCoV-OC43 IgG antibody levels was detected with SARS-CoV-2 seroconversion in a subset of subjects for whom early infection sera were available prior to their SARS-CoV-2 seroconversion, suggestive of an HCoV-OC43 memory response triggered by SARS-CoV-2 infection.

A betacoronavirus multiplex microsphere immunoassay detects early SARS-CoV-2 seroconversion and controls for pre-existing seasonal human coronavirus antibody cross-reactivity (preprint)

With growing concern of persistent or multiple waves of SARS-CoV-2 in the United States, sensitive and specific SARS-CoV-2 antibody assays remain critical for community and hospital-based SARS-CoV-2 surveillance. Here, we describe the development and application of a multiplex microsphere-based immunoassay (MMIA) for COVD-19 antibody studies, utilizing serum samples from non-human primate SARS-CoV-2 infection models, an archived human sera bank and subjects enrolled at five U.S. military hospitals. The MMIA incorporates prefusion stabilized spike glycoprotein trimers of SARS-CoV-2, SARS-CoV-1, MERS-CoV, and the seasonal human coronaviruses HCoV-HKU1 and HCoV-OC43, into a multiplexing system that enables simultaneous measurement of off-target pre-existing cross-reactive antibodies. We report the sensitivity and specificity performances for this assay strategy at 98% sensitivity and 100% specificity for subject samples collected as early as 10 days after the onset of symptoms. In archival sera collected prior to 2019 and serum samples from subjects PCR negative for SARS-CoV-2, we detected seroprevalence of 72% and 98% for HCoV-HKU1 and HCoV-0C43, respectively. Requiring only 1.25 uL of sera, this approach permitted the simultaneous identification of SARS-CoV-2 seroconversion and polyclonal SARS-CoV-2 IgG antibody responses to SARS-CoV-1 and MERS-CoV, further demonstrating the presence of conserved epitopes in the spike glycoprotein of zoonotic betacoronaviruses. Application of this serology assay in observational studies with serum samples collected from subjects before and after SARS-CoV-2 infection will permit an investigation of the influences of HCoV-induced antibodies on COVID-19 clinical outcomes.