Cross-Protection Induced by Highly Conserved Human B, CD4+, and CD8+ T Cell Epitopes-Based Coronavirus Vaccine Against Severe Infection, Disease, and Death Caused by Multiple SARS-CoV-2 Variants of Concern (preprint)

Background: The Coronavirus disease 2019 (COVID-19) pandemic has created one of the largest global health crises in almost a century. Although the current rate of SARS-CoV-2 infections has decreased significantly; the long-term outlook of COVID-19 remains a serious cause of high death worldwide; with the mortality rate still surpassing even the worst mortality rates recorded for the influenza viruses. The continuous emergence of SARS-CoV-2 variants of concern (VOCs), including multiple heavily mutated Omicron sub-variants, have prolonged the COVID-19 pandemic and outlines the urgent need for a next-generation vaccine that will protect from multiple SARS-CoV-2 VOCs. Methods: In the present study, we designed a multi-epitope-based Coronavirus vaccine that incorporated B, CD4+, and CD8+ T cell epitopes conserved among all known SARS-CoV-2 VOCs and selectively recognized by CD8+ and CD4+ T-cells from asymptomatic COVID-19 patients irrespective of VOC infection. The safety, immunogenicity, and cross-protective immunity of this pan-Coronavirus vaccine were studied against six VOCs using an innovative triple transgenic h-ACE-2-HLA-A2/DR mouse model. Results: The Pan-Coronavirus vaccine: (i) is safe; (ii) induces high frequencies of lung-resident functional CD8+ and CD4+ TEM and TRM cells; and (iii) provides robust protection against virus replication and COVID-19-related lung pathology and death caused by six SARS-CoV-2 VOCs: Alpha (B.1.1.7), Beta (B.1.351), Gamma or P1 (B.1.1.28.1), Delta (lineage B.1.617.2) and Omicron (B.1.1.529). Conclusions: A multi-epitope pan-Coronavirus vaccine bearing conserved human B and T cell epitopes from structural and non-structural SARS-CoV-2 antigens induced cross-protective immunity that cleared the virus, and reduced COVID-19-related lung pathology and death caused by multiple SARS-CoV-2 VOCs.

High Frequencies of PD-1+TIM3+TIGIT+CTLA4+ Functionally Exhausted SARS-CoV-2-Specific CD4+ and CD8+ T Cells Associated with Severe Disease in Critically ill COVID-19 Patients (preprint)

SARS-CoV-2-specific memory T cells that cross-react with common cold coronaviruses (CCCs) are present in both healthy donors and COVID-19 patients. However, whether these cross-reactive T cells play a role in COVID-19 pathogenesis versus protection remain to be fully elucidated. In this study, we characterized cross-reactive SARS-CoV-2-specific CD4+ and CD8+ T cells, targeting genome-wide conserved epitopes in a cohort of 147 non-vaccinated COVID-19 patients, divided into six groups based on the degrees of disease severity. We compared the frequency, phenotype, and function of these SARS-CoV-2-specific CD4+ and CD8+ T cells between severely ill and asymptomatic COVID-19 patients and correlated this with alpha-CCCs and beta-CCCs co-infection status. Compared with asymptomatic COVID-19 patients, the severely ill COVID-19 patients and patients with fatal outcomes: (i) Presented a broad leukocytosis and a broad CD4+ and CD8+ T cell lymphopenia; (ii) Developed low frequencies of functional IFN-gamma-producing CD134+CD138+CD4+ and CD134+CD138+CD8+ T cells directed toward conserved epitopes from structural, non-structural and regulatory SARS-CoV-2 proteins; (iii) Displayed high frequencies of SARS-CoV-2-specific functionally exhausted PD-1+TIM3+TIGIT+CTLA4+CD4+ and PD-1+TIM3+TIGIT+CTLA4+CD8+ T cells; and (iv) Displayed similar frequencies of co-infections with beta-CCCs strains but significantly fewer co-infections with alpha-CCCs strains. Interestingly, the cross-reactive SARS-CoV-2 epitopes that recalled the strongest CD4+ and CD8+ T cell responses in unexposed healthy donors (HD) were the most strongly associated with better disease outcome seen in asymptomatic COVID-19 patients. Our results demonstrate that, the critically ill COVID-19 patients displayed fewer co-infection with alpha-CCCs strain, presented broad T cell lymphopenia and higher frequencies of cross reactive exhausted SARS-CoV-2-specific CD4+ and CD8+ T cells. In contrast, the asymptomatic COVID-19 patients, appeared to present more co-infections with alpha-CCCs strains, associated with higher frequencies of functional cross-reactive SARS-CoV-2-specific CD4+ and CD8+ T cells. These findings support the development of broadly protective, T-cell-based, multi-antigen universal pan-Coronavirus vaccines.

A Tethered Ligand Assay to Probe the SARS-CoV-2 ACE2 Interaction under Constant Force (preprint)

The current COVID-19 pandemic has a devastating global impact and is caused by the SARS-CoV-2 virus. SARS-CoV-2 attaches to human host cells through interaction of its receptor binding domain (RBD) located on the viral Spike (S) glycoprotein with angiotensin converting enzyme-2 (ACE2) on the surface of host cells. RBD binding to ACE2 is a critical first step in SARS-CoV-2 infection. Viral attachment occurs in dynamic environments where forces act on the binding partners and multivalent interactions play central roles, creating an urgent need for assays that can quantitate SARS-CoV-2 interactions with ACE2 under mechanical load and in defined geometries. Here, we introduce a tethered ligand assay that comprises the RBD and the ACE2 ectodomain joined by a flexible peptide linker. Using specific molecular handles, we tether the fusion proteins between a functionalized flow cell surface and magnetic beads in magnetic tweezers. We observe repeated interactions of RBD and ACE2 under constant loads and can fully quantify the force dependence and kinetics of the binding interaction. Our results suggest that the SARS-CoV-2 ACE2 interaction has higher mechanical stability, a larger free energy of binding, and a lower off-rate than that of SARS-CoV-1, the causative agents of the 2002-2004 SARS outbreak. In the absence of force, the SARS-CoV-2 RBD rapidly (within [≤]1 ms) engages the ACE2 receptor if held in close proximity and remains bound to ACE2 for 400-800 s, much longer than what has been reported for other viruses engaging their cellular receptors. We anticipate that our assay will be a powerful tool investigate the roles of mutations in the RBD that might alter the infectivity of the virus and to test the modes of action of neutralizing antibodies and other agents designed to block RBD binding to ACE2 that are currently developed as potential COVID-19 therapeutics.

No evidence that plasmablasts transdifferentiate into developing neutrophils in severe COVID-19 disease (preprint)

A recent study by Wilk et al. of the transcriptome of peripheral blood mononuclear cells (PBMCs) in seven patients hospitalized with COVID-19 described a population of 'developing neutrophils' that were 'phenotypically related by dimensionality reduction' to plasmablasts, and that these two cell populations represent a 'linear continuum of cellular phenotype'. The authors suggest that, in the setting of acute respiratory distress syndrome (ARDS) secondary to severe COVID-19, a 'differentiation bridge from plasmablasts to developing neutrophils' connected these distantly related cell types. This conclusion is controversial as it appears to violate several basic principles in cell biology relating to cell lineage identity and fidelity. Correctly classifying cells and their developmental history is an important issue in cell biology and we suggest that this conclusion is not supported by the data as we show here that: (1) regressing out covariates such as unique molecular identifiers (UMIs) can lead to overfitting; and (2) that UMAP embeddings may reflect the expression of similar genes but not necessarily direct cell lineage relationships.

Genome-Wide Asymptomatic B-Cell, CD4+ and CD8+ T-Cell Epitopes, that are Highly Conserved Between Human and Animal Coronaviruses, Identified from SARS-CoV-2 as Immune Targets for Pre-Emptive Pan-Coronavirus Vaccines (preprint)

Over the last two decades, there have been three deadly human outbreaks of Coronaviruses (CoVs) caused by emerging zoonotic CoVs: SARS-CoV, MERS-CoV, and the latest highly transmissible and deadly SARS-CoV-2, which has caused the current COVID-19 global pandemic. All three deadly CoVs originated from bats, the natural hosts, and transmitted to humans via various intermediate animal reservoirs. Because there is currently no universal pan-Coronavirus vaccine available, two worst-case scenarios remain highly possible: (1) SARS-CoV-2 mutates and transforms into a seasonal "flu-like" global pandemic; and/or (2) Other global COVID-like pandemics will emerge in the coming years, caused by yet another spillover of an unknown zoonotic bat-derived SARS-like Coronavirus (SL-CoV) into an unvaccinated human population. Determining the antigen and epitope landscapes that are conserved among human and animal Coronaviruses as well as the repertoire, phenotype and function of B cells and CD4+ and CD8+ T cells that correlate with resistance seen in asymptomatic COVID-19 patients should inform in the development of pan-Coronavirus vaccines 1. In the present study, using several immuno-informatics and sequence alignment approaches, we identified several human B-cell, CD4+ and CD8+ T cell epitopes that are highly conserved in: (i) greater than 81,000 SARS-CoV-2 human strains identified to date in 190 countries on six continents; (ii) six circulating CoVs that caused previous human outbreaks of the "Common Cold"; (iii) five SL-CoVs isolated from bats; (iv) five SL-CoV isolated from pangolins; (v) three SL-CoVs isolated from Civet Cats; and (vi) four MERS strains isolated from camels. Furthermore, we identified cross-reactive asymptomatic epitopes that: (i) recalled B cell, CD4+ and CD8+ T cell responses from both asymptomatic COVID-19 patients and healthy individuals who were never exposed to SARS-CoV-2; and (ii) induced strong B cell and T cell responses in "humanized" Human Leukocyte Antigen (HLA)-DR/HLA-A*02:01 double transgenic mice. The findings herein pave the way to develop a pre-emptive multi-epitope pan-Coronavirus vaccine to protect against past, current, and potential future outbreaks.

Identification of TMEM106B as proviral host factor for SARS-CoV-2 (preprint)

The ongoing COVID-19 pandemic is responsible for worldwide economic damage and nearly one million deaths. Potent drugs for the treatment of severe SARS-CoV-2 infections are not yet available. To identify host factors that support coronavirus infection, we performed genome-wide functional genetic screens with SARS-CoV-2 and the common cold virus HCoV-229E in non-transgenic human cells. These screens identified PI3K type 3 as a potential drug target against multiple coronaviruses. We discovered that the lysosomal protein TMEM106B is an important host factor for SARS-CoV-2 infection. Furthermore, we show that TMEM106B is required for replication in multiple human cell lines derived from liver and lung and is expressed in relevant cell types in the human airways. Our results identify new coronavirus host factors that may potentially serve as drug targets against SARS-CoV-2 or to quickly combat future zoonotic coronavirus outbreaks.