Identification of differences in the magnitude and specificity of SARS-CoV-2 nucleocapsid antibody responses in naturally infected and vaccinated individuals.

As there are limited data on B cell epitopes for the nucleocapsid protein in SARS-CoV-2, we sought to identify the immunodominant regions within the N protein, recognized by patients with varying severity of natural infection with the Wuhan strain (WT), delta, omicron and in those who received the Sinopharm vaccines, which is an inactivated, whole virus vaccine.Using overlapping peptides representing the N protein, with an in-house ELISA, we mapped the immunodominant regions within the N protein, in seronegative (n=30), WT infected (n=30), delta infected (n=30), omicron infected+vaccinated (n=20) and Sinopharm (BBIBP-CorV) vaccinees (n=30). We then investigated the sensitivity and specificity of these immunodominant regions and analysed their conservation with other SARS-CoV-2 variants of concern, seasonal human coronaviruses and bat Sarbecoviruses. We identified four immunodominant regions aa 29-52, aa 155-178, aa 274 to 297 and aa 365 to 388, were highly conserved within SARS-CoV-2 and the bat coronaviruses. The magnitude of responses to these regions varied based on the infecting SARS-CoV-2 variants, >80% of individuals gave responses above the positive cut-off threshold to many of the four regions, with some differences with individuals who were infected with different VoCs. These regions were found to be 100% specific, as none of the seronegative individuals gave any responses. As these regions were highly specific with high sensitivity, they have a potential to be used to develop diagnostic assays and to be used in development of vaccines.

A Multitrait Locus Regulates Sarbecovirus Pathogenesis.

Infectious diseases have shaped the human population genetic structure, and genetic variation influences the susceptibility to many viral diseases. However, a variety of challenges have made the implementation of traditional human Genome-wide Association Studies (GWAS) approaches to study these infectious outcomes challenging. In contrast, mouse models of infectious diseases provide an experimental control and precision, which facilitates analyses and mechanistic studies of the role of genetic variation on infection. Here we use a genetic mapping cross between two distinct Collaborative Cross mouse strains with respect to severe acute respiratory syndrome coronavirus (SARS-CoV) disease outcomes. We find several loci control differential disease outcome for a variety of traits in the context of SARS-CoV infection. Importantly, we identify a locus on mouse chromosome 9 that shows conserved synteny with a human GWAS locus for SARS-CoV-2 severe disease. We follow-up and confirm a role for this locus, and identify two candidate genes, CCR9 and CXCR6, that both play a key role in regulating the severity of SARS-CoV, SARS-CoV-2, and a distantly related bat sarbecovirus disease outcomes. As such we provide a template for using experimental mouse crosses to identify and characterize multitrait loci that regulate pathogenic infectious outcomes across species. IMPORTANCE Host genetic variation is an important determinant that predicts disease outcomes following infection. In the setting of highly pathogenic coronavirus infections genetic determinants underlying host susceptibility and mortality remain unclear. To elucidate the role of host genetic variation on sarbecovirus pathogenesis and disease outcomes, we utilized the Collaborative Cross (CC) mouse genetic reference population as a model to identify susceptibility alleles to SARS-CoV and SARS-CoV-2 infections. Our findings reveal that a multitrait loci found in chromosome 9 is an important regulator of sarbecovirus pathogenesis in mice. Within this locus, we identified and validated CCR9 and CXCR6 as important regulators of host disease outcomes. Specifically, both CCR9 and CXCR6 are protective against severe SARS-CoV, SARS-CoV-2, and SARS-related HKU3 virus disease in mice. This chromosome 9 multitrait locus may be important to help identify genes that regulate coronavirus disease outcomes in humans.

Design, immunogenicity, and efficacy of a pan-sarbecovirus dendritic-cell targeting vaccine.

BACKGROUND: There is an urgent need of a new generation of vaccine that are able to enhance protection against SARS-CoV-2 and related variants of concern (VOC) and emerging coronaviruses. METHODS: We identified conserved T- and B-cell epitopes from Spike (S) and Nucleocapsid (N) highly homologous to 38 sarbecoviruses, including SARS-CoV-2 VOCs, to design a protein subunit vaccine targeting antigens to Dendritic Cells (DC) via CD40 surface receptor (CD40.CoV2). FINDINGS: CD40.CoV2 immunization elicited high levels of cross-neutralizing antibodies against SARS-CoV-2, VOCs, and SARS-CoV-1 in K18-hACE2 transgenic mice, associated with viral control and survival after SARS-CoV-2 challenge. A direct comparison of CD40.CoV2 with the mRNA BNT162b2 vaccine showed that the two vaccines were equally immunogenic in mice. We demonstrated the potency of CD40.CoV2 to recall in vitro human multi-epitope, functional, and cytotoxic SARS-CoV-2 S- and N-specific T-cell responses that are unaffected by VOC mutations and cross-reactive with SARS-CoV-1 and, to a lesser extent, MERS epitopes. INTERPRETATION: We report the immunogenicity and antiviral efficacy of the CD40.CoV2 vaccine in a preclinical model providing a framework for a pan-sarbecovirus vaccine. FUNDINGS: This work was supported by INSERM and the Investissements d'Avenir program, Vaccine Research Institute (VRI), managed by the ANR and the CARE project funded from the Innovative Medicines Initiative 2 Joint Undertaking (JU).

Exploring the Natural Origins of SARS-CoV-2 in the Light of Recombination.

The lack of an identifiable intermediate host species for the proximal animal ancestor of SARS-CoV-2, and the large geographical distance between Wuhan and where the closest evolutionary related coronaviruses circulating in horseshoe bats (members of the Sarbecovirus subgenus) have been identified, is fueling speculation on the natural origins of SARS-CoV-2. We performed a comprehensive phylogenetic study on SARS-CoV-2 and all the related bat and pangolin sarbecoviruses sampled so far. Determining the likely recombination events reveals a highly reticulate evolutionary history within this group of coronaviruses. Distribution of the inferred recombination events is nonrandom with evidence that Spike, the main target for humoral immunity, is beside a recombination hotspot likely driving antigenic shift events in the ancestry of bat sarbecoviruses. Coupled with the geographic ranges of their hosts and the sampling locations, across southern China, and into Southeast Asia, we confirm that horseshoe bats, Rhinolophus, are the likely reservoir species for the SARS-CoV-2 progenitor. By tracing the recombinant sequence patterns, we conclude that there has been relatively recent geographic movement and cocirculation of these viruses' ancestors, extending across their bat host ranges in China and Southeast Asia over the last 100 years. We confirm that a direct proximal ancestor to SARS-CoV-2 has not yet been sampled, since the closest known relatives collected in Yunnan shared a common ancestor with SARS-CoV-2 approximately 40 years ago. Our analysis highlights the need for dramatically more wildlife sampling to: 1) pinpoint the exact origins of SARS-CoV-2's animal progenitor, 2) the intermediate species that facilitated transmission from bats to humans (if there is one), and 3) survey the extent of the diversity in the related sarbecoviruses' phylogeny that present high risk for future spillovers.

Conserved molecular signatures in the spike protein provide evidence indicating the origin of SARS-CoV-2 and a Pangolin-CoV (MP789) by recombination(s) between specific lineages of Sarbecoviruses.

Both SARS-CoV-2 and SARS coronaviruses (CoVs) are members of the subgenus Sarbecovirus. To understand the origin of SARS-CoV-2, sequences for the spike and nucleocapsid proteins from sarbecoviruses were analyzed to identify molecular markers consisting of conserved inserts or deletions (termed CSIs) that are specific for either a particular clade of Sarbecovirus or are commonly shared by two or more clades of these viruses. Three novel CSIs in the N-terminal domain (NTD) of the spike protein S1-subunit (S1-NTD) are uniquely shared by SARS-CoV-2, Bat-CoV-RaTG13 and most pangolin CoVs (SARS-CoV-2r clade). Three other sarbecoviruses viz. bat-CoVZXC21, -CoVZC45 and -PrC31 (forming CoVZC/PrC31 clade), and a pangolin-CoV_MP789 also contain related CSIs in the same positions. In contrast to the S1-NTD, both SARS and SARS-CoV-2r viruses contain two large CSIs in the S1-C-terminal domain (S1-CTD) that are absent in the CoVZC/PrC31 clade. One of these CSIs, consisting of a 12 aa insert, is also present in the RShSTT clade (Cambodia-CoV strains). Sequence similarity studies show that the S1-NTD of SARS-CoV-2r viruses is most similar to the CoVZC/PrC31 clade, whereas their S1-CTD exhibits highest similarity to the RShSTT- (and the SARS-related) CoVs. Results from the shared presence of CSIs and sequence similarity studies on different CoV lineages support the inference that the SARS-CoV-2r cluster of viruses has originated by a genetic recombination between the S1-NTD of the CoVZC/PrC31 clade of CoVs and the S1-CTD of RShSTT/SARS viruses, respectively. We also present compelling evidence, based on the shared presence of CSIs and sequence similarity studies, that the pangolin-CoV_MP789, whose receptor-binding domain is most similar to the SARS-CoV-2 virus, has resulted from another independent recombination event involving the S1-NTD of the CoVZC/PrC31 CoVs and the S1-CTD of an unidentified SARS-CoV-2r related virus. The SARS-CoV-2 virus involved in this latter recombination event is postulated to be most similar to the SARS-CoV-2. Several other CSIs reported here are specific for other clusters of sarbecoviruses including a clade consisting of bat-SARS-CoVs (BM48-31/BGR/2008 and SARS_BtKY72). Structural mapping studies show that the identified CSIs form distinct loops/patches on the surface of the spike protein. It is hypothesized that these novel loops/patches on the spike protein, through their interactions with other host components, should play important roles in the biology/pathology of SARS-CoV-2 virus. Lastly, the CSIs specific for different clades of sarbecoviruses including SARS-CoV-2r clade provide novel means for the identification of these viruses and other potential applications.

Keep out! SARS-CoV-2 entry inhibitors: their role and utility as COVID-19 therapeutics.

The COVID-19 pandemic has put healthcare infrastructures and our social and economic lives under unprecedented strain. Effective solutions are needed to end the pandemic while significantly lessening its further impact on mortality and social and economic life. Effective and widely-available vaccines have appropriately long been seen as the best way to end the pandemic. Indeed, the current availability of several effective vaccines are already making a significant progress towards achieving that goal. Nevertheless, concerns have risen due to new SARS-CoV-2 variants that harbor mutations against which current vaccines are less effective. Furthermore, some individuals are unwilling or unable to take the vaccine. As health officials across the globe scramble to vaccinate their populations to reach herd immunity, the challenges noted above indicate that COVID-19 therapeutics are still needed to work alongside the vaccines. Here we describe the impact that neutralizing antibodies have had on those with early or mild COVID-19, and what their approval for early management of COVID-19 means for other viral entry inhibitors that have a similar mechanism of action. Importantly, we also highlight studies that show that therapeutic strategies involving various viral entry inhibitors such as multivalent antibodies, recombinant ACE2 and miniproteins can be effective not only for pre-exposure prophylaxis, but also in protecting against SARS-CoV-2 antigenic drift and future zoonotic sarbecoviruses.