Broadly neutralizing antibody induction by non-stabilized SARS-CoV-2 Spike mRNA vaccination in nonhuman primates (preprint)

Immunization with mRNA or viral vectors encoding spike with diproline substitutions (S-2P) has provided protective immunity against severe COVID-19 disease. How immunization with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) spike elicits neutralizing antibodies (nAbs) against difficult-to-neutralize variants of concern (VOCs) remains an area of great interest. Here, we compare immunization of macaques with mRNA vaccines expressing ancestral spike either including or lacking diproline substitutions, and show the diproline substitutions were not required for protection against SARS-CoV-2 challenge or induction of broadly neutralizing B cell lineages. One group of nAbs elicited by the ancestral spike lacking diproline substitutions targeted the outer face of the receptor binding domain (RBD), neutralized all tested SARS-CoV-2 VOCs including Omicron XBB.1.5, but lacked cross-Sarbecovirus neutralization. Structural analysis showed that the macaque broad SARS-CoV-2 VOC nAbs bound to the same epitope as a human broad SARS-CoV-2 VOC nAb, DH1193. Vaccine-induced antibodies that targeted the RBD inner face neutralized multiple Sarbecoviruses, protected mice from bat CoV RsSHC014 challenge, but lacked Omicron variant neutralization. Thus, ancestral SARS-CoV-2 spike lacking proline substitutions encoded by nucleoside-modified mRNA can induce B cell lineages binding to distinct RBD sites that either broadly neutralize animal and human Sarbecoviruses or recent Omicron VOCs.

Vaccine-mediated protection against merbecovirus and sarbecovirus challenge in mice (preprint)

The emergence of three distinct highly pathogenic human coronaviruses, SARS-CoV in 2003, MERS-CoV in 2012, and SARS-CoV-2 in 2019, underlines the need to develop broadly active vaccines against the Merbecovirus and Sarbecovirus betacoronavirus subgenera. While SARS-CoV-2 vaccines are highly protective against severe COVID-19 disease, they do not protect against other sarbecoviruses or merbecoviruses. Here, we vaccinate mice with a trivalent sortase-conjugate nanoparticle (scNP) vaccine containing the SARS-CoV-2, RsSHC014, and MERS-CoV receptor binding domains (RBDs), which elicited live-virus neutralizing antibody responses and broad protection. Specifically, a monovalent SARS-CoV-2 RBD scNP vaccine only protected against sarbecovirus challenge, whereas the trivalent RBD scNP vaccine protected against both merbecovirus and sarbecovirus challenge in highly pathogenic and lethal mouse models. Moreover, the trivalent RBD scNP elicited serum neutralizing antibodies against SARS-CoV, MERS-CoV and SARS-CoV-2 BA.1 live viruses. Our findings show that a trivalent RBD nanoparticle vaccine displaying merbecovirus and sarbecovirus immunogens elicits immunity that broadly protects mice against disease. This study demonstrates proof-of-concept for a single pan-betacoronavirus vaccine to protect against three highly pathogenic human coronaviruses spanning two betacoronavirus subgenera.

Impact of the COVID-19 Pandemic on Early Intervention Utilization and Need for Referral after NICU Discharge in VLBW Infants (preprint)

Objective: To examine the impact of COVID-19 pandemic on early intervention (EI) services in VLBW infants. Study Design: 208 VLBW infants seen in NICU FU pre-COVID-19 were compared to 132 infants seen during COVID-19 at 4, 8 and 20 months corrected age (CA) in terms of: enrollment in Child and Family Connections (CFC; intake agency for EI), EI therapies, need for CFC referral and Bayley scores. Results: Infants seen during COVID-19 at 4, 8 and 20 months CA were 3.4 (OR, 95% CI 1.64, 6.98), 4.0 (1.77, 8.95) and 4.8 (2.10, 11.08) times more likely to need CFC referral at FU based on severity of developmental delay. Infants followed during COVID-19 had significantly lower mean Bayley cognitive and language scores at 20 months CA. Conclusions: VLBW infants seen during COVID-19 had significantly higher odds of needing EI and significantly lower cognitive and language scores at 20 months CA.

Breadth of SARS-CoV-2 Neutralization and Protection Induced by a Nanoparticle Vaccine (preprint)

Coronavirus vaccines that are highly effective against SARS-CoV-2 variants are needed to control the current pandemic. We previously reported a receptor-binding domain (RBD) sortase A-conjugated ferritin nanoparticle (RBD-scNP) vaccine that induced neutralizing antibodies against SARS-CoV-2 and pre-emergent sarbecoviruses and protected monkeys from SARS-CoV-2 WA-1 infection. Here, we demonstrate SARS-CoV-2 RBD-scNP immunization induces potent neutralizing antibodies against all eight SARS-CoV-2 variants tested including the Beta, Delta, and Omicron variants in non-human primates (NHPs). The Omicron variant was neutralized by RBD-scNP-induced serum antibodies with a mean of 4.3-fold reduction of ID50 titers compared to SARS-CoV-2 D614G. Immunization with RBD-scNPs protected NHPs from SARS-CoV-2 WA-1, Beta, and Delta variant challenge, and protected mice from challenges of SARS-CoV-2 Beta variant and two other heterologous sarbecoviruses. These results demonstrate the ability of RBD-scNPs to induce broad neutralization of SARS-CoV-2 variants and to protect NHPs and mice from multiple different SARS-related viruses. Such a vaccine could provide the needed immunity to slow the spread of and reduce disease caused by SARS-CoV-2 variants such as Delta and Omicron.

The impact of the COVID-19 pandemic on children with medical complexity (preprint)

Background Descriptions of the COVID-19 pandemics indirect consequences on children are emerging. We aimed to describe the impacts of the pandemic on children with medical complexity (CMC) and their families. Methods A one-time survey of Canadian paediatricians using the Canadian Paediatric Surveillance Program (CPSP) was conducted in Spring 2021. Results A total of 784 paediatricians responded to the survey, with 70% (n=540) providing care to CMC. Sixty-seven (12.4%) reported an adverse health outcome due to a COVID-19 pandemic-related disruption in healthcare delivery. Disruption of the supply of medication and equipment was reported by 11.9% of respondents (n=64). Respondents reported an interruption in family caregiving (47.5%, n=252) and homecare delivery (40.8%, n=218). Almost 47% of respondents (n=253) observed a benefit to CMC due to COVID-19 related changes in healthcare delivery, including increased availability of virtual care and reduction in respiratory illness. Some (14.4%) reported that CMC were excluded from in-person learning when their peers without medical complexity were not. Conclusion Canadian paediatricians reported that CMC experienced adverse health outcomes during the COVID-19 pandemic, including disruptions to family caregiving and community supports. These results highlight the need for healthcare, community and education policymakers to collaborate with families to optimize their health.

SARS-CoV-2 vaccination induces neutralizing antibodies against pandemic and pre-emergent SARS-related coronaviruses in monkeys (preprint)

Betacoronaviruses (betaCoVs) caused the severe acute respiratory syndrome (SARS) and Middle East Respiratory Syndrome (MERS) outbreaks, and now the SARS-CoV-2 pandemic. Vaccines that elicit protective immune responses against SARS-CoV-2 and betaCoVs circulating in animals have the potential to prevent future betaCoV pandemics. Here, we show that immunization of macaques with a multimeric SARS-CoV-2 receptor binding domain (RBD) nanoparticle adjuvanted with 3M-052-Alum elicited cross-neutralizing antibody responses against SARS-CoV-1, SARS-CoV-2, batCoVs and the UK B.1.1.7 SARS-CoV-2 mutant virus. Nanoparticle vaccination resulted in a SARS-CoV-2 reciprocal geometric mean neutralization titer of 47,216, and robust protection against SARS-CoV-2 in macaque upper and lower respiratory tracts. Importantly, nucleoside-modified mRNA encoding a stabilized transmembrane spike or monomeric RBD protein also induced SARS-CoV-1 and batCoV cross-neutralizing antibodies, albeit at lower titers. These results demonstrate current mRNA vaccines may provide some protection from future zoonotic betaCoV outbreaks, and provide a platform for further development of pan-betaCoV nanoparticle vaccines.

The functions of SARS-CoV-2 neutralizing and infection-enhancing antibodies in vitro and in mice and nonhuman primates (preprint)

SARS-CoV-2 neutralizing antibodies (NAbs) protect against COVID-19, making them a focus of vaccine design. A safety concern regarding SARS-CoV-2 antibodies is whether they mediate disease enhancement. Here, we isolated potent NAbs against the receptor-binding domain (RBD) and the N-terminal domain (NTD) of SARS-CoV-2 spike protein from individuals with acute or convalescent SARS-CoV-2 or a history of SARS-CoV-1 infection. Cryo-electron microscopy of RBD and NTD antibodies demonstrated function-specific modes of antibody binding. Select RBD NAbs also demonstrated Fc receptor-{gamma} (Fc{gamma}R)-mediated enhancement of virus infection in vitro, while five non-neutralizing NTD antibodies mediated Fc{gamma}R-independent in vitro infection enhancement. However, both in vitro neutralizing and infection-enhancing RBD or infection-enhancing NTD antibodies protected from SARS-CoV-2 challenge in non-human primates and mice. One of 30 monkeys infused with enhancing antibodies had lung pathology and bronchoalveolar lavage cytokine evidence suggestive of enhanced disease. Thus, these in vitro assessments of enhanced antibody-mediated infection do not necessarily indicate biologically relevant in vivo infection enhancement.

Comprehensive analysis of the host-virus interactome of SARS-CoV-2 (preprint)

Host-virus protein-protein interaction is the key component of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lifecycle. We conducted a comprehensive interactome study between the virus and host cells using tandem affinity purification and proximity labeling strategies and identified 437 human proteins as the high-confidence interacting proteins. Functional characterization and further validation of these interactions elucidated how distinct SARS-CoV-2 viral proteins participate in its lifecycle, and discovered potential drug targets to the treatment of COVID-19. The interactomes of two key SARS-CoV-2 encoded viral proteins, NSP1 and N protein, were compared with the interactomes of their counterparts in other human coronaviruses. These comparisons not only revealed common host pathways these viruses manipulate for their survival, but also showed divergent protein-protein interactions that may explain differences in disease pathology. This comprehensive interactome of coronavirus disease-2019 provides valuable resources for understanding and treating this disease.

Unbuttoning the impact of N501Y mutant RBD on viral entry mechanism: A computational insight (preprint)

The ongoing coronavirus disease 2019 (COVID-19) pandemic has become a serious global threat. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the virus responsible for this pandemic has imposed a severe burden on the medical settings. The spike (S) protein of SARS-CoV-2 is an important structural protein playing a key role in the viral entry. This protein is responsible for the receptor recognition and cell membrane fusion process. The recent reports of the appearance and spread of new SARS-CoV-2 strain has raised alarms. It was reported that this new variant containing the prominent active site mutation in the RBD (N501Y) was rapidly spreading within the population. The reported N501Y mutation within the spike's essential part, known as the receptor-binding domain has raised several questions. Here in this study we have tried to explore the effect of N501Y mutation within the spike protein using several in silico approaches

Rapid inactivation of SARS-CoV-2 on copper touch surfaces determined using a cell culture infectivity assay (preprint)

COVID-19, caused by SARS-CoV-2, was first reported in China in 2019 and has transmitted rapidly around the world, currently responsible for 83 million reported cases and over 1.8 million deaths. The mode of transmission is believed principally to be airborne exposure to respiratory droplets from symptomatic and asymptomatic patients but there is also a risk of the droplets contaminating fomites such as touch surfaces including door handles, stair rails etc, leading to hand pick up and transfer to eyes, nose and mouth. We have previously shown that human coronavirus 229E survives for more than 5 days on inanimate surfaces and another laboratory reproduced this for SARS-CoV-2 this year. However, we showed rapid inactivation of Hu-CoV-229E within 10 minutes on different copper surfaces while the other laboratory indicated this took 4 hours for SARS-CoV-2. So why the difference? We have repeated our work with SARS-CoV-2 and can confirm that this coronavirus can be inactivated on copper surfaces in as little as 1 minute. We discuss why the 4 hour result may be technically flawed.

Alveolar type II cells harbouring SARS-CoV-2 show senescence with a proinflammatory phenotype (preprint)

SARS-CoV-2 infection of the respiratory system can evolve to a multi-system disease. Excessive levels of proinflammatory cytokines, known as a "cytokine storm" are associated with high mortality rates especially in the elderly and in patients with age-related morbidities. Senescent cells, characterized by secretion of such cytokines (Senescence Associated Secretory Phenotype - SASP), are known to occur in this context as well as upon a variety of stressogenic insults. Applying both: i) a novel "in house" antibody against the spike protein of SARS-CoV-2 and ii) a unique senescence detecting methodology, we identified for the first time in lung tissue from COVID-19 patients alveolar cells acquiring senescent features harboring also SARS-CoV-2. Moreover, using the same detection workflow we demonstrated the inflammatory properties of these cells. Our findings justify the application of senotherapeutics for the treatment or prevention of COVID-19 patients.

A Human-Immune-System mouse model for COVID-19 research(DRAGA mouse: HLA-A2.HLA-DR4.Rag1KO.IL-2Rγc KO.NOD) (preprint)

The current SARS-CoV-2 pandemic is accompanied by high morbidity and mortality rates, and there is a compelling need for effective vaccines and therapeutic agents to lessen the severity of COVID-19 disease. Appropriate animal models are essential for testing of vaccines and therapeutics and for mechanistic studies of infection and the host response. The Spike (S) protein of SARS-COV-2 has a high affinity for the human ACE2 receptor, which is expressed on multiple cell types including alveolar epithelial and vascular endothelial cells. Wild-type mice are not susceptible to developing coronavirus-mediated diseases. Accordingly, several human (h)ACE2 transgenic mouse models have been developed for coronavirus research. However, these mice have failed to closely mimic important aspects of the human immunopathological responses to SARS-CoV-2. We report herein that DRAGA (HLA-A2.HLA-DR4.Rag1KO.IL-2R{gamma}c KO.NOD) mice infused with human hematopoietic stem cells from cord blood reconstitute a fully functional human immune system, as well as engraft human epithelial and endothelial cells, sustain SARS-CoV-2 infection, and develop severe COVID-19-like symptoms. In pilot experiments, infected mice developed parenchymal and epithelial lung infiltrations with granzyme B+ and perforin+ CD8+ T cells and alveolar CD61+ microthrombi, mimicking human immunopathological responses to SARS-CoV-2. We propose the DRAGA mouse as a novel pre-clinical tool for studying COVID-19 immunopathology and human immune responses to SARS-CoV-2, including events leading to the cytokine storm and coagulopathies, as well as for testing of candidate vaccines and therapeutics.

Mapping the SARS-CoV-2 spike glycoprotein-derived peptidome presented by HLA class II on dendritic cells (preprint)

Understanding and eliciting protective immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an urgent priority. To facilitate these objectives, we have profiled the repertoire of human leukocyte antigen class II (HLA-II)-bound peptides presented by HLA-DR diverse monocyte-derived dendritic cells pulsed with SARS-CoV-2 spike (S) protein. We identify 209 unique HLA-II-bound peptide sequences, many forming nested sets, which map to sites throughout S including glycosylated regions. Comparison of the glycosylation profile of the S protein to that of the HLA-II-bound S peptides revealed substantial trimming of glycan residues on the latter, likely introduced during antigen processing. Our data also highlight the receptor-binding motif in S1 as a HLA-DR-binding peptide-rich region. Results from this study have application in vaccine design, and will aid analysis of CD4+ T cell responses in infected individuals and vaccine recipients.