Paridiprubart inhibits TLR4-dependant NF-κB activation by multiple pathogens (preprint)

Respiratory pathogens such as SARS-CoV-2 and influenza can activate an exaggerated inflammatory response (cytokine storm) in the lungs that may result in acute respiratory distress syndrome (ARDS), hospitalization, and death. Therapies that target a specific pathogen (i.e. anti-virals) must, by nature, be selected after a specific diagnosis and may become ineffective due to pathogen evolution. An alternate strategy is to counter the exaggerated innate immune response present in ARDS patients using host-directed drug therapies that are agnostic to the infectious agent to overcome both of these challenges. Originally described as the innate immune receptor for lipopolysaccharide (LPS), Toll-like receptor 4 (TLR4) is now understood to be an important mediator of inflammation caused by a variety of pathogen-associated molecular patterns (PAMPs) and host-derived damage-associated molecular patterns (DAMPs). Here we show that paridiprubart, a monoclonal antibody that prevents TLR4 dimer formation, inhibits the response to TLR4 agonists including LPS, the SARS-CoV-2 spike protein, the DAMP high mobility group box 1 (HMGB1), as well as the NF-{kappa}B response to infection by both viral and bacterial pathogens. Notable in this regard, we demonstrate that SARS-CoV-2 increases HMGB1 levels, and that paridiprubart inhibits both the SARS-CoV-2 and HMGB1-triggered NF-{kappa}B response, illustrating its potential to suppress this self-amplifying inflammatory signal. We also observed that the inhibitory effect of paridiprubart is apparent when cells are exposed to the SARS-CoV-2 spike protein, which is itself a direct TLR4 agonist. In the context of active infection, paridiprubart suppressed the NF-{kappa}B-dependent response elicited by infection with SARS-CoV-2, the seasonal coronavirus 229E, influenza A virus or Haemophilus influenzae, a gram-negative bacterial pathogen. Combined, these findings reinforce the central role played by TLR4 in the inflammatory response to infection by diverse pathogens, and demonstrates the protective potential of paridiprubart-dependent inhibition of pathogenic TLR4 responses.

Natural Language Processing for Clinical Laboratory Data Repository Systems: Implementation and Evaluation for Respiratory Viruses (preprint)

Background With the growing volume and complexity of laboratory repositories, it has become tedious to parse unstructured data into structured and tabulated formats for secondary uses such as decision support, quality assurance, and outcome analysis. However, advances in Natural Language Processing (NLP) approaches have enabled efficient and automated extraction of clinically meaningful medical concepts from unstructured reports. Objective In this study, we aimed to determine the feasibility of using the NLP model for information extraction as an alternative approach to a time-consuming and operationally resource-intensive handcrafted rule-based tool. Therefore, we sought to develop and evaluate a deep learning-based NLP model to derive knowledge and extract information from text-based laboratory reports sourced from a provincial laboratory repository system. Methods The NLP model, a hierarchical multi-label classifier, was trained on a corpus of laboratory reports covering testing for 14 different respiratory viruses and viral subtypes. The corpus included 85 k unique laboratory reports annotated by eight Subject Matter Experts (SME). The model’s performance stability and variation were analyzed across fine-grained and coarse-grained classes. Moreover, the model’s generalizability was also evaluated internally and externally on various test sets. Results The NLP model was trained several times with random initialization on the development corpus, and the results of the top ten best-performing models are presented in this paper. Overall, the NLP model performed well on internal, out-of-time (pre-COVID-19), and external (different laboratories) test sets with micro-averaged F1 scores >94% across all classes. Higher Precision and Recall scores with less variability were observed for the internal and pre-COVID-19 test sets. As expected, the model’s performance varied across categories and virus types due to the imbalanced nature of the corpus and sample sizes per class. There were intrinsically fewer classes of viruses being detected than those tested ; therefore, the model’s performance (lowest F1-score of 57%) was noticeably lower in the “ detected ” cases. Conclusions We demonstrated that deep learning-based NLP models are promising solutions for information extraction from text-based laboratory reports. These approaches enable scalable, timely, and practical access to high-quality and encoded laboratory data if integrated into laboratory information system repositories.

Use of whole genome sequencing to identify low-frequency mutations in COVID-19 patients treated with remdesivir (preprint)

Background: We investigate the effects of remdesivir (RDV) treatment on intra-host SARS-CoV-2 diversity and low-frequency mutations in moderately ill hospitalized COVID-19 patients and compare them to patients without RDV treatment. Methods: Sequential collections of nasopharyngeal and mid-turbinate swabs were obtained from 16 patients with and 31 patients without RDV treatment. A total of 113 samples were sequenced and mutation analyses were performed. Results: We did not identify any drug resistant mutations during RDV therapy. In genes encoding and associated with the replication complex, low-frequency minority variants that do not reach fixation within the sampling period were detected in 6/16 (37.5%) and 14/31 (45%) patients with and without RDV treatment respectively. We did not detect significant differences in within-host diversity and positive selection between the RDV-treated and untreated groups. Conclusions: Minimal intra-host variability and stochastic low-frequency variants detected in moderately ill patients suggests little selective pressure in patients receiving short courses of RDV. Patients undergoing short regimens of RDV therapy should continue to be monitored.

Antibody avidity and multi-specificity combined to confer protection against SARS-CoV-2 and resilience against viral escape (preprint)

SARS-CoV-2, the causative agent of COVID-19, has been responsible for a global pandemic. Monoclonal antibodies have been used as antiviral therapeutics, but have been limited in efficacy by viral sequence variability in emerging variants of concern (VOCs), and in deployment by the need for high doses. In this study, we leverage the MULTI-specific, multi-Affinity antiBODY (Multabody, MB) platform, derived from the human apoferritin protomer, to drive the multimerization of antibody fragments and generate exceptionally potent and broad SARS-CoV-2 neutralizers. CryoEM revealed a high degree of homogeneity for the core of these engineered antibody-like molecules at 2.1 [A] resolution. We demonstrate that neutralization potency improvements of the MB over corresponding IgGs translates into superior in vivo protection: in the SARS-CoV-2 mouse challenge model, comparable in vivo protection was achieved for the MB delivered at 30x lower dose compared to the corresponding IgGs. Furthermore, we show how MBs potently neutralize SARS-CoV-2 VOCs by leveraging augmented avidity, even when corresponding IgGs lose their ability to neutralize potently. Multiple mAb specificities could also be combined into a single MB molecule to expand the neutralization breadth beyond SARS-CoV-2 to other sarbecoviruses. Our work demonstrates how avidity and multi-specificity combined can be leveraged to confer protection and resilience against viral diversity that exceeds that of traditional monoclonal antibody therapies.

Surveillance for SARS-CoV-2 in Norway rats ( Rattus norvegicus ) from southern Ontario (preprint)

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from wildlife origins has raised concerns about spillover from humans to animals, the establishment of novel wildlife reservoirs, and the potential for future outbreaks caused by variants of wildlife origin. Norway rats ( Rattus norvegicus ) are abundant in urban areas and live in close proximity to humans, providing the opportunity for spillover of SARS-CoV-2. To date, there is no evidence of natural SARS-CoV-2 infection in rats and experimental studies suggest rats are likely not susceptible to ancestral SARS-CoV-2. However, as variants emerge, new species have been identified as competent hosts, as demonstrated by the susceptibility of rats to the SARS-CoV-2 Alpha variant of concern (VOC). We investigated SARS-CoV-2 infection and exposure in Norway rats from southern Ontario, Canada. From October 2019 to June 2021, 224 rats were submitted by collaborating pest control companies. The majority of samples were collected in Windsor (79.9%; n=179), Hamilton (13.8%; n=31), and the Greater Toronto Area (5.8%; n=13). Overall, 50.0% (n=112) were female and most rats were sexually mature (55.8%; n=125). Notably, 202 samples, including the two seropositive samples, were collected prior to the emergence of VOCs, and 22 were collected while the Alpha variant was the predominant circulating VOC in humans. Nasal turbinate (n=164) and small intestinal (n=213) tissue samples were analyzed for SARS-CoV-2 RNA by RT-PCR. Thoracic cavity fluid samples (n=213) were tested for neutralizing antibodies using a surrogate virus neutralization test (sVNT) (GenScript cPass); confirmatory plaque reduction neutralization test (PRNT) testing was conducted on presumptive positive samples. We did not detect SARS-CoV-2 RNA in any samples tested. Two out of eleven samples positive by sVNT had neutralizing antibodies by PRNT (1:40 and 1:320 PRNT70). It is imperative that efforts to control and monitor SARS-CoV-2 include surveillance of rats and other relevant wildlife species as novel variants continue to emerge.

Highly divergent white-tailed deer SARS-CoV-2 with potential deer-to-human transmission (preprint)

Wildlife reservoirs of SARS-CoV-2 can lead to viral adaptation and spillback from wildlife to humans (Oude Munnink et al., 2021). In North America, there is evidence of spillover of SARS-CoV-2 from humans to white-tailed deer (Odocoileus virginianus), but no evidence of transmission from deer to humans (Hale et al., 2021; Kotwa et al., 2022; Kuchipudi et al., 2021). Through a multidisciplinary research collaboration for SARS-CoV-2 surveillance in Canadian wildlife, we identified a new and highly divergent lineage of SARS-CoV-2. This lineage has 76 consensus mutations including 37 previously associated with non-human animal hosts, 23 of which were not previously reported in deer. There were also mutational signatures of host adaptation under neutral selection. Phylogenetic analysis revealed an epidemiologically linked human case from the same geographic region and sampling period. Together, our findings represent the first evidence of a highly divergent lineage of SARS-CoV-2 in white-tailed deer and of deer-to-human transmission.

Identification of a SARS-CoV-2 host metalloproteinase-dependent entry pathway differentially used by SARS-CoV-2 and variants of concern Alpha, Delta, and Omicron (preprint)

To infect cells, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) binds to angiotensin converting enzyme 2 (ACE2) via its spike glycoprotein (S), delivering its genome upon S-mediated membrane fusion. SARS-CoV-2 uses two distinct entry pathways: 1) a surface, serine protease-dependent or 2) an endosomal, cysteine protease-dependent pathway. In investigating serine protease-independent cell-cell fusion, we found that the matrix metalloproteinases (MMPs), MMP2/9, can activate SARS-CoV-2 S fusion activity, but not that of SARS-CoV-1. Importantly, metalloproteinases activation of SARS-CoV-2 S represents a third entry pathway in cells expressing high MMP levels. This route of entry required cleavage at the S1/S2 junction in viral producer cells and differential processing of variants of concern S dictated its usage. In addition, metalloproteinase inhibitors reduced replicative Alpha infection and abrogated syncytia formation. Finally, we found that the Omicron S exhibit reduced metalloproteinase-dependent fusion and viral entry. Taken together, we identified a MMP2/9-dependent mode of activation of SARS-CoV-2 S. As MMP2/9 are released during inflammation and severe COVID-19, they may play important roles in SARS-CoV-2 S-mediated cytopathic effects, tropism, and disease outcome.

First detection of SARS-CoV-2 infection in Canadian wildlife identified in free-ranging white-tailed deer (Odocoileus virginianus) from southern Quebec, Canada (preprint)

White-tailed deer are susceptible to SARS-CoV-2 and represent a relevant species for surveillance. We investigated SARS-CoV-2 infection in white-tailed deer in Quebec, Canada. In November 2021, 251 nasal swabs and 104 retropharyngeal lymph nodes from 258 deer were analyzed for SARS-CoV-2 RNA, whole genome sequencing and virus isolation and 251 thoracic cavity fluid samples were tested for neutralizing antibodies. We detected SARS-CoV-2 RNA in three nasal swabs from the Estrie region and virus was isolated from two samples; evidence of past exposure was detected among deer from the same region. Viral sequences were assigned to lineage AY.44, a sublineage of B.1.617.2. All deer sequences clustered with human GISAID sequences collected in October 2021 from Vermont USA, which borders the Estrie region. Mutations in the S-gene and a deletion in ORF8 encoding a truncated protein were detected. These findings underscore the importance of ongoing surveillance of key wildlife species for SARS-CoV-2.

Longitudinal Assessment of SARS-CoV-2 Specific T Cell Cytokine-Producing Responses for 1 Year Reveals Persistence of Multi-Cytokine Proliferative Responses, with Greater Immunity Associated with Disease Severity (preprint)

Cellular-mediated immunity is critical for long-term protection against most viral infections, including coronaviruses. We studied 23 SARS-CoV-2-infected survivors over a one year post symptom onset (PSO) interval by ex vivo cytokine ELISpot assay. All subjects demonstrated SARS-CoV-2-specific IFN-{gamma}, IL-2, and Granzyme B (GzmB) T cell responses at presentation, with greater frequencies in severe disease. Cytokines, mainly produced by CD4+ T cells, targeted all structural proteins (Nucleocapsid, Membrane, Spike) except Envelope, with GzmB > IL-2 > IFN-{gamma}. Mathematical modeling predicted that: 1) cytokine responses peaked at 6 days for IFN-{gamma}, 36 days for IL-2, and 7 days for GzmB, 2) severe illness was associated with reduced IFN-{gamma} and GzmB, but increased IL-2 production rates, 3) males displayed greater production of IFN-{gamma}, whereas females produced more GzmB. Ex vivo responses declined over time with persistence of IL-2 in 86% and of IFN-{gamma} and GzmB in 70% of subjects at a median of 336 days PSO. The average half-life of SARS-CoV-2-specific cytokine-producing cells was modelled to be 139 days (~4.6 months). Potent T cell proliferative responses persisted throughout observation, were CD4 dominant, and were capable of producing all 3 cytokines. Several immunodominant CD4 and CD8 epitopes identified in this study were shared by seasonal coronaviruses or SARS-CoV-1 in the Nucleocapsid and Membrane regions. Both SARS-CoV-2-specific CD4+ and CD8+ T cell clones were able to kill target cells, though CD8 tended to be more potent.

Immunogenicity of convalescent and vaccinated sera against clinical isolates of ancestral SARS-CoV-2, beta, delta, and omicron variants (preprint)

The omicron variant of concern (VOC) of SARS-CoV-2 was first reported in November 2021 in Botswana and South Africa. Omicron variant has evolved multiple mutations within the spike protein and the receptor binding domain (RBD), raising concerns of increased antibody evasion. Here, we isolated infectious omicron from a clinical specimen obtained in Canada. The neutralizing activity of sera from 65 coronavirus disease (COVID-19) vaccine recipients and convalescent individuals against clinical isolates of ancestral SARS-CoV-2, beta, delta, and omicron VOCs was assessed. Convalescent sera from unvaccinated individuals infected by the ancestral virus during the first wave of COVID-19 in Canada (July, 2020) demonstrated reduced neutralization against beta, delta and omicron VOCs. Convalescent sera from unvaccinated individuals infected by the delta variant (May-June, 2021) neutralized omicron to significantly lower levels compared to the delta variant. Sera from individuals that received three doses of the Pfizer or Moderna vaccines demonstrated reduced neutralization of both delta and omicron variants relative to ancestral SARS-CoV-2. Sera from individuals that were naturally infected with ancestral SARS-CoV-2 and subsequently received two doses of the Pfizer vaccine induced significantly higher neutralizing antibody levels against ancestral virus and all VOCs. Importantly, infection alone, either with ancestral SARS-CoV-2 or the delta variant was not sufficient to induce high neutralizing antibody titers against omicron. This data will inform current booster vaccination strategies and we highlight the need for additional studies to identify longevity of immunity against SARS-CoV-2 and optimal neutralizing antibody levels that are necessary to prevent infection and/or severe COVID-19.

SARS-CoV-2 wildlife surveillance in Ontario and Quebec, Canada (preprint)

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for the COVID-19 pandemic, is capable of infecting a variety of wildlife species. Wildlife living in close contact with humans are at an increased risk of SARS-CoV-2 exposure and if infected have the potential to become a reservoir for the pathogen, making control and management more difficult. Objective: To conduct SARS-CoV-2 surveillance in urban wildlife from Ontario and Quebec, Canada, increasing our knowledge of the epidemiology of the virus and our chances of detecting spillover from humans into wildlife. Methods: Using a One Health approach, we leveraged activities of existing research, surveillance, and rehabilitation programs among multiple agencies to collect samples from 776 animals from 17 different wildlife species between June 2020 and May 2021. Samples from all animals were tested for the presence of SARS-CoV-2 viral RNA, and a subset of samples from 219 animals across 3 species (raccoons, Procyon lotor; striped skunks, Mephitis mephitis; and mink, Neovison vison) were also tested for the presence of neutralizing antibodies. Results: No evidence of SARS-CoV-2 viral RNA or neutralizing antibodies was detected in any of the tested samples. Conclusion: Although we were unable to identify positive SARS-CoV-2 cases in wildlife, continued research and surveillance activities are critical to better understand the rapidly changing landscape of susceptible animal species. Collaboration between academic, public and animal health sectors should include experts from relevant fields to build coordinated surveillance and response capacity.

A "Made-in-Canada" serology solution for profiling humoral immune responses to SARS-CoV-2 infection and vaccination (preprint)

BACKGROUND: Testing for antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been instrumental in detecting previous exposures and analyzing vaccine-elicited immune responses. Here, we describe a scalable "Made-in-Canada" solution that can detect and quantify SARS-CoV-2 antibodies, discriminate between natural infection- and vaccination-induced responses, and assess antibody-mediated inhibition of the spike-angiotensin converting enzyme 2 (ACE2) interaction. METHODS: We developed a set of methods and reagents to detect SARS-CoV-2 antibodies by enzyme-linked immunosorbent assay (ELISA). The main assays focus on the parallel detection of immunoglobulin (Ig)Gs against the spike trimer, its receptor binding domain (RBD), and the nucleocapsid (N) protein. These antigens are complemented by a detection antibody (human anti-IgG fused to horseradish peroxidase (HRP)) and a positive control reference antibody (recombinant IgG against the RBD), permitting intra- and inter-laboratory comparisons. Using this toolkit and commercial reagents, we optimized automated ELISAs on two different high throughput platforms to measure antibody responses to SARS-CoV-2 antigens. The assays were calibrated to a reference standard from the World Health Organization. We also automated a surrogate neutralization (sn)ELISA that measures inhibition of ACE2-Spike or -RBD interactions by antibodies using biotinylated ACE2. RESULTS: Our individual IgG-based ELISAs measure antibody levels in single-point measurements in reference to a standard antibody curve to accurately distinguish non-infected and infected individuals (area under the curve > 0.96 for each assay). Positivity thresholds can be established in individual assays using precision-recall analysis (e.g., by fixing the false positive rate), or more stringently, by scoring against the distribution of the means of negative samples across multiple assays performed over several months. For seroprevalence assessment (in a non-vaccinated cohort), classifying a sample as positive if antibodies were detected for at least 2 of the 3 antigens provided the highest specificity. In vaccinated cohorts, increases in anti-spike and -RBD (but not -N) antibodies are observed. Here, we present detailed protocols to perform these assays using either serum/plasma or dried blood spots both manually and on two automated platforms, and to express the results in international units to facilitate data harmonization and inter-study comparisons. We also demonstrate that the snELISA can be performed automatically at single points, increasing the scalability of this functional assay for large seroprevalence studies. INTERPRETATION: The ability to measure antibodies to three viral antigens and identify neutralizing antibodies capable of disrupting spike-ACE2 interactions in high-throughput assays enables large-scale analyses of humoral immune responses to SARS-CoV-2 infection and vaccination. The "Made-in-Canada" set of protein reagents, produced at the National Research Council of Canada are publicly available to enable the up-scaling of standardized serological assays, permitting nationwide data comparison and aggregation.

A mucosal antibody response is induced by intra-muscular SARS-CoV-2 mRNA vaccination (preprint)

The first line of defense against SARS-CoV-2 is the upper respiratory tract, yet we know little about the amount, type, and kinetics of mucosal anti-Spike antibodies (Ab) in response to intramuscular (i.m.) COVID-19 vaccination. We analyzed salivary Ab against SARS-CoV-2 Spike following mRNA/mRNA and adenovirus (Ad)/mRNA regimes. While anti-Spike/RBD IgG was detected in the saliva and correlated with the systemic response, anti-Spike/RBD IgA associated with the secretory component (sIgA) was also detected, and did not necessarily correlate with serum Ab. Only modest levels of neutralizing capacity were observed in saliva at 2 weeks post-dose 2, and by 6 months, anti-Spike/RBD IgG were greatly diminished. In contrast, low levels of anti-Spike sIgA persisted up to 6 months after dose 2. Our results show that SARS-CoV-2 vaccination induces an IgG response in the saliva that decays over time and an sIgA response that does not necessarily correlate with systemic immunity. One-Sentence SummaryOur study delves into how intra-muscular mRNA/mRNA or mRNA/Ad COVID-19 vaccination regimes confer immunity in the oral cavity with important implications for understanding protection against breakthrough infections in healthy vaccinated people.

Systematic Genome-Scale Identification of Host Factors for SARS-CoV-2 Infection Across Models Yields a Core Single Gene Dependency; Ace2 (preprint)

SARS-CoV-2, depends on host cell components for replication, therefore the identification of virus-host dependencies offers an effective way to elucidate mechanisms involved in viral infection. Such host factors may be necessary for infection and replication of SARS-CoV-2 and, if druggable, presents an attractive strategy for anti-viral therapy. We performed genome wide CRISPR knockout screens in Vero E6 cells and 4 human cell lines including Calu-3, Caco-2, Hek293 and Huh7 to identify genetic regulators of SARS-CoV-2 infection. Our findings identified only ACE2, the cognate SARS-CoV-2 entry receptor, as a common host dependency factor across all cell lines, while all other host genes identified were cell line specific including known factors TMPRSS2 and CTSL. Several of the discovered host-dependency factors converged on pathways involved in cell signalling, lipid metabolism, immune pathways and chromatin modulation. Notably, chromatin modulator genes KMT2C and KDM6A in Calu-3 cells had the strongest impact in preventing SARS-CoV-2 infection when perturbed. Overall, the network of host factors that have been identified will be broadly applicable to understanding the impact of SARS-CoV-2 on human cells and facilitate the development of host-directed therapies.Funding Information: This work was supported by the University of Toronto COVID-19 Action Initiative Fund to J.M., B.J.B., S.G.O., J.G., K.M., and S.M.. Indirect support was also received from the University of Toronto and the Temerty Foundation to support enhanced capacity and operations of the Toronto Combined Containment Level 3 Facility during the COVID-19 pandemic. This work was also partially supported from a Canadian Institutes for Health Research Project Grant to J.M. (MOP142375). J.M. is a Tier 2 Canada Research Chair in Function Genomics.Declaration of Interest: None to declare.

Systematic genome-scale identification of host factors for SARS-CoV-2 infection across models yields a core single gene dependency; ACE2 (preprint)

SARS-CoV-2, depends on host cell components for replication, therefore the identification of virus-host dependencies offers an effective way to elucidate mechanisms involved in viral infection. Such host factors may be necessary for infection and replication of SARS CoV-2 and, if druggable, presents an attractive strategy for anti-viral therapy. We performed genome wide CRISPR knockout screens in Vero E6 cells and 4 human cell lines including Calu-3, Caco-2, Hek293 and Huh7 to identify genetic regulators of SARS-CoV-2 infection. Our findings identified only ACE2, the cognate SARS-CoV-2 entry receptor, as a common host dependency factor across all cell lines, while all other host genes identified were cell line specific including known factors TMPRSS2 and CTSL. Several of the discovered host-dependency factors converged on pathways involved in cell signalling, lipid metabolism, immune pathways and chromatin modulation. Notably, chromatin modulator genes KMT2C and KDM6A in Calu-3 cells had the strongest impact in preventing SARS CoV-2 infection when perturbed. Overall, the network of host factors that have been identified will be broadly applicable to understanding the impact of SARS-CoV-2 on human cells and facilitate the development of host directed therapies.

Exposure route, sex, and age influence disease outcome in a golden Syrian hamster model of SARS-CoV-2 infection (preprint)

The emergence of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the resultant pandemic of coronavirus disease 2019 (COVID-19) has led to over one hundred million confirmed infections, greater than three million deaths, and severe economic and social disruption. Animal models of SARS-CoV-2 are critical tools for the pre-clinical evaluation of antivirals, vaccines, and candidate therapeutics currently under urgent development to curb COVID-19-associated morbidity and mortality. The golden (Syrian) hamster model of SARS-CoV-2 infection recapitulates key characteristics of severe COVID-19, including high-titer viral replication in the upper and lower respiratory tract and the development of pathogenic lesions in the lungs. In this work we examined the influence of the route of exposure, sex, and age on SARS-CoV-2 pathogenesis in golden hamsters. We report that delivery of SARS-CoV-2 primarily to the nasal passages (low-volume intranasal), the upper and lower respiratory tract (high-volume intranasal), or the digestive tract (intragastric) results in comparable viral titers in the lung tissue and similar levels of viral shedding during acute infection. However, low-volume intranasal exposure results in milder weight loss during acute infection while intragastric exposure leads to a diminished capacity to regain body weight following the period of acute illness. Further, we examined both sex and age differences in response to SARS-CoV-2 infection. Male hamsters, and to a greater extent older male hamsters, display an impaired capacity to recover from illness and a delay in viral clearance compared to females. Lastly, route of exposure, sex, and age were found to influence the nature of the host inflammatory cytokine response, but they had a minimal effect on both the quality and durability of the humoral immune response as well as the susceptibility of hamsters to SARS-CoV-2 re-infection. Together, these data indicate that the route of exposure, sex, and age have a meaningful impact SARS-CoV-2 pathogenesis in hamsters and that these variables should be considered when designing pre-clinical challenge studies.

Immunity to SARS-CoV-2 persists 9 months post-symptoms with an altered T cell phenotype compared to influenza A virus-specific memory (preprint)

SARS-CoV-2 induces T cell, B cell and antibody responses that are detected for several months in recovered individuals. Whether this response resembles a typical respiratory viral infection is a matter of debate. Here we followed T cell and antibody responses in 24 mainly non-hospitalized SARS-CoV-2 recovered subjects at two time points (median of 45- and 145-days post-symptom onset). Antibody responses were detected in 95% of subjects, with a strong correlation between plasma and salivary anti-S and anti-RBD IgG, as well as a correlation between circulating T follicular helper cells and the SARS-CoV-2-specific IgG response. Based on intracellular cytokine production or proliferation, CD4+ T cell responses to SARS-CoV-2 were detected in all subjects, decaying with a half-life of 5-6 months for S-specific IL-2-producing cells. CD4+ responses were largely of the T helper 1 phenotype, but with a lower ratio of IFN-{gamma} : IL-2 producing cells and a lower frequency of CD8+: CD4+ T cells compared to influenza A virus-(IAV)-specific memory responses within the same subjects. Analysis of secreted molecules also revealed a lower ratio of IFN-{gamma}: IL-2 and IFN-{gamma}: IL-6 and an altered cytotoxic profile for S- and N-specific compared to IAV-specific responses. These data suggest that the memory T-cell phenotype after a single infection with SARS-CoV-2 persists over time, with an altered cytokine and cytotoxic profile compared to long term memory to IAV within the same subjects.

Surface and air contamination with SARS-CoV-2 from hospitalized COVID-19 patients in Toronto, Canada (preprint)

Background The aim of this prospective cohort study was to determine the burden of SARS-CoV-2 in air and on surfaces in rooms of patients hospitalized with COVID-19, and to identify patient characteristics associated with SARS-CoV-2 environmental contamination. Methods Nasopharyngeal swabs, surface, and air samples were collected from the rooms of 78 inpatients with COVID-19 at six acute care hospitals in Toronto from March to May 2020. Samples were tested for SARS-CoV-2 viral RNA and cultured to determine potential infectivity. Whole viral genomes were sequenced from nasopharyngeal and surface samples. Association between patient factors and detection of SARS-CoV-2 RNA in surface samples were investigated using a mixed-effects logistic regression model. Findings SARS-CoV-2 RNA was detected from surfaces (125/474 samples; 42/78 patients) and air (3/146 samples; 3/45 patients) in COVID-19 patient rooms; 14% (6/42) of surface samples from three patients yielded viable virus. Viral sequences from nasopharyngeal and surface samples clustered by patient. Multivariable analysis indicated hypoxia at admission, a PCR-positive nasopharyngeal swab with a cycle threshold of [≤]30 on or after surface sampling date, higher Charlson co-morbidity score, and shorter time from onset of illness to sample date were significantly associated with detection of SARS-CoV-2 RNA in surface samples. Interpretation The infrequent recovery of infectious SARS-CoV-2 virus from the environment suggests that the risk to healthcare workers from air and near-patient surfaces in acute care hospital wards is likely limited. Surface contamination was greater when patients were earlier in their course of illness and in those with hypoxia, multiple co-morbidities, and higher SARS-CoV-2 RNA concentration in NP swabs. Our results suggest that, while early detection and isolation of COVID-19 patients is important, air and surfaces may pose limited risk a few days after admission to acute care hospitals.

Preclinical evaluation of a SARS-CoV-2 mRNA vaccine PTX-COVID19-B (preprint)

Safe and effective vaccines are needed to end the COVID-19 pandemic caused by SARS-CoV-2. Here we report the preclinical development of a lipid nanoparticle (LNP) formulated SARS-CoV-2 mRNA vaccine, PTX-COVID19-B. PTX-COVID19-B was chosen among three candidates after the initial mouse vaccination results showed that it elicited the strongest neutralizing antibody response against SARS-CoV-2. Further tests in mice and hamsters indicated that PTX-COVID19-B induced robust humoral and cellular immune responses and completely protected the vaccinated animals from SARS-CoV-2 infection in the lung. Studies in hamsters also showed that PTX-COVID19-B protected the upper respiratory tract from SARS-CoV-2 infection. Mouse immune sera elicited by PTX-COVID19-B vaccination were able to neutralize SARS-CoV-2 variants of concern (VOCs), including the B.1.1.7, B.1.351 and P.1 lineages. No adverse effects were induced by PTX-COVID19-B in both mice and hamsters. These preclinical results indicate that PTX-COVID19-B is safe and effective. Based on these results, PTX-COVID19-B was authorized by Health Canada to enter clinical trials in December 2020 with a phase 1 clinical trial ongoing (ClinicalTrials.gov number: NCT04765436).