Omicron Breakthrough Infection Elicits Superior Humoral and Mucosal Immune Responses to SARS-CoV-2 Variants than an Intramuscular Booster Dose (preprint)

Our understanding of the quality of cellular and humoral immunity conferred by COVID-19 vaccination alone versus vaccination plus SARS-CoV-2 breakthrough (BT) infection remains incomplete. While the current (2023) SARS-CoV-2 immune landscape of Canadians is complex, in late 2021 most Canadians had either just received a third dose of COVID-19 vaccine, or had received their two dose primary series and then experienced an Omicron BT. Herein we took advantage of this coincident timing to contrast cellular and humoral immunity conferred by three doses of vaccine versus two doses plus BT. Our results show that mild BT infection induces cell-mediated immune responses to variants comparable to an intramuscular vaccine booster dose. In contrast, BT subjects had higher salivary IgG and IgA levels against the Omicron Spike and enhanced reactivity to the ancestral Spike for the IgA isotype, which also reacted with SARS-CoV-1. Serum neutralizing antibody levels against the ancestral strain and the variants were also higher after BT infection. Our results support the need for mucosal vaccines to emulate the enhanced mucosal and humoral immunity induced by Omicron without exposing individuals to the risks associated with SARS-CoV-2 infection.

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.

SPEEDS: A Portable Serological Testing Platform for Rapid Electrochemical Detection of SARS-CoV-2 Antibodies (preprint)

The COVID-19 pandemic has resulted in a worldwide health crisis. Rapid diagnosis, new therapeutics and effective vaccines will all be required to stop the spread of COVID-19. Quantitative evaluation of serum antibody levels against the SARS-CoV-2 virus provides a means of monitoring a patient's immune response to a natural viral infection or vaccination, as well as evidence of a prior infection. In this paper, a portable and low-cost electrochemical immunosensor is developed for the rapid and accurate quantification of SARS-CoV-2 serum antibodies. The immunosensor is capable of quantifying the concentrations of immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies against the SARS-CoV-2 spike protein in human serum. For IgG and IgM, it provides measurements in the range of 10.1 ng/mL--60 g/mL and 1.64 ng/mL -- 50 g/mL, respectively, and both antibodies can be assayed in 13 min. We also developed device stabilization and storage strategies to achieve stable performance of the immunosensor within 24-week storage at room temperature. We evaluated the performance of the immunosensor using COVID-19 patient serum samples collected at different time points after symptom onset. The rapid and sensitive detection of IgG and IgM provided by our immunosensor fulfills the need of rapid COVID-19 serology testing for both point-of-care diagnosis and population immunity screening.

Intranasal HD-Ad Vaccine Protects the Upper and Lower Respiratory Tracts of hACE2 Mice against SARS-CoV-2 (preprint)

The COVID-19 pandemic has affected more than 120 million people and resulted in over 2.8 million deaths worldwide. Several COVID-19 vaccines have been approved for emergency use in humans and are being used in many countries. However, all of the approved vaccines are administered by intramuscular injection and this may not prevent upper airway infection or viral transmission. Here, we describe intranasal immunization of a COVID-19 vaccine delivered by a novel platform, the helper-dependent adenoviral (HD-Ad) vector. Since HD-Ad vectors are devoid of adenoviral coding sequences, they have a superior safety profile and a large cloning capacity for transgenes. The vaccine (HD-Ad_RBD) codes for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein and intranasal immunization induced robust mucosal and systemic immunity. Moreover, intranasal immunization of K18-hACE2 mice with HD-Ad_RBD using a prime-boost regimen, resulted in complete protection of the upper respiratory tract against SARS-CoV-2 infection. As such, intranasal immunization based on the HD-Ad vector promises to provide a powerful platform for constructing highly effective vaccines targeting SARS-CoV-2 and its emerging variants.

Detection of SARS-CoV-2 Viral Particles using Direct, Reagent-Free Electrochemical Sensing (preprint)

This manuscript describes a new method that enables direct analysis of viral particles in unprocessed samples.Using an electrochemical readout method that requires no external reagents, we detect the SARS-CoV-2 virus in the saliva of infected patients.The approach relies on a molecular sensor tethered to the surface of a gold electrode that contains an antibody, specific to the targetof interest, which here is the SARS-CoV-2 S1 spike protein that is displayed on the viral capsule. The antibody is attached to the electrode using a negatively charged linker that is composed of DNA. When a positive potential is applied to the electrode, the sensor complex is attracted to the electrode surface. The kinetics of transport is measured using chronoamperometry and readout is possible based on the absense or precense of virus and its effect on the complex movevment on electrode surface.

Systematic examination of T cell responses to SARS-CoV-2 versus influenza virus reveals distinct inflammatory profile (preprint)

There is a pressing need for an in-depth understanding of immunity to SARS-CoV-2. Here we investigated T cell recall responses to fully glycosylated Spike trimer, recombinant N protein as well as to S, N, M and E peptide pools in the early convalescent phase. All subjects showed SARS-CoV-2-specific T cell responses to at least one antigen. SARS-CoV-2-specific CD4+ T cells were primarily of the central memory phenotype and exhibited a lower IFN-[gamma] to TNF-[alpha] ratio compared to influenza-specific responses of the same donors, independent of disease severity. SARS-CoV-2-specific T cells were less multifunctional than influenza-specific T cells, particularly in severe cases, potentially suggesting exhaustion. High IL-10 production was noted in response to N protein, possibly contributing to immunosuppression, with potential implications for vaccine design. We observed granzyme B+/IFN-[gamma] CD4+ and CD8+ proliferative responses to peptide pools in most individuals, with CD4+ responses predominating over CD8+ responses. Peripheral T follicular helper responses to S or N strongly correlated with serum neutralization assays as well as RBD-specific IgA. Overall, T cell responses to SARS-CoV-2 are robust, however, CD4+ Th1 responses predominate over CD8+ responses and are more inflammatory with a weaker Tfh response than influenza-specific CD4+ responses, potentially contributing to COVID-19 disease.

Evidence for sustained mucosal and systemic antibody responses to SARS-CoV-2 antigens in COVID-19 patients (preprint)

While the antibody response to SARS-CoV-2 has been extensively studied in blood, relatively little is known about the mucosal immune response and its relationship to systemic antibody levels. Since SARS-CoV-2 initially replicates in the upper airway, the antibody response in the oral cavity is likely an important parameter that influences the course of infection. We developed enzyme linked immunosorbent assays to detect IgA and IgG antibodies to the SARS-CoV-2 spike protein (full length trimer) and its receptor binding domain (RBD) in serum (n=496) and saliva (n=90) of acute and convalescent patients with laboratory-diagnosed COVID-19 ranging from 3-115 days post-symptom onset (PSO), compared to negative controls. Anti-CoV-2 antibody responses were readily detected in serum and saliva, with peak IgG levels attained by 16-30 days PSO. Whereas anti-CoV-2 IgA antibodies rapidly decayed, IgG antibodies remained relatively stable up to 115 days PSO in both biofluids. Importantly, IgG responses in saliva and serum were correlated, suggesting that antibodies in the saliva may serve as a surrogate measure of systemic immunity.

A simple protein-based SARS-CoV-2 surrogate neutralization assay (preprint)

With the COVID-19 pandemic surpassing 12M confirmed cases and 550K deaths worldwide, defining the key components of the immune response to SARS-CoV-2 infection is critical. Of particular importance is the identification of immune correlates of infection that would support public health decision-making on treatment approaches, vaccination strategies, and convalescent plasma therapy. While ELISA-based assays to detect and quantitate antibodies to SARS-CoV-2 in patient samples have been developed, the detection of neutralizing antibodies typically requires more demanding cell-based viral assays. Here, we present and validate a safe and efficient protein-based assay for the detection of serum and plasma antibodies that block the interaction of the SARS-CoV-2 spike (S) protein receptor binding domain (RBD) with its receptor, angiotensin converting-enzyme 2 (ACE2). This test is performed on the same platform and in parallel with an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against the RBD and serves as a surrogate neutralization assay.

Synthetic Antibodies neutralize SARS-CoV-2 infection of mammalian cells (preprint)

Coronaviruses (CoV) are a large family of enveloped, RNA viruses that circulate in mammals and birds. Three highly pathogenic strains have caused zoonotic infections in humans that result in severe respiratory syndromes including the Middle East Respiratory Syndrome CoV (MERS), Severe Acute Respiratory Syndrome CoV (SARS), and the ongoing Coronavirus Disease 2019 (COVID-19) pandemic. Here, we describe a panel of synthetic monoclonal antibodies, built on a human IgG framework, that bind to the spike protein of SARS-CoV-2 (the causative agent of COVID-19), compete for ACE2 binding, and potently inhibit SARS-CoV-2. All antibodies that exhibited neutralization potencies at sub-nanomolar concentrations against SARS-CoV-2/USA/WA1 in Vero E6 cells, also bound to the receptor binding domain (RBD), suggesting competition for the host receptor ACE2. These antibodies represent strong immunotherapeutic candidates for treatment of COVID-19.