Antibody and T cell responses to SARS-CoV-2 mRNA vaccines during maintenance therapy for immune-mediated inflammatory diseases

BackgroundLimited information is available on the impact of immunosuppressants on COVID-19 vaccination in patients with immune-mediated inflammatory diseases (IMID). MethodsThis observational cohort study examined the immunogenicity of SARS-CoV-2 mRNA vaccines in adult patients with inflammatory bowel disease, rheumatoid arthritis, ankylosing spondylitis, or psoriatic disease, with or without maintenance immunosuppressive therapies. Antibody and T cell responses to SARS-COV-2, including neutralization against SARS-CoV-2 variants were determined before and after 1 and 2 vaccine doses. ResultsWe prospectively followed 150 subjects, 26 healthy controls, 9 IMID patients on no treatment, 44 on anti-TNF, 16 on anti-TNF with methotrexate/azathioprine (MTX/AZA), 10 on anti-IL-23, 28 on anti-IL-12/23, 9 on anti-IL-17, and 8 on MTX/AZA. Antibody and T cell responses to SARS-CoV-2 were detected in all participants, increasing from dose 1 to dose 2 and declining 3 months later, with greater attrition in IMID patients compared to healthy controls. Antibody levels and neutralization efficacy against variants of concern were substantially lower in anti-TNF treated patients than in healthy controls and were undetectable against Omicron by 3 months after dose 2. ConclusionsOur findings support the need for a third dose of mRNA vaccine and for continued monitoring of immunity in these patient groups. FundingFunded by a donation from Juan and Stefania Speck and by Canadian Institutes of Health (CIHR) /COVID-Immunity Task Force (CITF) grants VR-1 172711 and VS1-175545 (T.H.W. and A.C.G); CIHR FDN-143250 (T.H.W.), GA2-177716 (V.C., A.C.G., T.W.), GA1-177703 (A.C.G.) and the CIHR rapid response network to SARS-CoV-2 variants, CoVaRR-Net (to A.C.G.).

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

OBJECTIVESAntibody testing 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 solution to 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. METHODSWe developed 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 nucleocapsid (N). We automated a surrogate neutralization (sn)ELISA that measures inhibition of ACE2-spike or -RBD interactions by antibodies. The assays were calibrated to a World Health Organization reference standard. RESULTSOur single-point IgG-based ELISAs accurately distinguished non-infected and infected individuals. For seroprevalence assessment (in a non-vaccinated cohort), classifying a sample as positive if antibodies were detected for [≥] 2 of the 3 antigens provided the highest specificity. In vaccinated cohorts, increases in anti-spike and -RBD (but not -N) antibodies are observed. We present detailed protocols for serum/plasma or dried blood spots analysis performed manually and on automated platforms. The snELISA can be performed automatically at single points, increasing its scalability. CONCLUSIONSMeasuring antibodies to three viral antigens and identify neutralizing antibodies capable of disrupting spike-ACE2 interactions in high-throughput enables large-scale analyses of humoral immune responses to SARS-CoV-2 infection and vaccination. The reagents are available to enable scaling up of standardized serological assays, permitting inter-laboratory data comparison and aggregation.

Neutralizing antibody responses to SARS-CoV-2 variants in vaccinated Ontario long-term care home residents and workers

Prioritizing Ontarios long-term care home (LTCH) residents for vaccination against severe acute respiratory syndrome coronavirus 2 has drastically reduced their disease burden; however, recent LTCH outbreaks of variants of concern (VOCs) have raised questions regarding their immune responses. In 198 residents, mRNA vaccine dose 1 elicited partial spike and receptor binding domain antibody responses, while the second elicited a response at least equivalent to convalescent individuals in most residents. Residents administered mRNA-1273 (Moderna) mounted stronger total and neutralizing antibody responses than those administered BNT162b2 (Pfizer-BioNTech). Two to four weeks after dose 2, residents (n = 119, median age 88) produced 4.8-6.3-fold fewer neutralizing antibodies than staff (n = 78; median age 47) against wild-type (with D614G) pseudotyped lentivirus, and residents administered BNT162b2 produced 3.89-fold fewer neutralizing antibodies than those who received mRNA-1273. These effects were exacerbated upon serum challenge with pseudotyped VOC spike, with up to 7.94-fold reductions in B.1.351 (Beta) neutralization. Cumulatively, weaker vaccine stimulation, age/comorbidities, and the VOC produced an [~]130-fold reduction in apparent neutralization titers in LTCH residents and 37.9% of BNT162b2-vaccinated residents had undetectable neutralizing antibodies to B.1.351. Continued immune response surveillance and additional vaccine doses may be required in this population with known vulnerabilities.

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

Although SARS-CoV-2 infects the upper respiratory tract, we know little about the amount, type, and kinetics of antibodies (Ab) generated at this site in response to intramuscular COVID-19 vaccination, and whether these Ab protect against subsequent "breakthrough" infections. We collected longitudinal serum and saliva samples from participants receiving two doses of mRNA COVID-19 vaccines over a 6-month period and measured the relative level of anti-Spike and anti-Receptor Binding Domain (RBD) Ab. We detected anti-Spike/RBD IgG and IgA and associated secretory component in the saliva of most participants receiving 1 dose of mRNA vaccine. Administration of a second dose of mRNA boosted the IgG but not the IgA response, with only 30% of participants remaining positive for IgA at this timepoint. At 6 months post-dose 2, these participants exhibited greatly diminished anti-Spike/RBD IgG and IgA levels concomitant with a reduction in neutralizing activity in the saliva, although the level of secretory component associated anti-Spike was less susceptible to decay. Examining two prospective cohorts of subjects that were monitored for infections post-vaccination, we found that participants who were subsequently infected with SARS-CoV-2 had lower levels of vaccine-induced serum anti-Spike/RBD IgA at 2-4 weeks post-dose 2 compared to participants who did not experience an infection, whereas IgG levels were comparable between groups. These data emphasize the importance of developing COVID-19 vaccines that elicit a durable IgA response. One-Sentence SummaryOur study delves into whether intra-muscular mRNA vaccination regimes confer a local IgA response in the oral cavity and whether the IgA response is associated with protection against breakthrough infection.

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

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. One Sentence SummaryImmunity to SARS-CoV-2 in a cohort of patients, mainly with mild COVID-19 disease, persists to 9 months with an altered T cell cytokine and cytotoxicity profile compared to influenza A virus-specific memory T cells from the same subjects.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seroprevalence: Navigating the absence of a gold standard

BackgroundMultiple anti-SARS-CoV-2 immunoassays are available, but no gold standard exists. We assessed four assays using various methodological approaches to estimate SARS-COV-2 seroprevalence during the first COVID-19 wave in Canada. MethodsThis serial cross-sectional study was conducted using plasma samples from healthy blood donors between April-September 2020. Qualitative assessment of SARS-CoV-2 IgG antibodies was based on four assays: Abbott Architect SARS-Cov-2 IgG assay (target nucleocapsid) (Abbott-NP) and three in-house IgG ELISA assays (target spike glycoprotein (Spike), spike receptor binding domain (RBD), and nucleocapsid (NP)). Seroprevalence was estimated using multiple composite reference standards (CRS) and by a series of Bayesian Latent Class Models (BLCM) (using uninformative, weakly, and informative priors). Results8999 blood samples were tested. The Abbott-NP assay consistently estimated seroprevalence to be lower than the ELISA-based assays. Discordance between assays was common, 13 unique diagnostic phenotypes were observed. Only 32 samples (0.4%) were positive by all four assays. BLCM using uninformative priors predicted seroprevalence increased from 0.7% (95% credible interval (CrI); 0.4, 1.0%) in April/May to 0.8% (95% CrI 0.5, 1.2%) in June/July to 1.1% (95% CrI 0.7, 1.6) in August/September. Results from CRS were very similar to the BLCM. Assay characteristics varied considerably over time. Overall spike had the highest sensitivity (89.1% (95% CrI 79.2, 96.9%), while the sensitivity of the Abbott-NP assay waned from 65.3% (95% CrI 43.6, 85.0%) in April/May to 45.9% (95% CrI 27.8, 65.6) by August/September. DiscussionWe found low SARS-CoV-2 seroprevalence rates at the end of the first wave and estimates derived from single assays may be biased. SummaryMultiple anti-SARS-CoV-2 immunoassays are available, but no gold standard exists. We used four unique assays to estimate very low SARS-COV-2 seroprevalence during the first COVID-19 wave in Canada. Caution should be exercised when interpretating seroprevalence estimates from single assays.

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

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). One Sentence SummaryPTX-COVID19-B is a SARS-CoV-2 mRNA vaccine that is highly immunogenic, safe, and effective in preventing SARS-CoV-2 infection in mice and hamsters and is currently being evaluated in human clinical trials.

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

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- 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}g+ 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

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, but how it correlates to the antibody response in serum is not known. Here, we profile by enzyme linked immunosorbent assays (ELISAs) IgG, IgA and IgM responses 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 and IgM antibodies rapidly decayed, IgG antibodies remained relatively stable up to 105 days PSO in both biofluids. In a surrogate neutralization ELISA (snELISA), neutralization activity peaks by 31-45 days PSO and slowly declines, though a clear drop is detected at the last blood draw (105-115 days PSO). Lastly, IgG, IgM and to a lesser extent IgA responses to spike and RBD in the serum positively correlated with matched saliva samples. This study confirms that systemic and mucosal humoral IgG antibodies are maintained in the majority of COVID-19 patients for at least 3 months PSO. Based on their correlation with each other, IgG responses in saliva may serve as a surrogate measure of systemic immunity. One Sentence SummaryIn this manuscript, we report evidence for sustained SARS-CoV-2-specific IgG and transient IgA and IgM responses both at the site of infection (mucosae) and systemically in COVID-19 patients over 3 months and suggest that saliva could be used as an alternative biofluid for monitoring IgG to SARS-CoV-2 spike and RBD antigens.

A simple protein-based SARS-CoV-2 surrogate neutralization assay

Most of the patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mount a humoral immune response to the virus within a few weeks of infection, but the duration of this response and how it correlates with clinical outcomes has not been completely characterized. 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 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 protein receptor binding domain (RBD) with its receptor, angiotensin converting-enzyme 2 (ACE2). The assay serves as a surrogate neutralization assay and is performed on the same platform and in parallel with an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against the RBD, enabling a direct comparison. The results obtained with our assay correlate with those of two viral based assays, a plaque reduction neutralization test (PRNT) that uses live SARS-CoV-2 virus, and a spike pseudotyped viral-vector-based assay.