Some SARS-CoV-2-exposed individuals develop immunity without overt infection. We identified 11 individuals who were negative by nucleic acid testing during prolonged close contact and with no serological diagnosis of infection. As this could reflect natural immunity, cross-reactive immunity from previous coronavirus exposure, abortive infection due to de novo immune responses, or other factors, our objective was to characterize immunity against SARS-CoV-2 in these individuals. Blood was processed into plasma and peripheral blood mononuclear cells (PBMC) and screened for IgG, IgA, and IgM antibodies (Ab) against SARS-CoV-2 and common β-coronaviruses OC43 and HKU1. Receptor blocking activity and interferon-alpha (IFN-α) in plasma were also measured. Circulating T cells against SARS-CoV-2 were enumerated and CD4⁺ and CD8⁺ T cell responses discriminated after in vitro stimulation. Exposed uninfected individuals were seronegative against SARS-CoV-2 spike (S) and selectively reactive against OC43 nucleocapsid protein (N), suggesting common β-coronavirus exposure induced Ab cross-reactive against SARS-CoV-2 N. There was no evidence of protection from circulating angiotensin-converting enzyme (ACE2) or IFN-α. Six individuals had T cell responses against SARS-CoV-2, with four involving CD4⁺ and CD8⁺ T cells. We found no evidence of protection from SARS-CoV-2 through innate immunity or immunity induced by common β-coronaviruses. Cellular immune responses against SARS-CoV-2 were associated with time since exposure, suggesting that rapid cellular responses may contain SARS-CoV-2 infection below the thresholds required for a humoral response.

Mucosal IgA is widely accepted as providing protection against respiratory infections, but stimulation of mucosal immunity, collection of mucosal samples and measurement of mucosal IgA can be problematic. The relationship between mucosal and circulating IgA responses is unclear, however, whole blood is readily collected and circulating antigen-specific IgA easily measured. We measured circulating IgA against SARS-CoV-2 spike (S) to investigate vaccine- and infection-induced production and correlation with protection. Circulating IgA against ancestral (Wuhan-Hu-1) and Omicron (BA.1) S proteins was measured at different time points in a total of 143 subjects with varied backgrounds of vaccination and infection. Intramuscular vaccination induced circulating anti-SARS-CoV-2 S IgA. Subjects with higher levels of vaccine-induced IgA against SARS-CoV-2 S (p = 0.0333) or receptor binding domain (RBD) (p = 0.0266) were less likely to experience an Omicron breakthrough infection. The same associations did not hold for circulating IgG anti-SARS-CoV-2 S levels. Breakthrough infection following two vaccinations generated stronger IgA anti-SARS-CoV-2 S responses (p = 0.0002) than third vaccinations but did not selectively increase circulating IgA against Omicron over ancestral S, indicating immune imprinting of circulating IgA responses. Circulating IgA against SARS-CoV-2 S following breakthrough infection remained higher than vaccine-induced levels for over 150 days. In conclusion, intramuscular mRNA vaccination induces circulating IgA against SARS-CoV-2 S, and higher levels are associated with protection from breakthrough infection. Vaccination with ancestral S enacts imprinting within circulating IgA responses that become apparent after breakthrough infection with Omicron. Breakthrough infection generates stronger and more durable circulating IgA responses against SARS-CoV-2 S than vaccination alone.

Dietary antigens are first encountered in the gut during early life, when the immune system and microbiota are still maturing. In healthy individuals, oral tolerance develops towards dietary antigens: an active process which results in local and systemic immune unresponsiveness to antigens first encountered in the gut. Despite a wealth of research describing mechanisms contributing to oral tolerance in adult rodent models, questions remain about how the early life environment impacts oral tolerance development. We set out to characterize the fate(s) of CD4 ⁺ T cells during oral tolerance development using a robust early life mouse model, where controlled oral doses of dietary antigen are given directly to pups during the pre-weaning period. Orally administering 2mg of ovalbumin (OVA) daily during the third week of life was sufficient to confer oral tolerance to OVA in female and male C57BL/6 and BALB/c mice. Following early life oral OVA exposure, a large proportion of OVA-specific CD4 ⁺ T cells acquired a Th2 phenotype, alongside some OVA-specific Tregs. Following systemic challenges of OVA with an adjuvant, both OVA-specific Tregs and Th lineage-negative cells expressing anergy markers were detectable in pups given early life oral OVA, while OVA-specific Th2 cells were suppressed in comparison to pups who never received early life oral OVA. These data highlight the diverse fates of CD4 ⁺ T cells during early life oral tolerance development and maintenance, and we present a model to study oral tolerance during the early life period when dietary antigens are first encountered.