Mucosal and Serum Neutralization Immune Responses Elicited by COVID-19 mRNA Vaccination in Vaccinated and Breakthrough-Infection Individuals: A Longitudinal Study from Louisville Cohort

Abstract

Background/Objectives: The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus type-2 (SARS-CoV-2), has resulted in 777 million cases worldwide. Various vaccines have been approved to control the spread of COVID-19, with mRNA vaccines (Pfizer and Moderna) being widely used in the USA. We conducted a prospective longitudinal study to analyze the immune response elicited by two/three and four doses of monovalent mRNA vaccines in both vaccinated individuals and those who experienced breakthrough infections. Participants were stratified into different age groups: 18-40, 41-60, and over 60 years. Methods: We assessed cross-variant neutralization responses in two cohorts-Cohort I: n = 167 (serum), Cohort II: n = 92 (serum and nasal swab) samples-using infectious virus microneutralization assay (MN) and antibody (IgG or IgA) binding ELISA titers to the spike protein receptor binding domain (RBD). Samples were collected from the Louisville Metro-Jefferson County Co-Immunity Project, a federally funded, population-based study for the surveillance of SARS-CoV-2 in Jefferson County, Kentucky during 2020-2022, involving both health care workers and a local community. Results: Individuals who received two doses of the mRNA vaccine exhibited reduced neutralization against Beta, Delta, and Omicron BA.1 variants compared to wildtype Wuhan, with further decline observed six months post-booster vaccination. However, individuals who experienced natural COVID-19 infection (breakthrough) after receiving two vaccine doses showed enhanced neutralization and antibody responses, particularly against Omicron BA.1. Following the 3rd dose, antibodies and neutralization responses were restored. Among triple-vaccinated individuals, reduced neutralization was observed against Omicron variants BA.1, BA.5, and BA.2 compared to Wuhan. Neutralization responses were better against BA.2 variant compared to BA.1 and BA.5. However, individuals who received three doses of vaccine and experienced a breakthrough infection (n = 45) elicited significantly higher neutralizing antibodies responses against all Omicron subvariants compared to vaccinated individuals. Interestingly, nasal swab samples collected from volunteers with breakthrough infection showed significantly elevated spike-reactive mucosal IgA antibodies and enhanced cross neutralization against BA.1, BA.2, and BA.5 compared to individuals who received only three vaccine doses. Conclusions: mRNA vaccination elicits a strong systemic immune response by boosting serum neutralizing antibodies (NAb), although this protection wanes over time, allowing new variants to escape neutralization. Breakthrough individuals have extra enrichment in nasal NAb offering protection against emerging variants. This longitudinal immune profiling underscores the strengthening of pandemic preparedness and supports the development of durable mucosal vaccines against respiratory infectious disease.

Keywords: COVID-19; IgA and IgG; Omicron; antibodies; breakthrough infections; hybrid immunity; mRNA vaccine; microneutralization (MN); mucosal response; systemic response.

Figure 1

Omicron BA.1 is more resistant to neutralization by sera from individuals vaccinated with two doses of Moderna or Pfizer vaccine. 50% SARS-CoV-2 neutralization titers (VNT50) of sera collected from 18–40 age group (A), 41–60 age group (B), >60 age group (C), vaccine recipients collected within or after 6 months post two doses of either Pfizer or Moderna vaccine. Sera from participants were tested for neutralization against ancestral Wuhan Strain, Beta, Delta, and Omicron BA.1. following 1 h incubation with indicated SARS-CoV-2 VOCs before adding to VeroE6/TMPRSS2 cell monolayers. Each serum was tested in duplicate, and geometric mean 50% SARS-CoV-2 virus neutralization titers (VNT50) or 50% inhibitory dilutions were plotted. Values above the dots represent group GMTs and fold reduction in neutralization of Beta, Delta, and Omicron relative to Wild type ancestral Wuhan strain. Descriptive statistical analysis was performed using two-sided Friedman test with Dunn’s multiple comparison using 3 variables (age group, vaccine duration, and different virus types) and 2 comparisons (age group vs. virus type and vaccine duration vs. virus type). The asterisks (*** p < 0.001, **** p < 0.0001) above the dots represents significant fold reduction in neutralization compared with ancestral Wuhan strain. All statistical results have been shown in Supplementary Tables S1–S3.

Figure 2

Reduced IgG binding titers against RBD protein of mutant SARS-CoV-2 VOCs from individuals vaccinated with the Moderna or Pfizer vaccine. ELISA IgG binding endpoint titers against different RBD of SARS-CoV-2 VOCs in sera collected from 18–40 age group (A), 41–60 age group (B), >60 age group (C), vaccine recipients collected within or after 6 months post two doses of either Pfizer or Moderna vaccine. Sera from participants were tested for binding interactions between RBD protein of different SARS-CoV-2 VOCs and antibody using endpoint titer ELISA. Serially diluted serum was added in duplicates to pre-coated RBD 96 well plates in duplicates, detected using anti-human IgG, and endpoint titers were calculated and plotted based on the cutoff value of naïve human sera (at 1:100 dilution). The fold reduction in neutralization of Beta, Delta, and Omicron relative to Wild type ancestral Wuhan strain. Descriptive statistical analysis was performed using two-sided Friedman test with Dunn’s multiple comparison using 3 variables (age group, vaccine duration, and virus type) and 2 comparisons (age group vs. virus type and vaccine duration vs. virus type). The asterisks (*** p < 0.001, ** p < 0.01) above the bars represent significant fold reduction in IgG binding titers to RBD protein compared with ancestral Wuhan strain. All statistical results have been shown in Supplementary Tables S4–S6.

Figure 3

Breakthrough infection of previously vaccinated (2 doses) individuals induced broad neutralization of mutant SARS-CoV-2 VOCs including Omicron BA.1. 50% SARS-CoV-2 neutralization titers (A) and ELISA IgG binding endpoint titers (B) against different RBD of SARS-CoV-2 VOCs in sera collected from Modena or Pfizer Vaccinated and naturally COVID-19 infected individuals. (A) Values above the dots represents group GMTs, fold reduction, and significant difference in neutralization of Beta, Delta, and Omicron relative to Wild type ancestral Wuhan Strain by serum samples. (B) IgG endpoint titers were calculated and plotted based on the cutoff value against naïve human sera (at 1:100 dilution). Values above the bars indicate GMTs, fold reduction, and significant difference in IgG binding titers to RBD protein of Beta, Delta, and Omicron relative to Wild type ancestral Wuhan strain. For both experiments, the serum was tested in duplicates, and in each panel, data are mean from 2 independent experiments. A two-sided Friedman test with Dunn’s multiple comparisons was performed (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 4

Omicron breakthrough infection of previously triple mRNA vaccinated individuals induces broad neutralization of Omicron sublineages. VNT50 titers of three-dose vaccinated (A) or three-dose vaccinated + infected sera samples (B), and IgG endpoint titers against RBD protein of three-dose vaccinated (C) or three-dose vaccinated + infected sera samples (D). Sera (n = 40) were collected from different age group—18–40 (n = 20), 41–60 (n = 15), and >60 (n = 5)—individuals who received three doses of vaccine (A) or Sera (n = 41) were collected from different age group—18–40 (n = 14), 41–60 (n = 21) and >60 (n = 6)—individuals who received three doses of vaccine and later were infected with Omicron (B) COVID-19 (breakthrough infections) were subjected to live-virus neutralization against ancestral Wuhan strain and recently emerged Omicron sublineages BA.1, BA.2, and BA.5. The fold reduction, GMTs, and significant difference in virus neutralization to BA.1, BA.2, and BA.5 relative to ancestral Wuhan strain were shown, and descriptive statistics using entire mixed model between each of the viral strains have been shown in Supplementary Tables S7 and S8. For (C,D), IgG binding ELISA was conducted against RBD proteins of ancestral Wuhan strain or Omicron sublineages BA.1, BA.2, and BA.5. The serum was tested in duplicates and in each panel, data are mean from 2 independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). The fold reduction, GMTs and significant difference in IgG binding titers against RBD proteins of BA.1, BA.2, and BA.5 relative to ancestral Wuhan strain were shown, and descriptive statistics using entire mixed model between each of the viral strains have been shown in Supplementary Tables S9 and S10.

Figure 5

Generation of mucosal cross-neutralizing antibodies at nasal mucosa following vaccination and Omicron breakthrough infection resulting in hybrid immunity. Nasal swabs were collected after 6 months post third dose from vaccinated only (n = 40) and vaccinated + Omicron infected (n = 41) individuals and assessed for microneutralization and IgA binding assay against Wuhan, BA.1, BA.2, and BA.5. VNT50 titers of three dose vaccinated (A) or 3-dose vaccinated + infected nasal swabs (B) and IgA endpoint titers against RBD protein of three-dose vaccinated (C) or three dose-vaccinated + infected swab samples (D). * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6

Reduced efficacy of neutralization against Omicron sublineages from sera and nasal swabs samples from quadruple mRNA vaccinated (double boosted) individuals. VNT50 titers from sera (A) and nasal swab (C); IgG endpoint titers against RBD (B) from sera and IgA endpoint titers against RBD from nasal swabs (D) of four dose vaccinated individuals (n = 11). Sera and nasal swabs were collected from >60 aged individuals who received four doses of vaccine and subjected to live-virus neutralization and RBD binding ELISA against ancestral Wuhan strain and recently emerged Omicron sublineages BA.1, BA.2, and BA.5. The fold reduction, GMTs and significant difference in virus neutralization to BA.1, BA.2, and BA.5 relative to ancestral Wuhan strain were shown from sera (A) and nasal swabs (C). The fold reduction, GMTs and significant difference in IgG from sera (B) and IgA from nasal swabs (D) binding titers against RBD proteins of BA.1, BA.2, and BA.5 were shown relative to ancestral Wuhan strain. The serum and swab samples were tested in duplicates and in each panel, data are mean from 2 independent experiments. (* p < 0.05, ** p < 0.01).