Immune Response in Moderate to Critical Breakthrough COVID-19 Infection After mRNA Vaccination

Abstract

SARS-CoV-2 variants of concern (VOCs) can trigger severe endemic waves and vaccine breakthrough infections (VBI). We analyzed the cellular and humoral immune response in 8 patients infected with the alpha variant, resulting in moderate to fatal COVID-19 disease manifestation, after double mRNA-based anti-SARS-CoV-2 vaccination. In contrast to the uninfected vaccinated control cohort, the diseased individuals had no detectable high-avidity spike (S)-reactive CD4+ and CD8+ T cells against the alpha variant and wild type (WT) at disease onset, whereas a robust CD4+ T-cell response against the N- and M-proteins was generated. Furthermore, a delayed alpha S-reactive high-avidity CD4+ T-cell response was mounted during disease progression. Compared to the vaccinated control donors, these patients also had lower neutralizing antibody titers against the alpha variant at disease onset. The delayed development of alpha S-specific cellular and humoral immunity upon VBI indicates reduced immunogenicity against the S-protein of the alpha VOC, while there was a higher and earlier N- and M-reactive T-cell response. Our findings do not undermine the current vaccination strategies but underline a potential need for the inclusion of VBI patients in alternative vaccination strategies and additional antigenic targets in next-generation SARS-CoV-2 vaccines.

Keywords: SARS-CoV-2; T cells; breakthrough infection; mRNA; neutralizing antibodies; vaccine.

Figure 1

Failure to develop S-reactive CD4+ T cells against the Alpha variant and WT S-reactive CD4+ T cells with high avidity at disease onset. Characterization of SARS-CoV-2 S-reactive CD4+ T cells in diseased and healthy subjects. Blood samples of 8 diseased patients with SARS-CoV-2 breakthrough infection and 5 healthy vaccinated patients were stimulated with SARS-CoV-2 S-WT, S-Alpha, M- and N-proteins, and analyzed by flow cytometry. (A, B) S-WT and S-Alpha reactive CD4+ T cells and their avidity between control at Tb and diseased at T1. Absence of Alpha reactive CD4+ T cells at disease onset, accompanied by presence of WT reactive CD4+ T cells with no avidity. (C, D) Quantification of S-WT, S-Alpha, M- and N-reactive CD4+ T cells among the diseased. Statistically significant strong frequencies of M- and N- reactive T cells compared to S-WT and S-Alpha reactive T cells at disease onset, with significant higher avidity. Generation of reactive CD4+ T cell response against all 4 SARS-CoV-2 proteins at T3. Reactive SARS-CoV-2 CD4+ T cells are defined as CD4+CD154+CD137+ cells. The avidity was determined by detecting CD3low+CD4+ T cells among CD4+CD154+CD137+. Negative controls were subtracted from reactive stimulated samples to exclude unreactive activation. Bars show mean and standard deviation. Paired data was compared with Wilcoxon matched pairs test, whereas unpaired data with Mann-Whitney-test. P<0.05 was considered significant, only significant p values are documented in the figures.

Figure 2

Failure to induce a strong SARS-CoV-2 reactive cytotoxic CD8+ response. (A) Correlation of S-WT and S-Alpha reactive CD8+ T cells between controls at Tb and VBI patients at T1. In the VBI cohort there is an absence of S-Alpha reactive CD8+ T cells at disease onset, accompanied by decreased frequencies of S-WT reactive CD4+ T cells, compared to the control. (B) Correlation of S-WT, S-Alpha, M- and N-reactive CD8+ T cells among VBI. There are statistically significant higher frequencies of M- and N- reactive T cells compared to S-Alpha reactive T cells on disease onset.

Figure 3

Insufficient WT and Alpha neutralizing antibodies production on disease onset. Anti-Sars-CoV-2 S protein and whole virus binding neutralizing antibodies assessed with WT virus hCoV-19 and VOC B.1.1.7 (Alpha). Comparison of the relative titers of SARS-CoV-2 neutralizing antibodies with the 50% neutralization dose among VBI, at T1 and T3, and the control cohort. (A) There is a significantly higher neutralizing potential of the control cohort against the Alpha whole virus, WT whole virus and (B) against the S-Alpha at disease onset, indicating failure of the anti-SARS-CoV-2 vaccination to generate protective humoral immunity among the diseased cohort. ⁎ (WT whole virus VBI T3 versus S-WT VBI T3), ⁎⁎ (Alpha whole virus VBI T3 versus S-Alpha T3). Increased neutralizing antibodies titers at T3, with Alpha and WT whole virus NAb titers are statistically significantly higher compared to S-WT and S-Alpha NAb at T3, implying reduced immunogenicity of the diseased cohort against the SARS-CoV-2 Spike (WT NAb versus S-WT NAb p=0.015, Alpha NAb versus S-Alpha NAb p=0.015). Paired data was compared with Wilcoxon matched pairs test, whereas unpaired data with Mann-Whitney-test. P<0.05 was considered significant, only significant p values are documented in the figures.

Figure 4

No vaccination advantage in VBI group compared to no-vac COVID group with severe and critical disease. Comparison of SARS-CoV-2 reactive CD4+ T cell response among the severe and critical VBI patients versus the severe and critical no-vac COVID-19 patients. (A) Frequencies of WT S- and M-reactive CD4+ T cells at T1 and T3 for the two cohorts are depicted (B) Comparison of CD3low populations of the unvaccinated COVID-19 cohort compared to the VBI patients.

Figure 5

Low frequencies of pre-existing SARS-CoV-2 reactive T cells with low avidity in the unvaccinated unexposed controls. PBMCs from 8 individuals, unexposed to SARS-CoV-2, were analyzed for pre-existing SARS-CoV-2 reactive CD4+ T cell response. (A) Low frequencies of pre-existing cross-reactive CD4 reactive T cells compared to VBI patients at T1. (B) The avidity of M- and N-CD3low populations is significantly lower among the no-vac unexposed cohort.