Immune responses in COVID-19 patients during breakthrough infection with SARS-CoV-2 variants Delta, Omicron-BA.1 and Omicron-BA.5

Abstract

Background: Breakthrough infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are increasingly observed in vaccinated individuals. Immune responses towards SARS-CoV-2 variants, particularly Omicron-BA.5, are poorly understood. We investigated the humoral and cellular immune responses of hospitalized COVID-19 patients during Delta and Omicron infection waves.

Methods: The corresponding SARS-CoV-2 variant of the respective patients were identified by whole genome sequencing. Humoral immune responses were analyzed by ELISA and a cell culture-based neutralization assay against SARS-CoV-2 D614G isolate (wildtype), Alpha, Delta (AY.43) and Omicron (BA.1 and BA.5). Cellular immunity was evaluated with an IFN-γ ELISpot assay.

Results: On a cellular level, patients showed a minor IFN-γ response after stimulating PBMCs with mutated regions of SARS-CoV-2 variants. Neutralizing antibody titers against Omicron-BA.1 and especially BA.5 were strongly reduced. Double-vaccinated patients with Delta breakthrough infection showed a significantly increased neutralizing antibody response against Delta compared to double-vaccinated uninfected controls (median complete neutralization titer (NT₁₀₀) 640 versus 80, p<0.05). Omicron-BA.1 infection increased neutralization titers against BA.1 in double-vaccinated patients (median NT₁₀₀ of 160 in patients versus 20 in controls, p=0.07) and patients that received booster vaccination (median NT₁₀₀ of 50 in patients versus 20 in controls, p=0.68). For boosted patients with BA.5 breakthrough infection, we found no enhancing effect on humoral immunity against SARS-CoV-2 variants.

Conclusion: Neutralizing antibody titers against Omicron-BA.1 and especially BA.5 were strongly reduced in SARS-CoV-2 breakthrough infections. Delta and Omicron-BA.1 but not Omicron-BA.5 infections boosted the humoral immunity in double-vaccinated patients and patients with booster vaccination. Despite BA.5 breakthrough infection, those patients may still be vulnerable for reinfections with BA.5 or other newly emerging variants of concern.

Keywords: COVID-19; Delta; Omicron; SARS-CoV-2; breakthrough infections.

Figure 1

Overview of the study. Blood samples and nasopharyngeal swabs were collected from hospitalized patients with SARS-CoV-2 breakthrough infection (Delta and Omicron). Blood samples were further analyzed with an enzyme-linked immunosorbent assay (ELISA), neutralization assay as well as enzyme-linked-immuno-spot (ELISpot) assay. Figure was created with BioRender.com.

Figure 2

Phylogenetic tree of assembled SARS-CoV-2 spike (S) region sequences of clinical isolates of patients with SARS-CoV-2 breakthrough infection. * S region extracted from GISAID reference genomes (GISAID).

Figure 3

Binding serum antibody levels in COVID-19 patients with SARS-CoV-2 breakthrough infection. IgG antibodies against the subunit 1 of spike protein (S1) (Wuhan-Hu-1 isolate) and IgG and IgM antibodies against the nucleocapsid protein (NCP) of all patients (n=48) (A) and double vaccinated patients (2x) with Delta (n=18) and BA.1 (n=8) infection and patients with booster vaccination with BA.1 (n=6) and BA.5 (n=12) infection (B). Binding serum antibodies were measured with an enzyme-linked immunosorbent assay (ELISA). An absorbance of <0.8 was regarded as negative (red dotted line), ≥0.8 to <1.1 borderline, and ≥1.1 positive (green dotted line). Differences between groups were analyzed by Kruskal-Wallis test with post-hoc Dunn’s multiple comparisons test (* p<0.05; ** p<0.01, **** p<0.0001). Horizontal lines indicate median values, while error bars represent the interquartile range.

Figure 4

Neutralizing antibody titers against SARS-CoV-2 variants of COVID-19 patients with SARS-CoV-2 breakthrough infection and uninfected, vaccinated controls. (A) Complete neutralization titer (NT₁₀₀) against clinical isolate with D614G mutation (wildtype), Alpha (B.1.1.7), Delta (AY.43) and Omicron (BA.1 and BA.5) of patients with SARS-CoV-2 breakthrough infection (n=50) compared to vaccinated uninfected control (n=28). (B) NT₁₀₀ against clinical isolate with D614G mutation (wildtype), Alpha (B.1.1.7), Delta (AY.43) and Omicron (BA.1 and BA.5) of double vaccinated patients (2x) with Delta (n=20) and BA.1 (n=8) infection and patients with booster vaccination with BA.1 (n=6) and BA.5 (n=12) infection compared to uninfected control with two vaccine doses (n=16) and booster vaccination (n=12). (A, B) Differences between groups were analyzed by Kruskal-Wallis test with post-hoc Dunn’s multiple comparisons test (* p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001). (C) NT₁₀₀ of sera from patients with breakthrough infection with Delta and Omicron (BA.1 and BA.5) against their equivalent clinical isolate compared to wildtype. Differences between groups were analyzed by Wilcoxon signed-rank test. (A–C) Horizontal lines indicate median values, while error bars represent the interquartile range.

Figure 5

Cellular response against SARS-CoV-2 variants in COVID-19 patients with SARS-CoV-2 breakthrough infection. Cellular immunity was assessed by an IFN-γ enzyme-linked-immuno-spot (ELISpot) assay using peripheral blood mononuclear cells (PBMCs) and is displayed for double vaccinated patients (2x) with Delta (n=18) and BA.1 (n=8) infection and patients with booster vaccination with BA.1 (n=5) and BA.5 (n=12) infection compared to uninfected control with two vaccine doses (n=15) and booster vaccination (n=12). PBMCs were stimulated with S protein of Wuhan wildtype, nucleocapsid protein (NCP) and selectively mutated regions Alpha (B.1.1.7), Delta (AY.1) and Omicron (B.1.1.529). A spot increment of three was considered positive (green dotted line). Differences between groups were analyzed by Kruskal-Wallis test with post-hoc Dunn’s multiple comparisons test (* p<0.05; *** p<0.001). Horizontal lines indicate median values, while error bars represent the interquartile range.

Figure 6

Correlation between serum antibody levels and neutralizing antibody titers against SARS-CoV-2 variants. Correlation coefficients (ρ) and p-values were calculated using Spearman’s rank analysis.

Figure 7

Correlation between serum antibody levels and cellular IFN-γ response after stimulation with SARS-CoV-2 variants. Correlation coefficients (ρ) and p-values were calculated using Spearman’s rank analysis.