Comparative immunogenicity and reactogenicity of heterologous ChAdOx1-nCoV-19-priming and BNT162b2 or mRNA-1273-boosting with homologous COVID-19…

Study population

The study was conducted among 331 healthy individuals mainly including health care personnel at Saarland University Medical Center, who either received homologous regimens with ChAdOx (n=62), BNT (n=43), or mRNA-1273 (n=59), or heterologous vaccinations with ChAdOx-priming followed by a boost with either the BNT (n=66) or the mRNA-1273 vaccine (n=101) (Fig.1 and Table1). Despite no known history of SARS-CoV-2 infection, one female was positive for nucleocapsid-specific IgG, and was excluded from further analysis. Due to convenience sampling based on current recommendations, the mean time between the two vaccinations was shorter for the homologous mRNA regimens (5.70.7 weeks) as compared to the vector-based regimens (11.90.9 weeks). In addition, the group showed some differences in age and gender (Table1). Blood sampling was carried out at a median of 14 (IQR 2) days after the second vaccination. In differential blood counts, leukocyte and granulocyte numbers differed between the groups with the highest numbers found after homologous mRNA-1273 vaccination. The numbers of monocytes, lymphocytes, and lymphocyte subpopulations such as B cells, CD4, and CD8 T cells did not differ. Among B cells, plasmablast numbers, which were identified as CD38 positive cells among IgD-CD27+ CD19-positive switched-memory B cells were also highest in individuals after homologous mRNA-1273 vaccination (Table1).

Schematic representation of the five vaccine regimens (three homologous: ChAdOx/ChAdOx n=62, BNT/BNT n=43, mRNA-1273/mRNA-1273 n=59; two heterologous: ChAdOx/BNT n=66, ChAdOx/mRNA-1273 n=101). Shown are the time frames between the first (prime) and the second (boost) vaccination, and between the boost vaccination and the day of blood analysis. #One individual of the mRNA-1273/mRNA-1273 group was excluded from further analysis due to detectable IgG towards the SARS-CoV-2 nucleocapsid.

Spike-specific IgG was detectable in all individuals, but their levels were significantly higher in individuals boosted with mRNA vaccines as compared to individuals after homologous ChAdOx vaccination (Fig.2a, p<0.0001). When comparing heterologous regimens, boosting with mRNA-1273 led to numerically higher IgG levels (6043 (IQR 4396) BAU/ml) than boosting with BNT (4275 (IQR 4080) BAU/ml). Likewise, among homologous regimens, IgG levels were higher after mRNA-1273 vaccination (5529 (IQR 5755) BAU/ml) than in BNT vaccinated individuals (3438 (IQR 3287) BAU/ml), although the differences did not reach statistical significance. As with IgG levels, neutralizing inhibitory capacity of spike-specific antibodies determined using a surrogate assay was high and reached a maximum of 100% in the majority of mRNA-boosted individuals, which contrasted with significantly lower neutralizing activity after homologous ChAdOx vaccination (median 77.8% (IQR 33.5%), p<0.0001, Fig.2a).

Cellular and humoral immune parameters were analyzed 1318 days post vaccination and compared between individuals with different homologous or heterologous COVID-19 vaccine regimens: homologous ChAdOx vaccination (n=62), heterologous ChAdOx/BNT vaccination (n=66), heterologous ChAdOx/mRNA-1273-vaccination (n=101), homologous BNT vaccination (n=43) or homologous mRNA-1273-vaccination (n=58). a ELISA and surrogate neutralization assays were performed to quantify levels of spike-specific IgG and neutralizing antibodies. Intracellular cytokine staining after antigen-specific stimulation of whole blood samples allowed for flow-cytometrical determination of SARS-CoV-2 spike-specific (b) and SEB-reactive (c) CD4 and CD8 T-cell levels. Reactive cells were identified by co-expression of CD69 and IFN among CD4 or CD8 T cells and subtraction of reactivity of respective negative control stimulations. CTLA-4 expression was determined on d spike-specific and e SEB-reactive CD4 and CD8 T cells in all samples with at least 20 cytokine-positive CD4 and CD8 T cells. f Correlation matrix of spike-specific T-cell and antibody responses among each group. Bars in ae represent medians with interquartile ranges. Differences between the groups were calculated using two-sided KruskalWallis test with Dunns multiple comparisons post-test. Correlations in f were analyzed according to two-tailed Spearman (see also Supplementary Table1). Dotted lines indicate detection limits for antibodies in a, indicating negative, intermediate, and positive levels or levels of inhibition, respectively as per manufacturers instructions, and detection limits for SARS-CoV-2-specific CD4 T cells in b and c. Source data are provided as a Source Data file. IFN Interferon, MFI median fluorescence intensitiy, SEB Staphylococcus aureus enterotoxin B.

Vaccine-induced CD4 and CD8 T cells were quantified after stimulation with overlapping peptides encompassing the spike protein. Activation-induced T cells were identified based on CD69 and IFN, TNF, and IL-2. A representative example of CD69-positive spike-specific CD4 and CD8 T cells producing IFN from a 49-year-old female after the second homologous mRNA-1273 vaccination is shown in Supplementary Fig.1, and data from all individuals analyzed after the second vaccination are summarized in Fig.2b. Spike-specific CD4 T-cell levels in the homologous ChAdOx vaccine group were significantly lower than in all other groups. Among mRNA-boosted regimens, median levels of spike-specific CD4 T cells were highest after heterologous ChAdOx1/mRNA-1273 vaccination (0.29% (IQR 0.23%)). Not only did a boost with mRNA-1273 outperform heterologous boosting with BNT after ChAdOx-priming (0.18% (IQR 0.17%), p<0.01), but CD4 T-cell levels were also higher after homologous vaccination with mRNA-1273 (0.24% (IQR 0.27%) than with BNT (0.10% (IQR 0.08%), p<0.0001). Interestingly, the two heterologous regimens also led to a strong induction of spike-specific CD8 T cells (0.29% (IQR 0.57%) for BNT and 0.40% (IQR 0.60%) for mRNA-1273), with significantly higher levels than all three homologous regimens (Fig.2b, p<0.0001). All vaccine-induced effects on CD4 and CD8 T cells were specific, as no differences in Staphylococcus aureus Enterotoxin B (SEB)-reactive CD4 and CD8 T cells were observed between the five groups (Fig.2c). Finally, in line with a pronounced induction of vaccine-induced T cells, CTLA-4 expression was strongly induced on spike-specific CD4 and CD8 T cells of all individuals after heterologous vaccination and in both homologous mRNA regimens, whereas CTLA-4 expression on specific T cells after homologous ChAdOx vaccination was significantly lower (Fig.2d). These differences in CTLA-4 expression were also spike-specific, as CTLA-4 expression on SEB-reactive CD4 and CD8 T cells were similarly low in all five groups (Fig.2e).

When analyzing correlations between spike-specific IgG levels, neutralizing activity, and spike-specific CD4 and CD8 T cells (Fig.2f and Supplementary Table1), neutralizing activity showed a strong correlation with IgG levels in each vaccine subgroup. Likewise, spike-specific CD4 and CD8 T cells showed a significant correlation. In line with the previous findings3, CD4 T cells correlated with IgG in ChAdOx/ChAdOx and ChAdOx/BNT vaccinated individuals only. In addition, it is interesting to note that IgG levels correlated with CD8 T-cell levels in the three homologous vaccine groups only, whereas no such correlation was found for the two heterologous vaccine groups, which may be a result of the exceptionally high CD8 T-cell response in these two groups (see Fig.2b).

As the five groups differed in age and gender due to convenience sampling and recruitment according to national recommendations (Table1), a subgroup analysis was performed among 40 individuals per vaccination regimen which were matched for age and gender (Supplementary Table2). As shown in Supplementary Fig.2, between-group differences in IgG levels, neutralizing activity and spike-specific T cells largely remain the same. In the whole cohort, adjusting for age and gender as confounders in a non-parametric regression analysis showed that both confounders did not have any significant effect on immunological parameters (Supplementary Table3). When testing for interactions of age within each vaccine group with the homologous ChAdOx group as a reference, age had no effect on T-cell levels and neutralizing antibody activity; the only effect of age was found for IgG levels within each of the two homologous regimens (p=0.003 for BNT/BNT and p=0.015 for mRNA-1273/mRNA-1273, Supplementary Table3).

Based on national recommendations, the interval between the first and the second dose was longer for ChAdOx-primed groups than for individuals on homologous mRNA regimens (see Table1). If within-group comparisons were restricted to regimens with the same interval, differences between the respective groups remain the same as those indicated in Fig.2.

Apart from IFN, we also analyzed spike-specific induction of the cytokines TNF and IL-2. As with IFN, differences between the groups were similar for CD4 T cells producing TNF or IL-2 (Fig.3a, b), or for cells producing any of the three cytokines alone or in combination (Fig.3c). This also held true for spike-specific CD8 T cells, except for IL-2 producing CD8 T cells, where levels were generally lower and only showed subtle differences between the groups (Fig.3b). To assess functionality on a single cell level, cytokine profiles of spike-specific CD4 and CD8 T cells were characterized after Boolean gating (Supplementary Fig.3). This allowed distinction of seven subpopulations including polyfunctional cells simultaneously expressing all three cytokines, two cytokines or one cytokine only (Fig.4). The cytokine-expression profiles showed significant differences between the vaccine regimens, and the highest percentage of polyfunctional CD4 T cells was observed for the three vector-primed regimens. These three regimens also showed the highest percentage of CD8 T cells expressing IFN and TNF, which was the dominant fraction among spike-specific CD8 T cells (Fig.4a). The differences in cytokine-expression profiles were spike-specific, as SEB-reactive cytokine expression did not differ among the groups (Fig.4b).

Levels of TNF and IL-2-expressing T cells and combined expression of either of the cytokine IFN, TNF and/or IL-2 were compared between individuals who either received homologous ChAdOx vaccination (n=62), heterologous ChAdOx/BNT vaccination (n=66), heterologous ChAdOx/mRNA-1273-vaccination (n=101), homologous BNT vaccination (n=43) or homologous mRNA-1273 vaccination (n=58). Percentages of CD69+ TNF+ (a), CD69+ IL-2+ b or CD69-positive cells co-expressing at least one of the cytokines TNF, IL-2, or IFN (c) among total CD4 (upper panel) or CD8 T cells (lower panel) were determined after stimulation of whole blood samples with overlapping peptides of SARS-CoV-2 spike protein and subtraction of background reactivity from negative control stimulations. Bars represent medians with interquartile ranges and two-sided KruskalWallis test with Dunns multiple comparisons post-test was used to calculate differences between the groups. Source data are provided as a Source Data file. IFN interferon, IL interleukin, SEB Staphylococcus aureus enterotoxin B, TNF tumor necrosis factor.

After antigen-specific stimulation (a) or polyclonal stimulation with Staphylococcus aureus enterotoxin B (SEB, b) of whole blood samples from individuals with different homologous or heterologous vaccination regimens, cytokine-expressing CD4 and CD8 T cells were subclassified into seven subpopulations according to single or combined expression of IFN, IL-2, and TNF. Blood samples from all individuals were analyzed. To ensure robust statistics, only samples with at least 30 cytokine-expressing CD4 or CD8 T cells after normalization to the negative control stimulation were considered (with the number of samples in each vaccine group indicated in the figures). Bars in a and b represent means and standard deviations, and ordinary one-way ANOVA tests were performed. Source data are provided as a Source Data file. IFN interferon, IL interleukin, TNF tumor necrosis factor.

Local and systemic adverse events within the first week after the first and the second vaccination were self-recorded using a questionnaire (Fig.5 and Supplementary Tables4 and 5). Irrespective of the vaccine type, local adverse events such as pain at the injection site were reported with similar frequency in individuals after the first vaccination. Swelling at the injection site was overall less frequently observed with the lowest percentage among BNT-primed individuals (Fig.5b). Systemic adverse events including fever, headache, fatigue, chills, gastrointestinal manifestations, myalgia, and arthralgia after priming were most frequent in individuals after ChAdOx vaccination, which also was associated with more frequent use of antipyretic medication (Fig.5c and Supplementary Table4). After the second vaccination, local adverse events were least frequent after homologous ChAdOx vaccination, and most frequent in both heterologous and in the homologous mRNA-1273 regimens. The occurrence of systemic adverse events clearly dominated in individuals after heterologous boosting with mRNA-1273, followed by homologous mRNA-1273 vaccination and heterologous BNT-boosting (Fig.5a, c, Supplementary Table5). Individual perception of severity was scored higher after secondary vaccination in both homologous mRNA regimens (Fig.5d). In contrast, more than 75% of subjects after both the homologous ChAdOx and heterologous BNT vaccination were more affected by the primary vaccination with the vector. Despite the strong reactogenicity after vector-priming, it was interesting to note that a sizable fraction of subjects after heterologous boosting with mRNA-1273 was more severely affected by the secondary vaccination, which contrasts with observations in the heterologous BNT vaccine group. Likewise, among individuals after homologous vaccination, the second vaccination with mRNA-1273 was more frequently perceived as more severe, although this vaccine was already strongly reactogenic after the primary vaccination. Overall, it therefore appeared that both the homologous and the heterologous regimens that included BNT were better tolerated than the respective mRNA-1273 regimens.

According to their COVID-19 vaccine regimens, individuals were classified into three groups after dose 1 (ChAdOx vector (n=229), BNT (n=43) or mRNA-1273 vaccine (n=58)) and five groups after dose 2 (homologous: ChAdOx/ChAdOx, n=62; BNT/BNT, n=43; mRNA-1273/mRNA-1273, n=58; heterologous: ChAdOx/BNT, n=66; ChAdOx/mRNA-1273, n=101). Self-reported reactogenicity within the first week after each vaccine dose was assessed using a standardized questionnaire. The presence of local or systemic adverse events in general (a), substantial local (b) or systemic adverse events (c), and individual perception of which of the two vaccinations affected more (d) are shown. Statistical analyses of differences between the groups after the first and the second vaccination are shown in Supplementary Tables2 and 3. Source data are provided as a Source Data file.

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Comparative immunogenicity and reactogenicity of heterologous ChAdOx1-nCoV-19-priming and BNT162b2 or mRNA-1273-boosting with homologous COVID-19...