Prime-boost vaccination elicited RBD-specific CD4
+
T and CD8
+
T cells
The RBD-specific CD4+T and CD8+T cells responses were measured with a flow cytometry T cell receptor (TCR) dependent activation-induced marker (AIM) assay using SARS-CoV-2 RBD protein. We found that a prominent increase in AIM+(CD137+OX40+) CD4+T cells on day 40 following prime vaccination and stronger on day 60 after booster vaccination (p = 0.0001, p < 0.0001, Fig. 1B, C). The frequency of AIM+CD4+T cells on day 180 (six months) after prime vaccination was significantly decreased (p = 0.02). We found a similar pattern with AIM+(CD69+CD137+) CD8+T cells after vaccination (p = 0.01, p = 0.03, Fig.1B, D). However, the frequency of the AIM+CD4+T cells at each study timepoint remained significantly higher than that of AIM+CD8+T cells. In agreement with previous studies [27, 28], we detected AIM+CD4+T and CD8+T cells in 6% and 10% of unvaccinated individuals, respectively, which may be attributed to cross-reactive T cells that were probably generated during previous encounters with seasonal coronaviruses. In general, CoronaVac vaccine induced the activities of RBD-specific CD4 and CD8+T cells following prime and boost vaccination.
CoronaVac vaccine triggered polyfunctional CD4
+
T cells response with a predominant Th1 and weak Th17 helper response against RBD protein
To assess functionality and polarization of the RBD-specific CD4 and CD8+T cells responses after prime and boost vaccination, we measured by intracellular cytokine staining (ICS) analysis in response to a 24 h stimulation of PBMC with RBD protein. For CD4+T cell polarization we observed dominant Th1 cells and modestly weaker induction of Th17 cells on day 40 after the prime two dose of CoronaVac vaccine (p < 0.000, p = 0.04, Fig. 2B1, B3). The same phenomenon was observed following booster vaccination on day 60 (p < 0.000, p = 0.04). However, we did not observed a polarization of CD8+T cells (p > 0.05, Fig. 2C, D). We measured the cytokines secreted into the supernatant by ELISA. The result showed that vaccine led to a robust increase in IFN-γ, IL-2 and TNF-α levels (p = 0.003, p < 0.0001, p = 0.0003, Fig. 2E1–E3) and modestly increased the level of IL-17A (p = 0.03, Fig. 2E4) on day 40 and 60 compared to controls. Cytokine IL-4 did not generated remarkable difference after vaccination (p > 0.05, Fig. 2E5). Additionally, we detected a few cells expressed TNF-α, IL-2, and IL-17A (p = 0.004, p = 0.03, Fig. 2E2–E4) in unvaccinated individuas, indicating may be exist the preexisting cross-reactive memory to SARS-CoV-2.
To qualitatively assess RBD-specific CD4 and CD8+T cells for polyfunctional responses after prime and boost vaccination, we performed coexpression analysis using Boolean gating. Dominant RBD-specific CD4+T and CD8+T cells on day 40 and 60 expressed IFN-γ, IL-2, or TNF-α alone or combination with each other (Fig. 2F, G). A small CD4+T cell groups expressed IL-17, IL-4, or both. On day 180, the proportion of CD4+T cells expressing one and two cytokines remarkable decreased. However, response on day 180 dominated by CD8+T cells expressing single cytokines. The function of coexpressing two cytokines was more stronger in the CD4+T cells than the CD8+T cells. In summary, these results demonstrated that CoronaVac vaccine mainly induced functional CD4+T cell responses in most vaccination individuals after prime and boost vaccination, with a predominant Th1 and a weak Th17 polarization of the helper response.
CoronaVac vaccine triggered predominant IgG1 antibody response and effectively recalled specific antibodies to RBD protein after booster vaccination
In order to study the SARS-CoV-2 RBD-specific antibodies responses following prime-boost CoronaVac vaccine. We detected RBD-specific IgG, IgM, IgA, and IgG subsets antibodies responses. Anti-RBD IgG and IgM antibodies were detected in 100% (29/29) (Fig. 3A) and 83% (24/29) (Fig. 3B) of subjects on day 40 and the levels were remarkable reduction up to 6 months after the 2nd dose vaccination, for 28% (8/29) and 38% (11/29) of subjects. These responses rates increased to 100% (29/29) and 93% (27/29) after booster vaccination. IgA antibody were detected in 83% (24/29) of subjects on day 40 after the 2nd dose vaccination (Fig. 3C). However, it's almost undetectable up to 6 months after 2nd dose vaccination. This response rate increased to 48% (14/29) after booster vaccination. In addition, we analyzed the IgG subclass against SARS-CoV-2 RBD protein (Fig. 3D). The IgG1 subclass was detected as the major antibody subclass after vaccination. The titers of RBD-IgG and IgM were categorized as 1:80, 1:160, 1:320, 1:640, 1:1280, 1:2560, and 1:5120. Titers less than 1:80 are considered as negative, 1:80–1:160 as low titers, 1:320–1:640 as moderate titers, and 1:1280 and ≥ 1:2560 as high titers [33]. As shown in Fig. 3E, F, the titers of RBD-IgG and IgM on day 40 after the 2nd dose vaccination displayed high titers (p < 0.0001). However, there was a remarkable reduction up to 6 months after the 2nd dose vaccination and the titers still presented high titers (p < 0.0001, p = 0.001). Booster vaccination obviously increased the IgG and IgM tilters (p < 0.0001, p = 0.001). These results demonstrated CoronaVac vaccine triggered predominant IgG1 antibody response and effectively recalled specific antibodies to RBD protein after booster vaccination.
Elicitation of robust memory B cell responses to SARS-CoV-2 RBD protein following booster vaccination
Since maintaining extensive protective antibodies and RBD-specific memory B cells are key features of long-term protective immunity, we evaluated whether vaccine-induced memory B cells can produce effective antibodies after activation. Peripheral blood mononuclear cells (PBMCs) were stimulated with human memory B-cell stimpack to differentiate memory B cells into antibody-secreting cells (ASCs).As the ELISPOT result, the frequency of RBD-specific memory B cells on day 40 following prime vaccination (p < 0.0001, Fig. 4B, C) presented a relative increase compared with the unvaccinated cohort and was still detected up to 6 months post-vaccination (p < 0.0001). The booster vaccination caused stranger memory B cells response than prime (p < 0.0001). We detected the antibody IgG secreted by RBD-specific memory B cells in the cultural supernatant (Fig. 4D). Anti-RBD IgG antibodies were detected in the cultural supernatant from prime (p = 0.001, p < 0.0001) and booster vaccination cohorts (p < 0.0001). This indicated that vaccine-induced memory B cells sustained at least six months after prime vaccination, and booster vaccination was imperative for long-term humoral immune memory to agaist SARS-CoV-2.
Vaccine-induced CD4
+
T cell responses correlated with CD8
+
T cell and humoral responses
CD4+T cells play an importantly auxiliary role in CD8+T and humoral responses. Th1 cells primarily promote CD8+T cell responses, whereas Th2 cells help foster humoral immune response. Therefore, we assessed the relationship among AIM+CD4, AIM+CD8, Th1, Th2, RBD-specific memory B cells, IgG, IgM, and IgA antibosies. We observed a strong correlation between total AIM+CD4 and AIM+CD8 cells after vaccination (r = 0.7147, 0.3258, p < 0.0001, p = 0.04, Fig. 4E–G). In addition, the frequency of RBD-specific memory B cells correlated with AIM+CD4 cells (r = 0.7083, p < 0.0001), however, were less well correlated with AIM+CD8 cells on day 40 after prime vaccination (r = 0.4345, p = 0.02, Fig. 4E). Notably, we observed that AIM+CD4 cells correlated with IgG and IgA (r = 0.6168, 0.5519, p = 0.0006, 0.003, Fig. 4E) and RBD-specific memory B cells correlated with IgG (r = 0.2775, p = 0.003, Fig. 4E). We also observed strong correlation among IgA, IgG, and IgM after booster vaccination (r = 0.4987, 0.6935, p = 0.007, p < 0.0001, Fig. 4G). These results indicated that vaccine-induced CD4+T cell responses may augment and coordinate the CD8+T and humoral responses.
Elicitation of broad and complicated cytokine immune profiles in plasma at early stage following prime and boost vaccination
Cytokine profiles in plasma from subjects after prime and booster vaccination were analysed using the Cytometric Bead Array (CBA). Th1 type cytokines IFN-γ, TNF-α, IL-12p70, IL-2, Th2 type cytokines IL-4, Th17 type cytokines IL-17A, immunoregulation cytokine IL-10, and proinflammatory cytokine IL-6, IL-8 and IL-1β were among the cytokines analysed. IL-8 and IL-12p70 were the most abundantly secreted cytokines from all individuals (Fig. 5A). TNF-α and IL-17A were abundantly secreted cytokines in the 40d dose2 cohort (p < 0.0001) and the 60d dose3 cohort (p = 0.02, p = 0.001), which was consistant with ICS result. IL-12p70, a key cytokine that initiated the Th1 response, was remarkable increased (p < 0.0001) following booster vaccination.
The cytokine profile correlation matrix of each group was established to obtain the following results: (1) In the unvaccinated cohort, we did not found correlations among these cytokines (Fig. 5B). (2) The cluster in the 40d dose2 cohort: cluster one consisted of IL-10, IL-6, IL-4, IL-17A, IFN-γ, and TNF-α, while cluster two consisted of IL-10, IL-6, IL-4, and IL-2 (Fig. 5C). (3) In the 180d dose2 cohort, we did not found correlations among these cytokines (Fig. 5D). (4) In the 60d dose3 cohort, a big cluster of cytokines showed correlations. A cluster consisting of IFN-γ, IL-10, IL-6, IL-4, IL-12p70, TNF-α, and IL-2 presented strong correlations (Fig. 5E). These findings indicated that CoronaVac vaccine induced a strong correlation among cytokines at the early stage after prime and booster vaccination, but the correlation and complexity among cytokines gradually decreases over time.