Heterologous prime-boost: breaking the protective immune response bottleneck of COVID-19 vaccine candidates

Abstract

COVID-19 vaccines emerging from different platforms differ in efficacy, duration of protection, and side effects. To maximize the benefits of vaccination, we explored the utility of employing a heterologous prime-boost strategy in which different combinations of the four types of leading COVID-19 vaccine candidates that are undergoing clinical trials in China were tested in a mouse model. Our results showed that sequential immunization with adenovirus vectored vaccine followed by inactivated/recombinant subunit/mRNA vaccine administration specifically increased levels of neutralizing antibodies and promoted the modulation of antibody responses to predominantly neutralizing antibodies. Moreover, a heterologous prime-boost regimen with an adenovirus vector vaccine also improved Th1-biased T cell responses. Our results provide new ideas for the development and application of COVID-19 vaccines to control the SARS-CoV-2 pandemic.

Keywords: COVID-19 vaccine; Heterologous prime-boost; T cell response; neutralizing antibody; th1/th2 balance.

Conflict of interest statement

No potential conflict of interest was reported by the author(s).

Figures

Figure 1.

Comparison of humeral immune responses induced by COVID-19 vaccines of different technology platforms and heterologous prime-boost regimens. (A). Schematic representation of experimental protocols and immunization groups. Mice in 9 groups were immunized with different COVID-19 vaccines or vaccine combinations: rAd, 2 × INA, 2 × rRBD,, rAd > INA, INA > rAd, rAd > rRBD, rRBD > rAd, INA > rRBD, rRBD > INA. (rAd: recombinant Ad5 vectored vaccine, INA: inactivated vaccine, rRBD: recombinant RBD vaccine), Mice in a blank control group were sham-vaccinated with PBS. For rAd group, mice were immunized with one dose of rAd vaccine and blood samples were collected 14 days post -vaccination; for other groups, blood samples were only collected 14 days after the second vaccine dose (B,C). Serum Nab levels measured by live SARS-CoV-2 virus (B) and pseudovirus (C). NAb titres are expressed as 50% inhibitory dilution (EC50) of serum. D. Spike-specific binding IgG titres were measured by ELISA (n = 8–10 per group, one spot represents one sample). Bars represent means ± SD, **p < 0.01, ****p < 0.0001, ns: p > 0.05.

Figure 1.

Comparison of humeral immune responses induced by COVID-19 vaccines of different technology platforms and heterologous prime-boost regimens. (A). Schematic representation of experimental protocols and immunization groups. Mice in 9 groups were immunized with different COVID-19 vaccines or vaccine combinations: rAd, 2 × INA, 2 × rRBD,, rAd > INA, INA > rAd, rAd > rRBD, rRBD > rAd, INA > rRBD, rRBD > INA. (rAd: recombinant Ad5 vectored vaccine, INA: inactivated vaccine, rRBD: recombinant RBD vaccine), Mice in a blank control group were sham-vaccinated with PBS. For rAd group, mice were immunized with one dose of rAd vaccine and blood samples were collected 14 days post -vaccination; for other groups, blood samples were only collected 14 days after the second vaccine dose (B,C). Serum Nab levels measured by live SARS-CoV-2 virus (B) and pseudovirus (C). NAb titres are expressed as 50% inhibitory dilution (EC50) of serum. D. Spike-specific binding IgG titres were measured by ELISA (n = 8–10 per group, one spot represents one sample). Bars represent means ± SD, **p < 0.01, ****p < 0.0001, ns: p > 0.05.

Figure 2.

Humeral immune responses induced by heterologous prime-boost regimen of adenovirus vectored and mRNA-based COVID-19 vaccine. (A). Schematic representation of experimental protocol and immunization groups. Mice in 4 groups were immunized with adenovirus vectored vaccine or mRNA vaccine: rAd, 2 × mRNA, rAd > mRNA, mRNA > rAd. (rAd: recombinant Ad5 vectored vaccine, RNA: mRNAbased vaccine). For the rAd group, mice were immunized with one dose of rAd vaccine and blood samples were collected 14 days post-vaccination; for other groups, bloods were collected 14 days post the second vaccine dose. (B). NAbs of serum measured by live SARS-CoV-2 virus and expressed as 50% inhibitory dilution (EC50) of serum. (C). Spike-specific binding IgG titres were measured by ELISA (n = 8–10 per group, one spot represents one sample). Bars represent means ± SD, **p < 0.01, ****p < 0.0001, ns: p > 0.05.

Figure 2.

Humeral immune responses induced by heterologous prime-boost regimen of adenovirus vectored and mRNA-based COVID-19 vaccine. (A). Schematic representation of experimental protocol and immunization groups. Mice in 4 groups were immunized with adenovirus vectored vaccine or mRNA vaccine: rAd, 2 × mRNA, rAd > mRNA, mRNA > rAd. (rAd: recombinant Ad5 vectored vaccine, RNA: mRNAbased vaccine). For the rAd group, mice were immunized with one dose of rAd vaccine and blood samples were collected 14 days post-vaccination; for other groups, bloods were collected 14 days post the second vaccine dose. (B). NAbs of serum measured by live SARS-CoV-2 virus and expressed as 50% inhibitory dilution (EC50) of serum. (C). Spike-specific binding IgG titres were measured by ELISA (n = 8–10 per group, one spot represents one sample). Bars represent means ± SD, **p < 0.01, ****p < 0.0001, ns: p > 0.05.

Figure 3.

T cell responses to SARS-CoV-2 spike peptides measured by IFN-γ ELISPOT. 6 mice in Figure 1 were sacrificed and T cell response were measured. Isolated lymphocytes were stimulated with 4 peptide pools spanning spike respectively, and the IFN-γ secreting cells were quantified by ELISPOT assay. (n = 6 per group, one spot represents one sample). Bars represent means ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 3.

T cell responses to SARS-CoV-2 spike peptides measured by IFN-γ ELISPOT. 6 mice in Figure 1 were sacrificed and T cell response were measured. Isolated lymphocytes were stimulated with 4 peptide pools spanning spike respectively, and the IFN-γ secreting cells were quantified by ELISPOT assay. (n = 6 per group, one spot represents one sample). Bars represent means ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 4.

Multiplex cytokine analysis for different immunization regimens. Isolated lymphocytes in 9 regimens identified in Figure 1 were stimulated with 4 spike peptide pools. Supernatants pooled by different groups (A) or different peptide pools (B) were collected. IL-2, IL-4, IL-10 and TNF-α levels in supernatants were measured by MSD; the concentration of each cytokine(pg/mL) is represented by histogram (A) or heatmap (B) (n = 6 per group). Bars represent means ± SEM, *p < 0.05, **p < 0.01, ****p < 0.0001.

Figure 4.

Multiplex cytokine analysis for different immunization regimens. Isolated lymphocytes in 9 regimens identified in Figure 1 were stimulated with 4 spike peptide pools. Supernatants pooled by different groups (A) or different peptide pools (B) were collected. IL-2, IL-4, IL-10 and TNF-α levels in supernatants were measured by MSD; the concentration of each cytokine(pg/mL) is represented by histogram (A) or heatmap (B) (n = 6 per group). Bars represent means ± SEM, *p < 0.05, **p < 0.01, ****p < 0.0001.