Heterologous prime-boost immunisation with mRNA- and AdC68-based 2019-nCoV variant vaccines induces broad-spectrum immune responses in mice

Abstract

The ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 or 2019-nCoV) variants has been associated with the transmission and pathogenicity of COVID-19. Therefore, exploring the optimal immunisation strategy to improve the broad-spectrum cross-protection ability of COVID-19 vaccines is of great significance. Herein, we assessed different heterologous prime-boost strategies with chimpanzee adenovirus vector-based COVID-19 vaccines plus Wuhan-Hu-1 (WH-1) strain (AdW) and Beta variant (AdB) and mRNA-based COVID-19 vaccines plus WH-1 strain (ARW) and Omicron (B.1.1.529) variant (ARO) in 6-week-old female BALB/c mice. AdW and AdB were administered intramuscularly or intranasally, while ARW and ARO were administered intramuscularly. Intranasal or intramuscular vaccination with AdB followed by ARO booster exhibited the highest levels of cross-reactive IgG, pseudovirus-neutralising antibody (PNAb) responses, and angiotensin-converting enzyme-2 (ACE2)-binding inhibition rates against different 2019-nCoV variants among all vaccination groups. Moreover, intranasal AdB vaccination followed by ARO induced higher levels of IgA and neutralising antibody responses against live 2019-nCoV than intramuscular AdB vaccination followed by ARO. A single dose of AdB administered intranasally or intramuscularly induced broader cross-NAb responses than AdW. Th1-biased cellular immune response was induced in all vaccination groups. Intramuscular vaccination-only groups exhibited higher levels of Th1 cytokines than intranasal vaccination-only and intranasal vaccination-containing groups. However, no obvious differences were found in the levels of Th2 cytokines between the control and all vaccination groups. Our findings provide a basis for exploring vaccination strategies against different 2019-nCoV variants to achieve high broad-spectrum immune efficacy.

Keywords: ARCoV; COVID-19; ChAdTS-S; SARS-COV-2 variants; heterologous prime-boost; intramuscular; intranasal.

Figure 1

Overall scheme of the immunisation strategies and experimental timeline using female BALB/c mice. (A) Mice in 15 groups were immunised with four COVID-19 vaccines using different protocols. Dashes indicate no booster vaccination. (B) Immunisation and immunological characterisation scheme. , vaccination; , bleeding; , spleen lymphocyte isolation. Im, intramuscular vaccination; in, intranasal vaccination.

Figure 2

IgG responses induced by AdW, AdB, ARW, and ARO vaccines that were administered using various protocols. All titres were measured on day 35 after primary immunisation. Serum IgG titres against (A) WH-1 and (B) B.1.1.529 spike protein (n = 4−5 per group; each spot represents one sample). Bars represent the geometric mean ± geometric SD; *P < 0.05; ***P < 0.001; ns, P > 0.05.

Figure 3

Pseudovirus-neutralising antibody (PNAb) titres were measured 35 days after primary immunisation. Serum NAb titres against (A) WH-1, (B) B.1.617.2, (C) B.1.1.529, and (D) BA.4/5 are expressed as 50% inhibitory dilutions (n = 4−5 per group; one spot represents one sample). Bars represent the geometric mean ± geometric SD; **P < 0.01; ***P < 0.001; ns, P > 0.05.

Figure 4

NAb titres were measured 49 days after primary immunisation. Serum PNAb titres against (A) WH-1, (B) B.1.617.2, (C) B.1.1.529, and (D) BA.4/5 are expressed as 50% inhibitory dilutions (n = 4−5 per group; one spot represents one sample). (E) SARS-CoV-2 NAb titration (n = 3 per group). Bars represent the geometric mean ± geometric SD; *P < 0.05; ***P < 0.001; ****P < 0.0001; ns, P > 0.05.

Figure 5

IgA responses induced by AdW, AdB, ARW, and ARO vaccines were administered using various protocols. All titres were measured on day 35 after primary immunisation. Serum IgA titres against (A) WH-1 and (B) B.1.1.529 spike protein (n = 4−5 per group; each spot represents one sample). Bars represent the geometric mean ± geometric SD; *P < 0.05; **P < 0.01; ***P < 0.001; ns, P > 0.05.

Figure 6

Neutralisation capacity of sera was observed by measuring the inhibition of binding between angiotensin-converting enzyme 2 (ACE2) and SARS-CoV-2 spike proteins on day 35 after primary immunisation. Spike proteins were from the SARS-CoV-2 prototype and B.1.1.7, B.1.351, B.1.526.1, B.1.617, B.1.617.1, B.1.617.2, B.1.617.3, P.1, and P.2 strains, respectively. Negative ACE2-binding inhibition rates are shown as zero (n = 5 per group). Bars represent the mean ± SD; numbers represent the mean of the corresponding group.

Figure 7

Neutralisation capacity of sera was observed by measuring the inhibition of binding between ACE2 and SARS-CoV-2 spike proteins on day 35 after primary immunisation. Spike proteins were from BA.2, BA.2.12.1, BA.2+L452M, BA.2+L452R, BA.3, BA.4, and BA.5 strains, respectively. Negative ACE2-binding inhibition rates are shown as zero (n = 5 per group). Bars represent the mean ± SD; numbers represent the mean of the corresponding group.

Figure 8

Cellular immune responses specific to SARS-CoV-2 spike proteins were measured 49 days after primary vaccination. Enzyme-linked immunospot (ELISpot) assays for IFN-γ after stimulation with the SARS-CoV-2 spike protein. Five mice per group were euthanised, and T-cell responses were measured. Lymphocytes were stimulated with (A) WH-1, (B) B.1.617.2, and (C) B.1.1.529 spike peptide pools spanning the entire spike protein sequence. Cells secreting IFN-γ were quantified using ELISpot assays (n = 5 per group; each point represents the mean number of spots from two wells per sample). Bars represent the geometric mean ± geometric SD; **P < 0.01; ****P < 0.0001; ns, P > 0.05.

Figure 9

Th1/Th2 skewing was detected via intracellular cytokine staining on day 49 after primary immunisation. Percentage of spike protein-specific IFN-γ-, IL-2-, and TNF-α-positive memory (A) CD4⁺ T and (B) CD8⁺ T cells was measured on day 49 after primary immunisation (n = 4 per group; each point represents one sample). Bars represent the geometric mean ± geometric SD; *P < 0.05; **P < 0.01; ***P < 0.001; ns, P > 0.05. The blue dashed lines represent the blank value.

Figure 10

Th1/Th2 skewing in immunised mice was measured using Meso Scale Discovery (MSD) cytokine profiling. Lymphocytes were stimulated with (A) WH-1, (B) B.1.617.2, and (C) B.1.1.529 spike peptide pools spanning the entire spike protein sequence for 24 h. IL-2 and TNF-α levels in supernatants were measured (n = 5 per group; each point represents one sample). Bars represent the geometric mean ± geometric SD; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, P > 0.05. The blue dashed lines represent the blank value.