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. 2023 Mar 10;4(2):e238.
doi: 10.1002/mco2.238. eCollection 2023 Apr.

Heterologous vaccination with subunit protein vaccine induces a superior neutralizing capacity against BA.4/5-included SARS-CoV-2 variants than homologous vaccination of mRNA vaccine

Dandan Peng  1 , Tingmei Zhao  1 , Weiqi Hong  1 , Minyang Fu  1 , Cai He  1 , Li Chen  1 , Wenyan Ren  1 , Hong Lei  1 , Jingyun Yang  1 , Aqu Alu  1 , Yanghong Ni  1 , Jian Liu  1 , Jiong Li  1 , Wei Wang  1 , Guobo Shen  1 , Zhiwei Zhao  1 , Li Yang  1 , Jinliang Yang  1 , Zhenling Wang  1 , Yoshimasa Tanaka  2 , Guangwen Lu  1 , Xiangrong Song  1 , Xiawei Wei  1
Affiliations

Heterologous vaccination with subunit protein vaccine induces a superior neutralizing capacity against BA.4/5-included SARS-CoV-2 variants than homologous vaccination of mRNA vaccine

Dandan Peng et al. MedComm (2020). .

Abstract

BA.4 and BA.5 (BA.4/5), the subvariants of Omicron, are more transmissible than BA.1 with more robust immune evasion capability because of its unique spike protein mutations. In light of such situation, the vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is in desperate need of the third booster. It has been reported that heterologous boosters might produce more effective immunity against wild-type SARS-CoV-2 and the variants. Additionally, the third heterologous protein subunit booster should be considered potentially. In the present study, we prepared a Delta full-length spike protein sequence-based mRNA vaccine as the "priming" shot and developed a recombinant trimeric receptor-binding domain (RBD) protein vaccine referred to as RBD-HR/trimer as a third heterologous booster. Compared to the homologous mRNA group, the heterologous group (RBD-HR/trimer vaccine primed with two mRNA vaccines) induced higher neutralizing antibody titers against BA.4/5-included SARS-CoV-2 variants. In addition, heterologous vaccination exhibited stronger cellular immune response and long-lasting memory response than the homologous mRNA vaccine. In conclusion, a third heterologous boosting with RBD-HR/trimer following two-dose mRNA priming vaccination should be a superior strategy than a third homologous mRNA vaccine. The RBD-HR/trimer vaccine becomes an appropriate candidate for a booster immune injection.

Keywords: SARS‐CoV‐2; heterologous vaccination; mRNA vaccine; recombinant RBD vaccine.

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Conflict of interest statement

This work was supported by the WestVac Biopharma Co. Ltd. Jiong Li, Wei Wang, Guobo Shen, Zhiwei Zhao, Li Yang, Jinliang Yang, Zhenling Wang, Guangwen Lu, and Xiawei Wei are also working at the WestVac Biopharma Co. Ltd. The remaining authors declare no conflict of interests.

Figures

FIGURE 1
FIGURE 1
Preparation and characterization of mRNA‐loaded liposome and recombinant receptor‐binding domain (RBD)–HR/trimer protein: (A) the schematic of Delta full‐length spike mRNA construct with mutations; (B) preparation of the mRNA vaccine. Alcohol phases formed by cholesterol, DMG‐PEG2000, DOPE, and E1‐1 lipid were mixed with mRNA aqueous phase at a proper ratio to formulate mRNA vaccine; (C) schematic of the recombinant RBD–HR/trimer protein construct. Our protein comprises an RBD derived from Delta with L452R and T478K mutations, HR1, and HR2 domains. Domains and elements are marked. HR1 and HR2, heptad repeats 1 and 2; NTD, N‐terminal domain; RBD, receptor binding domain. An N‐terminal GP67 signal peptide was designed for secretion. Parts (A) and (B) were created by BioRender.
FIGURE 2
FIGURE 2
The schematic diagram and anti‐IgG responses of heterologous and homologous immunization processes: (A) the schematic diagram of the immunization program. NIH mice were intramuscularly immunized with 1, 5, or 10 µg mRNA vaccine on days 0 and 21. A third heterologous booster, receptor‐binding domain (RBD)–HR/trimer (10 µg), or a homologous booster mRNA vaccine, was vaccinated on day 42, for the long‐term immunization, mRNA vaccine was injected on days 0 and 21, and RBD–HR/trimer booster was given on day 111. mRNA group: three doses of mRNA vaccine; RBD–HR/trimer group: three doses of recombinant RBD–HR/trimer vaccine; mRNA + RBD–HR/trimer group: two doses of mRNA vaccine followed by a heterologous booster with RBD–HR/trimer. The serum samples were collected on day 56 (B) and day 84 (C); (B) the endpoint titers of RBD‐specific IgG in sera collected on day 56 were assayed by enzyme‐linked immunosorbent assay (ELISA); (C) the detection of the titer of anti‐RBD IgG in serum samples collected 125 days after the third dose was performed by ELISA. Part (A) was created by BioRender. The significance of differences among groups was conducted by a One‐way ANOVA analysis followed by Tukey's multiple comparison post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.
FIGURE 3
FIGURE 3
Heterologous group induces stronger broad‐spectrum neutronization response against BA.4/5‐included Omicron variants: (A and B) broad‐spectrum neutronization responses against various pseudoviruses were determined; (C) neutralizing antibody titers against BA.4/5‐included Omicron variants were determined; (D and E) representative images (D) and analysis (E) of flow cytometry represent the blockade of receptor‐binding domain (RBD)‐Fc (Omicron) binding to human ACE2 receptor; (F and G) the flow cytometry images (F) and analysis (G) of the inhibition of RBD‐Fc (Omicron) binding to human ACE2 receptor. NIH mouse were intramuscularly injected with 1 µg mRNA in parts (A)–(C) and 5 µg mRNA in parts (D)–(G) in the homologous mRNA and heterologous groups. Serum samples in parts (A), (C), and (D) were collected on day 84, and serum samples in parts (B) and (F) were collected 153 days after the first dose (long‐term groups). One‐way ANOVA analysis followed by Tukey's multiple comparison post hoc test was conducted in parts (A)–(C), (E), and (G). All error bars represent SEM about the mean. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.
FIGURE 4
FIGURE 4
Heterologous vaccine elicits a stronger T cell immune response in vivo. Lymphocytes were collected from isolated spleens and stimulated with receptor‐binding domain (RBD) (10 µg/mL) proteins for 72 h: (A) representative flow cytometry dot plots of RBD‐specific IL‐4‐producing CD4+ (top) and CD8+ (bottom) memory T cells (CD44high B220−MHC‐II−CD4+ and CD44high B220−MHC‐II−CD8+); (B) the levels of IL‐4 in cell supernatant were analyzed by enzyme‐linked immunosorbent assay (ELISA); (C) the flow cytometry images of RBD‐specific CD4+IFN‐γ+ and CD8+ IFN‐γ+ memory T cells (CD44high B220−MHC‐II−CD4+IFN‐γ+ and CD44high B220−MHC‐II−CD8+IFN‐γ+); (D) the levels of RBD‐specific IFN‐γ‐producing CD4+ (top) and CD8+ (bottom) memory T cells; (E ‐ G) The percentage of RBD‐specific IL‐2‐producing memory T cells (CD44high B220−MHC‐II−CD4+IL‐2+ and CD44high B220−MHC‐II−CD8+ IL‐2+) in the spleen was detected by flow cytometry; (H and I) the percentage of CD4+ (G) or CD8+ (H) effect memory T cells (CD4+CD44+CD62L− or CD8+CD44+CD62L−) in lymph node was determined.
FIGURE 5
FIGURE 5
Heterologous vaccination induces further long‐lasting memory response in NIH mice: (A) typical pseudocolor flow cytometry plots showing Tfh cells; (B) typical pseudocolor flow cytometry plots showing plasmablast cells; (C–E) frequencies of the Tfh (C), plasmablast (D), and GC B (E) cells in lymph nodes were analyzed. The significance of differences among groups was conducted by a One‐way ANOVA analysis followed by Tukey's multiple comparison post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.
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