Development of Bivalent mRNA Vaccines against SARS-CoV-2 Variants

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected billions of individuals and is the cause of the current global coronavirus disease 2019 (COVID-19) pandemic. We previously developed an mRNA vaccine (LVRNA009) based on the S protein of the Wuhan-Hu-1 strain; the phases I and II clinical trials showed that LVRNA009 has a promising safety and immunogenicity profile. In order to counteract the immune escape by SARS-CoV-2 variants of concern, a panel of mRNA vaccines was developed based on the S proteins of the Wuhan-Hu-1, Delta, Omicron BA.1, BA.2, and BA.5 strains, and each vaccine’s protective potency against the virus variants was evaluated. Furthermore, to achieve excellent neutralization against SARS-CoV-2 variants, bivalent vaccines were developed and tested against the variants. We found that the monovalent Wuhan-Hu-1 or the Delta vaccines could induce high level of neutralization antibody and protect animals from the infection of the SARS-CoV-2 Wuhan-Hu-1 or Delta strains, respectively. However, serum samples from mice immunized with monovalent Delta vaccine showed relatively low virus neutralization titers (VNTs) against the pseudotyped virus of the Omicron strains. Serum samples from mice immunized with bivalent Delta/BA.1 vaccine had high VNTs against the pseudotyped Wuhan-Hu-1, Delta, and BA.1 strains but low VNTs against BA.2 and BA.5 (p < 0.05). Serum samples from mice immunized with Delta/BA.2 vaccine had high VNTs against the pseudotyped Wuhan-Hu-1, Delta, BA.1 and BA.2 strains but low VNTs against BA.5. Finally, serum samples from mice immunized with Delta/BA.5 vaccine had high VNTs against all the tested pseudotyped SARS-CoV-2 strains including the Wuhan-Hu-1, Delta, and Omicron variants (p > 0.05). Therefore, a bivalent mRNA vaccine with Delta/BA.5 combination is promising to provide broad spectrum immunity against all VOCs.

Keywords: SARS-CoV-2 variants; mRNA vaccine; neutralizing antibody; spike protein.

Figure 1

Design and characterization of the mRNA vaccines against different SARS-CoV-2 variants. (A) Schematic diagram of the target spike (S) protein-antigen encoded by mRNA vaccines against the Wuhan-Hu-1, Delta, Omicron BA.1, BA.2, and BA.5 variants. The mutation and/or deletion of amino acids are labeled. NTD, N-terminal domain. RBD, receptor-binding domain. FP, fusion peptide. HR1, heptad repeat 1. CH, central helix. (B) The expression of the S and S1 proteins in HEK293 cells transfected with IVT mRNA was detected by western blotting. (C) HEK293 cells were transfected with IVT mRNA, and hACE2-binding cells were analyzed by flow cytometry. (D) Spherical nanoparticles were visualized under a transmission electron microscope. Scale bar, 50 nm.

Figure 2

The Wuhan-Hu-1 and Delta vaccines exerted robust protection against SARS-CoV-2. (A) Experimental strategy. (B) Ad5-hACE2 mice (Wuhan-Hu-1; left panel) or Syrian hamsters (delta; right panel) were immunized twice with low-dose (5 μg/dose) or high-dose (15 or 25 μg/dose) of Wuhan-Hu-1 or Delta vaccine on day 0 and 14. The neutralizing antibody titers against SARS-CoV-2 in animals were tested by PRNT. Ad5-hACE2 mice, n = 4. Syrian hamsters, n = 6. *** p < 0.001 (C) Ad5-hACE2 mice or Syrian hamsters in the low- and high-dose groups were challenged with SARS-CoV-2 Wuhan-Hu-1 or Delta. The viral load was analyzed and expressed as FFU per gram of tissue in the lung (Ad5-hACE2 mice; at 4 dpi; left panel) or both lungs and trachea (Syrian hamsters; at 5 dpi; right panel). LOD, Low-limit of Detection. Ad5-hACE2 mice, n = 4. Syrian hamsters, n = 6. * p < 0.05, *** p < 0.001 (D) H&E staining was used to examine the histopathological changes in lung tissues in Ad5-hACE2 mice (Wuhan-Hu-1; at 4 dpi; left panel) or Syrian hamsters (Delta; at 5 dpi; right panel). Scale bar, 100 µm.

Figure 3

Profile of neutralizing antibodies produced by monovalent mRNA vaccines against SARS-CoV-2 variants in mice and Cynomolgus monkeys. (A) Cynomolgus monkeys were immunized twice with monovalent vaccines against Wuhan-Hu-1 strain, Delta strain or PBS on days 0 and 21. The VNTs were measured using a pseudovirus neutralization assay 35 days after the first immunization; n = 10. * p < 0.05; **** p < 0.0001; ns, no significance. (B) BALB/c mice were immunized twice with monovalent vaccines against Wuhan-Hu-1, Delta, Omicron BA.1, BA.2, BA.5 strain or PBS on days 0 and 14. The VNTs were measured using a pseudovirus neutralization assay 21 days after the first immunization; n = 3. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, no significance.

Figure 4

Bivalent vaccine produced high level neutralizing antibodies against the SARS-CoV-2 variants. BALB/c mice were immunized twice with bivalent vaccines (Delta/BA.1, Delta/BA.2, Delta/BA.5) or PBS on day 0 and 14. The VNTs were measured via the pseudovirus neutralization assay on day 21; n = 3. * p < 0.05; ** p < 0.01; ns, no significance.