Three SARS-CoV-2 spike protein variants delivered intranasally by measles and mumps vaccines are broadly protective

As the new SARS-CoV-2 Omicron variants and subvariants emerge, there is an urgency to develop intranasal, broadly protective vaccines. Here, we developed highly efficacious, intranasal trivalent SARS-CoV-2 vaccine candidates (TVC) based on three components of the MMR vaccine: measles virus (MeV), mumps virus (MuV) Jeryl Lynn (JL1) strain, and MuV JL2 strain. Specifically, MeV, MuV-JL1, and MuV-JL2 vaccine strains, each expressing prefusion spike (preS-6P) from a different variant of concern (VoC), were combined to generate TVCs. Intranasal immunization of IFNAR1−/− mice and female hamsters with TVCs generated high levels of S-specific serum IgG antibodies, broad neutralizing antibodies, and mucosal IgA antibodies as well as tissue-resident memory T cells in the lungs. The immunized female hamsters were protected from challenge with SARS-CoV-2 original WA1, B.1.617.2, and B.1.1.529 strains. The preexisting MeV and MuV immunity does not significantly interfere with the efficacy of TVC. Thus, the trivalent platform is a promising next-generation SARS-CoV-2 vaccine candidate.

The roles of T cell in protection against SARS-CoV-2 infection.Numerous studies have shown that T cell immune response play a critical roles in protection against SARS-CoV-2 infection 12,13 .
Antibodies derived from WA1 spike are not effective in neutralizing Omicron variants and subvariants.However, the conserved T cell epitopes in spikes may provide cross-protection against Omicron variant.For example, it was found that cynomolgus macaques immunized with the BNT162b2 and Ad.26.COV2.S had markedly lower Omicron-specific NAbs than WA1-specific NAbs, whereas Omicron-specific T cell responses were comparable to WA1/2020-specific T cell responses, indicating substantial cross-reactivity of cellular immune responses against SARS-CoV-2 BA1 14 .Moreover, it was reported that BNT162b2 and Ad.26.COV2.S provided 70% and 85% protection, respectively, against hospitalization with Omicron in South Africa, despite the absence of Omicron-specific NAbs 15 .In addition, Gao et et., showed SARS-CoV-2 S-specific CD4 + and CD8 + T cells elicited by BNT162b2 vaccination are also effective against B.1.1.529 16.
These data suggest that immune parameters other than NAb responses likely contribute to protection against severe disease.
Comparison of spleen T cell response with other studies.We also found that both i.n. and s.c.
immunization induced CD4 + and CD8 + T cells in splenocytes with a predominant Th1/17 response.
The immunization doses and animal models used in this study.Typically, an immunization dose of 10 5 -10 7 PFU or TCID50 was used for testing the efficacy of MeV and MuV-vectored vaccines in small animal models [17][18][19][20][21] .These doses used in small animal models seem higher than those (10 3 -10 4 PFU) used in MMR vaccine for humans.Since human is the natural host of MeV and MuV, a low dose in MMR formulation is sufficient to induce strong immune responses.The relatively higher dose used in rodent models is likely because the homology of viral entry receptors between rodents and humans is low 19 .Nonetheless, we found that 3×10 4 PFU of trivalent vaccine is sufficient to induce a strong immune response in hamsters (Fig. 8).In mouse experiment, a low dose (3×10 5 PFU) of trivalent vaccine included similar levels of T cell immune responses compared to the high dose (1.2×10 6 PFU) of trivalent vaccine (Fig. 5).A dose of 3×10 5 PFU is sufficient to induce a higher level of Trms (Fig. 5).It should be noted that neither MeV nor MuV infects immunocompetent mice efficiently.However, IFNAR -/-mice lacking type I interferon receptor are susceptible to infection by both MeV and MuV, and are the only mice models widely used for testing the efficacy of MeV-and MuV-based vaccines 22,23 .Despite the fact that these mice lack interferon responses, the trivalent vaccine induces robust antibody, T cell immune responses, and protection in these mice.Importantly, the immune responses and protective efficacy are verified in hamsters, the gold standard small animal model for testing SARS-CoV-2 vaccines.
Multivalent vaccine may generate broader immunity.The multivalent vaccine approach has been successful in preventing human papillomavirus virus (HPV).They protect against several HPV serotypes that can infect the genital area, as well as the mouth and throat 24 .The two main HPV vaccines currently in use are Gardasil 9 and Cervarix.Gardasil 9 protects against nine HPV types, including those that are associated with cervical, vulvar, vaginal, and anal cancers, as well as genital warts.The effectiveness of the HPV vaccines is notably high, up to 90% effective in preventing HPV-related cancers and diseases 25,26 .Using the MMR vaccine platform, trivalent or quadrivalent vaccines covering relevant circulating VoCs, updated each year, would likely provide the necessary depth and breadth of immune response for contemporary protection.

Fig. S15 .
Fig.S15.Comparison of NAb between TVC-VII and TVC-VIII.The serum NAb data in Fig.8F

Fig. S17 .
Fig.S17.Gating strategy for flow cytometry analysis of spleen T cells.The gating was set up to

Fig. S18 .
Fig.S18.Gating strategy for flow cytometry analysis of lung CD4 + T cells stimulated by S peptides.

Although
MeV and MuV are the respiratory viruses, they have different cell tropism and infect different cell types.The combination of MeV and MuV may provide have synergistic effects.This probably explains the observation that trivalent vaccines TVC-I and II induced higher serum IgG than rMuV-JL2-WA1 alone and that both low and high doses of TVC-IV induced higher lung Trm immune responses than rMuV-JL2-WA1 alone.Interestingly, we found that serum IgG, IgA, and NAb induced by TVC-VIII (3 viruses expressing 3 different spikes) were not significantly different from those of TVC-VII (3 viruses all expressing the same WA1 spike).These results also indicate that 3 viruses expressing WA1 spike is more immunogenic than rMuV-JL2-WA1 alone because of the synergistic effects of the vectors.Both MeV and MuV are excellent viral vectors.Insertion of foreign antigens into MeV and MuV further attenuates the vaccine viruses, thereby enhancing their safety as vaccines and as vaccine vectors.Rapid update of MeV and MuV-based SARS-CoV-2 vaccines.It should be noted that our TVC containing preS-6P protein of WA1, Alpha, Beta, or Delta VoC are not able to induce sufficient