A next-generation intranasal trivalent MMS vaccine induces durable and broad protection against SARS-CoV-2 variants of concern

Abstract

As SARS-CoV-2 variants of concern (VoCs) that evade immunity continue to emerge, next-generation adaptable COVID-19 vaccines which protect the respiratory tract and provide broader, more effective, and durable protection are urgently needed. Here, we have developed one such approach, a highly efficacious, intranasally delivered, trivalent measles-mumps-SARS-CoV-2 spike (S) protein (MMS) vaccine candidate that induces robust systemic and mucosal immunity with broad protection. This vaccine candidate is based on three components of the MMR vaccine, a measles virus Edmonston and the two mumps virus strains [Jeryl Lynn 1 (JL1) and JL2] that are known to provide safe, effective, and long-lasting protective immunity. The six proline-stabilized prefusion S protein (preS-6P) genes for ancestral SARS-CoV-2 WA1 and two important SARS-CoV-2 VoCs (Delta and Omicron BA.1) were each inserted into one of these three viruses which were then combined into a trivalent "MMS" candidate vaccine. Intranasal immunization of MMS in IFNAR1^-/- mice induced a strong SARS-CoV-2-specific serum IgG response, cross-variant neutralizing antibodies, mucosal IgA, and systemic and tissue-resident T cells. Immunization of golden Syrian hamsters with MMS vaccine induced similarly high levels of antibodies that efficiently neutralized SARS-CoV-2 VoCs and provided broad and complete protection against challenge with any of these VoCs. This MMS vaccine is an efficacious, broadly protective next-generation COVID-19 vaccine candidate, which is readily adaptable to new variants, built on a platform with a 50-y safety record that also protects against measles and mumps.

Keywords: MMR vaccine; SARS-CoV-2; intranasal trivalent vaccine.

Fig. 1.

Recovery and characterization of rMuV-JL1-Delta-preS-6P expressing the six proline-stabilized prefusion spike of SARS-CoV-2 Delta variant. (A) Strategy for insertion of preS-6P of the Delta variant into the P and M gene junction in the MuV-JL1 genome. (B) The plaque morphology of rMuV-JL1 and rMuV-JL1-Delta-preS-6P in Vero CCL81 cells at day 5. (C) rMuV-JL1-Delta-preS-6P exhibits delayed syncytia formation in Vero CCL81 cells (MOI of 0.1). (D) Replication kinetics of recombinant viruses in Vero CCL81 cells at an MOI of 0.1. (E) Expression of preS-6P in rMuV-JL1-Delta-preS-6P (Left) or rMuV-JL2-WA1-preS-6P (Right)-infected Vero CCL81 cells. An MOI of 0.1 was used for infection, and 10 μL of cell lysate (from total 200 μL) and 10 μL (from total 1 mL) of supernatant were used for Western blot.

Fig. 2.

Recovery and characterization of rMeV-BA.1-preS-6P expressing the six proline-stabilized prefusion spike of SARS-CoV-2 BA.1. (A) Strategy for insertion of preS-6P of Omicron BA.1 into the P and M gene junction in the MeV genome. (B) The plaque morphology of rMeV-preS-BA.1 in Vero CCL81 cells at day 5. (C) rMeV-BA.1-preS-6P exhibits delayed syncytia formation in Vero CCL81 cells (MOI of 0.1). (D) Replication kinetics of recombinant viruses in Vero CCL81 cells at an MOI of 0.1. (E) Expression of preS-6P of Omicron BA.1 by the MeV vector in Vero CCL81 cells. An MOI of 0.1 was used for infection, and 10 μL of cell lysate (from total 200 μL) and 10 μL (from total 1 mL) of supernatant were used for Western blot.

Fig. 3.

Immunogenicity of monovalent and trivalent vaccines in IFNAR1^−/− mice at doses of 1.5 × 10⁶ PFU. (A) Immunization schedule. IFNAR1^−/− mice (n = 5) were immunized with a high dose (1.5 × 10⁶ PFU) of monovalent or trivalent vaccine via a combination of i.n. and s.c. routes and were boosted three weeks later. Sera were collected for determination of SARS-CoV-2 WA1 (B), Delta (C), or BA.1 (D) S-specific IgG titer measured by ELISA, and SARS-CoV-2 WA1 (E), Delta (F), or BA.1 (G) S-specific IgA titer measured by ELISA. (H) NAbs against different SARS-CoV-2 VoCs. Sera at week 7 were used for pseudotype neutralization assay against SARS-CoV-2 D614G, Delta, Omicron BA.1, or BA.4/5 spike. Data are expressed as the mean of five mice ± SD. Dotted line indicates the limit of detection (LOD). Data were analyzed using Student’s t-test (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

Fig. 4.

Durability of antibody responses of monovalent and trivalent vaccines in IFNAR1^−/− mice. (A) Immunization schedule. IFNAR1^−/− mice (n = 5) were immunized with a low (5 × 10⁵ PFU) or high (1.5 × 10⁶ PFU) dose of monovalent or trivalent vaccine via a combination of i.n. and s.c. routes and were boosted three weeks later. (B) Dynamic of WA1 S-specific IgG titers. (C) Dynamic of BA.1 S-specific IgG titers. (D and E) Comparison of WA1 (D) and BA.1 (E) S-specific IgG between low and high dose immunization. Data are expressed as the mean of five mice ± SD. Dotted line indicates the limit of detection (LOD). Data were analyzed using two-way ANOVA (*P < 0.05).

Fig. 5.

Monovalent and trivalent vaccines induce S-specific tissue-resident CD4⁺ and CD8⁺ T cell immune responses in the lungs. At week 7, immunized IFNAR1^−/− mice (n = 5) from Fig. 3 were injected with CD45-PE antibody 10 min before the mice were killed in order to separate the resident (CD45⁻) and circulating (CD45⁺) T cells. Lung CD45⁻ T cell suspensions were stimulated with a WA1 S-specific peptide pool. Cells were surface stained with antibodies specific for CD4 or CD8, CD62L, CD44, and CD69, then fixed, permeabilized, and stained with anti-IFNγ, anti-IL-17, and anti-IL-5 for CD4⁺ T cells. The percent and number of S-specific CD45⁻ CD4⁺CD44⁺CD62L⁻CD69⁺ T cells (A and B), IFNγ⁺ (C and D), IL-17⁺ (E and F), and IL-5⁺ (G and H) producing CD4⁺ T cells are shown in A–H. The CD8⁺CD69⁺ T cells in the lungs were stimulated a WA1 S-specific peptide pool (I–L). The percent and number of S-specific CD45^-CD8⁺CD44⁺CD62L⁻CD69⁺ T cells (I and J) and IFNγ⁺-producing CD8⁺ T cells (K and L) are shown. One-way ANOVA with Tukey’s multiple comparisons was used to detect differences among groups (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

Fig. 6.

Monovalent and trivalent vaccines induce systemic T cell response. At week 7, IFNAR1^−/− mice (n = 5) from Fig. 3 were killed, and splenocyte suspension was stimulated with 20 µg/mL of WA1 preS protein for 5 d. The frequencies and number of S-specific IFN-γ⁺CD4⁺ (A and B), TNF-α⁺CD4⁺ (C and D), IL-4⁺CD4⁺ (E and F), IL-10⁺CD4⁺ (G and H), IL-17⁺ CD4⁺ (I and J), and IL-21⁺ CD4⁺ (K and L) cells were determined by flow cytometry after intracellular staining with the corresponding anti-cytokine antibody. Data are expressed as mean of five mice ± SD. Data were analyzed using one-way ANOVA (*P < 0.05; **P < 0.01).

Fig. 7.

Characterization of immune responses following monovalent and trivalent immunization in golden Syrian hamsters. (A) Immunization schedule in hamsters. Sera were collected for detection of SARS-CoV-2 WA1 (B), Delta (C), or BA.1 (D) S-specific serum IgG titer by ELISA, and SARS-CoV-2 WA1 (E), Delta (F), or BA.1 (G) S-specific serum IgA titer by ELISA. (H) BA.1 and WA1 S-specific IgA titer in nasal wash (week 7) measured by ELISA. (I) NAbs against different SARS-CoV-2 VoCs. Sera at week 7 were used for the pseudotype neutralization assay against SARS-CoV-2 D614G, Delta, Omicron BA.1, or BA.4/5 spike. Data are the mean of fifteen hamsters ± SD. Dotted line indicates the limit of detection (LOD). Data were analyzed using Student’s t-test (ns > 0.05, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

Fig. 8.

The trivalent vaccine provides broader protection against SARS-CoV-2 challenge in golden Syrian hamsters than the monovalent vaccine. Hamsters (n = 5) from Fig. 7. were challenged with 2 × 10⁴ PFU SARS-CoV-2 WA1 (A–D), or Delta variant (E–H), or 7 × 10⁵ PFU of Omicron BA.1 (I–L). (A) Body weight changes in hamsters. (B and C) SARS-CoV-2 titer in the lung (B) and nasal turbinate (C) at day 4. (D) Lung pathology score after challenge with SARS-CoV-2 WA1. Each lung section was scored based on the severity of histologic changes. Score 4, extremely severe; score 3, severe; score 2, moderate; score 1, mild; score 0, no pathological changes. (E) Body weight changes in hamsters. (F and G) SARS-CoV-2 titer in the lung (F) and nasal turbinate (G) at day 4. (H) Lung pathology score. (I) Body weight changes in hamsters. (J and K) SARS-CoV-2 titers in the lung (J) and nasal turbinate (K) at day 3. (L) Lung pathology score. Dotted line indicates the limit of detection (LOD). Data were analyzed using two-way ANOVA and one-way ANOVA (ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

Fig. 9.

Monovalent and trivalent vaccine immunization protects against lung pathology after challenge with Omicron BA.1. Hamsters were killed at day 3 after challenge with the SARS-CoV-2 BA.1 variant. Lung tissue was stained with hematoxylin/eosin and examined histologically. Micrographs with 1×, 4×, and 10× magnification of a representative lung section from each group are shown.

Fig. 10.

Intranasal immunization of trivalent vaccine provides complete protection against SARS-CoV-2 challenge in golden Syrian hamsters. Hamsters immunized i.n. with 1.5 × 10⁶ PFU of trivalent vaccine or parental MMM vector and were boosted i.n. 2 wk later. SARS-CoV-2 WA1 (A), Delta (B), or BA.1 (C) S-specific IgG was determined by ELISA. SARS-CoV-2 WA1 (D), Delta (E), or BA.1 (F) S-specific IgA was determined by ELISA. At week 7, hamsters were challenged with 2 × 10⁴ PFU SARS-CoV-2 WA1 (G–I), or Delta variant (J–L), or 7 × 10⁵ PFU of Omicron BA.1 (M–O). (G) Body weight changes, SARS-CoV-2 titer in the lung (H), and nasal turbinate (I) in hamsters after challenge with SARS-CoV-2 WA1. (J) Body weight changes, SARS-CoV-2 titer in the lung (K), and nasal turbinate (L) in hamsters after challenge with the Delta variant. (M) Body weight changes, SARS-CoV-2 titer in the lung (N), and nasal turbinate (O) in hamsters after challenge with Omicron BA.1 variant. Dotted line indicates the limit of detection (LOD). Data were analyzed using two-way ANOVA and one-way ANOVA (ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

Fig. 11.

Comparison of S-specific IgG and IgA responses following intranasal or subcutaneous immunization of trivalent vaccine. (A) Experimental schema. (B) Serum WA1 S-specific IgG titers. (C) Serum Delta S-specific IgG titer. (D) Serum BA.1 S-specific IgG titer. (E) WA1, Delta, and BA.1 S-specific IgG in the BAL at week 6 postimmunization. (F) Serum WA1 S-specific IgA titers. (G) Serum Delta S-specific IgA titers. (H) Serum BA.1 S-specific IgA titers. (I) WA1, Delta, and BA.1 S-specific IgA titer in the BAL at week 6 postimmunization. Dotted line indicates the limit of detection (LOD). Data are expressed as the mean of ten mice ± SD. Statistical differences were determined by Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).