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. 2024 Apr 3;16(4):559.
doi: 10.3390/v16040559.

Measles Virus-Based Vaccine Expressing Membrane-Anchored Spike of SARS-CoV-2 Inducing Efficacious Systemic and Mucosal Humoral Immunity in Hamsters

Zhi-Hui Yang  1 , Yan-Li Song  1 , Jie Pei  1 , Song-Zhuang Li  1 , Rui-Lun Liu  1 , Yu Xiong  1 , Jie Wu  1 , Yuan-Lang Liu  1 , Hui-Fen Fan  1 , Jia-Hui Wu  1 , Ze-Jun Wang  1 , Jing Guo  1 , Sheng-Li Meng  1 , Xiao-Qi Chen  1 , Jia Lu  1 , Shuo Shen  1
Affiliations

Measles Virus-Based Vaccine Expressing Membrane-Anchored Spike of SARS-CoV-2 Inducing Efficacious Systemic and Mucosal Humoral Immunity in Hamsters

Zhi-Hui Yang et al. Viruses. .

Abstract

As SARS-CoV-2 continues to evolve and COVID-19 cases rapidly increase among children and adults, there is an urgent need for a safe and effective vaccine that can elicit systemic and mucosal humoral immunity to limit the emergence of new variants. Using the Chinese Hu191 measles virus (MeV-hu191) vaccine strain as a backbone, we developed MeV chimeras stably expressing the prefusion forms of either membrane-anchored, full-length spike (rMeV-preFS), or its soluble secreted spike trimers with the help of the SP-D trimerization tag (rMeV-S+SPD) of SARS-CoV-2 Omicron BA.2. The two vaccine candidates were administrated in golden Syrian hamsters through the intranasal or subcutaneous routes to determine the optimal immunization route for challenge. The intranasal delivery of rMeV-S+SPD induced a more robust mucosal IgA antibody response than the subcutaneous route. The mucosal IgA antibody induced by rMeV-preFS through the intranasal routine was slightly higher than the subcutaneous route, but there was no significant difference. The rMeV-preFS vaccine stimulated higher mucosal IgA than the rMeV-S+SPD vaccine through intranasal or subcutaneous administration. In hamsters, intranasal administration of the rMeV-preFS vaccine elicited high levels of NAbs, protecting against the SARS-CoV-2 Omicron BA.2 variant challenge by reducing virus loads and diminishing pathological changes in vaccinated animals. Encouragingly, sera collected from the rMeV-preFS group consistently showed robust and significantly high neutralizing titers against the latest variant XBB.1.16. These data suggest that rMeV-preFS is a highly promising COVID-19 candidate vaccine that has great potential to be developed into bivalent vaccines (MeV/SARS-CoV-2).

Keywords: Omicron BA.2; SARS-CoV-2; SP-D; measles virus; mucosal immunity; spike protein; trimerization tag.

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Conflict of interest statement

All authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Rescue and characterization of rMeV expressing the stabilized prefusion full-length spike or secretory ectodomain of SARS-CoV-2. (A) Experimental flow chart. (B) Strategy for insertion of preFS and S+SPD of Omicron BA.2 into the MeV genome. The stabilized, prefusion preFS and S+SPD genes were inserted into the gene junction between the H and L genes in the genome of the MeV-S191 vaccine strain. The domains of the spike protein are shown. SP, signal peptide; RBM, receptor binding motif; RBD, receptor binding domain; HR, heptad repeat; FP, fusion peptide; CH, central helix; CT, cytoplasmic tail; TM, transmembrane domain; SPD, human pulmonary surfactant protein-D sequence. The organization of the genes in the negative-sense MeV genome is shown. N, nucleoprotein gene; P, phosphoprotein gene; M, membrane protein gene; F, fusion protein gene; H, hemagglutinin gene; L, large polymerase gene. (C) The expression diagram of SARS-CoV-2 spike proteins in rMeV-preFS- and rMeV-S+SPD-infected cells. (D) The plaque morphology of rMeV expressing preFS or S+SPD proteins and parental strain were observed on day 6 post-infection. (E) The average diameters of 18 randomly selected plaques from each measles virus were compared. (F,G) Multistep growth curves were determined in confluent monolayers of Vero cells in six-well plates. Cells were infected with individual viruses at a multiplicity of infection (MOI) of 0.001 at 34 °C (F) or 37 °C (G). From day 1 to day 6, viral titers in supernatants were determined by a CCID50 assay. (H) The peak titers in supernatants of individual recombinants within 6 days at 34 °C or 37 °C were compared. (I) The titers in supernatants and cell pellets of individual recombinants were titrated in Vero cells in T25 flasks infected at an MOI of 0.001 at 34 °C. The infected cells were frozen and thawed twice, and cell pellets were resuspended in DMEM in the same volume as the harvested supernatants. (J) The morphology of syncytia formed by rMeVs at 34 °C or 37 °C was observed on day 5 post-infection. Statistical assay: *, p < 0.05; **, p < 0.01; ***, 0.001; ****, p < 0.0001; ns, not significant.
Figure 2
Figure 2
Analysis of spike proteins expressed by rMeVs using IFA and Western blot assays. (A) Analysis of preFS and S+SPD protein expression by IFA. Vero cells in 6-well plates were infected with each virus at an MOI of 0.001 at 34 °C. At 72 h post-infection, the cells were fixed with 80% pre-cooled acetone and stained with anti-SARS-CoV-2 RBD chimeric mAb. (B) Analysis of Omicron BA.2 preFS and S+SPD protein expression in supernatants and cell lysates by Western blotting. Vero cells in T25 flasks were infected or mock-infected with individual recombinants at an MOI of 0.001 at 34 °C. At 96 h post-infection, medium supernatants were removed and cells were lysed in 500 μL of SDS lysis buffer and diluted tenfold to the same volume of the harvested supernatants. Proteins in cell lysates and supernatants were treated without BME (a) or with BME (b) and separated by SDS-PAGE. (C,D) Time courses of preFS protein (C) and S+SPD protein (D) expression in cells and media by Western blotting. The same volumes of lysates or supernatants harvested on days 1, 2, 3, 4, 5, and 6 post-infection were analyzed. Membrane-blotted proteins were probed with mouse anti-Omicron RBD protein monoclonal antibody, rabbit anti-MeV-N3 antiserum, and mouse anti-β-tubulin antibody in a Western blotting assay.
Figure 3
Figure 3
Expression analysis of spike proteins by rMeVs through 10–60% sucrose density gradient. Vero cells were infected with the recombinants at an MOI of 0.01 at 34 °C, and supernatants were harvested on day 4 post-incubation. The supernatants were centrifuged through a 10–60% sucrose gradient. The viral proteins in individual fractions derived from supernatants of infected cells with rMeV-preF S (A) and rMeV-S+SPD (B) were treated without BME (a) or with BME (b) and separated by SDS-PAGE and analyzed by Western blotting. Anti-Omicron RBD monoclonal antibodies, rabbit anti-MeV-N3 antiserum, and anti-MeV-P antibodies were used for the detection of viral proteins, respectively.
Figure 4
Figure 4
Mucosal immune responses in immunized golden Syrian hamsters by rMeV-preFS and rMeV-S+SPD through intranasal and subcutaneous routes. (A) Immunization schedule of golden Syrian hamsters. Groups of four-week-old female hamsters (n = 6) were immunized intranasally (i.n.) or subcutaneously (s.c.) at a dose of 1 × 106 CCID50 of parental rMeV, S recombinants rMeV-preFS, and rMeV-S+SPD or mock-immunized with DMEM. Hamsters were boosted 3 weeks post-priming. (B) IgA levels in sera via s.c. and i.n. routes. (C) IgA levels in the BALF via s.c. and i.n. routes. (D) IgA levels in the NAL via s.c. and i.n. routes. (E) Fecal IgA levels via s.c. and i.n. routes. Samples were collected on days 14, 28, and 35 post-priming. Data were analyzed using a two-way ANOVA test (**, p < 0.01, ***, p < 0.001; ****, p < 0.0001, ns, not significant) and presented as the GMT of six hamsters ± SD.
Figure 5
Figure 5
Tolerance and immunogenicity of rMeVs expressing Omicron BA.2 spikes in golden Syrian hamsters against different strains. (A) Immunization schedule of golden Syrian hamsters. Four-week-old female hamsters (n = 8) were immunized intranasally with 1 × 106 CCID50 of parental rMeV, rMeV-preFS, or rMeV-S+SPD or mock-immunized with DMEM and boosted 3 weeks post-priming. On days 14, 28, and 35, the sera were collected for antibody detection. On day 35, hamsters were intranasally challenged with 1 × 106 CCID50 of Omicron BA.2. Unimmunized, unchallenged animals in the control group were inoculated with DMEM. (B,C) Evaluation of tolerance of each recombinant post-priming. The temperatures (B) and changes in body weight (C) of individual hamsters were monitored at intervals of two days post-priming. (D) Serum ELISA-specific IgG levels against Omicron BA.2 S protein. (E) Serum NAbs levels against Omicron BA.2 strain. (F) Serum NAbs levels against SARS-CoV-2 prototype strain. (G) Serum NAbs levels against SARS-CoV-2 XBB.1.16 strain. (H) Serum NAbs levels against measles virus. Data are the GMT of eight animals ± SD pre-challenge or five animals ± SD post-challenge. Data were analyzed using a two-way ANOVA test. (**, p < 0.01; ****, p < 0.0001).
Figure 6
Figure 6
Body weight changes and viral loads post-challenge against Omicron BA.2 in golden Syrian hamsters vaccinated by rMeV-preFS and rMeV-S+SPD via the intranasal immunization route. (A) Dynamics of hamster body weight changes post-challenge with Omicron BA.2. The body weight for each hamster was recorded from day 0 to day 5. On day 5 post-challenge, all hamsters were euthanized. The viral titers of Omicron BA.2 in the lungs (B) and nasal turbinate (C). Viral titers are the GMT of five animals ± SD. The limit of detection (LoD) is 2.7 log10 CCID50/mL per gram of tissue (dotted lines). (D,E) Omicron BA.2 subgenomic RNA copies of the N gene in lungs (D) and nasal turbinates (E). (F,G) Omicron BA.2 genomic RNA copies of the ORF1ab gene in lungs (F) and nasal turbinates (G). The dotted lines indicate the limit of detection. Data were analyzed using a two-way ANOVA test (*, p < 0.05; **, p < 0.01; ****, p < 0.0001).
Figure 7
Figure 7
Lung pathology and virus replication post-challenge of Omicron BA.2 for hamsters immunized with rMeV-preFS and rMeV-S+SPD. (A) The lung tissues were fixed and embedded in paraffin on day 5 post-challenge. Following samples being sectioned, deparaffinized, and rehydrated, hematoxylin-eosin staining was performed for the examination of histological changes under microscopy. Lung histopathological changes were indicated by arrows. Micrographs of 1×, 5×, and 10× magnifications of a representative lung section from each group were shown, and scale bars were indicated at the left corner of each image. (B) Lung pathology scores post-challenge with Omicron BA.2. Each slide was evaluated based on the severity of histological changes, including edema, hyaline membrane, fragments of necrotic cell, neutrophil infiltration, monocyte, and thrombus. Scores 6–8, extremely severe pathological changes; scores 4–6, severe pathological changes; scores 2–4, moderate pathological changes; scores 0–2, mild pathological changes; score 0, no pathological changes. Data were analyzed using a two-way ANOVA test (*, p < 0.05; **, p < 0.01; ****, p < 0.0001).
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