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. 2024 Dec;11(47):e2404197.
doi: 10.1002/advs.202404197. Epub 2024 Nov 11.

Assessment of Single-Cycle M-Protein Mutated Vesicular Stomatitis Virus as a Safe and Immunogenic Mucosal Vaccine Platform for SARS-CoV-2 Immunogen Delivery

En Zhang  1   2 , Yong Ke  3 , Weihong Ran  1   2 , Yu Zhang  1   2 , Ruihang Li  1   2 , Xinkui Fang  1 , Lei Wang  3 , Baohong Zhang  3 , Tao Sun  1   2
Affiliations

Assessment of Single-Cycle M-Protein Mutated Vesicular Stomatitis Virus as a Safe and Immunogenic Mucosal Vaccine Platform for SARS-CoV-2 Immunogen Delivery

En Zhang et al. Adv Sci (Weinh). 2024 Dec.

Abstract

The goal of the next-generation COVID-19 vaccine is to provide rapid respiratory tract protection with a single dose. Circulating antibodies do not protect the olfactory mucosa from viral infection, necessitating localized mucosal immunization. Live attenuated vesicular stomatitis virus (VSVMT)-based COVID-19 vaccines effectively stimulate mucosal immunity in animals, though safety concerns remain, particularly in immunocompromised populations. A viral vector capable of single-cycle replication may face less stringent regulatory requirements. A replication-defective VSVMT is developed with its G protein replaced by a SARS-CoV-2 spike protein (S) mutant, where residues K986 and V987 are substituted by prolines (S2P). This studies show that single-cycle VSVMT encoding Omicron subvariant S2P (VSVMT-S2P) is safe in both healthy and immunocompromised animals treated with cyclophosphamide (CP). Significant antibody and T-cell responses against the spike protein are observed in VSVMT-S2P vaccinated healthy animals. Intramuscular VSVMT-S2P administration induces neutralizing antibody responses comparable to those from replication-competent VSVMT-S. In immunocompromised animals, lower and delayed immune responses are observed. Thus, single-cycle M-protein mutated VSV offers a safe and effective platform for SARS-CoV-2 immunogen delivery. Remarkably, replication-competent VSVMT-S caused no pathogenicity and elicited potent mucosal immunity via intranasal administration, highlighting its potential as a mucosal COVID-19 vaccine.

Keywords: COVID‐19; Spike protein; mucosal immunity; replication defective; vesicular stomatitis virus.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Generation of a non‐propagating VSVMT vector expressing the prefusion stabilized spike protein of SARS‐CoV‐2. The spike protein (S) was engineered with two proline substitutions at residues K986 and V987. Additionally, 21 amino acids were deleted from the C‐terminal region of the spike protein (S∆212P). A) Schematic representation of the recombinant VSV constructs expressing the spike (S) protein. The parental VSVMT genome encodes nucleoprotein (N), phosphoprotein (P), glycoprotein (G), matrix protein (M), and RNA polymerase (L). The mutant M protein (MT) carries three specific mutations (M51 deletion, V221F, and S226R). By utilizing MluI and XhoI restriction enzyme digestion, the VSV G protein gene was replaced with either the S∆21 protein or the S∆212P mutant from the Omicron subvariants BA.1 or BA.5. The GFP reporter gene was inserted between the G and L genes in the VSV genome. B and C) Intact antigenicity of the S∆212P proteins. S∆212P of both BA.1 and BA.5 subvariants were expressed in BHK21 cells, with the full‐length S proteins from BA.1 and BA.5 serving as controls. The binding activity of human ACE2 to the S∆212P and full‐length S proteins was assessed across a concentration range of 5–200 µg mL−1 using a cell‐based ELISA assay B). The antigenicity of S∆212P and full‐length S proteins was further characterized using the BA.1‐specific antibody 8G3 C). D and E) Syncytia formation mediated by the S or S∆212P proteins. 293T cells, transfected to express the GFP reporter along with S∆212P or full‐length S protein from BA.1 or BA.5, were cocultured with cells expressing hACE2 and mCherry, with or without TMPRSS2. Cells were stained with Hoechst (blue), and 24 h later, syncytia (appearing yellow or orange) were visualized using confocal microscopy E); The average syncytium area (µm2) was quantified using Image J software D). F) Western blot analysis of rVSV viruses using a spike protein‐specific antibody. VeroE6 cells were infected with VSVMT‐S2P or VSVMT‐S at an MOI of 1. Cells were harvested and lysed 24 h post‐infection. Lane 1: protein ladder; lane 2–5: lysates from cells infected with VSVMT‐S2PBA.1, VSVMT‐SBA.1, VSVMT‐S2PBA.5, and VSVMT‐SBA.5 respectively; lane 6: lysates from cells infected with VSV; lane 7: mock control. (G and H) Cellular tropism of VSVMT‐S2P and VSVMT‐S. hACE2‐BHK21 or BHK21 cells were infected with rVSVMT‐S2P, VSVMT‐S, or rVSV‐GFP viruses at an MOI of 1. 24 h post‐infection, the supernatants were collected, and the viral progeny were used to infect fresh hACE2‐BHK21 or BHK21 cells. Infected cells were observed under a fluorescent microscope G). H) Summary of viral tropism and replication of rVSVs in cells with or without hACE2 expression. “+”: fluorescence positive; “‐”: fluorescence negative. P1 and P2 represent the first and second rounds of infection, respectively. All the above data are presented as mean ± SD (n = 3). Statistical significance was determined using an unpaired two‐tailed t‐test: ns, p > 0.05; ***p < 0.001.
Figure 2
Figure 2
Safety evaluation of VSVMT‐S2P and VSVMT‐S viruses in hamsters. A) Experimental design for safety evaluation. Female hamsters were divided into groups as outlined in Table 1 (n = 12 each group) and inoculated with either VSVMT‐S2P or VSVMT‐S at doses of 1 × 107 or 1 × 106 PFU per hamster via intranasal administration. A PBS buffer served as the control. Body weight loss was monitored daily for up to 28 days post‐inoculation (d.p.i.). Sera and organs, including nasal turbinates, lungs, and brains, were collected on 2, 4, and 6 d.p.i. for further analysis (n = 3 each group). B) Body weight changes in inoculated hamsters over the course of the study. C) Total white blood cell (WBC) counts in inoculated hamsters. D) Ratio of large white blood cells (WBC‐large cell ratio, W‐LCR). (E‐G) VSV N gene expression was quantified by RT‐qPCR in the nasal turbinates E), lungs F), and brains G) collected on days 2, 4, and 6 d.p.i. All the above data are presented as mean ± SD (n = 3). Statistical significance was assessed using two‐way ANOVA with multiple comparisons: ns, p > 0.05.
Figure 3
Figure 3
Immunogenicity of VSVMT‐S2P and VSVMT‐S in healthy hamsters. A) Schematic representation of the vaccination protocol in healthy female hamsters (n = 3 each group). Hamsters were inoculated with VSVMT‐S2P or VSVMT‐S at doses of 1 × 106 and 1 × 105 PFU per hamster via intranasal or intramuscular administration, with PBS buffer serving as the control. Serum samples were collected every 7 days, and bronchoalveolar lavage fluids (BALF) was collected at 28 days post‐inoculation (d.p.i.). Neutralizing antibody (NAb) titers in the serum and BALF were measured using VSV‐SBA.1 and VSV‐SBA.5 respectively. The titers were expressed as the reciprocal of the highest antibody dilution that achieved 100% inhibition of the cytopathic effect. B and C) Kinetic curves of NAb titers in the sera of hamsters inoculated with VSVMT‐S2PBA.1 or VSVMT‐SBA.1 via intranasal B) or intramuscular routes C). D and E) Kinetic curves of NAb titers in the sera of hamsters inoculated with VSVMT‐S2PBA.5 or VSVMT‐SBA.5 via intranasal D) or intramuscular routes E). F) NAb titers in BALF from hamsters immunized with VSVMT‐S2PBA.1 or VSVMT‐SBA.1 via intranasal or intramuscular routes. G) NAb titers in BALF from hamsters immunized with VSVMT‐S2PBA.5 or VSVMT‐SBA.5 via intranasal or intramuscular routes. All the above data are presented as mean ± SD (n = 3). Statistical significance was determined using two‐way ANOVA with multiple comparisons. ns, p > 0.05; *p < 0.05; ***p < 0.001. UD: undetectable.
Figure 4
Figure 4
Safety and immunogenicity of VSVMT‐S2P in healthy Balb/c mice. A) Experimental design for safety and immunogenicity evaluation in female Balb/c mice. For the safety assessment, adult Balb/c mice were inoculated with VSVMT‐S2P at doses of 2.5 × 106 and 2.5 × 105 PFU per mouse via the intranasal route, with PBS as the control (n = 12 each group, 3 female mice in each group at indicated times were euthanized for tissues collecting). Body weight loss was monitored daily for 28 days post‐infection (d.p.i.). Sera were collected at 2, 4, and 6 d.p.i. B) Body weight changes in inoculated mice. C) Total white blood cell (WBC) counts. D) WBC‐large cell ratio (W‐LCR). To assess viral neurovirulence, sixteen‐day‐old suckling mice were intracranially injected with 1500 PFU of VSVMT‐S2PBA.5 or VSV‐GFP, with PBS as the mock control (n = 15 each group). Mice were monitored daily. E) Survival curve of sucking mice. F) Kinetics of viral replication in the brain. G) IVIS imaging for viral detection. For the immunogenicity assessment, female Balb/c mice were inoculated with VSVMT‐S2P at doses of 2.5 × 105 and 2.5 × 104 PFU via intranasal or intramuscular routes, with PBS as the control (n = 3 each group). Sera were collected every 7 days. Bronchoalveolar lavage fluids (BALF), nasal wash, and spleen samples were collected at 28 d.p.i. Neutralizing antibody (NAb) titers were measured using VSVMT‐SBA.1 and VSVMT‐SBA.5, expressed as the reciprocal of the highest antibody dilution yielding 100% inhibition of cytopathic effect. (H and I) NAb titers of sera samples from mice inoculated with VSVMT‐S2PBA.1 H) or VSVMT‐S2PBA.5 I). J and K) Anti‐spike IgA antibody assay. IgA antibodies in nasal wash or BALF samples from mice vaccinated with VSVMT‐S2PBA.1 J) or VSVMT‐S2PBA.5 K) were detected using ELISA. For cellular‐mediated immune responses (CMI), interferon‐gamma (IFN‐γ) levels were measured using an ELISPOT assay as described in the “Methods” section. Splenectomies were performed on mice sacrificed at 28 d.p.i following intranasal or intramuscular vaccination with VSVMT‐S2P. CMI responses induced by VSVMT‐S2PBA.1 L) or VSVMT‐S2PBA.5 M) were tested using a panel of epitopes from the S1 or S2 regions of the SARS‐CoV‐2 spike protein. Results are presented as mean ELISPOTs per million splenocytes, subtracting any background ELISPOTs from unpulsed mock controls. All the above data are shown as mean ± SD (n = 3). Statistical significance was determined using two‐way ANOVA with multiple comparisons: ns, p > 0.05; 0.01 < **p < 0.05; ***p < 0.001. The arrow points to the fluorescent region of the brain. VSV: VSV‐GFP, S2P:VSVMT‐S2PBA.5.
Figure 5
Figure 5
Safety and immunogenicity of VSVMT‐S2P compared to VSVΔG‐S in cyclophosphamide‐induced immunocomprised LVG hamsters. A) Experimental design for the safety and immunogenicity evaluations in cyclophosphamide (CP)‐induced immunocompromised female hamsters. Safety assessments were conducted via intranasal administration. Hamsters were inoculated with VSVMT‐S2P or VSVMT‐S viruses at doses of 1 × 107 and 1 × 106 PFU/hamster, with VSVΔG‐S and PBS serving as controls (n = 3 per group). Body weight loss was monitored daily until 20 days post‐inoculation (d.p.i.). Blood samples were collected on ‐5, 0, 7, 14, 21 and 28 d.p.i. B and E) Body weight changes in immunocompromised hamsters inoculated with rVSVs encoding either the S or S2P proteins of the BA.5 B) or BA.1 E) variants. C and F) White blood cell (WBC) counts in immunocompromised hamsters inoculated with rVSVs encoding either the S or S2P proteins of the BA.5 C) or BA.1 F) variants. D and G) WBC‐large cell ratio (W‐LCR) values in immunocompromised hamsters inoculated with rVSVs encoding either the S or S2P proteins of the BA.5 D) or the BA.1 G) variants. For immunogenicity evaluations, immunocompromised hamsters were inoculated with VSVMT‐S2P or VSVMT‐S at doses of 1 × 106 and 1×105 PFU/hamster via the intranasal route (n = 3 each group). Serum samples were collected every 7 days, and bronchoalveolar lavage fluids (BALF) was collected at 28 d.p.i. Neutralizing antibody titers in serum and BALF were quantified using VSVMT‐SBA.1 and VSVMT‐SBA.5, respectively. Titers are expressed as the reciprocal of the highest antibody dilution yielding 100% inhibition of the cytopathic effect. (H) Neutralizing antibody titers in serum from immunocompromised hamsters vaccinated with VSVMT vectors encoding either the S or S2P proteins of BA.5 via the intranasal route. I) Neutralizing antibody titers in serum from immunocompromised hamsters vaccinated with VSVMT vectors encoding either the S or S2P proteins of BA.1. All the above data are presented as mean ± SD (n = 3). Statistical significance was determined using two‐way ANOVA with multiple comparisons. ns, p > 0.05; 0.01 <**p < 0.05; ***p < 0.001.
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