Enterovirus D68 (EV-D68), a pathogen that causes respiratory symptoms, mainly in children, has been implicated in acute flaccid myelitis, which is a poliomyelitis-like paralysis. Currently, there are no licensed vaccines or treatments for EV-D68 infections. Here, we investigated the optimal viral inactivation reagents, vaccine adjuvants, and route of vaccination in mice to optimize an inactivated whole-virion (WV) vaccine against EV-D68. We used formalin, β-propiolactone (BPL), and hydrogen peroxide as viral inactivation reagents and compared their effects on antibody responses. Use of any of these three viral inactivation reagents effectively induced neutralizing antibodies. Moreover, the antibody response induced by the BPL-inactivated WV vaccine was enhanced when adjuvanted with cytosine phosphoguanine oligodeoxynucleotide (CpG ODN) or AddaVax (MF59-like adjuvant), but not with aluminum hydroxide (alum). Consistent with the antibody response results, the protective effect of the inactivated WV vaccine against the EV-D68 challenge was enhanced when adjuvanted with CpG ODN or AddaVax, but not with alum. Further, while the intranasal inactivated WV vaccine induced EV-D68-specific IgA antibodies in the respiratory tract, it was less protective against EV-D68 challenge than the injectable vaccine. Thus, an injectable inactivated EV-D68 WV vaccine prepared with appropriate viral inactivation reagents and an optimal adjuvant is a promising EV-D68 vaccine.

Vaccinations are ideal for reducing the severity of clinical manifestations and secondary complications of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); however, SARS-CoV-2 continues to cause morbidity and mortality worldwide. In contrast to parenteral vaccines such as messenger RNA vaccines, nasal vaccines are expected to be more effective in preventing viral infections in the upper respiratory tract, the primary locus for viral infection and transmission. In this study, we examined the prospects of an inactivated whole-virion (WV) vaccine administered intranasally against SARS-CoV-2.
Mice were immunized subcutaneously (subcutaneous vaccine) or intranasally (nasal vaccine) with the inactivated WV of SARS-CoV-2 as the antigen.
The spike protein (S)-specific IgA level was found to be higher upon nasal vaccination than after subcutaneous vaccination. The level of S-specific IgG in the serum was also increased by the nasal vaccine, although it was lower than that induced by the subcutaneous vaccine. The nasal vaccine exhibited a stronger defense against viral invasion in the upper respiratory tract than the subcutaneous vaccine and unimmunized control; however, both subcutaneous and nasal vaccines provided protection in the lower respiratory tract. Furthermore, we found that intranasally administered inactivated WV elicited robust production of S-specific IgA in the nasal mucosa and IgG in the blood of mice previously vaccinated with messenger RNA encoding the S protein.
Overall, these results suggest that a nasal vaccine containing inactivated WV can be a highly effective means of protection against SARS-CoV-2 infection.