3.4 Protective efficacy of VSV/RABV-GP vaccination
To determine the protective efficacy of VSV/RABV-GP vaccination, the neutralizing activity of the sera samples was analyzed by FAVNT. The average serum neutralization titers of the inactivated RABV and VSV/RABV-GP groups were 241.41 and 103.37 IU/mL, respectively (Figure 4).
DISCUSSION
Rabies was, after smallpox, the second human disease for which an efficacious vaccine was developed, by Pasteur in 1885. Although it is eminently preventable, with highly efficacious vaccines available for both humans and animals, it still causes considerable mortality in low and middle-income countries (Hampson et al., 2015) . Primary prevention is the best defense against rabies. It includes the following: elimination of animal rabies through dog vaccination campaigns; promoting awareness of the disease and available prevention; and encouraging responsible dog ownership and vaccination, particularly in endemic areas, where existing cultural practices might need to be adapted to integrate these interventions (Banyard, Horton, Freuling, Muller, & Fooks, 2013) . Where possible, rabies pre-exposure prophylaxis (PrEP) via immunization is recommended for travelers to endemic regions, although such advice might not always be followed(Fooks, Johnson, Brookes, Parsons, & McElhinney, 2003) . However, in the absence of infrastructure to support such initiatives, secondary prevention attempts to prevent the onset of clinical disease through post-exposure prophylaxis (PEP). Most human cases of rabies are the result of dog bites (Fooks et al., 2017) . There is a strong argument for investment in dog vaccination: the feasibility and cost-effectiveness of rabies control and human rabies elimination by dog vaccination has been demonstrated in some countries (Wang, C. et al., 2018) . Several inactivated preparations of RABV are available as vaccines to immunize humans and domestic animals. For wildlife vaccination, both live-attenuated and subunit vaccines are available(Rupprecht, Nagarajan, & Ertl, 2016) . Licensed human rabies vaccines are all based upon an inactivated rabies virus and may be used either for PrEP as well as PEP (Chulasugandha, Khawplod, Havanond, & Wilde, 2006) . PrEP regimes have until recently involved three doses of inactivated rabies virus vaccine spread over 28 days with typical costs (Chulasugandha et al., 2006) . Not only inactivated vaccines, but also other recombinant vaccines trials are reported elsewhere. Recently, a simian adenovirus-vectored candidate showed that single-dose vaccination induced strong neutralizing antibodies in mice(Wang, C. et al., 2018) . Also, other rabies vaccine candidates using viral vectors have been reported (Abreu-Mota et al., 2018; Chen et al., 2019; Ramya et al., 2011; Wang, X. et al., 2019) . Very recently, RNA vaccines for rabies were reported (Armbruster, Jasny, & Petsch, 2019) .
The importance of rabies immunoglobulin (RIG) has been studied. PEP can include both the administration of RIG and vaccination, although individuals who receive adequate PrEP should receive a booster vaccination but not RIG. The application of RIG directly to the wound aims to neutralize any live virus in the immediate wound area and prevent the spread of the virus in the time that it takes to develop sufficient immunity in response to vaccination.
In the present study, we generated VSV/RABV-GP and investigated its immunogenicity in mice. The results described above demonstrate that the VSV/RABV-GP immunization can elicit a strong, specific humoral immune response in mice. The average neutralizing titer in immunized mice was 103 IU/ml, which is higher than the recommended lowest titers by the WHO. VSV/RABV-GP did not induce cytotoxic effects in transduced cells or the immunized mice. Collectively, the data suggest that VSV/RABV-GP is a promising candidate for rabies vaccine.
Vaccination is considered the best way to combat a rabies infection before and after exposure, as there is no treatment available once the symptoms have appeared. Since the nerve tissue vaccine was first developed by Pasteur in 1885, various rabies vaccines for animal and human use have been developed (Ertl, 2019; Nandi & Kumar, 2010) . Inactivated nerve tissue vaccines are economically beneficial but can cause serious side effects, such as autoimmune encephalomyelitis in inoculated animals (Cliquet & Picard-Meyer, 2004) . Inactivated cell cultures and embryonated egg vaccines have replaced nerve tissue vaccines and are considered safe and are well-tolerated(Kamoltham, Khawplod, & Wilde, 2002; Kamoltham et al., 2007) . However, these vaccines are expensive and unaffordable for people and animals living in developing countries. Attenuated live vaccines can efficiently elicit a protective immune response without the use of adjuvants. In contrast, there is a potential risk for rabies in the inoculated animals due to their residual virulence or pathogenic mutation during viral propagation (Nandi & Kumar, 2010) .
The replication-deficient VSV-vectored vaccine is a promising vaccine platform that is safe and effective. Single-cycle VSV-based vectors lacking the VSV G protein can infect cells but cannot produce infectious particles (Schnell, Johnson, Buonocore, & Rose, 1997) . Therefore, there are no potential safety issues associated with infectious vaccines. In addition, VSV proteins have adjuvant-like properties, thereby eliciting robust adaptive immune responses to the vaccine immunogen (Cobleigh, Bradfield, Liu, Mehta, & Robek, 2012; Tober et al., 2014; Wu, Fan, Yue, Xiong, & Dong, 2014) . Nevertheless, although the VSV protein(s) is important for this enhancing effect, it has not been fully identified.
IgM possesses multiple functions that promote effective antimicrobial properties, including particle agglutination (direct neutralization), complement activation, and enhanced phagocytosis. In addition, IgM has the ability to influence the generation of adaptive immunity (Boes et al., 1998) and to influence the magnitude of virus-specific IgG antibody responses (Baumgarth et al., 2000) . Natural IgM is present in the blood at high concentrations, while immune IgM is the first antibody isotype induced upon infection or immunization, suggesting that vaccine-induced IgM may help to improve the efficacy of RABV vaccination in the context of PEP (Racine & Winslow, 2009) . IgM plays critical roles in limiting virus dissemination for some neurotropic and non-neurotropic viruses, including West Nile virus(Diamond et al., 2003) , influenza virus (Baumgarth et al., 2000; Kopf, Brombacher, & Bachmann, 2002) , and vesicular stomatitis virus (Hangartner, Zinkernagel, & Hengartner, 2006) . The role of IgM generated from RABV vaccination and its protection have been studied elsewhere. Dorfmeier et al. reported that antibody subtype and subclass revealed the potential for early protective IgM antibodies in recombinant RABV-M-immunized mice (Dorfmeier, Lytle, Dunkel, Gatt, & McGettigan, 2012) . One IgM antibody is needed to cover 9 or 10 glycoprotein spikes on the surface of RABV particles for neutralization, compared to one or two IgG antibodies to cover only three glycoprotein spikes (Flamand, Raux, Gaudin, & Ruigrok, 1993) . Very recently, Dorfmeier et al. reported that IgM showed a very important role in postexposure vaccination (Dorfmeier, Shen, Tzvetkov, & McGettigan, 2013) . Taken together, the information from all reference studies suggests that immune IgM is induced in response to vaccination, and that the high valency and lower number of IgM than IgG needed to cover the RABV spike glycoproteins make induced IgM attractive from a PEP vaccination standpoint.
In conclusion, replication-deficient VSVs expressing RABV-GP displayed well-folded RABV-GP on their surface and elicited potent humoral immune responses. To our knowledge, no VSV-based vaccines against RABV have been reported. Our study provides a novel and efficient strategy for developing vaccines against this specific viral infection. However, the significant differences between mice, dogs, and humans should not be overlooked. Therefore, confirming the effectiveness and safety of vaccines in humans and dogs is an important topic for future research on vaccines against rabies. In particular, based on high level of IgM induced, our vaccine might be useful for postexposure vaccination.