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.