During the past decade, a number of recombinant rabies vaccine candidates based on live attenuated RABV or recombinant viruses expressing RABV G (such as V-RG) have been developed as potential alternatives to current rabies vaccines (17
). While some of the vaccine candidates generated protective immunity when administered via the i.m. route, data on the efficacy of oral immunization with these candidates are lacking. Of those reported, oral immunization with canine adenovirus carrying the rabies virus G gene did not confer protection against rabies virus infection in mice (32
). As a safer alternative to V-RG, the recombinant MVA vaccine expressing a rabies virus glycoprotein gene was generated and tested in mice, and the result showed that protection was observed only in mice vaccinated with a dose as high as 109
PFU by a peripheral route (17
). In this work, to demonstrate the proof of principle, we inserted the G gene of RABV between the HN and L genes of PIV5 and found that this vaccine candidate was effective via oral immunization as well as i.n. and i.m. immunization. To our knowledge, this is the first demonstration of oral immunization efficacy in mice for a rabies virus vaccine using paramyxovirus as a vaccine vector. The results also suggest that PIV5 can be used as a vector for developing vaccines for which oral delivery is essential. Further development of the PIV5-based RABV vaccine may lead to a novel RABV vaccine for humans and for animals.
We tested three immunization routes in mice. Of those, i.n. inoculation gave the best immune responses and protection, demonstrating that rPIV5-RV-G can elicit a protective immune response against rabies. i.n. immunization has been used in many pet dogs in the United States for many years, such as for kennel cough vaccination. The fact that PIV5-RV-G was effective via i.n. immunization suggests that it is possible to incorporate it into the existing canine vaccination program. We have found that the i.m. route of immunization was more effective at higher doses. In this work, we detected incorporation of RABV G into rPIV5-RV-G virions (). It has been reported before that there are about 1,200 RABV G proteins (400 trimers) in RABV virions (36
). Thus, there were about 150 to 180 G proteins in rPIV5-RV-G. We speculate that the higher that the dose of PIV5-RV-G was, the more G protein was injected in the i.m. immunization. In our recent work, highly efficacious immunization with a PIV5-based vaccine via the i.m. route does require virus to be live, since the inactivated PIV5-based vaccine provides only partial immunity when it is administered via the i.m. route (38
). While a single-dose inoculation provided immunity against lethal rabies virus challenge in mice, a boost further enhanced the efficacy of rPIV5-RV-G: prime-boost with 105
PFU of rPIV5-RV-G provided 100% protection, whereas 77% protection was afforded by a single dose of 105
PFU via the i.n. route of inoculation. Average VNA titers increased from 2.76 IU with one dose of 105
PFU to 7.94 IU with the two-dose vaccination. Most remarkably, VNAs in mice were increased from 4.73 IU with one dose to 62.96 IU with two doses of 106
PFU of rPIV5-RV-G via i.n. inoculation. The anamnestic immune response was elicited in mice vaccinated with rPIV5-RV-G, suggesting that prior exposure to PIV5 did not prevent a robust immune response to PIV5-vectored antigen. One of the concerns over using an rPIV5-RV-G vaccine is whether preexisting anti-PIV5 immunity will negatively influence the efficacy of the PIV5-vectored vaccine. The robust immune responses from the boost with live vaccine suggest that preexisting immunity to the viral vector did not affect the efficacy of the PIV5-based vaccine. Recently, we have found that dogs with neutralizing antibodies against PIV5 generated a protective immune response against influenza virus after immunization with PIV5 expressing HA of influenza A virus, demonstrating that a PIV5-based vaccine is effective in dogs with prior exposure (39
). The use of rabies vaccines, especially live attenuated ones, in newborn dogs is limited due to maternal antirabies antibody, which can last as long as 6 months. The PIV5-based rabies vaccine provides an alternative that can effectively be used to vaccinate newborn dogs, further demonstrating the benefit of using a PIV5-based rabies vaccine for dogs.
While i.n. immunization provided the best protection for rPIV5-RV-G, to vaccinate stray dogs or wild animals, oral immunization will be the best approach. Oral vaccine has advantages over traditional vaccines, such as its ease of use, compatibility with mass immunization campaigns, and ability to reach hard-to-reach species (40
). Although only half of the mice in our study were protected with one-dose vaccination, the result is encouraging. It is comparable to the rate achieved with the current live rabies vaccine. The protection mechanism of oral vaccination is poorly understood. The VNA titer of serum from peripheral blood from mice vaccinated orally indicated a systemic immune response. The range of VNA titers from the oral inoculation was from 0.1 IU to 3.8 IU, with the average titer being 1.5 IU. We speculate that PIV5 was able to deliver antigen to mucosal cells by the oral route, which resulted in specific immune responses, including a systemic response to the PIV5-vectored antigen. It is possible to further increase the protection efficacy of oral inoculation by using a prime-boost regimen. In addition, modification of the PIV5 vector may increase the efficacy of the PIV5-vectored rabies vaccine. In our recent work, we have found that the insertion site of a foreign gene within PIV5 affects the immunogenicity of the PIV5-based vaccine (41
). For instance, insertion of HA of H5N1 between SH and HN within PIV5 results in a vaccine candidate that generates better immunity against H5N1 challenge than a vaccine with the insertion of HA of H5N1 between HN and L within PIV5 in mice (41
). We can further improve the efficacy of the PIV5-based vaccine for oral immunization by inserting the G gene in different places within the PIV5 genome. In addition, expression of additional rabies virus antigens may enhance the potency of the PIV5-based vaccine. It has been reported before that the rabies RNP can be protective (42
). For example, expression of one of the rabies virus proteins N, P, or L together with G using PIV5 may enhance immune protection efficacy.
The fact that the PIV5-based rabies vaccine generated a robust immune response that protected against a lethal challenge from rabies virus infection demonstrates the potential of PIV5 as a vaccine that may be used to control rabies virus infection in dogs as well as its potential to be a vector for other infectious diseases in dogs and other animals as well as in humans. Because live PIV5 has been used in dogs for many years without safety concerns for dogs and humans, incorporating a PIV5-based vaccine into existing vaccination programs for dogs should be feasible.