Basic Research Of Oral Rabies Virus Vaccine By Using Attenuated Salmonella

Rabies is a highly fatality rate zoonosis in human and animals.Rabies virus (RV) is mainly transmitted to people by dogs/animal bites. Large scale of vaccination rabies vaccine in dogs and other animals is the best way for controlling rabies. Currently, reversion to virulence in animal– attenuated, live rabies virus vaccine is a public safety hazard. The inactivated rabies vaccines currently used in China are imported. However, high cost and complex vaccination procedures of these vaccines are out-of-step with the rabies control strategy in China. A rabies oral vaccine is recommended for its facility, simple vaccination procedure and its suitability for large-scale rabies vaccination campaigns.Salmonella has more than 2,000 serotypes. In general,salmonellae serovars are able to infect a wide range of hosts from cold-blooded animals to humans. Mice and dogs are well known to be highly susceptible to Salmonella enterica serovar Typhimurium (S. Typhimurium). As an invasive, intracellular pathogen, Salmonella has an affinity for mucous membranes and antigen presenting cells (APCs). Attenuated Salmonella can deliver foreign antigens directly to APCs by natural mucosal infection, and as a result, stimulate antigen-specific mucosal, humoral and cellular immune responses. CpG motifs and outer membrane proteins, natural adjuvants, are present at high quantity in Salmonella. In addition, Salmonella pathogenesis has been studied in depth, and attenuated mutants have been widely used as candidate live bacterial vaccine vectors. To date, attenuated Salmonella have been employed to mediate the presentation of more than 40 kinds of foreign antigens from various viruses, bacteria, and parasites. Also, attenuated Salmonella have been used to deliver anti-tumor therapies.RV glycoprotein (GP gene) is the only antigen effective in inducing virus-neutralizing antibodies (VNA) and immune function equals that of whole RV vaccines. Nucleoprotein (NP gene) can stimulate cell-mediated immunity (CMI) and augment protection. With these facts in mind, we have undertaken the following experiments:1. Using PCR and cloning techniques, we inserted the rop gene regulation fragment of pBR322 into the pVAX1 (i.e.pV) carrier to successfully construct a low-copy eukaryotic expression vector pR. RT-PCR was employed to amplify the GP and NP genes of the Chinese human rabies virus vaccine strain CTN-1. Single gene expression vectors pV-G and pV-N, including the GP or NP gene, respectively, were constructed by recombinant DNA technology. Overlapping PCR was used for gene splicing, and the fusion expression vectors pV-GT and pR-GT were constructed in which the tetanus toxin fragment C (TTc) and GP gene were connected by a flexible linker. Indirect immunofluorescence assay(IFA)and Western blot demonstrated that recombinant proteins were successfully expressed. The RV gene-expressing plasmids were electroporated into an attenuated S. Typhimurium strain. PCR, gram-stain, and salmonella O-antigen agglutination tests were used to characterize the final constructs. After 100 passages with no antibiotic selective pressure, the single-copy pR-GT was more stable than the multicopy pV-GT, as expected. To examine vaccine efficacy, 10¹⁰ CFU recombinant bacteria were administered orally to female 11-13 gram Kunming mice on day (d) 1, 3, and 14, respectively. All control mice were of the same age and gender as the vaccinated animals, and were inoculated orally with the Salmonella phoP/phoQ vaccine strain (i.e.PQ). The protection rate in mice immunized with Salmonella expressing both GT and NP was greater than 65%, compared to groups immunized with a single RV gene or other mixed RV genes. Importantly, the control mice demonstrated signs of rabies between 3 d and 14 d after challenge, and all died.2.The DNA sequence and derived amino-acid sequence of a 179-281 aa region from the GP gene was used to perform codon-optimization,and was named Gopt. Using the low-copy prokaryotic plasmid vector pGB2, we constructed a plasmid carrier capable of anaerobically-inducing expression of any inserted antigenic gene via control of the nirB gene promoter. Exploiting the surface display properties of the ice nucleation protein, a plasmid encoding surface-expressed Gopt was constructed (i.e. pGnirB-I-Gopt). We used attenuated Salmonella carrying the constructed plasmid in a heterologus prime-boost immunization, in which attenuated Salmonella expressing the RGT+NP (i.e. PQ-RGT + PQ-VN ) were used to prime and the constructed recombinant carrier PQ-IGO was used to boost the immune response. We measured the titer of rabies VNA, IgG against Salmonella and tetanus toxin, the level of IL2 and INFγstimulated by prime-boost in the immunized groups. The results demonstrated that the recombinant DNA constructs delivered by the attenuated Salmonella carrier could induce immune responses against rabies, tetanus toxin and Salmonella. When PQ-IGopt was used for prime-boost immunization, the protection was elevated 10%, just a little lower than current commercial vaccine used for heterologous prime-boost imunization.In summary, our results indicated that the oral attenuated Salmonella vector carrying the G-TTc and NP genes, after heterologous prime-boost, could induce stronger HMI and CMI,also produced high levels of rabies VNA, and protected mice against lethal intracerebral challenge with CVS-24.There is an ever-increasing amount of domestic pets in China, commercialized trading of feral dogs, and the difficulty of controlling RV infection in wild animal populations. In addition, the existing veterinary rabies vaccines used in China suffer problems of reversion to virulence, difficult immunization regimens and high cost. To address these issues, we used the attenuated S.Typhimurium as a simple-to-use oral delivery system to express the rabies virus GP and NP (combined with TTc and codon-optimized for maximal expression in Salmonella) to construct a multivalent oral vaccine against rabies. These studies demonstrate that attenuated Salmonella can be used to develop a safe, easy to use, and low cost new rabies vaccine.