Modified Rabies Virus ERA Strain And Recombinant ERA Vaccine Strain Exspressing Glycoprotein Of Ebola Or Marburg Virus

Ⅰ: Modified rabies virus ERA strainRabies is a zoonotic infectious disease caused by the rabies virus(RABV), leading to almost 100% acute fatal encephalitis once the neurologic symptoms appear. Rabies is responsible for approximately 60,000 human deaths per year every year, more than 95% in Africa and Asia. The exposure to rabid dogs is still the main cause of almost all of human deaths worldwide. For preventing human rabies, the most efficient strategy is to eliminate the disease through vaccination. However, the increasing number of stray dogs, and even some free-range livestock and wildlife rabies is also increased the risk of human rabies in rabies epidemic region. Oral immunization for free-range animals and wild animal species, especially canine, is one of the most practical approaches protecting human from rabies. Therefore, the safe and effective oral rabies vaccines are still being sought.In this study, the biological characteristic and feasibility of a modified ERA vaccine strain(r ERAG333E) containing a substitution of Arg with Glu at position 333 in glycoprotein to serve as an oral rabies vaccine against rabies in dog was evaluated. r ERAG333 E and the parent strain r ERA grew to similar levels in BSR cells and reached peak titers of approximately 8.2 lg FFU/m L at 96 h post-infection. The genetic stability of the G333 E mutation in the genome background of the ERA strain was confirmed by RT-PCR and sequencing with r ERAG333 E from the 5th passage in suckling mouse brains and the 20 th passage in NA cells. The in vitro neurotropism index of r ERA and r ERAG333 E was 0.4 and 0, respectively. As expected, the r ERAG333 E lost the in vitro neurotropic characteristics. To investigate the pathogenicity of r ERAG333 E in mammals, three-week-old juvenile mice and three-day-old sulking mice were inoculated intracerebrally(i.c.) with r ERAG333 E or r ERA. All juvenile mice inoculated with r ERAG333 E survived at 4 weeks post-inoculation and had no significant difference in the body weight changes. In the contrast, all juvenile mice inoculated with r ERA showed typical clinical signs of rabies, significant body weight changes and died within 14 days of inoculation, which indicated that the G333 E mutation lost the virulence of the ERA strain in adult mice. Furthermore, the death date of suckling mice i.c. inoculated with r ERAG333 E was postponed 2 days compared to that with r ERA, which indicated that the G333 E mutation decreases the virulence of the ERA strain in suckling mice. To assess the immunogenicity of r ERAG333 E, mice and dogs vaccinated intramuscularly(i.m.) with r ERAG333 E developed a strong RABV neutralizing antibody(VNA) response, and completely protected mice from challenge with a lethal dose of street virus GX/09 strain at 3 weeks post-vaccination(p.v.). These results demonstrated that the r ERAG333 E is safe and immunogenic in mice and dogs.To investigate the safety and efficacy of r ERAG333 E as an oral vaccine against rabies, mice vaccinated orally with 100 μL PBS contains 106, 107 or 108 FFU dose of r ERAG 333E developed a strong RABV VNA response, which completely protected mice from challenge with a lethal dose of street virus GX/09 strain at 52 weeks p.v.. Meanwhile, dogs were distributed randomly to be vaccinated orally with 1,000 μL PBS contains 108 or 109 FFU dose of r ERAG 333E one dose or two doses of 55 weeks interval. The one dose vaccinated dogs developed a long-lasting for a period of 156 weeks RABV VNA response, which the mean titers of VNAs to RABV in 109 FFU dose-vaccinated dogs was 1.24 IU/m L, whereas that of 108 FFU dose-vaccinated dogs was 0.34 IU/m L(2/4 > 0.5 IU/m L). The two doses both in 108 FFU and 109 FFU vaccinated dogs developed strong, stable VNAs to RABV with 2.71 IU/m L or 1.54 IU/m L of mean titer VNAs to RABV at 55 weeks p.v., respectively. After receiving the booster vaccination at 55 weeks p.v., the dogs, both in 109 FFU dose and 108 FFU dose groups, showed substantial boost responses, and induce strong and long-lasting VNAs to RABV with 1.80 IU/m L or 1.61 IU/m L of the mean titers at 104 weeks post boost vaccination, respectively. Furthermore, mod ERAte levels of saliva RABV-specific Ig A were detected both in109 FFU and 108 FFU dose-vaccinated dogs, and the positive conversion rate were 50%(5 of 10) and 60%(6 of 10) at 4 weeks p.v., respectively. Of note, no abnormal clinical signs were observed during the course of the dog study.These results demonstrate that r ERAG333 E is safe and induced long-lasting for a period of 3 years protective RABV VNA response in dogs, resulting a candidate of oral rabies vaccine for dogs. Ⅱ: Recombinant ERA vaccine strain exspressing glycoprotein of Ebola or Marburg virusEbola hemorrhagic fever(EHF) and Marburg hemorrhagic fever(MHF) are zoonosis diseases caused by the Ebola virus(EBOV) or Marburg virus(MBGV) respectively, cause acute hemorrhagic fever and high case-fatality rate. Because of potential for human-to-human transmission and observed infection of fruit bats or other wild animals and dogs, pigs or other free-ranging livestock, EHF and MHF are still serious worldwide public health problem once epidemic outbreaks. Unfortunately, there is no vaccine with official license for EHF and MHF prevention or treatment. Of particular concern, it is extremely difficult to eliminate the source disease from reservoir and cut off the transmission from free-range animals through the injected vaccine. Therefore, the development of efficacious vaccine strategies to protect humans and animals against EHF and MHF has always been of importance and urgency.Therefore, we gen ERAted recombinant RABVs, r ERAG333E/ZGP, r ERAG333E/ZGPGCD, r ERAG333E/SGP, r ERAG333E/SGPGCD, r ERAG333E/MGP and r ERAG333E/MGPGCD expressing the ZEBOV, SEBOV or MBGV wild-type G protein(wt-G) and codon-optimized G protein(co-G) by using the reverse genetics based on the candidate oral rabies vaccine strain r ERAG333 E. The recombinant RABVs express the expected G protein, respectively, and have the similar biological characteristics, excellent adaptability and genetic stability in BSR cells compared with parental r ERAG333 E, and avirulent in adult mice. To investigate the pathogenicity of the recombinant RABVs, adult and suckling mice i.c. inoculated with the EBOV relative recombinant RABVs and adult mice orally or i.m. inoculated with the MBGV relative recombinant RABVs showed that all infected adult mice had no signs of rabies and infected suckling mice had the similar death pattern compared with the vector. These results demonstrated that the insertion and expression of EBOV or MBGV G gene did not increase the virulence in mice.To assess the immunogenicity of the EBOV relative recombinant RABVs, mice were orally or i.m inoculated with live virus for one dose or i.m inoculated with BPL-inactive virus for two doses, respectively. All mice inoculated recombinant RABVs and the vector virus by different routes or types of virus developed strong and lasting for a period of 13 weeks RABV, ZEBOV or SEBOV VNA response and high level Ig G and Ig G2 a to ZEBOV or SEBOV. Orally inoculated-mice developed relatively lower level of Ig G but similar level of Ig G2 a to ZEBOV or SEBOV at different time points compared to mice i.m. inoculated with live or inactive viruses. There is no significant different between mice inoculated with wt-G or co-G inserted recombinant RABVs both in VNAs to RABV, ZEBOV, SEBOV and the level of Ig G and Ig G2 a. These results demonstrated that the recombinant RABVs r ERAG333E/ZGP, r ERAG333E/ZGPGCD, r ERAG333E/SGP and r ERAG333E/SGPGCD are immunogenic in mice.To assess the immunogenicity of the MBGV relative recombinant RABVs, mice were orally or i.m inoculated with live virus for one dose or i.m inoculated with BPL-inactive virus for two doses, respectively. All mice inoculated recombinant RABVs by different routes or types of virus developed strong RABV VNA response, which were completely protected mice from challenge with a lethal dose of street virus GX/09 strain at 5 weeks post-vaccination(p.v.), and mod ERAte MBGV VNA response. Meanwhile, there is no significant different between mice inoculated with wt-G or co-G inserted recombinant RABVs both in VNAs to RABV and MBGV. These results demonstrated that the recombinant RABVs r ERAG333E/MGP and r ERAG333E/MGPGCD are immunogenic in mice.These results demonstrate that the recombinant RABVs r ERAG333E/ZGP, r ERAG333E/ZGPGCD, r ERAG333E/SGP, r ERAG333E/SGPGCD, r ERAG333E/MGP and r ERAG333E/MGPGCD are safe and immunogenic in mice, and have potential to be animal oral vaccine or human inactive vaccine against rabies and Zaire EHF, Sudan EHF or MHF.