The Construction And Immunogenicity Studies Of DNA Vaccine And Recombinant Rabies Virus-Based Vectored MERS Vaccine

The Construction and Immunogenicity Studies of DNA Vaccine and Recombinant Rabies Virus-Based Vectored MERS VaccineMiddle East Respiratory Syndrome（MERS）-Coronavirus（MERS-CoV）,a highly lethal respiratory tract virus,is the causative agent of MERS.In many patients,the disease caused by the MERS-CoV unusually follows an aggressive clinical course,with rapid deterioration and high mortality.MERS-CoV was first identified in Saudi Arabia in 2012,since then,laboratory-confirmed MERS-CoV infection cases have been reported in 27 countries.At the end of April 2018,World Health Organization（WHO）has been officially notified of 2 206 MERS cases with at least 787 related deaths（³5.7%mortality）.What’s more,confirmed cases of MERS-CoV have also spread from the Middle East to Africa,Aisa,Europe and North America.The high MERS-CoV infection case fatality rate,growing geographic distribution,human-to-human transmission,as well as the household clusters and nosocomial outbreaks have raised the concern of a global pandemic and placed MERS disease as one of the 11 diseases prioritized in the WHO R&D Blueprint for Action to Prevent Epidemics.Currently,no licensed vaccines or therapeutics are available for MERS-CoV infection,moreover,the quarantine and biosecurity measures in place to reduce the exposure of humans to the virus are also limited.Several studies have shown camels especially dromedary camels serve as an important reservoir for the maintenance and diversification of the MERS-CoVs and are the main source of human infections with this virus.As highlighted in the ongoing WHO-led consultation on an ideal target product profile for MERS vaccines,a camel vaccine strategy was probably for the best way to prevent human MERS-CoV infections,moreover,may have the faster development and licensing pathway.Therefore,the development of safe and effective MERS vaccines to contain the threat is of great significant.Furthermore,central Asia is serve as an important bridge of the Silk Road Economic Belt and the 21st-Century Maritime Silk Road（the Belt and Road）.The development of MERS-CoV efficient countermeasures and vaccines will actively promote the joint prevention and control among China and many countries along the road,enhancing China’s international status.For all this,we conduct the construction and immunogenicity studies of DNA vaccine and recombinant rabies virus-based vectored MERS vaccine.1.The construction and immunogenicity study of DNA vaccineS protein has been considered as the most immunogenic antigen of MERS-CoV.It the major target for current vaccines development to protect against MERS-CoV infection.Comparison of the immunogenicity of different S gene fragments will provides valuable reference for MERS vaccine development.Here,the synthetic S,SΔCD（S without the cytoplasmic domain）,and S1 subunit were respectively subcloned into the mammalian expression vector pcDNA3.1（+）to generate the recombinant plasmids pcDNA3.1-S,pcDNA3.1-SΔCD,and pcDNA3.1-S1.Expression of MERS-CoV spike protein in 293T cells respectively transfected with the above recombinant plasmids was confirmed by Western blot.The expression level of S1protein was significantly higher than S and SΔCD.Of the three DNA vaccines constructed,pcDNA3.1-S1 DNA vaccine elicited the highest anti-MERS-CoV antibody titers in immunized mice and thus was selected for further experiments.This is the first study compared the immunogenicity of different S gene fragments in MERS-CoV DNA vaccines（published in Vaccine）.We evaluated the antigen-specific humoral and cellular immune responses induced by MERS DNA vaccine in mice,and further investigated the protective efficacy of pcDNA3.1-S1 DNA vaccine in an Ad5-hDPP4-transduced mouse model following MERS-CoV challenge.Our data show that high levels of anti-MERS-CoV neutralizing antibodies were generated following three immunizations,the serum samples neutralized 50%MERS-CoV in vitro even after 1:10⁴ dilution.The pcDNA3.1-S1 DNA vaccine significantly enhanced the frequencies of antigen-specific CD4⁺T cells and CD8⁺T cells,and markedly increased the secretion of IL-2,IL-4,IL-10 and IFN-γin splenocytes.DNA vaccine immunization induced a balanced Th1/Th2 immune responses in BALB/c mice.Furthermore,both vaccine immunization and passive transfer of immune serum from vaccinated mice accelerated virus clearance in the lungs.Our results clearly indicate that the pcDNA3.1-S1 DNA vaccine induced both antigen-specific humoral immune responses as well as cellular immune responses in mice,able to well protected h DPP4-transduced mice from MERS-CoV challenge.Of note,our results strongly support the use of S1 protein of MERS-CoV as an effective target for gene-based MERS vaccine development,which provides important reference for the further construction of the recombinant rabies virus-based vectored MERS vaccine.2.The construction and identification of the recombinant RABV expressing MERS-CoV S1 proteinRecombinant rabies virus-based vectored vaccines present a range of unique advantages such as high virus titer,suitable for large-scale preparation,high levels of exogenous protein expression,which makes the rescued recombinant rabies virus able to be used as a candidate vaccine strain.In the current study,a recombinant cDNA clone expressing MERS-CoV S1 gene was generated based on our previously constructed rabies virus strain SRV9 reverse-genetic system.To rescue the recombinant rabies virus,the full-length recombinant cDNA clone and four helper plasmids expressing the N,P,G,and L genes of SRV9 parent virus were co-transfected into BSR cells.The insertion and expression of exogenous genes were identified from both genomic and protein analysis.Moreover,the growth kinetics,genetic stability as well as neurovirulence were assessed through in vitro and in vivo studies.Our results show that the recombinant rabies virus(rSRV9-MERS_S1)was recovered from BSR cells as confirmed by direct immunofluorescence assay.The morphology of rSRV9-MERS_S11 was examined by transmission electron microscopy,which was bullet shaped as the morphological features of the typical rabies virus.RT-PCR detections demonstrate that MERS-S1 gene were inserted into the genome of rSRV9-MERS_S11 and maintained genetic stability.The expression of MERS-CoV S1 protein was identified by indirect immunofluorescence detection assay,SDS-PAGE as well as Western Blot analysis.As shown by the immunoelectron microscopy evaluation,MERS-CoV S1 protein and RABV G protein were both incorporation into recombinant rabies virus.The constitutive expression of exogenous gene laid the foundation for further virus inactivation.Growth curves showed that the new virus expressing MERS-CoV S1 grew to similar titers as rSRV9parent virus on BSR cells,the highest titer of rSRV9-MERS_S11 was up to 2×10^8.5TCID₅₀/mL.The results of intracerebral challenge in suckling mice and adult mice demonstrate that the neurovirulence of rSRV9-MERS_S11 was reduced compared to the parental virus rSRV9.Overall,we constructed the recombinant cDNA clone expressing MERS-CoV S1 gene and rescued a recombinant rabies virus expressing MERS-CoV S1protein with high titers and genetic stability,as well as the S1 protein of MERS-CoV were successfully incorporated into the RABV virions.The recovered recombinant rabies virus rSRV9-MERS_S11 can be used as a candidate vaccine strain for further immunogenicity study in animal models.3.The immunogenicity study of recombinant rabies virus-based vectored MERS-CoV vaccineThe development of safe and effective dromedary camel vaccine for the prevention of transmission of MERS-CoV among camels and from camels to humans is of great significant.In the current study,the recombinant rabies virus-based vectored MERS vaccine were developed based on the rescued rSRV9-MERS_S1,then the immunogenicity of the recombinant rabies virus-based vectored MERS vaccine was investigated in mice with different forms（live,inactivated）and delivery doses（one dose,two doses）.The inactivated rabies virus-based vectored MERS vaccine with two immunizations elicited the most robust immune response in immunized mice and thus was selected for further experiments in C57BL/6 mice,alpacas and camels.The dynamic changes and duration of serum antibody,neutralizing antibodies titer as well as antigen-specific T cells responses of the immunized mice were evaluated.The levels of neutralizing antibodies against both MERS-CoV and RABV were respectively detected in the serum samples from the immunized alpacas and camels.Our data show that the total anti-MERS-CoV IgG antibody titers peaked at 5 weeks post the first immunization and were still detectable 10 months post the first immunization.The serum samples from inactivated vaccine immunized mice were able to neutralize the MERS pseudoviruses expressing S proteins of human MERS-CoV KOR/HIN strain and dromedary camel MERS-CoV D1271 strain.Besides,the inactivated rabies virus-based vectored MERS vaccine markedly increased the antigen-specific CD8⁺T cells responses in mice.Furthermore,potent neutralizing antibodies cross-neutralizing representative human and camel MERS-CoV strains were induced in the immunized alpacas and camels.High levels of anti-RABV neutralizing antibodies（above 0.5 IU/m L）were generated in both immunized alpacas and camels,higher than the standards 0.5-IU level considered protective by WHO and World Organisation for Animal Health（OIE）.Above all,our results demonstrate that the inactivated rabies virus-based vectored MERS vaccine induced antigen-specific humoral immune responses and T cell immune responses,would provide a novel technical storage for the control of camel MERS-CoV.4.Evaluation of DNA vaccine and inactivated recombinant rabies virus rSRV9-MERS_S1 vaccine combination strategies for MERS-CoVSeveral studies have shown that for many vaccines combination approach is more effective than the homologous multiple immunizations against infection,with eliciting higher frequency of antigen-specific memory T cell immune responses.To optimize and promote the efficacy of our immunization strategy for MERS vaccines,here,a heterologous MERS DNA vaccine prime-inactivated recombinant rabies virus vaccine boost immunization approach was assessed,and compared with the strategy using either pcDNA3.1-S1 DNA vaccine or inactivated rSRV9-MERS_S1 vaccine for homologous multiple immunizations.Additionally,considering the coinoculation strategy is preferred to the prime-boost strategy since administration of a single vaccine formulation rather than different vaccines for primary and booster immunizations is ideally suited for use in vaccination regimens involving multiple immunizations,a combination MERS vaccine containing a DNA vaccine and inactivated recombinant rabies virus rSRV9-MERS_S1 vaccine was further valuated in mice.Both the induction of antigen-specific humoral immunity and cellular immunity were evaluated in the above strategies.Our results demonstrate that the heterologous prime-boost vaccination was more immunogenic than the homologous immunizations using either DNA vaccine or inactivated recombinant rabies virus rSRV9-MERS_S1 vaccine alone,with significantly higher levels of MERS-CoV neutralizing antibodies and frequencies of CD8⁺T cell immune responses.Furthermore,our results show that mice received coinoculation developed higher total anti-MERS-CoV IgG antibody titers,as well as significantly higher neutralizing antibodies and antigen-specific CD8⁺T cell immune responses.Taken together,our current data demonstrate that immunization with the combination strategies using MERS DNA vaccine and inactivated recombinant rabies virus rSRV9-MERS_S1 vaccine were able to significantly enhance the immunogenicity than the homologous multiple immunizations with the above vaccines alone.Moreover,the combination strategies used in the current study can be applied for enhancing the potency of MERS vaccines and may provide reference for the development of immunization strategies as well as novel and cost-effective MERS vaccines in the future.