Construction Of Rabies Virus Oral Vaccine Strain Containing LTB Gene And Immunization In Mice

Rabies is caused by the rabies virus?RABV?which leads to fatal and highly neurotropic zoonotic infectious diseases.Its mortality is almost 100%and itIt has become a major public health issue.There is no effective therapy for this disease,however,complete prevention can be achieved through pre-or post-exposure prophylaxis.Therefore,it is critical to explore the replication and pathogenesis of the RABV,and develop the affordable and efficient vaccines.Mucosal immunity is an important goal of vaccine development because it can not only resist pathogens that invade through the mucosa,but also may be related to optimizing immunotherapy for tumors located on the mucosa.Oral vaccines are effective in inducing immune responses and immune tolerance in the body.It is an ideal way of immunity because Its operation is simple and quick.Oral antigenic protein alone does not produce good immunogenicity,and it is usually combined with a certain adjuvant or carrier to produce the desired,ideal immune effect.The Enterotoxin coli heat-labile enterotoxin B subunit?LTB?has proven to be a good mucosal immune adjuvant molecule.This study on the established reverse genetic operating platform in laboratory,the recombinant pfull-length cDNA lasmids pHEP-Get-LTB with Get gene and LTB gene have been constructed and successfully rescuing the recombined virus HEP-Get-LTB.The results of the biological characters study of the recombined virus HEP-Get-LTB indicated that the recombined virus HEP-Get-LTB could stably proliferate in NA cells.There was no significant difference of the virus titer with rHEP-Flury,both of which could reach higher levels.The growth curve showed that HEP-Get-LTB had similar growth characteristics with rHEP-Flury.At MOI of 0.01 and 3,the titers of three virus all peakde at72 h.Animal experiments showed that the LD₅₀ of recombinant virus HEP-Get-LTB on the neonatal 10^-4.33/0.03 mL,slightly lower than rHEP-Flury.Imtracranial vaccination 5⁶weeks of adult mice,shows no fatal.Through oral immunization and intranasal immunization,effect of HEP-Get-LTB on the weight of mice higher than the parent strain rHEP-Flury.However,There are no significant differences when through intramuscular injections and intracranial injections.The results of immunogenicity indicated that the recombined virus HEP-Get-LTB can induced the higher production of the rabies antibody in mice than the parent rHEP-Flury through oral immunization and intranasal immunization.Through Intramuscular injection,the recombined HEP-Get-LTB and the parent rHEP-Flury can Induce mice to produce neutralizing antibodies above 0.5 IU/mL and their protection rate is over 80%.It shows that HEP-Get-LTB is similar to rHEP-Flury when through conventional immunization that can induce mice to produce neutralizing antibodies with effective protective level.Moreover,It has better immunogenicity through oral immunization.In summary,the recombinant virus HEP-Get-LTB has good virus replication and Its pathogenicity,immunogenicity,and immune protection vary under different modes of immunization.There is a clear advantage over rHEP-Flury in oral immunization.It is necessary to further study its toxicity and adjust its immunization dose and immunization program to improve its applicability.