Construction And Immunoprotective Evaluation Of A Recombination Toxoplasma Gondii Strain Expressing The Spike Protein Fragments Of The Feline Infectious Peritonitis Virus

Feline Infectious Peritonitis(FIP)is a highly lethal disease in felines caused by the abnormal immune response to Feline Infectious Peritonitis(FIPV).Cats infected with FIPV develop purulent granulomatous lesions in multiple organs throughout the body or produce large effusions in the thoracic and abdominal cavities,with a mortality rate approaching 100%.Toxoplasma gondii,another feline pathogen,is a specialized intracellular parasite prevalent worldwide.The oocysts excreted by cats infected with Toxoplasma gondii,cause long-term infection in the environment,resulting in a widespread epidemic of Toxoplasmosis.There is still no ideal FIPV vaccine or Toxoplasma gondii,vaccine for clinical application.Although the currently reported FIPV vaccine candidates are able to induce high titers of neutralizing antibodies in the host,stimulation by humoral immunity alone still did not provide effective immune protection in cats,suggesting that the key for FIPV vaccine design may lie in the induction of more robust cellular immunity.Toxoplasma gondii,infection can effectively induce a cellular immune response in the host,and using Toxoplasma gondii,as a vector provides a new idea for the design of FIPV vaccines from the level of enhancing cellular immunity.The Toxoplasma gondii gene deletion strain ME49ΔompdcΔldh1 constructed in our laboratory by Taifang Zhou had superior live attenuated vaccine potential and provided better immune protection for intermediate hosts,but its immune protectiveness against Terminal hosts was unknown.The spike S(Spike,S)protein is a key protein for FIPV infection of the host.Its fragment S1 subunit is mainly responsible for recognizing the host receptor and contains a Receptor Binding Domain(RBD)that mediates receptor binding of the virus to the host cell and is an important target antigen for subunit vaccine design and induction of immune protection in the host.In this study,we used ME49ΔompdcΔldh1 as a vector to construct a recombinant worm strain expressing the S1 subunit and RBD region of the FIPV sting protein,and evaluated its in vitro growth phenotype and in vivo pathogenicity,safety,and immune protection against Toxoplasma gondii and feline infectious peritonitis virus infection.1.Construction of recombinant strains expressing FIPV S1 subunit and RBD region and in vitro growth phenotype studyThe FIPV S1 and RBD genes were inserted into the genome of Toxoplasma gondii ME49ΔompdcΔldh1 by CRISPR/Cas9,and the FIPV S1 and RBD genomes were integrated into the genome of Toxoplasma gondii and the S1 and RBD proteins were stably expressed in the Toxoplasma gondii vesicle space by PCR and IFA.The in vitro growth phenotypes of the recombinant strain ME49ΔompdcΔldh1 Com FIPV S1 and ME49ΔompdcΔldh1 Com FIPV RBD were not significantly different from those of the parent strain ME49ΔompdcΔldh1 and the complementation of FIPV genes did not affect their growth stability.2.In vivo pathogenicity and safety evaluation of recombinant strains expressing FIPV S1 subunit and RBD regionThe recombinant strain was inoculated intraperitoneally with 10~4tachyzoa in female Kunming mice,and the survival rate of the mice was 100%after 30 days without the formation of brain encysts,indicating that the recombinant strain had little virulence to the Toxoplasma intermediate host and did not cause chronic infection;Healthy adult cats were inoculated subcutaneously with 10~6tachyzoites.After inoculation,body temperature and body weight were normal,and there were no adverse effects such as depression,loss of appetite and hair loss,and no oocysts were excreted.The results showed that the recombinant strain was not pathogenic to the intermediate and final hosts of Toxoplasma gondii and had a good safety profile.3.Evaluation of immune protection of recombinant strains expressing FIPV S1 subunit and RBD regionIn a protection test against Toxoplasma gondii infection,a model of Toxoplasma gondii ME49 oocyst infection was established to successfully simulate Toxoplasma gondii infection in cats under natural conditions.Oocysts were detected in the feces of the non-immunized cats on day 4-5 after infection and continued to be discharged for about 10-15 days,with a maximum of 1.2×10~6oocysts per day,while no oocysts were discharged in the feces of the immunized cats.This indicated that the immunized recombinant strain was effective against wild-type ME49 oocysts(800 oocysts/cat)and provided better immune protection against Toxoplasma gondii infection in the Terminal-host cats.In the FIPV QS-79 attack protection test,cats in the unimmunized group showed a rapid increase in body temperature,clinical signs such as wasting,shortness of breath and ocular jaundice within 1 week after the attack,and 100%mortality;in contrast,the immunized group delayed the onset of symptoms by 1-2 weeks,and the cat survival cycle was extended by 2-3 weeks.This indicates that immunization with recombinant strains ME49ΔompdcΔldh1 Com FIPV S1 and ME49ΔompdcΔldh1 Com FIPV RBD delayed the onset of symptoms in affected cats,and bought more valuable treatment time for cats with FIP in clinical practice.In conclusion,the two recombinant Toxoplasma gondii strains ME49ΔompdcΔldh1 Com FIPV S1 and ME49ΔompdcΔldh1 Com FIPV RBD,which express the S1 subunit of FIPV stinging protein and RBD region,constructed in this study,can provide better immune protection against Toxoplasma gondii infection in cats after immunization,and can prolong the survival cycle of cats with FIP,and provide a reference for the prevention and control of FIP and toxoplasmosis,which are more dangerous in companion animals cats.