Construction And Preliminary Immune Efficacy Studies Of Recombinant Lactobacillus Expressing SARS-CoV-2 RBD And H1N1 Influenza Virus HA

Oral vaccines can stimulate humoral and cellular immune responses at the systemic and mucosal levels,and have the advantages of low cost and convenient administration.Lactic acid bacteria are food-grade probiotics that can reach the intestinal tract alive and colonize its surface.It can be used as a carrier for oral vaccine delivery and exert its probiotic function while stably expressing foreign antigens.Therefore,the development of Lactobacillus genetically engineered oral vaccine is of great significance to the development of new vaccines.The novel coronavirus(SARS-Co V-2)is a newly emerged respiratory virus with high contagiousness and high pathogenicity.It is very important to develop new vaccines to control the spread and infection of the virus.Swine influenza is one of the most important respiratory diseases in pigs,and vaccination is the main strategy to prevent and control influenza virus infection.Therefore oral vaccines that induce mucosal immune responses have the potential to be developed as new vaccines to prevent new coronavirus and influenza virus infections.This study analyzed the biological characteristics in vitro and vivo of Lactobacillus rhamnosus LR16.Then using LR16 as the lactobacillus genetic engineering vaccine carrier,constructed the recombinant lactobacillus expressing SARS-Co V-2 RBD dimer and influenza virus HA and preliminaryly studied its immune protection efficacy.The specific research results are as follows:1.Biological characteristics studies of Lactobacillus rhamnosus LR16 in vitro and vivoFirstly,the biological characteristics of Lactobacillus rhamnosus LR16 preserved in our laboratory were studied.It was found that its growth and proliferation rate in vitro was fast,and it was easy to be cultured in large quantities.Evaluated the stress resistance and probiotic properties of LR16.It was found that LR16 still had a survival rate of more than 60%at p H 3～9,partially survived at 0.1%bile salts.It was resistant to 1%pancreatin and heat treatment at 55°C and had antibacterial effect.The results show that LR16 had certain stress resistance of acid and alkali resistance,bile salt resistance,pancreatin resistance and heat resistance,and probiotic properties.In order to analyze the intestinal residence characteristics of LR16,analyzed quantitatively by RT-PCR the distribution and residence of LR16-GFP recombinant strain in the mouse intestine.It was found that the recombinant strain can reside in the ileum,cecum and colon of mice for more than 72 hours,and have good intestinal colonization ability.The results indicate that LR16 can be used as a lactobacillus genetic engineering vaccine vector to colonize the gut to deliver exogenous proteins.2.Construction and preliminary immune efficacy studies of recombinant lactobacillus expressing SARS-Co V-2 RBD2To analyze whether LR16 strain can be used as the carrier of RBD domain of SARS Co V-2 S protein.In this study,the SARS Co V-2 RBD dimer was linked respectively in tandem with CTA1-DD,DCpep,Fc,and CTB.They were inserted into the genome of LR16.Through PCR identification and sequencing,it was determined that four recombinant lactobacillus were successfully constructed.To preliminaryly study the immune protection effect of them,divided the mice into two groups:the gavage group and the free-eating group.When 14 days post 4 weeks,infected all mice with 10~3 LD50 of SARS-Co V-2(IVCAS 6.7512,Chinese Academy of Sciences)in the P4 laboratory.The viral load in the lungs of the infected mice was detected by RT-PCR.It was found that the viral load of the LR16-RBD2-CTA1-DD group was significantly lower than that of PBS control group and LR16 control group(P<0.01).The viral load of the LR16-RBD2-DCpep group was also significantly lower than that of PBS and LR16 control groups(P<0.05),while the viral load of LR16-RBD2-Fc and LR16-RBD2-CTB groups has no significant difference with LR16 control group(P>0.05).In the free-eating group,the viral load of the LR16-RBD2-CTA1-DD group was also lower than that of the LR16 control group,but the difference was not significant(P>0.05).The results show that the LR16-RBD2-CTA1-DD recombinant strain can significantly inhibit the proliferation of SARS-Co V-2 in the lungs of mice,among the four recombinant lactobacillus expressing the RBD2 of SARS-Co V-2.It had the potential to be used as the SARS-Co V-2 oral vaccine.3.Construction and preliminary immune efficacy studies of recombinant lactobacillus expressing influenza virus HATo analyze whether the LR16 strain can be used as the delivery vector of influenza virus HA.The HA1 and HA2 were linked respectively in tandem with CTA1-DD and inserted into the LR16 genome.PCR identification and sequencing confirmed that the two recombinant lactobacillus were successfully constructed.To preliminaryly study the immune protection effect of them,weekly gavage the mice for four consecutive weeks.When 7 days post gavage for 4 weeks,detected the titer of the antibody by indirect ELISA.It was found that the level of specific Ig G antibody of the LR16-HA1-CTA1-DD group was significantly higher than that of the LR16 control group(P<0.01).The mice were infected with 10~3 LD50 A/WSN/1933(H1N1)when 14 days post gavage.On the 3rd day after infection,the weight of the LR16 control group and LR16-HA2-CTA1-DD group decreased significantly.The survival rate of the LR16 control group was 40%,all mice died in the LR16-HA2-CTA1-DD group,and the survival rate of the LR16-HA1-CTA1-DD group was 90%.The results show that LR16-HA1-CTA1-DD recombinant lactobacillus expressing influenza virus HA had better immune protection effect in mice.Therefore,analyzed the biological characteristics in vitro and genetic stability of the LR16-HA1-CTA1-DD recombinant strain.And it was found that the LR16-HA1-CTA1-DD recombinant strain can grow rapidly and proliferate in vitro,and has a certain stress resistance of acid and alkali resistance,bile salts resistance,pancreatic enzymes resistance and heat resistance,and the genetic stability is good.In conclusion,analyzed the growth properties in vitro,stress resistance,probiotic properties and intestinal residence ability of LR16,then it was found that it has properties as the probiotic carrier for oral vaccines.The recombinant lactobacillus expressing respectively RBD2 of SARS-Co V-2 and HA of influenza virus had a certain immune protective effect on mice.Recombinant Lactobacillus rhamnosus has the potential to be developed as a genetic engineering oral vaccine for lactobacillus.