Enhanced Protective Early Immunity To Escherichia Coli Vaccine By Protamine Protein

Pathogenic Escherichia coli(E.coli)can infect people and other animals,newborn and young animals are more susceptible,and the clinical manifestations are acute infectious diseases.Currently,pathogenic E.coli has become resistance to most antibiotics,which has brought great difficulties in the prevention and treatment of E.coli infection.At present,the prevention and control of E.coli disease in farms mainly depends on vaccination,but the existing vaccines have many disadvantages,such as high side effects,slow induction of immune protection,and unsatisfactory immune effect.Therefore,it has great clinical significance to improve the immune efficacy of E.coli vaccine by adding adjuvants.Protamine protein(PP)is an alkaline protein mainly existing in the nucleus of mature fish sperm.It is widely used in cardiac surgery,anti-thrombosis,antitumor,food antisepsis and antigenic carrier.Previous studies in our laboratory have proved that PP can significantly enhance the early humoral immune response and protective immunity induced by E.coli vaccine,with good clinical application potential.In order to further explore the adjuvant effect of PP on E.coli vaccine and its mechanism,this paper will carry out experimental exploration from the following four parts.1.To investigate the enhancd efficacy of E.coli vaccine by PP.Methods 24 female ICR mice were randomly divided into 3 groups,and were injected subcutaneously with E.coli inactivated vaccine(1×10~7CFU/mouse)(the same below),PP(100?g)+E.coli inactivated vaccine and saline.7 days later,mice were challenged with pathogenic E.coli 0.2mL(containing 1×10~7 CFU of live bacteria).The clinical symptoms and mortality were recorded.Results PP,as an adjuvant,use to immunize mice with E.coli vaccine,making the survival rate increase from 12.5%to 87.5%,significantly improving the early immune protection induced by vaccine.2.To investigate the enhanced protective early immunity mechanism of E.coli vaccine by PP.Methods 32 female ICR mice were randomly divided into 4 groups,and were injected subcutaneously with E.coli inactivated vaccine,aluminum(200?g)+E.coli inactivated vaccine,PP(100?g)+E.coli inactivated vaccine and saline.3 days later,mice were challenged with pathogenic E.coli(containing 2×10~6 CFU of live bacteria).The clinical symptoms and mortality were recorded,specific antibody level was measured 48 hours after immunization,and blood bacterial burden,blood physiological index were measured after challenge.Results The survival rate of PP group was 100%,which was significantly different from that of blank group(50%),aluminum group(25%)and control group(37.5%)(P<0.01).At 48h after immunization,PP can induce the body to produce relatively high levels of specific IgM and IgG antibodies.In addition,it can promote the clearance of bacteria by the body after challenge(P<0.05),increase the amount of PLT in blood circulation(P<0.01),and alleviate the reduction of WBC,LYM~#and GRAN~#caused by E.coli sepsis(P<0.05).It suggested that PP,as an adjuvant,could activate the early immune response 3 days after immunization,possibly enhancing the body's protection against E.coli attack by producing higher levels of specific antibodies and increasing the amount of PLT in the blood circulation.3.To investigate the correlativity between the anti-E.coli challenge effect of PP and TLR4 signaling pathway.Methods(1)Female ICR mice were injected subcutaneously with PP(100?g)or saline.7 days later,mice were challenged with lethal dose or sub-dose pathogenic E.coli.The lethal dose group's clinical symptoms and mortality were recorded after challenge,blood LPS,ALT concentration and inflammatory factor transcription level of mice after subdose challenge were measured.Another 8 untreated mice were taken and sampled as a blank group after execution.(2)24 female ICR mice were randomly divided into 2 groups,and injected with PP(100?g)or normal saline.Mice were sacrificed with half of each group at 2d and 7d after administration to measure the effect of PP on the expression of TLR4-related cytokines mRNA.Results(1)PP injection alone(7d)increased the survival rate of mice from 0%to 80%,indicating that PP had significant resistance to E.coli invasion.PP can reduce the blood LPS,ALT concentration,down-regulate the transcription level of TLR4,NF-?B,IL-1?,IL-6,IRF7,IFN-?.It may has certain inhibitory effect on MyD88-dependent and TRIF-dependent TLR4 signaling pathway initiation to avoid the excessive development of inflammatory response and the occurrence of immune disorder in the early acute infection.(2)After 7 days of PP injection alone,the mRNA expression levels of TLR4,IFN-?and IL-6 were up-regulate to some extent,a tentative inference on this result is that PP may enhance the body's resistance to E.coli by promoting the inflammatory response in advance.4.To investigate the enhanced early immune response of PP on E.coli vaccine in rabbitsMethods 12 rabbits were randomly divided into 2 groups,and were injected subcutaneously with E.coli inactivated vaccine and PP(100?g)+E.coli inactivated vaccine.7 days later,rabbits were challenged with pathogenic E.coli 0.6mL(containing 3×10~7 CFU of live bacteria).The clinical symptoms and mortality were recorded,6 untreated rabbits were used as blank groups,measured blood physiological index,LPS concentration and blood biochemistry.Results PP combined with E.coli inactivated vaccine immunized rabbits can increase the amount of PLT,WBC in blood.Compared with the control group,PP group after challenge can significantly reduce the increase of LPS,ALT and AST caused by challenge,and alleviate the decrease of PLT and WBC caused by challenge.The results showed that PP,as an adjuvant,could effectively enhance the immune efficacy of E.coli vaccine in rabbits.In summary,PP,as an adjuvant of E.coli vaccine,can rapidly activate the body's early immune response and immune protection.Mechanism studies have shown that PP has an immunomodulatory effect,which can not only improve the immune effect of the vaccine by promoting the body's inflammatory response,but also inhibit the excessive development of the inflammatory response,to ensure the body's disease resistance and maintain the stability of the internal environment.PP can rapidly enhance the resistance of mice to E.coli,which may be related to the regulation of inflammatory factors related to the LPS-TLR4 signaling pathway.Therefore,PP,as a candidate adjuvant for E.coli vaccine,has potential clinical application value.