Comparative Study On Three Of Adjuvanticities Of TLR Agonist

Subunit vaccine such as live-vectored vaccines are the important direction for vaccine development,and adjuvants play the key role in the vaccine efficacy.The agonists of Toll-like receptors(TLRs),such as Ag473 lipoprotein from Neisseria meningitides,the major flagellin FlaB from Vibrio vulnificus and heat shock protein 70 from Mycobacterium tuberculosis(mHsp70),are promising molecular adjuvants,but lack of systemic comparison study.In this study,by using infectious bursal disease virus(IBDV)VP2 as the indicator antigen,the adjuvanticities of the three TLR agonists were compared in mice.Then,by using African swine fever virus(ASFV)CD2v-p30-p54 fusion protein(called F1)as the indicator antigen,the mucosal adjuvanticities of FlaB and mHsp70 were compared by intranasal immunization of pigs with recombinant adenovirus(rAd)vectors,aiming at providing the rationale for use of adjuvants for vaccine development.Expression and identification of TLR agonists and recombinant viral antigens in E.coliThe expressed recombinant proteins included TLR agonists Ag473,FlaB and mHsp70,IBDV VP2 protein and its TLR agonist fusion proteins D1-VP2,FlaB-VP2,VP2-Hsp70,and ASFV CD2v,p30 and p54 proteins.Each coding sequence was cloned into pET-30(+)vector and transformed into E.coli strain BL21(DE3).The protein expression was induced with IPTG and purified using nickel affinity chromatography.The purified proteins were identified by Western blotting using His tag-specific antibody or ASFV antibodies.The lipid modification of D1-VP2 fusion protein was identified with MALDI TOF/TOF mass spectrometry.SDS-PAGE analysis showed that the expressed seven single proteins had the expected molecular weights,among which Ag473,mHsp70,CD2v,p30 and p54 were expressed as soluble proteins,while FlaB and VP2 were expressed as insoluble inclusion bodies.The expressed three fusion proteins also had the expected molecular weights,among which VP2-Hsp70 was expressed as a soluble protein,while D1-VP2 and FlaB-VP2 were expressed as insoluble inclusion bodies.All recombinant proteins were purified to more than 94%purities and recognized by His tag-specific antibody or ASFV antibody.After cleaving off the signal peptide,the N-terminal peptide of D1-VP2 fusion protein was 6-aa long with the same lipid modification as Ag473 lipoprotein.These data suggest that the prepared TLR agonists and recombinant antigens could be used for comparative study on adjuvanticities of TLR agonists.Comparison of adjuvanticities of three TLR agonists administered in two different waysFifty-one BALB/c mice were assigned into 8 experimental groups and one control group.The mice in experimental groups were intramuscularly injected with the same mole of VP2,D1-VP2,FlaB-VP2,VP2-mHsp70,VP2+ Ag473,VP2+FlaB or VP2+mHsp70,and boosted with the same antigen formulation at day 14 after primary immunization.The mice in control group were injected with the same volume of PBS.At different days after immunization,serum samples were collected for antibody detection by ELISA.On day 28 after immunization,all mice were sacrificed and spleen cells were isolated.After stimulation with IBDV VP2 antigen,cytokine expression was measured with ELISA kits.Among the three co-administration groups,the VP2-specific antibody level of Ag473+VP2 group was significantly higher than that of FlaB+VP2 or mHsp70+VP2 group.After stimulation of splenic cells with VP2 antigen,IL-4 and IL-12 expression levels of Ag473+VP2 group were higher than that of other two groups,while IFN-y and TNF-? expression levels of FlaB+VP2 group were higher than of other two groups.Among the three genetic fusion groups,the VP2-specific antibody level of D1-VP2 group was significantly higher than that of FlaB-VP2 and VP2-mHsp70 groups not only,but Ag473+VP2 group as well.After stimulation of splenic cells with VP2 antigen,IL-4,IL12 and IFN-y expression levels of D1-VP2 group were higher than that of other two groups,while TNF-? expression level of VP2-mHsp70 groups was higher than that of other two groups.These data suggest that Ag473 or its D1 domain had the strongest humoral and cellular adjuvanticities among the three co-administered or genetically fused TLR agonists.Overall adjuvanticities of genetically fused TLR agonists were stronger than that of co-administered ones.Generation of rAds expressing TLR agonists fused with ASFV antigensThe coding sequence for mHsp70 or FlaB was amplified from the pET-30(+)vector by PCR and cloned into pShuttle-CMV vector with restriction digestion.The synthetic sequence for ASFV CD2v-p30-p54 fusion protein(called F1)was cloned downstream of FlaB or upstream of mHsp70 in the pShuttle-CMV vector.The Ad transfer vectors were called pShuttle-FlaB-F1 and pShuttle-Fl-Hsp70,respectively.The F1 gene was also cloned into pShuttle-CMV and the pShuttle-F1 vector was used as the control.The three transfer vectors were transformed into E.coli strain BJ1583-AD-1 and the recombinant plasmids were transfected into HEK293A cells.The resultant rAds were called rAd-F1 rAd-FlaB-F1 and rAd-Fl-Hsp70,respectively.After 3 cycles of amplification on HEK293A cells,the three rAds had titers of 8 × 108?1 × 109 ? 5 × 108TCID50/mL,respectively.Immunofluorescent test showed that all of three rAd-infected cells reacted positively with ASFV antibodies.Western blotting showed that three proteins with molecular weight of 125,140 or 170kDa were expressed in rAd-F1-,rAd-FlaB-F1-or rAd-F1-Hsp70-infected cells.The detected molecular weight of the three proteins were significantly larger than the predicted molecular weights,which may be due to glycosylation of CD2v and p54 proteins.These data suggest the usefulness of three rAds for adjuvanticity comparison of the two TLR agonists.Comparison of mucosal adjuvanticity of two TLR agonists on ASFV fusion antigenEleven piglets were assigned into three experimental groups(n=3)one control group(n=2).The pigs in experimental groups 1-3 were intranasally immunized with the same dose(108 TCID50/nostril)of rAd-Fl,rAd-FlaB-Fl or rAd-Fl-Hsp70.The pigs in control group were intranasally administered with the same volume of PBS.On day 21 after primary immunization,the pigs in experimental groups were boosted with the same dose rAd.At different days after immunization,serum samples were collected for antigen-specific IgG and IgA antibody detection by ELISA.At the same times,nasal swabs were collected for IgA antibody detection.The blood samples were collected for PBMC isolation.After stimulation ASFV antigens,cytokine expression in PBMCs was detected using ELISA kits.On day 35 after immunization,all pigs were sacrificed.Both tracheal washes and lung lavages were collected for IgA antibody detection.PAMs were isolated and stimulated with ASFV antigens for cytokine detection.The results showed that all immunized pigs were positive for the antigen-specific IgG and IgA antibodies from day 7 after immunization,which were significantly boosted by secondary immunization.The antibody levels in rAd-FlaB-Fl-immunized pigs were significantly higher than that in rAd-Fl-immunized pigs,but slightly lower than that in rAd-Fl-Hsp70-immunized pigs.The IgA antibody patterns in the nasal fluids,tracheal washes and lung lavages were similar to that in the serum samples.For cytokine expression in antigen-stimulated PBMCs,IL-2,Il4,IL-10 and IFN-y expression levels of rAd-FlaB-F1 immunization were higher than that of rAd-F1 immunization,but lower than that of rAd-Fl-Hsp70 immunization.For cytokine expression in antigen-stimulated PAMs,IL-4 and IFN-? expression levels of rAd-FlaB-F1 immunization were significantly higher than that of rAd-F1 immunization,but significantly lower-than that of rAd-F1-Hsp70 immunization.IL-10 expression level of rAd-FlaB-Fl immunization was significantly higher than that of rAd-F1 immunization,but slightly lower than that of rAd-F1-Hsp70 immunization.IL-1? expression level of rAd-FlaB-Fl immunization was slightly higher than that of rAd-F1 immunization,but significantly lower than that of rAd-Fl-Hsp70 immunization.The three immunization groups had no significant difference in IL-12 expression.These data suggest that the mucosal adjuvanticity of mHsp70 was stronger than FlaB,which could be explained by its stronger ability to stimulate IL-4,IL-10 and IFN-? expression.