Preliminary Study On Recombinant EV71and CA16HFMD VLP Bivalent Vaccines

Objectives Enterovirus71and Coxsackievirus A Group16are virusescausing hand-foot-mouth disease. In this study, we expressed EV71and CA16VLPs by Bac-to-Bac (r) Baculovirus Expression System, and evaluated theirimmunogenicity.Methods （1） I n t h e f i r s t p a r t， We p r o d u c e d the recombinant EV71VLPand CA16VLP i n i n s e c t c e l l s b y B a c-t o-B a c b a c ulo v i r u s e x p r e s s i o ns y s t e m s.Optimization codon of EV71P1、3CD and CA16P1、3CD in insect cellswere cloned into pFastBacDual.The pFastBacDual EV71P1-3CD Bacmid andpFastBacDual CA16P1-3CD Bacmid were constructed by homologousrecombination, Bacmid DNA were transfected into Sf9cells. Structural proteincapsid P1was cut by virus-encoded protease3CD in Bac-to-Bac, the recombinantEV71VLP and CA16VLP were assembled and purified by sucrose densitygradient centrifugation. The expressed proteins were detected b y n e g a t i v es t a i n i n g e l e c t r o n m i c r o s c o p y, IFA,SDS-PAGE and We s t e r n b l o t；(2）Preliminary evaluation of immunogenicity and protection efficiency of universalinfluenza vaccines on4-6w e e k s o l d ICR mice. Mice for each group werevaccinated intraperitoneal injection of immune with the recombinant EV71V L Pmonovalent vaccine、 CA16VLP monovalent vaccine and the EV71、 CA16VLPsb i v a l e n t vaccines with Al(OH)3adjuvant in2weeks apart. By indirect ELISA, theantibody subtype distribution， micro neutralization， ELISPOT, the vaccine immunogenicity in ICR mice was evaluated.Results (1)The expression of EV71、 CA16VLPs were identified bySDS-PAGE, WB and indirect immunofluoresence. Virus-like particles wereobserved in the cytoplasm. These particles measured25-30nm in diameter andappeared icosahedral, thus resembling the authentic enterovirus in size andappearance. EV71、CA16VLPs with more than95%purification were obtained bysucrose density gradient centrifugation；(2)R e s ult s o f E L I S A s h o w e d t h a t therecombinant EV71、CA16VLPs b i v a l e n t vaccines c o uld o b v i o u s l y e l i c i t s t r o n gi m m u n e r e s p o n s e s t h a n the blank control group (PBS)(p <0.05),and theantibody subtype distribution were mainly IgG1,while the neutralization antibodyc o uld o b v i o u s l y e l i c i t s t r o n g i m m u n e r e s p o n s e s t h a n the blank control group(PBS)(p <0.05).The micro neutralization results showed that the recombinantEV71、 CA16VLPs b i v a l e n t vaccines c o uld o b v i o u s l y e l i c i t s t r o n g i m m u n er e s p o n s e s t h a n monovalent vaccine groups (p <0.05).The ELISPOT results toldus that t h e n u m b e r o f c y t o k i n e s I F N-γ w e r e m o r e t h a n I L-4i n s p l e e n c e l l s(p <0.05).T h e recombinant EV71VLP and CA16VLP b i v a l e n t v a c c i n e s w e r ei n d u c e d h u m o r a l a n d c e l l μLa r m a i n l y c a u s e d h u m o r a l i m m u n er e s p o n s e. expressed by Bac-to-Bac (r) Baculovirus Expression System, andimmunogenicity of EV71and CA16VLPs bivalent v a c c i n e s are preliminaryevaluated, which are helpful for the researches of EV71or CA16subunit vaccine.Conclusion T h e recombinant EV71and CA16VLPs bivalent vaccines areprepared, they are able to induce better humoral immune response, also inducedcertain cellular immune response. T h e recombinant EV71and CA16VLPsbivalent vaccines are constructed,expressed and purified.