Studies On The Formation Of Virus-like Particles Of Highly Pathogenic Picornaviruses And Immunization Of A Part Of Particles

The family Picornaviridae consists of17genera, such as aphthovirus,enterovirus, rhinovirus and hepatovirus. Picornaviruses are non-enveloped withanicosahedral capsid. Viral capsid is composed of4polypeptides known as VP (viralprotein)1,2,3and4. The most common highly pathogenic viruses of this familyinclude enterovirus71(EV71), coxsackievirus, poliovirus and hepatitis A virus (HAV).EV71and coxsackievirus A16(CVA16) are etiological agents of hand, foot andmouth disease, brainstem encephalitis and aseptic meningitis. Poliovirus and HAV arecausative agents of poliomyelitis and hepatitis, respectively. There are no vaccinesavailable to prevent the infection of EV71and CVA16. Inactivated and live-attenuatedvaccines are available in commercial market against poliovirus and hepatitis A virus.However, live-attenuated of oral polio vaccine (OPV) revert to neurovirulence andcause vaccine-associated paralytic poliomyelitis in vaccinees and inactivated vaccinesare restricted by their high-costs.Engineered vaccines are representing the trend of the development of vaccinefollowing the rapid improvement of biotechnologies. Vaccines based on virus-likeparticle (VLP) attracted great attentions by its similar structure with wild-type virus,which harbors both conformational and sequential epitopes. The formation of theVLPs of EV71, CVA16, poliovirus and HAV was investigated in our current studies,which could provide the new insights in the development of engineered vaccines ofvirus of the family Picornaviridae.Saccharomy cescerevisiae was used to produce the VLPs, which is characterizedby the high productivities and low costs. In our studies, the genes encoding EV71structural protein P1and protease3CD were initially optimized to increase proteinexpression by efficiently usage of the internal transcription and translation system ofsaccharomy cescerevisiae. After codon optimization, the EV71VLPs expressionvectors was constructed and confirmed by sequencing. Saccharomy cescerevisiaecells were transfected with validated vectors and the expression of P1and3CD genesof EV71was induced. Yeast cells were harvested and lysed and the cell lysis wasprimarily purified by ultracentrifugation. The expression of EV71proteins in purifiedproducts was detected by Western Blot, and the formation of EV71VLPs with the sizesimilar to that of EV71was observed by transmission electron microscope. In order toverify the stability of vectors in saccharomyces cerevisiae, Real-Time PCR was usedto detect the copy numbers of plasmid. The experimental results show that theplasmid expression vector has a low lossing rate during culturing. Furthermore, inorder to improve the yield of EV71VLPs, the culture conditions of saccharomyces cerevisiae was optimized. The result shows that after the optimization of cultureconditions, the yield of saccharomyces cerevisiae increased by30%than before andthe expression of EV71VLPs could be successfully improved.Furthermore, the gene of structural protein P1and protease was optimized andthe VLPs expression vector of CVA16, poliovirus I were constructed and validated bysequencing. Saccharomy cescerevisiae cells were transfected with validated vectorsand the expression of viral proteins was induced. After purification, the viral proteinsof CVA16and poliovirus I were detected by Western Blot, and the formation of VLPswas observed by transmission electron microscope. The formation of the VLPs ofHAV was observed by similar method. But, the expression of viral proteins was lowerwithout codon optimizations, indicating that the codon optimization is necessary forefficient expression of foreign proteins.The purified EV71VLPs was used to immune BALB/c mouse by muscleinjection and boosted at2nd week. Blood was collected and separated. The specificIgG antibody titers and neutralization titers in serum was measured. Our resultsshowed that EV71VLPs was capable of eliciting neutralization response in mice,indicating that EV71VLPs are highly immunogenic.In conclusion, the VLPs of highly pathogenic picornavirus, such as EV71,CVA16, poliovirus and HAV were successfully produced in yeast, and theimmunogenicity of EV71VLPs was preliminarily evaluated, which could eliciteneutralizing immunity againt EV71. Thus, EV71VLPs expressed by saccharomycescerevisiae system have good immunogenicity and safety. Our current studies havea great potential for engineered vaccine design against the virus of the familyPicornaviridae.