Development Of A Broad-spectrum Virus-like Particle Vaccine Against Enterovirus D68 And Evaluation Of Its Immune Effect

Enterovirus D68(EVD68),a member of the species Enterovirus D of the Enterovirus genius.Besides the prototype Fermon strain,EVD68 can be divided into three main clades: A(A1-A2),B(B1-B3),and C.Since 2014,EVD68 has erupted in many places around the world and caused a series of respiratory-related diseases,including bronchitis,pneumonia,respiratory failure and even death.At the same time,the recently epidemic EVD68 subclade B1/B3 strains have a causal association with acute flaccid myelitis.So far,there are no effective therapeutic drugs or preventive vaccines on the market.EVD68 is still a hidden threat to public health at present and in the future.Development of EVD68 vaccine,especially the vaccine covering EVD68 B clade,will greatly reduce the financial burden and suffering of patients caused by EVD68 infection.In our previous work,we have developed an EVD68-US/MO/14-18947 strain(designated as 18947 strain)inactivated vaccine and a virus-like particle(VLP)vaccine against EVD68-US/MO/14-18950 strain(designated as 18950 strain).Based on the early research,this experiment aims to develop a VLP vaccine that can broadly cover EVD68 B clade epidemic strains.We first took EVD68 B clade P1 structural protein sequence as the principal part,referenced the P1 sequences of EVD68 A and C clade strains and Fermon,and selected the consensus sequence to design the EVD68 Consensus P1 sequence.Then draw the phylogenic tree in order to compare the evolutionary differences between Consensus P1 and several EVD68 virus strains' P1 sequences.Next,Consensus P1 sequence and EVD68-18947 strain P1 structural protein sequence were expressed in Pichia pastoris using the "P1 + 3CD protease" strategy,respectively.Western Blot,SDS-PAGE and negative staining electron microscope shown that both P1 structural proteins were effectively cleaved into VP0,VP1,and VP3 and spontaneously assembled into virus-like particles(designated as ConsensusVLP / 18947VLP).Consensus VLP expressed by Pichia pastoris can stimulate antibody responses in mice,and its antiserum has a good binding capacity to 18947 VLP and ConsensusVLP.At the same time,the antiserum has a high level of neutralization titer for the EVD68 B1 subclade 18947 strain virus,and has a low neutralization response to the A clade EVD68-US/KY/14-18953 strain and Fermon strain.The 18947 VLP expressed by Pichia pastoris has good immunogenicity and antigenicity.18947 VLP can elicit antibody responses in mice,and the antiserum can bind the 18947 VLP.At the same time,the antiserum shown a high level of neutralization response to EVD68 B1 subclade 18947 strain virus and A clade 18953 strain virus,but only a low neutralization response to Fermon strain.These results proves that the two VLPs have good immunogenicity and antigenicity,which provides a basis for the development of a broad-spectrum VLP vaccine against EVD68 B clade viruses in the future.