Respiratory Syncytial Virus G Protein Subunit Mucosal Vaccine Research

Respiratory syncytial virus(RSV) is one of major pathogens of infection lower respiratory tract of infant in the world. Every year, almost 4 million children were infected with RSV. Up to date, there is no effective vaccine against RSV infection. To enhance RSV subunit vaccine efficiency, decrease the number of eosinophile granulocyte and improve mucosa immunity in respiratory tract, replacing the CX3C motif in G protein of RSV, and immunization mice with the mixture of G protein and enterotoxigenic Escherichia heat labile enterotoxin (LT) were used in the present study.Overlapping PCR was used to replace the CX3C motif nucleotides in G protein partly synthesize artificially with one of CTL epitope nucleotides from M protein of RSV. Recombinant plasmids G/pET22b and G(CTL)/pET22b were constructed and transformed into E. coli BL21(DE3) for expression. G protein and G(CTL) were successfully expressed in E. coli, and the amount of the recombinant proteins were estimated more than 30% among total E. coli proteins. The purity of recombinant proteins is above 92% after Co²⁺ and His-tag affinity chromatography.Four group mice were vaccinated at 0, 1 and 4 weeks with different solutions, group A was injected with PBS; group B with LT; group C with G(CTL) protein + LT and Group D with G protein. There were analyzed IgG, IgA and eosinophile granulocyte in serum, and IgA and eosinophile granulocyte in respiratory tract. The results showed that the amount of IgG in serum in group C and D were higher than group A, and B, and the geometrical mean titer (GMT) were 1995.26 and 1584.89, respectively. IgA only appeared in serum and respiratory tract in group C with the GMT of 1781.23 and 3.167, respectively, but not in group A, B and D. The number of eosinophile granulocyte in group C was as the same level as group A and B, but that in group D is magnificently higher than other groups.The recombinant proteins were successfully expressed and purified, and the immunogenicity of G subunit vaccine was assayed. Replacement of CX3C in G protein led to decrease the amount of eosinophile granulocyte in respiratory tract. Immunization mice with the mixture G(CTL) protein with LT, one of mucosa adjuvants obtained not only immunity in serum, but also improvement of IgA in serum and respiratory tract, the immunity of mucosa in respiratory tract. The result provides valuable information in study of G protein in RSV pathogenic mechanism and a good strategy in development of newgeneration of RSV vaccine.