Construction, Expression And Immunogenicity Analysis Of A Fusion Protein Containing M2e Of Influenza A Virus Fused To A Modified Pseudomonas Aeruginosa Exotoxin A

Influenza is one of the major respiratory viral diseases in human, accompanied by coughing, high fever and myalgia. The incidents of influenza is about 10%-20% of the worldwide popuLation during seasonal epidemics, resulting in 3-5million cases of severe illness and 250000-500000 deaths per year. Immunization was proved to be the most effective measure in preventing influenza. Contemporary vaccines typically contain strains representative of two influenza A subtypes (A/H3N2 and A/H1N1) and one influenza B virus. The currently licensed vaccines are designed to stimulate antibodies primarily against hemagglutinin (HA) and neuroaminadase (NA), the major neutralizing antigen of the influenza virus. However, as these antigens frequently changes by mutations (drift) and gene re-assortment(shift), these antibodies loose their efficacy to prevent disease. Therefore, influenza infections can occur repeatedly throughout life and protection by vaccination requires annual administration with updated vaccines. Unlike HA and NA, the membrane protein M2e which is a 97 aa long non-glycosylated transmembrane protein is very conserved in its 23 aa ectodomain. Many results demonstrated that antibody to M2 can restrict influenza virus replication in cell culture and in infected mice. Therefore, the extra-domain of influenza M2 protein(M2e) is considered as a promising candidate for universal influenza vaccine.PEA is secreted by Pseudomonas aeruginosa as a 67-kDa protein composed of three prominent globular domains (Ⅰa,Ⅱ, andⅢ) and one small subdomain (Ⅰb) connecting domainsⅡandⅢ. Domain la of PE binds to the low density lipoprotein receptor-related protein (LRP), also known as the a2-macroglobulin receptor. LRP is expressed on the surface of most mammalian cells and tissues, including those of the immune system. DomainⅡmediates translocation to the cytosol, and domainⅢhas ADP-ribosylating activity. Once bound to LRP, the toxin traffics via coated pits to an endosomal compartment, where it is cleaved by the protease, furin, to generate a 37-kDa C-terminal fragment composed of domainsⅡ,Ⅰb, andⅢ. This fragment translocates to the cell cytosol ADP-ribosylates elongation factor 2, and shuts down protein synthesis. When glutamic acid 553 in domainⅢis deleted (DE553), the toxin still gains access to the cytosol but is rendered nontoxic since this mutation eliminates ADP-ribosylating activity.M2e protein of type A influenza virus is a good candidate for universal influenza vaccine, exotoxin A of Pseudomonas aeruginosa may facilitate the immunogenicity of M2e protein.In our study, two prokaryotic expression plasmids containing M2e and PEA fusion gene was constructed, and expressed in E.Coli respectively. The purified fusion proteins were inoculated into Balb/c mice, their immunoprotection was observed.The major results composed of five parts.1. Cloning of Pseudomonas aeruginosa exotoxin A (PEA) and generation of PEA non-toxic mutant (ntPE).The PEA gene was amplified using PCR and confirmed by DNA sequencing. Then, Glu553 of domainⅢwas deleted by site directed mutagenesis so the ADP-ribosylating activity was eliminated, and the PEA non-toxic mutant (ntPE) was achieved. 2. Construction and expression of the plasmid pET/ntPE-M2e and pET/ntPE-3M2eThe gene sequence of influenza virus M2e was generated by chemical synthesis, the eight amino acid Ib loop of ntPE was replaced with the M2e gene. The fusion gene ntPE-M2e was then inserted into prokaryotic expression plasmid pET-30(a). In addition, the regionⅠandⅡof PEA gene was amplified by PCR, and ligated with three copies of M2e in tandem linked by linker peptide.The plasmid of pET/ntPE-M2e and pET/ntPE-3M2e were transformed into E.Coli BL21(DE3) strain and induced with 1mM IPTG. SDS-PAGE showed the fusion protein was expressed as inclusion body.3 Immunization of the two fusion proteins in Balb/c miceWe purified the two fusion proteins using SDS-PAGE, and immunized six weeks old female Balb/c mice via subcutaneouLy route with Incomplete Freund's adjuvant (100ug per mice) at day 0, day 21 and day 42. Mice were devided into three groups: PBS, ntPE-M2e and ntPE-3M2e. Blood samples were taken from mice, and the ELISA results showed that the specific IgG Abs against M2e was induced and the titers increased with boosting. Spleen cells were collected for ELISPOT, and results showed that the M2e can induce mice splenocyte secreting IFN-y upon M2e stimutation. Forteen days after the final injection, the mice were challenged by influenza virus A/PR/8/34(H1N1), survival and weight change of the immunized mice were observed for 14 days after challenging. Results showed that the fusion proteins protected mice form the influenza virus lethal challenge. Five mice were sacrificed on the fifth day after virus challenge to detect virus load of lung tissue. The results showed that the replication of virus in the lungs was inhibited.4. Construction, expression and purification of the two fusion proteins with His tagHis tag was connected to the ntPE-M2e and ntPE-3M2e gene by PCR, which made purification easier. The new plasmids was transformed into BL21(DE3) and expressed in E.Coil with 1mM IPTG induction. SDS-PAGE showed the proteins were expressed as inclusion body. The inclusion body was denatured with 8M Urea and purified with immobilized metal affinity chromatography. The purity of the eluted proteins was up to 90%. Western blot confirmed their antigenecity. 5. Immunization of mice with the fusion proteins containing His tagWe compared two routes of administration:intranasally and subcutaneouly.6 weeks old female BALB/c mice were used in this study. The dosage of the fusion proteins were kept at 40 ug per immunization via either intranasal (i.n.) or the subcutaneous route. Control mice received only PBS. 100uL proteins were mixed with aluminium adjuvants (1mg/mL) for subcutaneous immunization. No adjuvant was used for intranasal groups. Primary immunization was followed by one booster dose 2 weeks later. Blood samples were obtained at one day before each immunization and stored at-20℃until analysis.ELISA and ELISPOT results demonstrated that high cellular and humoral responses were induced by each immunized group.14 days after second immunization, the mice were infected with influenza virus A/PR/8/34(25uL for each nostril,40 LD50. They were followed up for 14 days after the infection; survival and weight loss were monitored. The results showed that the groups with subcutaneouly immunization could protect mice against the lethal challenge of influenza virus, However, the intranasally immunization groups failed.5 mice were sacrificed on the fifth day after virus challenge to detect virus load in lung tissue. The results showed that the replication of virus in the lungs of the immunized mice was inhibited.We further investigated inoculate mice intranasally with different dosage of the fusion protein. Each fusion protein were inoculated at 0.4,2, 10ug respectively,and boosted after two weeks.ELISA and ELISPOT results demonstrated that high cellular and humoral immune responses were induced by intranasal route.14 days after 2nd immunization, the mice were infected with influenza virus A/PR/8/34(25uL for each nostril,4LD50). They were followed up for 14 days after infection; survival and weight loss were monitored. The results showed that the group ntPE-M2eH/2ug and ntPE-3M2eH/0.4ug could partially protect mice from influenza virus challenge while others failed.In summary, we constructed and expressed a series of prokaryotic expression plasmids containing fusion gene between M2 extracellular coding region and partial PEA gene, expressed and purified from E.coli and observed the immunoprotection in BALB/c mice vaccinated with the fusion proteins. The fusion proteins were administered to BALB/c mice by intranasal (i.n.) and subcutaneouly route. The immunized mice were challenged with influenza virus strain A/PR/8/3414 days after immunization. The results demonstrated that the fusion proteins with subcutaneouly route can induce a strong systemic immune response against synthetic M2e peptide, and virus replication in the lungs of mice was inhibited, while the groups with intranasal route can relieve symptom caused by influenza virus. This study provides foundation for developing broad-spectrum vaccines against type A influenza viruses.