Immunological Study Of Branched M2e Polypeptides Linked To Tuftsin: A Novel Vaccine Against Influenza A Virus

Influenza has become a serious global health threat. Vaccination is the most effective means for preventing influenza-associated morbidity and mortality. Since the HA, NA genes of influenza virus mutate frequently.The virus strains for new vaccine production should be changed according to predicted epidemic strains. Recent research focused on the conserved epitopes of the influenza virus proteins, which may protect against a much wider range of virus strains when used as vaccines.Matrix protein 2 (M2) is a 97 aa-long transmembrane protein of IAV. The extracellular domain of M2 (M2e) is 23 amino acids long. The M2e is highly conserved and M2e-specific Abs has shown to display significant protective activity in animal models. There has been growing interest in M2 as a "universal" vaccine that may protect against a much wider range of IAVs than current vaccines.In view of the relatively small size of M2e (23aa), most efforts to produce an M2e vaccine have made use of chemical or genetic M2e fusion constructs to a variety of carriers. While one drawback of the fusion protein is that owing to adding irrelevant protein, the composition and structure of them are ambiguous.In 1988 James Tam first reported the multiple antigen peptide system, which is based on a small immunologically inert core molecule of radially branching lysine dendrites onto which a number of peptide antigens are anchored. The MAP system does not require a carrier protein for conjugation. The high molar ratio and dense packing of multiple copies of the antigenic epitope in a MAP has shown to produce strong immunogenic response.Tuftsin is a tetrapeptide (Thr-Lys-Pro-Arg) produced by enzymatic cleavage of the Fc-domain of the heavy chain of IgG. It can be recognized by specific receptors on macrophages, and is capable of targeting protein and peptides to these sites. Tuftsin has been one of the promising carrier due to its immunomodulatory role in the immune system. Several studies indicated that conjugates with tuftsin increase the epitope specific antibody production.In this study we formulated a novel construction of influenza vaccine based on M2e protein. The research is composed of four parts:1. Appraisal of physicochemical and biological properties of synthetic peptidesThe M2e peptide, (M2e)4-Tuftsin and (M2e)4-G4 peptides were synthesized using the standard solid-phase synthesizing methods. These peptides were analysed by-RP-HPLC, showing their purity were 93%,99% and 98% respectively. MALDI-TOF-MS spectrum of M2e peptide revealed the component with estimated masses of 2625.77(single M2e),11,558 Da for (M2e)4-Tuftsin and 11,114 Da for (M2e)4-G4.These results strongly suggest that the synthetic peptides are in accordance with the expected composition. Western blot and ELISA results showed that the synthetic peptides could recognize by M2 mAb specifically.The branched peptides were labelled with FITC, the FITC-(M2e)4-Tuftsin was incubated with mouse peritoneal macrophage (MPM) at 4℃.The green fluorescent could be seen on the surface of MPM. However, this phenomenon was not seen when FITC-(M2e)4-G4 was incubated with MPM. This indicated that the Tuftsin attached to M2e MAP contributes to the binding and capturing of peptide by macrophage.2. Immunostimulation and protection of mice by intramuscular vaccination with synthetic peptidesSix week old female BALB/c mice were devided into four groups, each group immunized intramuscularly (i.m.) with 10μg of one of synthetic peptides or PBS plus aluminum adjuvant, boosted at 2 week intervals. Two weeks post final immunization, anesthetized mice were challenged intranasally (i.n.) with 10 X LD50 of influenza virus PR8 strain. The mice were monitored for body weight and death for 14 days.M2e-specific IgG antibodies were determined at 2 weeks after boost immunization by ELISA using the M2e peptide as a coating antigen. The M2e-specific IgG titers in (M2e)4-Tuftsin and (M2e)4-G4 groups were 89144 and 29407 respectively, which were significantly higher than that of M2e peptide group(10), and the antibody levels between the two M2e MAP groups were also significantly different from each other.These results indicate that the (M2e)4-Tuftsin induce highest level of M2 specific antibody.The enzyme-linked immunospot (ELISPOT) assays were performed using commercial ELISPOT set (BD Biosciences). IFN-γproducing cell spots were detected in all groups, and the spot numbers were 46,254 and 156 respectively. As control, PBS plus adjuvant could not induce T cell response. The spot numbers of two M2e MAP groups were both more than M2e group. Furthermore, spot numbers of IFN-γsecreting cells were higher in (M2e)4-Tuftsin group than in (M2e)4-G4 group. These results provide evidence that (M2e)4-Tuftsin was the most effective in stimulating T cell responses.Forteen days after challenge with influenza virus, mice in PBS group were not protected (10% survival). Survival rates of M2e group and (M2e)4-G4 group were 30% and 44% respectively, which were significantly lower than (M2e)4-Tuftsin group (80% survival).These results clearly demonstrate that the (M2e)4-Tuftsin could provide the most efficient protective immunity among synthetic peptides.3. Immunostimulation and protection of mice by intranasal vaccination with synthetic peptidesSix week old female BALB/c mice were devided into four groups, each group were immunized intranasally (i.m.) with 10μg of one of synthetic peptides plus JY adjuvant, boosted twice at 2 week intervals. JY adjuvant is the compound of positively charged chitosan and negatively charged IL-2.Two weeks post final immunization, anesthetized mice were challenged intranasally (i.n.) with 10×LD50 of influenza virus PR8 strain.The mice were monitored for body weight and death for 14 days.M2e-specific IgG antibodies were determined by ELISA using the M2e peptide as a coating antigen. After final immunization, the M2e-specific IgG titers in (M2e)4-Tuftsin and (M2e)4-G4 groups were 4526 and 3592 respectively, which were significantly higher than that in the M2e peptide group(8). These results indicate that the (M2e)4-Tuftsin induce highest level of M2 specific antibody. Because intranasal immunization mainly induced local mucosal immunity, the IgG titers of mice immunized by intramuscular injection were much lower than that of mice immunized by intramuscular injection.Measurement of T cell responses by ELISPOT.IFN-Y producing cell spots were detected in all PBS, M2e, (M2e)4-Tuftsin and (M2e)4-G4 groups, and the spot numbers were 22,49,371 and 112 respectively. The spot numbers of two M2e MAP group were both more than M2e peptide. Furthermore, spot numbers of IFN-γsecreting cells were higher in (M2e)4-Tuftsin group than in (M2e)4-G4 group. These results provide evidence that (M2e)4-Tuftsin was the most effective in stimulating T cell responses.Four day after challenging with influenza virus, mice in PBS group were not protected (0% survival).Survival rates of M2e group, (M2e)4-Tuftsin group and (M2e)4-G4 group were all 40%. The results revealed that all these synthetic peptides had protective effect, but the protective effect of intranasal immunization was much lower than intramuscular immunization.4. Establishment of a stable and inducible mammalian cell line expressing influenza virus A M2 proteinBy insertion of M2 gene into mammalian cell expression vectors and transfed into a mammalian cell line, we establish a stable 293 cell line that express M2 protein under the control of the tetracycline operator. The expression of M2 protein from hygromycin -resistant cell was induced by addition of tetracycline into the cell culture media, and then tested by indirect immunofluorescence assay.16 strains with high expression of M2 were selected. After subculturing for more than ten passages, the cell lines still stably expressed M2 protein. No M2 protein could be detected without tetracycline induction, suggesting that the expression was strictly controlled by tetracycline operator.The cell lines expressing M2 will be useful for further functional studies of M2 protein, detection of immune response against natural structure M2 protein and development of live attenuated influenza virus vaccine with reverse genetics technique.In summary, the three kinds peptides including M2e,(M2e)4-Tuftsin and (M2e)4-G4 were synthesized using the standard solid-phase methods. The purity and integrity of synthetic peptides was confirmed by HPLC and MS. The accuracy and antigenicity of synthetic peptides were analysed by SDS-PAGE, Western blot and ELISA. The rusults showed that the structure of synthetic peptides were inconsistent with design. The peptides mixed with aluminum adjuvant or JY adjuvant respectively, and were administered to BALB/c mice by intramuscular or intranasal route.The ability of induction of humoral and cellular immune response in (M2e)4-Tuftsin and (M2e)4-G4 group were greater than that in M2e group, and this ability in (M2e)4-Tuftsin group were greater than (M2e)4-G4 group. Coincidently, the protective effect in (M2e)4-Tuftsin and (M2e)4-G4 group were superior to that in the M2e group, and the protective effect in (M2e)4-Tuftsin group were greater than (M2e)4-G4 group. When comparing the two immunization approach, the protective effects of intramuscular immunization were superior to intranasal immunization.This work has built a solid foundation for development of branched M2e peptide as a novel influenza vaccine.