Vaccination with recombinant 4 × M2e.HSP70c fusion protein as a universal vaccine candidate enhances both humoral and cell-mediated immune responses and decreases viral shedding against experimental challenge of H9N2 influenza in chickens.

As cellular immunity is essential for virus clearance, it is commonly accepted that no adequate cellular immunity is achieved by all available inactivated HA-based influenza vaccines. Thus, an improved influenza vaccine to induce both humoral and cell-mediated immune responses is urgently required to control LPAI H9N2 outbreaks in poultry farms. M2e-based vaccines have been suggested and developed as a new generation of universal vaccine candidate against influenza A infection. Our previous study have shown that a prime-boost administration of recombinant 4×M2e.HSP70c (r4M2e/H70c) fusion protein compared to conventional HA-based influenza vaccines provided full protection against lethal dose of influenza A viruses in mice. In the present study, the immunogenicity and protective efficacy of (r4M2e/H70c) was examined in chickens. The data reported herein show that protection against H9N2 viral challenge was significantly increased in chickens by injection of r4M2e/H70c compared with injection of conventional HA-based influenza vaccine adjuvanted with MF59 or recombinant 4×M2e (r4M2e) without HSP70c. Oropharyngeal and cloacal shedding of the virus was detected in all of the r4M2e/H70c vaccinated birds at 2 days after challenge, but the titer was low and decreased rapidly to reach undetectable levels at 7 days after challenge. Moreover, comparison of protective efficacy against LPAI H9N2 in birds intramuscularly immunized with r4M2e/H70c likely represented the ability of the M2e-based vaccine in providing cross-protection against heterosubtypic H9N2 challenge and also allowed the host immune system to induce HA-homosubtype neutralizing antibody against H9N2 challenge. This protective immunity might be attributed to enhanced cell-mediated immunity, which is interpreted as increased lymphocytes proliferation, increased levels of Th1-type (IFN-γ) and Th2-type (IL-4) cytokines production and increased CD4(+) to CD8(+) ratios, resulting from the injection of four tandem repeats of the ectodomain of the conserved influenza matrix protein M2 (4×M2e) genetically fused to C-terminus of Mycobacterium tuberculosis HSP70 (mHSP70c).