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

- Protection against H9N2 viral challenge was significantly increased in birds by injection of recombinant 4 × M2e.HSP70c fusion protein compared with injection of Conventional HA-based vaccine.
- A prime-boost administration of recombinant 4 × M2e.HSP70c fusion protein formulated in F105 buffer enhanced humoral and cell-mediated immune responses in chickens with significant lower viral shedding.
- Recombinant 4 × M2e.HSP70c fusion protein compared with r4M2e alone, increased lymphocytes proliferation, increased levels of Th1-type (IFN-γ) and Th2-type (IL-4) cytokines production and increased CD4+ to CD8+ ratios 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).