Hemagglutinin based vaccinal and therapeutic approaches against Influenza viruses

Abstract

Influenza viruses, in seasonal and pandemic forms, cause the most severe respiratory infections in humans. The currently licensed, egg-based vaccines fail to meet the global vaccine demand during a pandemic. Moreover, there is a constant need to reformulate these vaccines on annual basis according to the circulating viral strains. In order to develop efficient prophylactic measures, alternate vaccine strategies and production platforms need to be exploited. Subunit vaccines, like recombinant hemagglutinin, offer an alternative over conventional vaccines. Current study was designed to clone and express soluble influenza hemagglutinin (HA) of H9 and/or H7, which could be used as a vaccine candidate. To achieve high level of secretory expression and stabilized native trimer structure, HA was assembled with sequence for the tissue plasminogen activator signal peptide (tPA-SP) and foldon domain of bacteriophage T4 fibritin (FT) respectively by employing splicing by overlap extension amplification. The amplified product was cloned in pCMVR-8kb having human cytomegalovirus (CMV) immediate-early enhancer and promoter for transient expression of HA in 293F cells. SDS PAGE analysis confirmed a band of ≃ 70 kDa corresponding to H9HA and H7HA which was further confirmed by Western blot analysis and purified by Ni-NTA agarose column. The purified recombinant protein was found biologically active. This expression and purification process can be scaled up easily and recombinant protein can be produced rapidly which can then be used for further studies on virus-host interactions, viral pathogenesis and development of vaccines. The failure of currently available antiviral agents, due to high mutation rate of the influenza virus, calls for employing rigorous strategies to develop safe and potent inhibitory strategies against the virus. Nanoparticles have been studied extensively as potential antimicrobials, their applications recently extended to development of antivirals. In the present study, silver nanoparticles were successfully developed by green synthetic approach using bark extract of Cinnamomum cassia (Cinnamon) and leaves extract of Thymus vulgaris (Thyme). The synthesized nanoparticles were characterized using UV-Vis absorption spectroscopy, scanning electron microscopy (SEM) and fourier transform infrared spectroscopy (FTIR). Plant extracts (Cinnamon and Thyme) and their corresponding nanoparticles were tested against avian influenza virus subtype H7N3 in Vero cells and the viability of cells was determined by tetrazolium dye (MTT) assay. The silver nanoparticles derived from plant extracts enhanced the antiviral activity and were found to be effective in both treatments, when incubated with the virus prior to infection and introduced to cells after infection. Moreover, the safety profile of the extract and the nanoparticles showed that they were non-toxic to Vero cells even at a concentration of 500 μg/ml. The biosynthesized nanoparticles may, hence, be a promising approach to provide treatment against influenza virus infections. Further research on characterization and mechanism of action of nanoparticles is required to develop better antiviral therapeutics for highly mutating viruses.