Somayeh Zamani, Fatemeh Fotouhi Chahouki, Zahra Nourmohammadi , Saeideh Sadeghi Neshat, Vahideh Mazaheri , Ali Torabi , Behrokh Farahmand ,
Volume 73, Issue 7 (10-2015)
Abstract
Background: The influenza virus is one of the most important factors for higher morbidity and mortality in the world. Recently, researchers have been focused on influenza conserved antigenic proteins such as hemagglutinin stalk domain (HA2) for vaccine production and serological studies. The HA2 plays a major role in the fusion of the virus with host cells membrane. The immunity system enables to produce antibody against HA2. The aim of this study is polyclonal antibody production against influenza HA2. Methods: This study was done in the Influenza Research Lab, Pasteur Institute of Iran, Tehran for one year from September 2013 to October 2014. In the present study, recombinant HA2 protein was produced in prokaryotic system and purified using Nickel affinity chromatography. The purified HA2 was mixed with Freund’s adjuvant (complete and incomplete) and injected into two New Zealand white rabbits by intramuscularly and subcutaneously routes. Immunization was continued for several months with two weeks interval. Before each immunization, blood was drawn by venous puncture from the rabbit ear. Function of rabbit's sera was evaluated using radial immunodiffusion (RID) in both forms, Single RID (SRID) and Double RID (DRID). Finally, antiserum activity against HA2 was evaluated using western blotting as serological assay. Results: Sedimentary line and zone was observed in RID assays (SRID and DRID) represent interaction between HA2 protein and anti- HA2 antibody. As well as, western blotting results was positive for HA2 protein. Therefore, these results showed that polyclonal antibody produced against HA2 protein can identify HA2 protein antigenic sites. Conclusion: These findings show that humoral immune responses have properly been stimulated in rabbits and these antibodies can identify HA2 protein and may be suitable for other serological methods.
Sina Soleimani , Shahla Shahsavandi , Omid Madadgar ,
Volume 74, Issue 8 (11-2016)
Abstract
Background: Problems of live and inactivated influenza vaccines such as, increasing emerge and re-emerge viruses with high human mortality, current epidemics of influenza and direct transmission of avian viruses to human, affect the vaccination program. DNA vaccines as third generation of vaccines is specially considered for control of influenza in human and poultry. The main advantage of these vaccines is humoral and cellular immune responses and broad spectrum of using these vaccines for control of circulating strains of influenza. In this study the conserved fragment of HA2 to form of DNA vaccine was designed to induce immunity against influenza viruses and its heterologous protective immunity against these viruses was evaluated.
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Methods: The experimental study was performed in Razi Vaccine and Serum Research Institute from December 2014 to July 2015 in Iran. The HA2 was cloned into pcDNA3.1 to assess the HA2 DNA vaccine and mice were immunized with the generated constructs in a DNA prime-DNA boost regimen in 4 groups. The humoral immune responses were analyzed at defined intervals by VN tests. The safety of the vaccine was evaluated by daily inspection and histopathological examination. For evaluation of cellular immunity, proliferation assay was used.
Results: The antibody titre and cellular immunity of immunized mice was significantly higher than control group for two serotypes and the highest responses was in the group with two-time boosting (P<0.01). There were no any local, general and histopathology reactions in immunized mice.
Conclusion: The HA2 DNA vaccine significantly enhanced circulatory antibody responses and cellular immunity against influenza current serotypes. This study showed the highest immune responses were in the group that immunized with HA2 in prime and two boosts. Besides that, this construct did not have any local and general reaction and any side effects in treated mice. So, this construct was introduced as candidate for control of influenza virus serotypes.
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