Inhibition of Influenza M2-Induced Cell Death Alleviates Its Negative Contribution to Vaccination Efficiency

The effectiveness of recombinant vaccines encoding full-length M2 protein of influenza virus or its ectodomain (M2e) have previously been tested in a number of models with varying degrees of success. Recently, we reported a strong cytotoxic effect exhibited by M2 on mammalian cells in vitro. Here we demonstrated a decrease in protection when M2 was added to a DNA vaccination regimen that included influenza NP. Furthermore, we have constructed several fusion proteins of conserved genes of influenza virus and tested their expression in vitro and protective potential in vivo. The four-partite NP-M1-M2-NS1 fusion antigen that has M2 sequence engineered in the middle part of the composite protein was shown to not be cytotoxic in vitro. A three-partite fusion protein (consisting of NP, M1 and NS1) was expressed much more efficiently than the four-partite protein. Both of these constructs provided statistically significant protection upon DNA vaccination, with construct NP-M1-M2-NS1 being the most effective. We conclude that incorporation of M2 into a vaccination regimen may be beneficial only when its apparent cytotoxicity-linked negative effects are neutralized. The possible significance of this data for influenza vaccination regimens and preparations is discussed

Expression of recombinant multi-partite fusion proteins based on conserved genes of influenza in vitro.

<p>Lanes 1–4 - cells transfected with pNPM1NS1, lanes 5–8 - with pNPM1M2NS1. A - anti-Flag-tag antibodies used for protein detection; B - anti-HA-tag antibodies used. Lanes 1, 2, 5 and 6 - starting point of chase (0 hours). Chase time: 3.5 hours (panel A, lanes 3 and 7), 7 hours (panel A, lanes 4 and 8), 8 hours (panel B, lanes 3, 4, 7 and 8). Proteosome inhibitor MG132 added to samples in panel B, lanes 4 and 8. Lanes 1 and 5 contain ⅓ of total protein loaded to lanes 2–4 and 6–8, correspondingly. Position of molecular weight markers is shown.</p

Absence of cytotoxicity induced by multi-partite fusion proteins based on conserved genes of influenza containing and not containing M2 sequence (as measured by green fluorescence).

<p>HEK cells were co-transfected with 0.2 µg of pGFP and 0.8 µg of the following plasmids: A - pCAGGS, B - pM2, C - pNPM1NS1, D - pNPM1M2NS1. Images were taken 64 hours after transfection.</p

Survival of mice vaccinated with pNPM1NS1 and pNPM1M2NS1 after challenge with 5 LD50 of H5N2 influenza virus strain A/Mallard/Pennsylvania/10218/84.

<p>Animals were immunized and challenged as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001417#s2" target="_blank">Materials and Methods</a>.</p

Survival of mice vaccinated with the combinations of pNP and pM2 after challenge with 100 LD50 of H5N2 influenza virus strain A/Mallard/Pennsylvania/10218/84.

<p>Animals were immunized and challenged as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001417#s2" target="_blank">Materials and Methods</a>.</p

Lung pathology (day 6 after infection) in vaccinated and experimentally infected mice from the same experimental groups described in the legend to

<p><b>Copyright information:</b></p><p>Taken from "Protection against mouse and avian influenza A strains via vaccination with a combination of conserved proteins NP, M1 and NS1"</p><p></p><p>Influenza and Other Respiratory Viruses 2007;1(2):71-79.</p><p>Published online Jan 2007</p><p>PMCID:PMC2040185.</p><p>© 2007 Cure Lab, Inc. Journal Compilation 2007 Blackwell Publishing Ltd</p> Haemorrhagic inflammation areas are shown by arrows

Survival of mice vaccinated with the combination of pNP, pM1 and pNS1 after challenge with 5 LD of H5N2 influenza virus strain A/Mallard/Pennsylvania/10218/84

<p><b>Copyright information:</b></p><p>Taken from "Protection against mouse and avian influenza A strains via vaccination with a combination of conserved proteins NP, M1 and NS1"</p><p></p><p>Influenza and Other Respiratory Viruses 2007;1(2):71-79.</p><p>Published online Jan 2007</p><p>PMCID:PMC2040185.</p><p>© 2007 Cure Lab, Inc. Journal Compilation 2007 Blackwell Publishing Ltd</p