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Inhibition of influenza M2-induced cell death alleviates its negative contribution to vaccination efficiency.

Ilyinskii PO, Gambaryan AS, Meriin AB, Gabai V, Kartashov A, Thoidis G, Shneider AM - PLoS ONE (2008)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Cure Lab, Canton, Massachusetts, United States of America. ilyinskii@curelab.com

ABSTRACT
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.

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Related in: MedlinePlus

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).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.
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pone-0001417-g003: 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).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.

Mentions: Plasmids encoding fusion genes, pNPM1NS1 and pNPM1M2NS1, that contained full sequences of conserved influenza NP, M1 and NS1 genes (with or without M2) were constructed and shown to express the proteins of predicted size (Fig. 2). They were tested for their expression in vitro using antibodies to N-terminal Flag-tag (Fig. 2A) or C-terminal HA-tag (Fig. 2B). It was clear that inclusion of M2-encoding sequence resulted in dramatic impairment of expression of the fusion proteins, with NPM1NS1 construct expressing at least 10 times more efficiently than NPM1M2NS1 (Fig. 2A). Higher levels of expression of the three-partite over four-partite fusion protein was also seen when antibody to C-terminal HA-tag was used (Fig. 2B), although the overall difference was less. Neither of the fusion proteins exhibited any cytotoxicity similar to that induced by wild-type M2 (Fig. 3). Specifically, for NPM1M2NS1 no signs of cytotoxicity were seen up to 96 hours after transfection even when its efficiency of transfection was 90–100% (not shown).


Inhibition of influenza M2-induced cell death alleviates its negative contribution to vaccination efficiency.

Ilyinskii PO, Gambaryan AS, Meriin AB, Gabai V, Kartashov A, Thoidis G, Shneider AM - PLoS ONE (2008)

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).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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2175529&req=5

pone-0001417-g003: 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).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.
Mentions: Plasmids encoding fusion genes, pNPM1NS1 and pNPM1M2NS1, that contained full sequences of conserved influenza NP, M1 and NS1 genes (with or without M2) were constructed and shown to express the proteins of predicted size (Fig. 2). They were tested for their expression in vitro using antibodies to N-terminal Flag-tag (Fig. 2A) or C-terminal HA-tag (Fig. 2B). It was clear that inclusion of M2-encoding sequence resulted in dramatic impairment of expression of the fusion proteins, with NPM1NS1 construct expressing at least 10 times more efficiently than NPM1M2NS1 (Fig. 2A). Higher levels of expression of the three-partite over four-partite fusion protein was also seen when antibody to C-terminal HA-tag was used (Fig. 2B), although the overall difference was less. Neither of the fusion proteins exhibited any cytotoxicity similar to that induced by wild-type M2 (Fig. 3). Specifically, for NPM1M2NS1 no signs of cytotoxicity were seen up to 96 hours after transfection even when its efficiency of transfection was 90–100% (not shown).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Cure Lab, Canton, Massachusetts, United States of America. ilyinskii@curelab.com

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus