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Long-Lasting Cross-Protection Against Influenza A by Neuraminidase and M2e-based immunization strategies.

Schotsaert M, Ysenbaert T, Smet A, Schepens B, Vanderschaeghe D, Stegalkina S, Vogel TU, Callewaert N, Fiers W, Saelens X - Sci Rep (2016)

Bottom Line: There is mounting evidence that in the absence of neutralizing antibodies cross-reactive T cells provide protection against pandemic influenza viruses.HA-vaccinated mice were fully protected against challenge with homologous H1N1 2009 virus, failed to mount cross-reactive CD8+ T cells and succumbed to the second challenge with heterologous H3N2 virus.In summary, NA- and M2e-based immunity can protect against challenge with (homologous) virus without compromising the induction of robust cross-reactive CD8+ T cell responses upon exposure to virus.

View Article: PubMed Central - PubMed

Affiliation: Medical Biotechnology Center, VIB, Ghent, 9052, Belgium.

ABSTRACT
There is mounting evidence that in the absence of neutralizing antibodies cross-reactive T cells provide protection against pandemic influenza viruses. Here, we compared protection and CD8+ T cell responses following challenge with H1N1 2009 pandemic and H3N2 viruses of mice that had been immunized with hemagglutinin (HA), neuraminidase (NA) and the extracellular domain of matrix protein 2 (M2e) fused to a virus-like particle (VLP). Mice were challenged a first time with a sublethal dose of H1N1 2009 pandemic virus and, four weeks later, challenged again with an H3N2 virus. Mice that had been vaccinated with HA, NA, NA + M2e-VLP and HA + NA + M2e-VLP were protected against homologous H1N1 virus challenge. Challenged NA and NA + M2e-VLP vaccinated mice mounted CD8+ T cell responses that correlated with protection against secondary H3N2 challenge. HA-vaccinated mice were fully protected against challenge with homologous H1N1 2009 virus, failed to mount cross-reactive CD8+ T cells and succumbed to the second challenge with heterologous H3N2 virus. In summary, NA- and M2e-based immunity can protect against challenge with (homologous) virus without compromising the induction of robust cross-reactive CD8+ T cell responses upon exposure to virus.

No MeSH data available.


Related in: MedlinePlus

Design and characterization of recombinant soluble trimeric hemagglutinin (triHA) and tetrameric neuraminidase (tetNA) antigens used for vaccination.(a) Schematic representation of triHA and tetNA expression constructs. Amino acid residues separating the building blocks of the constructs are shown in single letter code. CD5-SS: secretion signal of CD5; triGCN4: trimerization leucine zipper derived from Saccharomyces cerevisae GCN4; Tag: Strep-Tag; tetrabrachion: tetramerization coiled coil derived from Staphylothermus marinus tetrabrachion. (b,c) UV absorption profile after size exclusion chromatography of triHA (b) and tetNA (c). (d) Enzymatic activity of tetNA in culture medium from pEF-NA transfected HEK293T cells (closed symbols) or culture medium only (open symbols) as measured by kinetic release of fluorescent 4-Methylumbelliferone from the 2′-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic substrate. (e) Fluorescence calibration curve for calculation of released 4-Methylumbelliferone. (f) Neuraminidase activity of tetNA results in desialylation of triHA N-glycans when mixed under conditions of vaccine formulation. Top panel shows a dextran ladder as a reference for the glycan structures, numbers represent glucose units. Other panels show N-linked glycan profiles for triHA (HA), tetNA (NA), triHA + tetNA in the presence of 8 M Urea (HA + NA (denatured)) and triHA + tetNA under vaccine formulation conditions (30 min at 4 °C) (HA + NA). Peaks in red indicate glycans on triHA that are desialylated in the presence of native tetNA.
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f1: Design and characterization of recombinant soluble trimeric hemagglutinin (triHA) and tetrameric neuraminidase (tetNA) antigens used for vaccination.(a) Schematic representation of triHA and tetNA expression constructs. Amino acid residues separating the building blocks of the constructs are shown in single letter code. CD5-SS: secretion signal of CD5; triGCN4: trimerization leucine zipper derived from Saccharomyces cerevisae GCN4; Tag: Strep-Tag; tetrabrachion: tetramerization coiled coil derived from Staphylothermus marinus tetrabrachion. (b,c) UV absorption profile after size exclusion chromatography of triHA (b) and tetNA (c). (d) Enzymatic activity of tetNA in culture medium from pEF-NA transfected HEK293T cells (closed symbols) or culture medium only (open symbols) as measured by kinetic release of fluorescent 4-Methylumbelliferone from the 2′-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic substrate. (e) Fluorescence calibration curve for calculation of released 4-Methylumbelliferone. (f) Neuraminidase activity of tetNA results in desialylation of triHA N-glycans when mixed under conditions of vaccine formulation. Top panel shows a dextran ladder as a reference for the glycan structures, numbers represent glucose units. Other panels show N-linked glycan profiles for triHA (HA), tetNA (NA), triHA + tetNA in the presence of 8 M Urea (HA + NA (denatured)) and triHA + tetNA under vaccine formulation conditions (30 min at 4 °C) (HA + NA). Peaks in red indicate glycans on triHA that are desialylated in the presence of native tetNA.

Mentions: We produced and purified recombinant, soluble oligomeric HA (triHA) and NA (tetNA) derived from H1N1v. To ensure correct oligomerisation and stability in the absence of the transmembrane domains, we genetically fused the extracellular domain of HA (HA 18–522) at the C-terminus to a trimerization GCN4-derived leucine zipper, and the extracellular domain of NA (NA 65–469), without the stalk, at the N-terminus to the tetramerization tetrabrachion coiled coil (Fig. 1a). TriHA and tetNA were produced in HEK293T cells and affinity purified from the cleared culture supernatant followed by a polishing step by size exclusion chromatography. The predominant peak of triHA eluted with a retention volume corresponding to glycosylated trimeric HA (Fig. 1b). A minor fraction, which was discarded, eluted faster and likely corresponded to higher order complexes of triHA (Fig. 1b). Gel filtration analysis of tetNA revealed a single peak that corresponded to the predicted molecular weight of soluble tetrameric NA (Fig. 1c). TetNA was enzymatically active and had a specific activity of 11,000 pmole/min/μg as determined by a fluorimetric assay using 2′-4-Methylumbelliferyl-α-D-N-acetylneuraminic acid as substrate (Fig. 1d,e). We also analyzed the N-glycan composition of purified triHA and tetNA using DNA-sequencer-assisted fluorophore-assisted carbohydrate electrophoresis38. Recombinant triHA contained sialylated N-glycans (Fig. 1f). In contrast, purified tetNA only contained neutral, non-sialylated N-glycans (Fig. 1f), most probably because of self desialylation. We also analyzed the N-glycan profiles of triHA and tetNA mixed together in equal amounts (mass) in phosphate buffered saline and incubated at 4 °C for 30 min. Even though incubation was at 4 °C, HA was almost completely desialylated by tetNA under these conditions (Fig. 1f). Prior denaturation of tetNA with 8 M urea did not affect the sialylation status of co-incubated triHA, indicating that enzymatically active tetNA was responsible for the desialylation of triHA. In summary, we obtained pure, soluble HA and NA with a natural oligomeric conformation. Furthermore, tetNA was enzymatically active.


Long-Lasting Cross-Protection Against Influenza A by Neuraminidase and M2e-based immunization strategies.

Schotsaert M, Ysenbaert T, Smet A, Schepens B, Vanderschaeghe D, Stegalkina S, Vogel TU, Callewaert N, Fiers W, Saelens X - Sci Rep (2016)

Design and characterization of recombinant soluble trimeric hemagglutinin (triHA) and tetrameric neuraminidase (tetNA) antigens used for vaccination.(a) Schematic representation of triHA and tetNA expression constructs. Amino acid residues separating the building blocks of the constructs are shown in single letter code. CD5-SS: secretion signal of CD5; triGCN4: trimerization leucine zipper derived from Saccharomyces cerevisae GCN4; Tag: Strep-Tag; tetrabrachion: tetramerization coiled coil derived from Staphylothermus marinus tetrabrachion. (b,c) UV absorption profile after size exclusion chromatography of triHA (b) and tetNA (c). (d) Enzymatic activity of tetNA in culture medium from pEF-NA transfected HEK293T cells (closed symbols) or culture medium only (open symbols) as measured by kinetic release of fluorescent 4-Methylumbelliferone from the 2′-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic substrate. (e) Fluorescence calibration curve for calculation of released 4-Methylumbelliferone. (f) Neuraminidase activity of tetNA results in desialylation of triHA N-glycans when mixed under conditions of vaccine formulation. Top panel shows a dextran ladder as a reference for the glycan structures, numbers represent glucose units. Other panels show N-linked glycan profiles for triHA (HA), tetNA (NA), triHA + tetNA in the presence of 8 M Urea (HA + NA (denatured)) and triHA + tetNA under vaccine formulation conditions (30 min at 4 °C) (HA + NA). Peaks in red indicate glycans on triHA that are desialylated in the presence of native tetNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4829898&req=5

f1: Design and characterization of recombinant soluble trimeric hemagglutinin (triHA) and tetrameric neuraminidase (tetNA) antigens used for vaccination.(a) Schematic representation of triHA and tetNA expression constructs. Amino acid residues separating the building blocks of the constructs are shown in single letter code. CD5-SS: secretion signal of CD5; triGCN4: trimerization leucine zipper derived from Saccharomyces cerevisae GCN4; Tag: Strep-Tag; tetrabrachion: tetramerization coiled coil derived from Staphylothermus marinus tetrabrachion. (b,c) UV absorption profile after size exclusion chromatography of triHA (b) and tetNA (c). (d) Enzymatic activity of tetNA in culture medium from pEF-NA transfected HEK293T cells (closed symbols) or culture medium only (open symbols) as measured by kinetic release of fluorescent 4-Methylumbelliferone from the 2′-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic substrate. (e) Fluorescence calibration curve for calculation of released 4-Methylumbelliferone. (f) Neuraminidase activity of tetNA results in desialylation of triHA N-glycans when mixed under conditions of vaccine formulation. Top panel shows a dextran ladder as a reference for the glycan structures, numbers represent glucose units. Other panels show N-linked glycan profiles for triHA (HA), tetNA (NA), triHA + tetNA in the presence of 8 M Urea (HA + NA (denatured)) and triHA + tetNA under vaccine formulation conditions (30 min at 4 °C) (HA + NA). Peaks in red indicate glycans on triHA that are desialylated in the presence of native tetNA.
Mentions: We produced and purified recombinant, soluble oligomeric HA (triHA) and NA (tetNA) derived from H1N1v. To ensure correct oligomerisation and stability in the absence of the transmembrane domains, we genetically fused the extracellular domain of HA (HA 18–522) at the C-terminus to a trimerization GCN4-derived leucine zipper, and the extracellular domain of NA (NA 65–469), without the stalk, at the N-terminus to the tetramerization tetrabrachion coiled coil (Fig. 1a). TriHA and tetNA were produced in HEK293T cells and affinity purified from the cleared culture supernatant followed by a polishing step by size exclusion chromatography. The predominant peak of triHA eluted with a retention volume corresponding to glycosylated trimeric HA (Fig. 1b). A minor fraction, which was discarded, eluted faster and likely corresponded to higher order complexes of triHA (Fig. 1b). Gel filtration analysis of tetNA revealed a single peak that corresponded to the predicted molecular weight of soluble tetrameric NA (Fig. 1c). TetNA was enzymatically active and had a specific activity of 11,000 pmole/min/μg as determined by a fluorimetric assay using 2′-4-Methylumbelliferyl-α-D-N-acetylneuraminic acid as substrate (Fig. 1d,e). We also analyzed the N-glycan composition of purified triHA and tetNA using DNA-sequencer-assisted fluorophore-assisted carbohydrate electrophoresis38. Recombinant triHA contained sialylated N-glycans (Fig. 1f). In contrast, purified tetNA only contained neutral, non-sialylated N-glycans (Fig. 1f), most probably because of self desialylation. We also analyzed the N-glycan profiles of triHA and tetNA mixed together in equal amounts (mass) in phosphate buffered saline and incubated at 4 °C for 30 min. Even though incubation was at 4 °C, HA was almost completely desialylated by tetNA under these conditions (Fig. 1f). Prior denaturation of tetNA with 8 M urea did not affect the sialylation status of co-incubated triHA, indicating that enzymatically active tetNA was responsible for the desialylation of triHA. In summary, we obtained pure, soluble HA and NA with a natural oligomeric conformation. Furthermore, tetNA was enzymatically active.

Bottom Line: There is mounting evidence that in the absence of neutralizing antibodies cross-reactive T cells provide protection against pandemic influenza viruses.HA-vaccinated mice were fully protected against challenge with homologous H1N1 2009 virus, failed to mount cross-reactive CD8+ T cells and succumbed to the second challenge with heterologous H3N2 virus.In summary, NA- and M2e-based immunity can protect against challenge with (homologous) virus without compromising the induction of robust cross-reactive CD8+ T cell responses upon exposure to virus.

View Article: PubMed Central - PubMed

Affiliation: Medical Biotechnology Center, VIB, Ghent, 9052, Belgium.

ABSTRACT
There is mounting evidence that in the absence of neutralizing antibodies cross-reactive T cells provide protection against pandemic influenza viruses. Here, we compared protection and CD8+ T cell responses following challenge with H1N1 2009 pandemic and H3N2 viruses of mice that had been immunized with hemagglutinin (HA), neuraminidase (NA) and the extracellular domain of matrix protein 2 (M2e) fused to a virus-like particle (VLP). Mice were challenged a first time with a sublethal dose of H1N1 2009 pandemic virus and, four weeks later, challenged again with an H3N2 virus. Mice that had been vaccinated with HA, NA, NA + M2e-VLP and HA + NA + M2e-VLP were protected against homologous H1N1 virus challenge. Challenged NA and NA + M2e-VLP vaccinated mice mounted CD8+ T cell responses that correlated with protection against secondary H3N2 challenge. HA-vaccinated mice were fully protected against challenge with homologous H1N1 2009 virus, failed to mount cross-reactive CD8+ T cells and succumbed to the second challenge with heterologous H3N2 virus. In summary, NA- and M2e-based immunity can protect against challenge with (homologous) virus without compromising the induction of robust cross-reactive CD8+ T cell responses upon exposure to virus.

No MeSH data available.


Related in: MedlinePlus