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A) Sequence alignments of the ectodomain of M2 proteins (M2e) from different subtypes of influenza A and B. Each M2e encoded gene sequence was fused in frame to the transmembrane domain and the cytoplasmic region of an H5N1 M2 expressing plasmid (on the top of right panel). The variable amino acids in M2e, which are different from H5N1 (human, 2005), are marked in green. B) Each M2 plasmid encodes M2e from different subtypes of influenza (as indicated) was transfected into 293T cells. After 48 hours, the cells were lysed and run on a 12% SDS-PAGE gel, followed by immunoblotting with a pool of sera from V-EtM2e/H5 22 immunized mice and an anti-actin antibody. C) The anti-HA <t>ADCC</t> activity induced by V-EtM2e/H5 05 or V-EtM2e/H5 22 immunized mice pooled sera (3×serially dilution) was detected by measuring the Luciferase activity from activated effector <t>Jurkat</t> cells and calculated as fold change against the no-serum control. D) Comparison of the anti-H5 22 (group1), -M2e H5 (group2), and - M2e H7 (group 3)-ADCC activity levels mediated by pooled sera (1:810 dilution) from V-EtM2e/H5 05 - or V-EtM2e/H5 22 -immunized mice. E) V-EtM2e/H5 22 -immunized mice (1:810 dilution) mediated broad ADCC activities against each M2e from variable subtypes of influenza A and B, which was expressed on the transfected target 293T cells (as indicated).
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A) Sequence alignments of the ectodomain of M2 proteins (M2e) from different subtypes of influenza A and B. Each M2e encoded gene sequence was fused in frame to the transmembrane domain and the cytoplasmic region of an H5N1 M2 expressing plasmid (on the top of right panel). The variable amino acids in M2e, which are different from H5N1 (human, 2005), are marked in green. B) Each M2 plasmid encodes M2e from different subtypes of influenza (as indicated) was transfected into 293T cells. After 48 hours, the cells were lysed and run on a 12% SDS-PAGE gel, followed by immunoblotting with a pool of sera from V-EtM2e/H5 22 immunized mice and an anti-actin antibody. C) The anti-HA <t>ADCC</t> activity induced by V-EtM2e/H5 05 or V-EtM2e/H5 22 immunized mice pooled sera (3×serially dilution) was detected by measuring the Luciferase activity from activated effector <t>Jurkat</t> cells and calculated as fold change against the no-serum control. D) Comparison of the anti-H5 22 (group1), -M2e H5 (group2), and - M2e H7 (group 3)-ADCC activity levels mediated by pooled sera (1:810 dilution) from V-EtM2e/H5 05 - or V-EtM2e/H5 22 -immunized mice. E) V-EtM2e/H5 22 -immunized mice (1:810 dilution) mediated broad ADCC activities against each M2e from variable subtypes of influenza A and B, which was expressed on the transfected target 293T cells (as indicated).
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A) Sequence alignments of the ectodomain of M2 proteins (M2e) from different subtypes of influenza A and B. Each M2e encoded gene sequence was fused in frame to the transmembrane domain and the cytoplasmic region of an H5N1 M2 expressing plasmid (on the top of right panel). The variable amino acids in M2e, which are different from H5N1 (human, 2005), are marked in green. B) Each M2 plasmid encodes M2e from different subtypes of influenza (as indicated) was transfected into 293T cells. After 48 hours, the cells were lysed and run on a 12% SDS-PAGE gel, followed by immunoblotting with a pool of sera from V-EtM2e/H5 22 immunized mice and an anti-actin antibody. C) The anti-HA ADCC activity induced by V-EtM2e/H5 05 or V-EtM2e/H5 22 immunized mice pooled sera (3×serially dilution) was detected by measuring the Luciferase activity from activated effector Jurkat cells and calculated as fold change against the no-serum control. D) Comparison of the anti-H5 22 (group1), -M2e H5 (group2), and - M2e H7 (group 3)-ADCC activity levels mediated by pooled sera (1:810 dilution) from V-EtM2e/H5 05 - or V-EtM2e/H5 22 -immunized mice. E) V-EtM2e/H5 22 -immunized mice (1:810 dilution) mediated broad ADCC activities against each M2e from variable subtypes of influenza A and B, which was expressed on the transfected target 293T cells (as indicated).

Journal: bioRxiv

Article Title: A VSV-vector vaccine simultaneously targeting H5N1 hemagglutinin (HA) and matrix protein 2 (M2) induces robust neutralizing and ADCC antibody responses and provides full protection against lethal H5N1 infection in a mouse model

doi: 10.64898/2025.12.26.696590

Figure Lengend Snippet: A) Sequence alignments of the ectodomain of M2 proteins (M2e) from different subtypes of influenza A and B. Each M2e encoded gene sequence was fused in frame to the transmembrane domain and the cytoplasmic region of an H5N1 M2 expressing plasmid (on the top of right panel). The variable amino acids in M2e, which are different from H5N1 (human, 2005), are marked in green. B) Each M2 plasmid encodes M2e from different subtypes of influenza (as indicated) was transfected into 293T cells. After 48 hours, the cells were lysed and run on a 12% SDS-PAGE gel, followed by immunoblotting with a pool of sera from V-EtM2e/H5 22 immunized mice and an anti-actin antibody. C) The anti-HA ADCC activity induced by V-EtM2e/H5 05 or V-EtM2e/H5 22 immunized mice pooled sera (3×serially dilution) was detected by measuring the Luciferase activity from activated effector Jurkat cells and calculated as fold change against the no-serum control. D) Comparison of the anti-H5 22 (group1), -M2e H5 (group2), and - M2e H7 (group 3)-ADCC activity levels mediated by pooled sera (1:810 dilution) from V-EtM2e/H5 05 - or V-EtM2e/H5 22 -immunized mice. E) V-EtM2e/H5 22 -immunized mice (1:810 dilution) mediated broad ADCC activities against each M2e from variable subtypes of influenza A and B, which was expressed on the transfected target 293T cells (as indicated).

Article Snippet: The ADCC reporter assay was performed by using ADCC Bioassay effector Jurkat cell line which expresses firefly luciferase under the control of NFAT (nuclear factor of activated T cells) response elements, and mouse FcγRIV (BPS Bioscience).

Techniques: Sequencing, Expressing, Plasmid Preparation, Transfection, SDS Page, Western Blot, Activity Assay, Luciferase, Control, Comparison