complete recombinant ha pr8 protein  (Sino Biological)

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: A DNA Vaccine That Encodes an Antigen-Presenting Cell-Specific Heterodimeric Protein Protects against Cancer and Influenza

doi: 10.1016/j.omtm.2020.01.007

Figure Lengend Snippet: MHCII-Targeting A/B Heterodimeric and C H 3 Homodimeric DNA Vaccines Both Confer Complete Protection against Influenza Infection In (A)–(D), BALB/c mice were vaccinated with 100 μg of (two plasmids) DNA i.m./EP with the indicated A/B heterodimeric vaccines expressing HA from PR8 (H1N1). In (E) and (F), A/B heterodimeric and C H 3-based homodimeric vaccines were compared, using 100 μg (A/B heterodimer, two plasmids) and 50 μg (C H 3 homodimer, one plasmid) of DNA i.m./EP. (A, B, E, and F) Levels of HA-specific IgG1 (A and E) and IgG2a (B and F) serum antibodies 6 weeks after immunization (mean ± SEM, **p < 0.01, Mann-Whitney test; ND, not detectable). (C and G) 15 (C) or 4 (G) weeks after vaccination, the mice were infected with a lethal dose of PR8 influenza virus (7.5 × LD 50 ). Weight was followed for 10 days (mean ± SEM; *p < 0.05, **p < 0.01, Mann-Whitney test, comparing targeting to non-targeting vaccines). (D and H) Survival curves of the vaccinated mice. Non-significant (ns, p = 0.0549) or significant p values are indicated (**p < 0.01, Mantel-Cox test). n = 6/group in (A)–(D), n = 8/group in (E)–(H). Weight loss of 20% was defined as the humane endpoint, at which time point the mice were euthanized.

Article Snippet: Spleens were harvested after 14 days, plated in triplicates (5 × 10 5 cells/well), and stimulated with 2 μg/mL MHCII (SVSSFERFEIFPK and HNTNGVTAACSHEG)- or MHCI (IYSTVASSL, both InvivoGen)-restricted HA PR8 peptides, MHCII-restricted OVA peptide (amino acids 323–339, ISQAVHAAHAEINEAGR, InvivoGen), or complete recombinant HA PR8 protein (2 μg/mL, Sino Biological) or OVA protein (5 μg/mL, InvivoGen).

Techniques: Infection, Expressing, Plasmid Preparation, MANN-WHITNEY

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: A DNA Vaccine That Encodes an Antigen-Presenting Cell-Specific Heterodimeric Protein Protects against Cancer and Influenza

doi: 10.1016/j.omtm.2020.01.007

Figure Lengend Snippet: Diverse Targeting Units and Antigenic Units Are Efficiently Expressed on Both the A and B Arm of the Heterodimeric Protein Vaccine In Vitro (A–D) Targeting units, dimerization units, and antigenic units are indicated by symbols (given in boxes). HEK293E cells were transiently transfected with combinations of various DNA vaccine plasmids, theoretically yielding the vaccine molecules depicted in the top rows. Supernatants were analyzed in sandwich ELISAs as indicated (mean + SD). Expected binding of coat mAb (green) or detection mAb (red) to protein vaccine units (squares) is indicated by colors. Positive or negative signal obtained in sandwich ELISAs are indicated by + or −. (A) The targeting units, Xcl1 chemokine and scFv αNIP , were genetically fused to either A or B chains in combination with OVA and mCherry as antigenic units, as indicated. Homodimeric scFv αNIP -C H 3-scFv 315 was included as a negative control. (B) The targeting units, Xcl1 and scFv αNIP , were genetically fused to either A or B chains in combination with antigenic units OVA and mCherry, as indicated. (C) MIP1α and scFv αNIP were expressed in all possible combinations in the targeting units of A/B heterodimers and C H 3-based homodimers, as indicated. The antigenic unit, scFv 315 , was invariable. (D) The targeting units, scFv αMHCII and scFv αNIP , were expressed with antigenic units HA from PR8 and Cal07 influenza strains in A/B heterodimers, as indicated.

Article Snippet: Spleens were harvested after 14 days, plated in triplicates (5 × 10 5 cells/well), and stimulated with 2 μg/mL MHCII (SVSSFERFEIFPK and HNTNGVTAACSHEG)- or MHCI (IYSTVASSL, both InvivoGen)-restricted HA PR8 peptides, MHCII-restricted OVA peptide (amino acids 323–339, ISQAVHAAHAEINEAGR, InvivoGen), or complete recombinant HA PR8 protein (2 μg/mL, Sino Biological) or OVA protein (5 μg/mL, InvivoGen).

Techniques: In Vitro, Transfection, Binding Assay, Negative Control

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: A DNA Vaccine That Encodes an Antigen-Presenting Cell-Specific Heterodimeric Protein Protects against Cancer and Influenza

doi: 10.1016/j.omtm.2020.01.007

Figure Lengend Snippet: Antigen-Specific Responses Are Induced Irrespective of Whether the Antigen Is Fused to the A or B Arm in the Heterodimeric Vaccine Protein (A) BALB/c mice were vaccinated with 100 μg (two plasmids) of DNA i.m./EP with A/B heterodimers expressing influenza HA antigen from Cal07 (H1N1) and PR8 (H1N1) connected to either the A or B arm (n = 5-6/group). 14 days after vaccination, the mice were bled and Cal07- and PR8-specific IgGs were analyzed in the sera by ELISA (mean ± SEM; Mann-Whitney test, ns, not significant; ND, not detectable). (B) BALB/c mice were vaccinated with 50 μg (two plasmids) of DNA i.d./EP with A/B heterodimers expressing influenza HA antigen from PR8 (H1N1) and OVA connected to either the A or the B arm (n = 4/group, analyzed as biological replicates). After 11 days, spleens were harvested and analyzed for IFN-γ-secreting T cells (ELISPOT) after stimulation with HA whole protein, MHCII-restricted HA peptides (HNTNGVTAACSHEG or SVSSFERFEIFPK), or MHCI-restricted HA peptide (IYSTVASSL), OVA whole protein, MHCII-restricted OVA peptide (ISQAVHAAHAEINEAGR), or irrelevant peptide (λ2 315 CDR3 peptide, ALWFRNHFVFGGGTKVT). Mann-Whitney test, two-tailed; ns, not significant.

Article Snippet: Spleens were harvested after 14 days, plated in triplicates (5 × 10 5 cells/well), and stimulated with 2 μg/mL MHCII (SVSSFERFEIFPK and HNTNGVTAACSHEG)- or MHCI (IYSTVASSL, both InvivoGen)-restricted HA PR8 peptides, MHCII-restricted OVA peptide (amino acids 323–339, ISQAVHAAHAEINEAGR, InvivoGen), or complete recombinant HA PR8 protein (2 μg/mL, Sino Biological) or OVA protein (5 μg/mL, InvivoGen).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Enzyme-linked Immunospot, Two Tailed Test

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: A DNA Vaccine That Encodes an Antigen-Presenting Cell-Specific Heterodimeric Protein Protects against Cancer and Influenza

doi: 10.1016/j.omtm.2020.01.007

Figure Lengend Snippet: Comparison of A/B Heterodimeric and C H 3-Homodimeric DNA Vaccines Expressing Two Different HAs BALB/c mice were vaccinated i.m./EP with 100 μg of DNA (two plasmids) encoding either A/B heterodimeric or C H 3 homodimeric vaccine proteins that express HA from both PR8(H1N1) and Cal07(H1N1) as indicated. Mice were boosted after 5 weeks. (A and B) Levels of PR8-specific IgG (A) and Cal07-specific IgG (B) serum antibodies 2 weeks after the boost (mean ± SEM; **p < 0.01 Mann Whitney; ND, not detectable). (C and E) 2 weeks after the boost, the mice were infected with a lethal dose of PR8 (C, 5 × LD 50 ), or Cal07 (E, 5 × LD 50 ) influenza virus. Weight was followed for 10 days. Weight loss of 20% was defined as the humane endpoint, at which time point the mice were euthanized (n = 8/group, mean ± SEM; Mann-Whitney test, *p < 0.05, **p < 0.01; ns, not significant). (D and F) Survival curves of the vaccinated mice after challenge with either PR8 (D) or Cal07 (F) influenza virus.

Article Snippet: Spleens were harvested after 14 days, plated in triplicates (5 × 10 5 cells/well), and stimulated with 2 μg/mL MHCII (SVSSFERFEIFPK and HNTNGVTAACSHEG)- or MHCI (IYSTVASSL, both InvivoGen)-restricted HA PR8 peptides, MHCII-restricted OVA peptide (amino acids 323–339, ISQAVHAAHAEINEAGR, InvivoGen), or complete recombinant HA PR8 protein (2 μg/mL, Sino Biological) or OVA protein (5 μg/mL, InvivoGen).

Techniques: Expressing, MANN-WHITNEY, Infection