A universal vaccine is in high demand to address the uncertainties of antigenic drift and the reduced effectiveness of current influenza vaccines.In this study,a strategy called computationally optimized broadly react...A universal vaccine is in high demand to address the uncertainties of antigenic drift and the reduced effectiveness of current influenza vaccines.In this study,a strategy called computationally optimized broadly reactive antigen(COBRA)was used to generate a consensus sequence of the hemagglutinin globular head portion(HA1)of influenza virus samples collected from 1918 to 2021 to trace evolutionary changes and incorporate them into the designed constructs.Constructs carrying different HA1regions were delivered into eukaryotic cells by Salmonella-mediated bactofection using a Semliki Forest virus RNA-dependent RNApolymerase(RdRp)-based eukaryotic expression system,pJHL204.Recombinant protein expression was confirmed by Western blot and immunofluorescence assays.Mice immunized with the designed constructs produced a humoral response,with a significant increase in immunoglobulin G(IgG)levels,and a cell-mediated immune response,including a 1.5-fold increase in CD4^(+) and CD8^(+)T cells.Specifically,constructs #1 and #5 increased the production of interferon-γ(IFN-γ)producing CD4^(+)and CD8^(+)T cells,skewing the response toward the T helper type 1 cell(Th1)pathway.Additionally,interleukin-4(IL-4)-producing T cells were upregulated 4-fold.Protective efficacy was demonstrated,with up to 4-fold higher production of neutralizing antibodies and a hemagglutination inhibition titer>40 against the selected viral strains.The designed constructs conferred a broadly protective immune response,resulting in a notable reduction in viral titer and minimal inflammation in the lungs of mice challenged with the influenza A/PR8/34,A/Brisbane/59/2007,A/California/07/2009,KBPV VR-92,and NCCP 43021 strains.This discovery revolutionizes influenza vaccine design and delivery;Salmonella-mediated COBRA-HA1 is a highly effective in vivo antigen presentation strategy.This approach can effectively combat seasonal H1N1 influenza strains and potential pandemic outbreaks.展开更多
Aim To study whether antivenom from laying hens can be used for the detection of venom antigens, Methods Chickens （white Leghorn） were immunized with detoxicated king cobra venom by formaldehyde and egg yolk immunog...Aim To study whether antivenom from laying hens can be used for the detection of venom antigens, Methods Chickens （white Leghorn） were immunized with detoxicated king cobra venom by formaldehyde and egg yolk immunoglobulin （IgY） isolated from yolk; IgY was labelled with the horseradish peroxidase （HRP）. Experimental condition and parameters were determined by chessboard test. The specificity, sensitivity, precision, and stability of this method were assayed in the experiment. Results This method could detect as low as 32 μg· L^-1 of the king cobra antigens. A good linear relation was found within 32 ～ 750 μg· L^-1 of king cobra venom concentrations （ r = 0. 963）. There was no cross reactivity for the reagents with Agkistrodon acutus Guenther venom or Vipera russelli siamensis Smith venom;slight cross reactivity .with Bungarus multicinctus Blyth venom or Bungarus fasciatus Chmeider venom; and notable cross reactivity with cobra venom. The average intra-assay relative standard deviation （RSD） was 1% - 3%, and the inter-assay RSD was less than 8%. The reagents （including IgY and HRP-IgY） were stable; no differences （P 〉 0.05） were observed for the detection of venom antigens when the reagents were stored at 37 ℃ up to 6 d. Conclusion IgY is a good reagent for diagnosis of snakebite after eliminating the genus cross reactivity.展开更多
基金Basic Science Research Program through the National Research Foundation of Korea(NRF)funded by the Ministry of Education(2019R1A6A1A03033084).
文摘A universal vaccine is in high demand to address the uncertainties of antigenic drift and the reduced effectiveness of current influenza vaccines.In this study,a strategy called computationally optimized broadly reactive antigen(COBRA)was used to generate a consensus sequence of the hemagglutinin globular head portion(HA1)of influenza virus samples collected from 1918 to 2021 to trace evolutionary changes and incorporate them into the designed constructs.Constructs carrying different HA1regions were delivered into eukaryotic cells by Salmonella-mediated bactofection using a Semliki Forest virus RNA-dependent RNApolymerase(RdRp)-based eukaryotic expression system,pJHL204.Recombinant protein expression was confirmed by Western blot and immunofluorescence assays.Mice immunized with the designed constructs produced a humoral response,with a significant increase in immunoglobulin G(IgG)levels,and a cell-mediated immune response,including a 1.5-fold increase in CD4^(+) and CD8^(+)T cells.Specifically,constructs #1 and #5 increased the production of interferon-γ(IFN-γ)producing CD4^(+)and CD8^(+)T cells,skewing the response toward the T helper type 1 cell(Th1)pathway.Additionally,interleukin-4(IL-4)-producing T cells were upregulated 4-fold.Protective efficacy was demonstrated,with up to 4-fold higher production of neutralizing antibodies and a hemagglutination inhibition titer>40 against the selected viral strains.The designed constructs conferred a broadly protective immune response,resulting in a notable reduction in viral titer and minimal inflammation in the lungs of mice challenged with the influenza A/PR8/34,A/Brisbane/59/2007,A/California/07/2009,KBPV VR-92,and NCCP 43021 strains.This discovery revolutionizes influenza vaccine design and delivery;Salmonella-mediated COBRA-HA1 is a highly effective in vivo antigen presentation strategy.This approach can effectively combat seasonal H1N1 influenza strains and potential pandemic outbreaks.
文摘Aim To study whether antivenom from laying hens can be used for the detection of venom antigens, Methods Chickens （white Leghorn） were immunized with detoxicated king cobra venom by formaldehyde and egg yolk immunoglobulin （IgY） isolated from yolk; IgY was labelled with the horseradish peroxidase （HRP）. Experimental condition and parameters were determined by chessboard test. The specificity, sensitivity, precision, and stability of this method were assayed in the experiment. Results This method could detect as low as 32 μg· L^-1 of the king cobra antigens. A good linear relation was found within 32 ～ 750 μg· L^-1 of king cobra venom concentrations （ r = 0. 963）. There was no cross reactivity for the reagents with Agkistrodon acutus Guenther venom or Vipera russelli siamensis Smith venom;slight cross reactivity .with Bungarus multicinctus Blyth venom or Bungarus fasciatus Chmeider venom; and notable cross reactivity with cobra venom. The average intra-assay relative standard deviation （RSD） was 1% - 3%, and the inter-assay RSD was less than 8%. The reagents （including IgY and HRP-IgY） were stable; no differences （P 〉 0.05） were observed for the detection of venom antigens when the reagents were stored at 37 ℃ up to 6 d. Conclusion IgY is a good reagent for diagnosis of snakebite after eliminating the genus cross reactivity.
