Journal: NPJ Vaccines

Article Title: Systemic prime mucosal boost significantly increases protective efficacy of bivalent RSV influenza viral vectored vaccine

doi: 10.1038/s41541-024-00912-1

Figure Lengend Snippet: a Vaccination schematic for the assessment of the immunogenicity of ChAdOx1-NP + M1-RSVF administered through different regimens in outbred, 5-week-old, CD-1 mice ( n = 6). Mice were culled on day 50, with sera, lungs, spleens, nasal-associated lymphoid tissue (NALT) and bronchioalveolar lavage fluid (BALF) harvested. b IgG, IgA and IgM responses against RSV-F, influenza A NP and M1 (H1N1) in sera three weeks post-final vaccination, measured by ELISA. Values are displayed as ELISA units (EUs) (log 10 ). Individual mouse values are represented as symbols. Values were analysed using non-parametric Kruskal-Wallis tests to assess for statistically significant differences between groups, which are then expressed as p values (*= p < 0.05, **= p < 0.01, ***= p < 0.001). c Levels of IgA specific to influenza A NP (H1N1) and RSVF in NALT, BALF and lung homogenate supernatant (LHS) collected from mice three weeks post-final vaccination as measured by ELISAs (*= p < 0.05, **= p < 0.01, ***= p < 0.001). d Relative levels of anti-influenza A NP (H1N1) and anti-RSVF IgG subclasses in serum (IgG1, IgG2a, IgG2b, IgG2c and IgG3), as measured by tIgG-normalised indirect ELISA. Relative IgG subclass levels following each regimen are presented as individual doughnut charts, with each section of the doughnut representing the median IgG subclass OD 405nm response. Bar charts with individual sample responses per subclass per regimen are present in Supplementary Fig. . e Serum antigen-specific IgG2a to IgG1 subclass ratios (IgG2a OD 405nm /IgG1 OD 405nm ). For all boxplots, whisker endings represent upper and lower extremes, the box bounds represent upper and lower quartiles, respectively, and the central line represents the group median.

Article Snippet: 96 well Nunc TM MaxiSorp TM plates were separately coated with 50μL/well of 2μg/mL (for tIgG and IgG subclass detection) or 5μg/mL (for IgA and IgM detection) recombinant Influenza A H1N1 (A/Puerto Rico/8/34/Mount Sinai) Nucleoprotein/NP I116M (ECD, His Tag), Influenza A H1N1 (A/Puerto Rico/8/34/Mount Sinai) Matrix protein 1 / M1 Protein (His Tag), Influenza A H3N2 (A/Hong Kong/2671/2019) Nucleoprotein / NP Protein (His Tag) Influenza A H3N2 (A/Aichi/2/1968) Matrix protein 1 / M1 Protein (His Tag) or Human respiratory syncytial virus (RSV) (A2) Fusion glycoprotein/RSV-F protein (ECD, His Tag) (all SinoBiological), overnight at 4 °C.

Techniques: Enzyme-linked Immunosorbent Assay, Indirect ELISA, Whisker Assay

Journal: NPJ Vaccines

Article Title: Systemic prime mucosal boost significantly increases protective efficacy of bivalent RSV influenza viral vectored vaccine

doi: 10.1038/s41541-024-00912-1

Figure Lengend Snippet: a Cytokine responses in CD4 + and CD8 + splenocytes that were separately stimulated with influenza A (H1N1) NP + M1- and RSVF-spanning peptides; % cytokine + T cells were determined through intracellular staining. Basal frequencies of cytokine + T cells (unstimulated sample) were subtracted from stimulated sample frequencies. Median responses in each group are displayed as the top lines of bars on graphs on the left-hand side of the figure, with symbols representing individual mice, grey-shaded bars prime-boost regimens and white bars prime-only regimens. b Cytokine responses in CD4 + and CD8 + lung cells harvested from mice, separately stimulated with influenza A (H1N1) NP + M1- and RSVF-spanning peptides. c Vaccination schematic for the continued assessment of the cellular immunogenicity of ChAdOx1-NP + M1-RSVF. d IFNγ responses in splenocytes stimulated with NP + M1- and RSVF-spanning peptides (IFNγ spot-forming cells (SFCs)/million splenocytes), as measured by IFNγ enzyme-linked immunosorbent spot (ELISpot) assay. e Total counts of CD8 + T RM cells (left), as well as relative counts of CD8 + T EM and T RM cells (right) in lungs post-vaccination (all non-antigen-specific). T RM cells were defined as CD8 + CD44 + CD62L - CD103 + CD69 + , and negative for intravenous (IV) circulatory CD3 + T cell stain. T EM cells were defined as CD8 + CD44 + CD62L - CD127 + , and negative for IV circulatory CD3 + T cell stain. Group differences of data in ( a ), ( b ), ( d ) and ( e ) were analysed using non-parametric Kruskal-Wallis tests (*= p < 0.05, **= p < 0.01, ***= p < 0.001). For all boxplots, whisker endings represent upper and lower extremes, the box bounds represent upper and lower quartiles, respectively, and the central line represents the group median.

Article Snippet: 96 well Nunc TM MaxiSorp TM plates were separately coated with 50μL/well of 2μg/mL (for tIgG and IgG subclass detection) or 5μg/mL (for IgA and IgM detection) recombinant Influenza A H1N1 (A/Puerto Rico/8/34/Mount Sinai) Nucleoprotein/NP I116M (ECD, His Tag), Influenza A H1N1 (A/Puerto Rico/8/34/Mount Sinai) Matrix protein 1 / M1 Protein (His Tag), Influenza A H3N2 (A/Hong Kong/2671/2019) Nucleoprotein / NP Protein (His Tag) Influenza A H3N2 (A/Aichi/2/1968) Matrix protein 1 / M1 Protein (His Tag) or Human respiratory syncytial virus (RSV) (A2) Fusion glycoprotein/RSV-F protein (ECD, His Tag) (all SinoBiological), overnight at 4 °C.

Techniques: Staining, ELISpot Assay, Enzyme-linked Immunospot, Whisker Assay

Journal: NPJ Vaccines

Article Title: Systemic prime mucosal boost significantly increases protective efficacy of bivalent RSV influenza viral vectored vaccine

doi: 10.1038/s41541-024-00912-1

Figure Lengend Snippet: a Vaccination and challenge schematic for the assessment of the protective capacity of ChAdOx1-NP + M1-RSVF against X31 (H3N2) infection and subsequent disease in mice. Mice were prime-boost-vaccinated, then challenged with X31, and culled 6 days later with tissues and fluids harvested. Blood sampling was performed 4 weeks post-prime and 3 weeks post-boost. b Weight change in mice over time post-challenge, measured as % of pre-challenge weight. Significant differences at timepoints between IM-IN and control mouse groups are represented with *, between IM-IN and IM-IM as # and between IN-IN and unvaccinated as @ (*, @ or # = p < 0.05, **= p < 0.01). c Viral load in lungs 6 days post-challenge (M gene copies/μg lung RNA (log 10 )). d H3N2 NP-specific IgG and IgA levels in serum, NWs, BALF and LHS post-challenge, as measured by ELISA (log 10 EU). Median negative control values are displayed as dashed lines. e Levels of antigen-specific CD8 + T RM cells, and relative levels of antigen-specific CD8 + T EM and T RM , in BAL and lungs post-challenge. T RM and T EM cells were defined as CD3 + CD8 + CD44 + CD62L - CD103 + CD69 + , and CD3 + CD8 + CD44 + CD62L - , respectively, and positive for influenza pentamer H-2Kd TYQRTALV. In boxplots, a “+” symbol represents the group mean. One mouse in group IM-IN did not have detectable Lung T RM . Group differences for all data were analysed using non-parametric Kruskal-Wallis tests (*= p < 0.05, **= p < 0.01). For all boxplots, whisker endings represent upper and lower extremes, the box bounds represent upper and lower quartiles, respectively, and the central line represents the group median.

Article Snippet: 96 well Nunc TM MaxiSorp TM plates were separately coated with 50μL/well of 2μg/mL (for tIgG and IgG subclass detection) or 5μg/mL (for IgA and IgM detection) recombinant Influenza A H1N1 (A/Puerto Rico/8/34/Mount Sinai) Nucleoprotein/NP I116M (ECD, His Tag), Influenza A H1N1 (A/Puerto Rico/8/34/Mount Sinai) Matrix protein 1 / M1 Protein (His Tag), Influenza A H3N2 (A/Hong Kong/2671/2019) Nucleoprotein / NP Protein (His Tag) Influenza A H3N2 (A/Aichi/2/1968) Matrix protein 1 / M1 Protein (His Tag) or Human respiratory syncytial virus (RSV) (A2) Fusion glycoprotein/RSV-F protein (ECD, His Tag) (all SinoBiological), overnight at 4 °C.

Techniques: Infection, Sampling, Control, Enzyme-linked Immunosorbent Assay, Negative Control, Whisker Assay

Journal: NPJ Vaccines

Article Title: Systemic prime mucosal boost significantly increases protective efficacy of bivalent RSV influenza viral vectored vaccine

doi: 10.1038/s41541-024-00912-1

Figure Lengend Snippet: a Vaccination and challenge schematic for the assessment of the protective capacity of ChAdOx1-NP + M1-RSVF against H1N1 infection and subsequent disease in mice. Mice were prime-boost-vaccinated, then challenged with H1N1, and culled 5 days later with tissues and fluids harvested. Blood sampling was performed 4 weeks post-priming and 3 weeks post-boosting. b Weight change in mice over time post-challenge, as measured by % of pre-challenge weight. The significant difference between IM-IN and IM-IM mouse groups is represented with ## (##= p < 0.01). c Viral load in lungs 5 days post-challenge (number of M gene copies/μg lung RNA (log 10 )). d H1N1 NP-specific IgG and IgA levels in serum, NWs, BALF and LHS post-challenge, as measured ELISA (log 10 EU). Median control values are displayed as dashed lines on graphs. e Levels of antigen-specific CD8 + T RM cells , and relative levels of antigen-specific CD8 + T EM and T RM , in BAL and lungs post-challenge. T RM and T EM cells were defined as CD3 + CD8 + CD44 + CD62L - CD103 + CD69 + , and CD3 + CD8 + CD44 + CD62L - , respectively, and positive for influenza pentamer H-2Kd TYQRTALV. In boxplots, a “+” symbol represents the group mean. Two mice in group IM-IM did not have detectable BAL T RM , and one mouse in group IM-IM did not have detectable Lung T RM . Group differences for all data were analysed using non-parametric Kruskal-Wallis tests (*= p < 0.05, ** =p < 0.01, ***= p < 0.001). For all boxplots, whisker endings represent upper and lower extremes, the box bounds represent upper and lower quartiles, respectively, and the central line represents the group median.

Article Snippet: 96 well Nunc TM MaxiSorp TM plates were separately coated with 50μL/well of 2μg/mL (for tIgG and IgG subclass detection) or 5μg/mL (for IgA and IgM detection) recombinant Influenza A H1N1 (A/Puerto Rico/8/34/Mount Sinai) Nucleoprotein/NP I116M (ECD, His Tag), Influenza A H1N1 (A/Puerto Rico/8/34/Mount Sinai) Matrix protein 1 / M1 Protein (His Tag), Influenza A H3N2 (A/Hong Kong/2671/2019) Nucleoprotein / NP Protein (His Tag) Influenza A H3N2 (A/Aichi/2/1968) Matrix protein 1 / M1 Protein (His Tag) or Human respiratory syncytial virus (RSV) (A2) Fusion glycoprotein/RSV-F protein (ECD, His Tag) (all SinoBiological), overnight at 4 °C.

Techniques: Infection, Sampling, Enzyme-linked Immunosorbent Assay, Control, Whisker Assay

Journal: NPJ Vaccines

Article Title: Systemic prime mucosal boost significantly increases protective efficacy of bivalent RSV influenza viral vectored vaccine

doi: 10.1038/s41541-024-00912-1

Figure Lengend Snippet: a Vaccination and challenge schematic for the assessment of the protective capacity of ChAdOx1-NP + M1-RSVF against RSV-A2 infection. Mice were prime-boost-vaccinated, then challenged with RSV-A2, and culled 7 days later with tissues and fluids harvested. Blood sampling was performed 4 weeks post-prime and 3 weeks post-boost. b Weight change in mice over time post-challenge, measured as % of pre-challenge weight. At each timepoint, lines represent group medians, and bars represent group bodyweight ranges. Significant differences between IM-IN and control groups are represented with *, between IM-IM and control as & (* or & = p < 0.05, **= p < 0.01). c Viral load in lungs 7 days post-challenge, measured as number of RSV L gene copies/μg lung RNA (log 10 ). d RSVF-specific IgG and IgA levels in serum, NWs, BALF and LHS post-challenge, measured by ELISA. Median negative control values are displayed as dashed lines. e Levels of antigen-specific CD8 + T RM , and relative levels of antigen-specific CD8 + T EM and T RM , in BAL and lungs post-challenge. T RM and T EM cells were defined as CD3 + CD8 + CD44 + CD62L - CD103 + CD69 + , and CD3 + CD8 + CD44 + CD62L - , respectively, and positive for RSV pentamer H-2Kd KYKNVTEL. In boxplots, a “+” symbol represents the group mean. Two mice from the IM-IM-vaccinated group did not have detectable BAL or lung T RM . Group differences for all data were analysed using non-parametric Kruskal-Wallis tests (*= p < 0.05, **= p < 0.01). For all figure boxplots, whisker endings represent upper and lower extremes, the box bounds represent upper and lower quartiles, respectively, and the central line represents the group median.

Article Snippet: 96 well Nunc TM MaxiSorp TM plates were separately coated with 50μL/well of 2μg/mL (for tIgG and IgG subclass detection) or 5μg/mL (for IgA and IgM detection) recombinant Influenza A H1N1 (A/Puerto Rico/8/34/Mount Sinai) Nucleoprotein/NP I116M (ECD, His Tag), Influenza A H1N1 (A/Puerto Rico/8/34/Mount Sinai) Matrix protein 1 / M1 Protein (His Tag), Influenza A H3N2 (A/Hong Kong/2671/2019) Nucleoprotein / NP Protein (His Tag) Influenza A H3N2 (A/Aichi/2/1968) Matrix protein 1 / M1 Protein (His Tag) or Human respiratory syncytial virus (RSV) (A2) Fusion glycoprotein/RSV-F protein (ECD, His Tag) (all SinoBiological), overnight at 4 °C.

Techniques: Infection, Sampling, Control, Enzyme-linked Immunosorbent Assay, Negative Control, Whisker Assay

Journal: Frontiers in Veterinary Science

Article Title: Single Dose of Bivalent H5 and H7 Influenza Virus-Like Particle Protects Chickens Against Highly Pathogenic H5N1 and H7N9 Avian Influenza Viruses

doi: 10.3389/fvets.2021.774630

Figure Lengend Snippet: The bivalent H5+H7 VLP vaccine induces efficient HI and VN antibodies. (A) To assess the immunogenicity of the vaccine candidate, chickens were intramuscularly (i.m.) immunized with 15 μg of the bivalent H5+H7 VLP or 0.3 mL of the commercial trivalent (H5: Re-11; H5: Re-12; H7: Re-3) vaccine. (B) HI titers at Week 2 and 3 p.v. against H5N1 TT3 virus. (C) HI titers at Week 2 and 3 p.v. against H7N9 GD15 virus. (D) VN antibody against 100 TCID 50 of H5N1 TT3 virus. (E) VN antibody against 100 TCID 50 of H7N9 GD15 virus. The detection limit was below 4 log 2 for the HI assay and below 10 for the VN assay.

Article Snippet: In brief, the flat-bottomed 96-well microplate plates were coated with 250 ng of the purified HA protein of H7N9 A/Anhui/1/2013 (AH13) virus (Sino Biological) or the purified TT3 HA protein expressed in Sf9 insect cells at 4°C overnight.

Techniques: Virus, HI Assay

Journal: Frontiers in Veterinary Science

Article Title: Single Dose of Bivalent H5 and H7 Influenza Virus-Like Particle Protects Chickens Against Highly Pathogenic H5N1 and H7N9 Avian Influenza Viruses

doi: 10.3389/fvets.2021.774630

Figure Lengend Snippet: The bivalent H5+H7 VLP vaccine confers good protection against the lethal H7N9 virus. Chickens were i.m. immunized with 15 μg of the bivalent H5+H7 VLP or 0.3 mL of the commercial trivalent (H5: Re-11; H5: Re-12; H7: Re-3) vaccine. (A) Survival rates of the birds after challenged with 10 6.0 EID 50 of the H7N9 GD15 virus. (B) Virus titration in laryngotracheal swabs on Day 2 and 4 p.c. (C) Virus titration in cloacal swabs on Day 2 and 4 p.c. Viral titers in the heart (D) , cecum (E) , lung (F) , and spleen (G) of the birds on Day 3 and 5 p.c. Viral titers were determined by measuring EID 50 . The detection limit was below 10 1.0 EID 50 /0.1mL for swabs and 10 1.48 EID 50 /g for organs.

Article Snippet: In brief, the flat-bottomed 96-well microplate plates were coated with 250 ng of the purified HA protein of H7N9 A/Anhui/1/2013 (AH13) virus (Sino Biological) or the purified TT3 HA protein expressed in Sf9 insect cells at 4°C overnight.

Techniques: Virus, Titration

Journal: Frontiers in Veterinary Science

Article Title: Single Dose of Bivalent H5 and H7 Influenza Virus-Like Particle Protects Chickens Against Highly Pathogenic H5N1 and H7N9 Avian Influenza Viruses

doi: 10.3389/fvets.2021.774630

Figure Lengend Snippet: Virus shedding of the birds challenged by H5N1 or H7N9 virus.

Article Snippet: In brief, the flat-bottomed 96-well microplate plates were coated with 250 ng of the purified HA protein of H7N9 A/Anhui/1/2013 (AH13) virus (Sino Biological) or the purified TT3 HA protein expressed in Sf9 insect cells at 4°C overnight.

Techniques: Virus

Journal: Frontiers in Veterinary Science

Article Title: Single Dose of Bivalent H5 and H7 Influenza Virus-Like Particle Protects Chickens Against Highly Pathogenic H5N1 and H7N9 Avian Influenza Viruses

doi: 10.3389/fvets.2021.774630

Figure Lengend Snippet: The bivalent H5+H7 VLP vaccine alleviates lung injury after the lethal H7N9 virus challenge. (A) H & E staining results of the bird's lung on Day 2 and 4 post-H7N9 virus challenge. Black triangle indicates dilatation in the pulmonary chamber or bronchial; white triangle arrow stands for infiltration of fiber cells, detached epithelial cell mucus, and erythrocytes in the parabrochus; quad star means lymphocytes infiltration in the lung chamber. (B) Scores of the overall histopathologic changes in the bird's lung post-H7N9 virus challenge. The specific information concerning the scoring criteria was listed in the Materials and Method section.

Article Snippet: In brief, the flat-bottomed 96-well microplate plates were coated with 250 ng of the purified HA protein of H7N9 A/Anhui/1/2013 (AH13) virus (Sino Biological) or the purified TT3 HA protein expressed in Sf9 insect cells at 4°C overnight.

Techniques: Virus, Staining