Journal: Emerging Microbes & Infections

Article Title: Optimized production and immunogenicity of an insect virus-based chikungunya virus candidate vaccine in cell culture and animal models

doi: 10.1080/22221751.2021.1886598

Figure Lengend Snippet: Production and purification of EILV/CHIKV in mosquito cells. (A) C6/36 cells and C7/10 cells were infected with EILV/CHIKV (MOI 0.05) and then incubated at 28°C. At the indicated time points, media were replaced, and titers were determined by plaque assay on C7/10 cells. This experiment was reproducibly repeated 2 times. The results of one of them are presented. (B) C7/10 cells were infected with EILV/CHIKV at different MOIs. Media were harvested at the indicated time-points, and infectious titers were determined by plaque assay on C7/10 cells. (C,D). EILV/CHIKV samples were produced and purified as described in Materials and Methods. Samples were collected from different stages to determine the viral titers and purity. C. Comparative analysis of 6 different batches of purified viruses at different stages of purification. (D) Viral presence was detected by Coomassie staining in the harvested media.

Article Snippet: Millipore ELISPOT plates (Millipore Ltd, Darmstadt, Germany) were coated with EILV/CHIKV (1 × 10 8 PFU/ well), CHIKV VLP (The Native Antigen Company, Oxford, UK) 15 μg/ml, or anti-human Ig capture Ab (Mabtech In).

Techniques: Purification, Infection, Incubation, Plaque Assay, Produced, Staining

Journal: Emerging Microbes & Infections

Article Title: Optimized production and immunogenicity of an insect virus-based chikungunya virus candidate vaccine in cell culture and animal models

doi: 10.1080/22221751.2021.1886598

Figure Lengend Snippet: Hypersensitivity study in mice. B6 mice were sensitized by exposure to female Ae. albopictus mosquitoes three times during a 14-day period. On day 43, mice were injected i.d. on footpad with 10 µg SGE protein, 10 8 PFU of EILV/CHIKV or PBS (mock). 24 h after challenge, mouse tissues were harvested to assess allergy responses. (A) Scheme of sensitization study. ( B) Splenocytes were stimulated with 20 μg/ml SGE for 48 h and culture supernatants were collected to measure cytokine production by Bioplex. n = 5. * P< 0.05 compared to mock group. (C) H &E staining of footpad inflammation. Mock: no inflammation. EILV/CHIKV: minor inflammation in the subcutaneous tissue with mononuclear cells, eosinophils and few neutrophils. SGE: right panel shown enlarged image of the black rectangular box marked in the left panel. Severe inflammation in the subcutaneous tissue with large amounts of eosinophils (red arrows) and neutrophils (blue arrows) as well as mononuclear cells.

Article Snippet: Millipore ELISPOT plates (Millipore Ltd, Darmstadt, Germany) were coated with EILV/CHIKV (1 × 10 8 PFU/ well), CHIKV VLP (The Native Antigen Company, Oxford, UK) 15 μg/ml, or anti-human Ig capture Ab (Mabtech In).

Techniques: Injection, Staining

Journal: Emerging Microbes & Infections

Article Title: Optimized production and immunogenicity of an insect virus-based chikungunya virus candidate vaccine in cell culture and animal models

doi: 10.1080/22221751.2021.1886598

Figure Lengend Snippet: A minimal dose of 10 8 PFU EILV/CHIKV is required to induce strong CHIKV-specific IgM and IgG production, and memory B cell responses in mice. (A) 4-week-old B6 mice were vaccinated with various doses of EILV/CHIKV or PBS (mock). CHIKV- specific IgM and IgG antibodies in sera were detected by ELISA. n = 4–5. (B) 4-week-old B6 mice were vaccinated with 10 8 PFU EILV/CHIKV or PBS (mock). At different time post-vaccination, CHIKV- specific IgM and IgG antibodies in sera were detected by ELISA. n = 6–9. C-D. 4-week-old B6 mice were vaccinated with 10 8 PFU EILV/CHIKV, 5.5 x10 5 PFU CHIKV 181/25, or PBS (mock). At day 28, CHIKV- specific MBC responses were determined by ELISPOT analysis. (C) Images of wells from MBC culture. (D) Frequencies of CHIKV antibody secreting cells per 10 7 input cells in MBC cultures from the subject. n = 5–8. ** P < 0.01 compared to mock group.

Article Snippet: Millipore ELISPOT plates (Millipore Ltd, Darmstadt, Germany) were coated with EILV/CHIKV (1 × 10 8 PFU/ well), CHIKV VLP (The Native Antigen Company, Oxford, UK) 15 μg/ml, or anti-human Ig capture Ab (Mabtech In).

Techniques: Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

Journal: Emerging Microbes & Infections

Article Title: Optimized production and immunogenicity of an insect virus-based chikungunya virus candidate vaccine in cell culture and animal models

doi: 10.1080/22221751.2021.1886598

Figure Lengend Snippet: EILV/CHIKV triggers long-lasting CD8 + T cell responses compared to CHIKV/IRES in mice. 4-week-old B6 mice were vaccinated with 10 8 PFU non-purified EILV/CHIKV, 5log 10 PFU CHIKV/IRES, 8.3 log 10 PFU formalin-inactivated wild-type CHIKV, or PBS (mock). At day 7 (A,B) and day 55 (C,D) post-vaccination, splenocytes were cultured ex vivo with PMA and ionomycin for 4 h, and stained for IFN-γ, CD3, and CD4 or CD8. Total T cells were gated. Total number of IFN-γ + T cell subsets per spleen is shown. n = 5–7. ** P < 0.01 compared to mock group. # P < 0.01 or ## P < 0.01 compared to EILV/CHIKV vaccinated group. One experiment was shown here.

Article Snippet: Millipore ELISPOT plates (Millipore Ltd, Darmstadt, Germany) were coated with EILV/CHIKV (1 × 10 8 PFU/ well), CHIKV VLP (The Native Antigen Company, Oxford, UK) 15 μg/ml, or anti-human Ig capture Ab (Mabtech In).

Techniques: Purification, Cell Culture, Ex Vivo, Staining

Journal: Emerging Microbes & Infections

Article Title: Optimized production and immunogenicity of an insect virus-based chikungunya virus candidate vaccine in cell culture and animal models

doi: 10.1080/22221751.2021.1886598

Figure Lengend Snippet: A single dose of 10 8 PFU of purified EILV/CHIKV elicits strong CD8 + T cell responses in mice. 4-week-old B6 mice were vaccinated with EILV/CHIKV, CHIKV 181/25 or PBS (mock). At indicated time points post vaccination, splenocytes were harvested cultured ex vivo with CHIKV specific peptide for 5 h, and stained for IFN-γ, TNF-α, CD3, and CD8. Total T cells were gated. (A,B) Day 28 post vaccination. Total number of IFN-γ + (A) and IFNγ + TNFα + (B) T cell subsets per spleen is shown. C.Day (D) 4, 8 and 28 post vaccination. Total T cells were gated. Percentage of IFN-γ + T cell subsets is shown. n = 2–4. D. Total number of IFN-γ + T cell subsets per spleen at day 28 post vaccination is shown. n = 5–7. ** P < 0.01, * P < 0.05 compared to mock group. ## P < 0.01 compared to EILV/CHIKV vaccinated group.

Article Snippet: Millipore ELISPOT plates (Millipore Ltd, Darmstadt, Germany) were coated with EILV/CHIKV (1 × 10 8 PFU/ well), CHIKV VLP (The Native Antigen Company, Oxford, UK) 15 μg/ml, or anti-human Ig capture Ab (Mabtech In).

Techniques: Purification, Cell Culture, Ex Vivo, Staining

Journal: Emerging Microbes & Infections

Article Title: Optimized production and immunogenicity of an insect virus-based chikungunya virus candidate vaccine in cell culture and animal models

doi: 10.1080/22221751.2021.1886598

Figure Lengend Snippet: EILV/CHIKV induces potent antiviral cytokine responses and activation of APCs in vivo, but does not induce APCs alone upon in vitro exposure. 4-week-old B6 mice were infected with 1× 10 8 PFU EILV/CHIKV or PBS (mock). (A) On days 1, 4 and 8 post-vaccination, blood cytokines levels were determined by Q-PCR assay ( n = 4–11) or Bioplex assay. Q-PCR results were presented as the fold increase compared to mock- infected. Data are presented as means ± SE and are representative of at least 2 similar experiments. (B) The percentages of CD80 + CD11c + , CD86 + CD11c + , or MHCII + CD11c + for DCs and CD80 + F4/80 + , CD86 + F4/80 + , or MHCII + F4/80 + for macrophages. B6 mice were vaccinated with 10 8 PFU EILV/CHIKV or PBS (mock). At days 3 (D3) and 6 (D6) post vaccination, splenocytes were stained for cell surface markers, and analyzed by flow cytometry ( n = 5). Total splenocytes were gated. (C) Bone marrow- derived DCs and macrophages were treated in vitro with EILV/CHIKV (MOI = 5). Cells were harvested at day 4 to measure cytokine levels by Q-PCR assay ( n = 4–10). ** P < 0.01 or * P < 0.05 compared to mock group. # P < 0.01 or ## P < 0.01 compared to day 3.

Article Snippet: Millipore ELISPOT plates (Millipore Ltd, Darmstadt, Germany) were coated with EILV/CHIKV (1 × 10 8 PFU/ well), CHIKV VLP (The Native Antigen Company, Oxford, UK) 15 μg/ml, or anti-human Ig capture Ab (Mabtech In).

Techniques: Activation Assay, In Vivo, In Vitro, Infection, BioPlex Assay, Staining, Flow Cytometry, Derivative Assay