Journal: Signal Transduction and Targeted Therapy

Article Title: CHIKV mRNA vaccines encoding conserved structural/envelope proteins confer broad cross-lineage protection against infection

doi: 10.1038/s41392-025-02182-2

Figure Lengend Snippet: Vaccine design, preparation, and in vitro expression. a Vaccine design. A total of 769 strains were identified from the NCBI database, including 14 from the West African lineage, 335 from the Asian lineage, 121 from the East, Central, and South African lineage, and 299 from the Indian Ocean lineage. Conserved sequences were selected for further analysis; b Utilize Alpha-Fold3 for structural prediction and receptor interaction prediction of the E protein in vaccine sequences and viral sequences (MH670649.1); c Dynamic light scattering (DLS) particle size distribution; d Transmission electron microscopy (TEM) images; e E1, E2, C protein, and β-actin Western blot images; (−) indicates the negative control, and M indicates the control transfected with a commercial kit; f Immunofluorescence experiments were conducted to detect the distribution of the E1 protein, using nontransfected cells as the mock condition

Article Snippet: E2 protein (Sino Biological, #40440-V08B, 1 μg/ml, 100 μl/well) was coated in a 96-well plate (442404, Thermo Fisher Scientific) overnight at 4 °C.

Techniques: In Vitro, Expressing, Transmission Assay, Electron Microscopy, Western Blot, Negative Control, Control, Transfection, Immunofluorescence

Journal: Signal Transduction and Targeted Therapy

Article Title: CHIKV mRNA vaccines encoding conserved structural/envelope proteins confer broad cross-lineage protection against infection

doi: 10.1038/s41392-025-02182-2

Figure Lengend Snippet: Cellular immune response assessment. a – f Elispot experiments to enumerate specific spots indicating cytokine secretion by splenocytes stimulated with the E2 protein for IFN-γ ( a ), IL-2 ( c ), and IL-4 ( e ) and stimulated with the inactivated virus for IFN-γ ( b ), IL-2 ( d ), and IL-4 ( f ); g – j Flow cytometry analysis of the percentages of CD4+ T cells producing IL-2 ( g ), IL-4 ( h ), IFN-γ ( i ), and TNF-α ( j ); k – m The proportions of CD8+ T cells producing IL-2 ( k ), IFN-γ ( l ), and TNF-α ( m ). The data are presented as the mean ± SEM ( n = 5 or 3). Statistical analysis was conducted using two-way ANOVA and Tukey’s multiple comparison test for bar graphs; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns not significant

Article Snippet: E2 protein (Sino Biological, #40440-V08B, 1 μg/ml, 100 μl/well) was coated in a 96-well plate (442404, Thermo Fisher Scientific) overnight at 4 °C.

Techniques: Enzyme-linked Immunospot, Virus, Flow Cytometry, Comparison

Journal: Signal Transduction and Targeted Therapy

Article Title: CHIKV mRNA vaccines encoding conserved structural/envelope proteins confer broad cross-lineage protection against infection

doi: 10.1038/s41392-025-02182-2

Figure Lengend Snippet: mCV-1 and mCV-2 protect A129 mice from mortality due to CHIKV infection. a The vaccination and sample collection timeline, Serum collection, and determination of binding antibody titers and neutralizing antibody levels were performed on days 21 and 28 after initial immunization, followed by challenge experiments on day 42. Body temperature, body weight, and joint changes were monitored daily after the challenge until all mice in the placebo group died, and survival curves were plotted; b The titers of binding antibodies at days 21 and 28; c Neutralizing antibody titers at 21, 28, and 56 days post-immunization; d Variation in viremia within 21 days post-challenge; e and f Changes in body weight ( e ) and the degree of swelling in the right hind limb joints ( f ) of A129 mice post-challenge; g Survival rate of mice post-challenge; h Viral loads in the spleen and joint tissues of mice on day 21 post-challenge; i Elispots experiments to enumerate specific spots indicating cytokine secretion by splenocytes stimulated with the E2 protein for IFN-γ, IL-2, and IL-4; j-k, Joint tissue pathological sections ( k ) and pathological scores ( j ). Statistical analysis was conducted using one-way ANOVA and Tukey’s multiple comparison tests for bar graphs; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns not significant

Article Snippet: E2 protein (Sino Biological, #40440-V08B, 1 μg/ml, 100 μl/well) was coated in a 96-well plate (442404, Thermo Fisher Scientific) overnight at 4 °C.

Techniques: Infection, Binding Assay, Comparison

Journal: Signal Transduction and Targeted Therapy

Article Title: CHIKV mRNA vaccines encoding conserved structural/envelope proteins confer broad cross-lineage protection against infection

doi: 10.1038/s41392-025-02182-2

Figure Lengend Snippet: mCV-1 and mCV-2 protect rhesus macaques from CHIKV challenge. a The immunization schedule for rhesus macaques is depicted in the flowchart, with vaccinations administered on Day 0 and Day 21, at a dosage of 300 μg per animal, using physiological saline as the placebo group. Serum samples were collected on Days 7, 14, 21, 28, and 35 post-primary immunization, followed by a challenging experiment on Day 42 ( n = 3); b and c The titers of binding antibodies ( b ) and neutralizing antibodies against adapted strains ( c ) at different time points; d GMTs of pseudovirus neutralizing antibodies against various strains at 35-day post-immunization for mCV-1 and mCV-2; e Elispot experiments to enumerate specific spots indicating cytokine secretion by splenocytes stimulated with the E2 protein for IFN-γ, IL-2, and IL-4; f Changes in viremia within 7 days post-immunization with mCV-1 and mCV-2; g Tissue viral loads in rhesus macaques immunized with mCV-1, mCV-2 or physiological saline on day 7 post-challenge, LN: Hilar lymph nodes, Kid: Kidney, PA: Pancreas, ILN: Inguinal lymph nodes, MLN: mesenteric lymph nodes, SMG: submandibular lymph node; h and i pathological scores ( h ) and pathological sections ( i ). The data are presented as The mean ± SEM ( n = 3), and each symbol represents a mouse. Statistical analysis was conducted using one-way ANOVA and Tukey’s multiple comparison tests for bar graphs; *** p < 0.001; **** p < 0.0001; ns not significant

Article Snippet: E2 protein (Sino Biological, #40440-V08B, 1 μg/ml, 100 μl/well) was coated in a 96-well plate (442404, Thermo Fisher Scientific) overnight at 4 °C.

Techniques: Saline, Binding Assay, Enzyme-linked Immunospot, Comparison

Journal: NPJ Vaccines

Article Title: A safe insect-based chikungunya fever vaccine affords rapid and durable protection in cynomolgus macaques

doi: 10.1038/s41541-024-01047-z

Figure Lengend Snippet: NHPs were vaccinated with a low (LD) or high doses (HD) of EILV/CHIKV, CHIKV 181/25 or PBS (mock). At day 350, all animals were implanted with electronic data loggers as temperature sensor and challenged subcutaneously with 1.0 × 10 5 PFU of WT La Reunion strain of CHIKV at day 370. A Study design and vaccination timeline [Created with BioRender.com Wang, T. (2023) BioRender.com/o29z240]. B Body temperature changes were recorded every 15 min and reported as mean ± standard error of the mean (SEM) starting 7 days before to 14 days after infection with WT CHIKV strain La Réunion. C Viremia were measured by plaque assays at indicated days post-challenge (PC). UD: Undetectable. Data are presented as means ± SEM.

Article Snippet: Millipore ELISPOT plates (Millipore Ltd, Darmstadt, Germany) were coated with CHIKV peptide pools (15 µg/ml), or CHIKV E2 recombinant protein (SinoBiological, USA, 15 ug/ml), 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).

Techniques: Infection

Journal: NPJ Vaccines

Article Title: A safe insect-based chikungunya fever vaccine affords rapid and durable protection in cynomolgus macaques

doi: 10.1038/s41541-024-01047-z

Figure Lengend Snippet: A – E Transcriptional changes of PBMCs of vaccinated macaques and mock controls at day 4 PC. A Principal component analysis (PCA) of all normalized transcripts to visualize the relatedness of samples via dimensional reduction. Each dot represents an individual RNA sample for mock (pink; n = 3), EILV/CHIKV (gray; n = 3), and CHIKV 181/25 (aqua; n = 3) groups. Samples that cluster are considered overall more similar, whereas distant samples are considered overall more distinct. B Heatmap of the most differentially expressed mRNAs in each group. Transcripts with a Benjamini–Hochberg adjusted p -value < 0.05 were sorted by log fold change values for EILV/CHIKV-vaccinated compared to CHIKV 181/25-immunized subjects. Red indicates increased expression; blue indicates decreased expression. C Bar plot of the most significant signaling pathways in EILV/CHIKV-vaccinated compared to mock-immunized subjects. Any differentially expressed transcripts with a Benjamini–Hochberg corrected P < 0.1 were deemed significant for enrichment. Pathways were sorted by statistical significance (a higher -log ( P ) indicates higher significance). Red indicates increased expression; blue indicates decreased expression. D Heatmap of the most upregulated pathways in each group; samples were sorted by z-score for EILV/CHIKV-vaccinated compared to CHIKV 181/25-immunized subjects. Yellow/green indicates increased expression, blue/purple indicates decreased expression. E Dot plots depicting predicted cell type trend scores for mock (pink; n = 3), EILV/CHIKV (gray; n = 3), and CHIKV 181/25 (aqua; n = 3) groups. Values were derived from transcriptional signatures indicative for a particular cell subset. F , G PBMCs of day 350 vaccinated NHPs were cultured ex vivo with CHIKV capsid, E3, E2 and E1 peptide pools for 6 h, and stained for IFN-γ, CD3, CD4, or CD8. Total number of IFN-γ + CD4 + and CD8 + T cells is shown. *** P < 0.001, ** P < 0.01, or * P < 0.05 compared to mock. #### P < 0.001, ## P < 0.01, or # P < 0.05 compared to CHIKV 181/25.

Article Snippet: Millipore ELISPOT plates (Millipore Ltd, Darmstadt, Germany) were coated with CHIKV peptide pools (15 µg/ml), or CHIKV E2 recombinant protein (SinoBiological, USA, 15 ug/ml), 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).

Techniques: Expressing, Derivative Assay, Cell Culture, Ex Vivo, Staining

Journal: NPJ Vaccines

Article Title: A safe insect-based chikungunya fever vaccine affords rapid and durable protection in cynomolgus macaques

doi: 10.1038/s41541-024-01047-z

Figure Lengend Snippet: A – C CHIKV-specific MBC responses by ELISPOT analysis. PBMCs of day 30 ( A ) and day 350 ( B - C ) vaccinated NHPs were stimulated for 5 d with R848 plus rIL-2 and seeded onto ELISPOT plates coated with CHIKV capsid, E3, E2 and E1 peptide pools ( A ), total IgG or CHIKV recombinant E2 protein ( B , C ). A – C Frequencies of CHIKV-specific antibody secreting cells (ASC)s per 10 6 input cells in MBC cultures from the subject. B Images of total IgG-ASCs, CHIKV peptide-specific or CHIKV E2 protein- specific MBCs, are shown. D Serum neutralizing activity against CHIKV 181/25 was measured by a plaque reduction neutralization test (PRNT). E CHIKV E2 binding IgG responses at indicate time points by ELISA. F , G Passive immunization study. Pooled sera collected at day 350 PV of macaques were diluted 1:2 in PBS and transferred to 6 week-old AB6 mice 24 h before infection with a LD100 dose of WT CHIKV. Mice were monitored daily for morbidity. F Survival rate. G Percent weight loss compared to prior infection. *** P < 0.001, ** P < 0.01, or * P < 0.05 compared to mock. #### P < 0.001, ## P < 0.01, or # P < 0.05 compared to CHIKV 181/25.

Article Snippet: Millipore ELISPOT plates (Millipore Ltd, Darmstadt, Germany) were coated with CHIKV peptide pools (15 µg/ml), or CHIKV E2 recombinant protein (SinoBiological, USA, 15 ug/ml), 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).

Techniques: Enzyme-linked Immunospot, Recombinant, Activity Assay, Plaque Reduction Neutralization Test, Binding Assay, Enzyme-linked Immunosorbent Assay, Infection

Journal: NPJ Vaccines

Article Title: A safe insect-based chikungunya fever vaccine affords rapid and durable protection in cynomolgus macaques

doi: 10.1038/s41541-024-01047-z

Figure Lengend Snippet: NHPs were implanted with electronic data loggers as temperature sensor and bled 14 days before vaccination. NHPs were vaccinated with 1.3 × 10 8 PFU EILV/CHIKV ( n = 3), 5 × 10 5 PFU inactivated WT CHIKV ( n = 3) or PBS (mock, n = 2). At day 6 PV, NHPs were challenged subcutaneously with 10 5 PFU of WT La Reunion strain of CHIKV. A Study design and vaccination timeline [Created with BioRender.com Wang, T. (2023) BioRender.com/o29z240]. B Body temperature changes were recorded every 15 min and reported as hourly mean ± SEM starting 8 days before to 14 days after the challenge with WT CHIKV. C , D Viremia were measured by plaque assays ( C ) or Q-PCR ( D ) at indicated days PC. Undetectable: UD. ** P < 0.01 compared to mock group. Unpaired, 2-tailed Student’s t -test was used to determine the differences. Data are presented as means ± SEM.

Article Snippet: Millipore ELISPOT plates (Millipore Ltd, Darmstadt, Germany) were coated with CHIKV peptide pools (15 µg/ml), or CHIKV E2 recombinant protein (SinoBiological, USA, 15 ug/ml), 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).

Techniques:

Journal: NPJ Vaccines

Article Title: A safe insect-based chikungunya fever vaccine affords rapid and durable protection in cynomolgus macaques

doi: 10.1038/s41541-024-01047-z

Figure Lengend Snippet: A – D Transcriptional changes of PBMCs in EILV/CHIKV- vaccinated macaques and mock controls at day 7 PV. A PCA of all normalized transcripts to visualize the relatedness of samples via dimensional reduction. Each dot represents an individual RNA sample for mock (pink; n = 3) and EILV/CHIKV (gray; n = 3) groups. Samples that cluster are considered overall more similar, whereas distant samples are considered overall more distinct. B Heatmap of the most differentially expressed mRNAs in EILV/CHIKV-vaccinated compared to the mock-immunized subjects; sorted by statistical significance (Benjamini–Hochberg adjusted P < 0.05). Red indicates increased expression, white indicates no change in expression, blue indicates decreased expression. C Bar plot of the most significant signaling pathways in EILV/CHIKV-vaccinated compared to mock-immunized subjects. Any differentially expressed transcripts with a Benjamini–Hochberg corrected P < 0.1 were deemed significant for enrichment purposes. Pathways are sorted by statistical significance (a higher -log( p -value) indicates higher significance). Red indicates increased expression, blue indicates decreased expression. D Dot plots depicting predicted cell type trend scores for mock (pink; n = 3) and EILV/CHIKV (gray; n = 3) groups. Values were derived from transcriptional signatures indicative for a particular cell subset. E – G Sera cytokines were measured by nonhuman primate inflammation 13-plex kits at indicated time points PC. Data are presented as fold increases compared to the mock group.

Article Snippet: Millipore ELISPOT plates (Millipore Ltd, Darmstadt, Germany) were coated with CHIKV peptide pools (15 µg/ml), or CHIKV E2 recombinant protein (SinoBiological, USA, 15 ug/ml), 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).

Techniques: Expressing, Derivative Assay

Journal: NPJ Vaccines

Article Title: A safe insect-based chikungunya fever vaccine affords rapid and durable protection in cynomolgus macaques

doi: 10.1038/s41541-024-01047-z

Figure Lengend Snippet: A , B NHPs were vaccinated with EILV/CHIKV, inactivated WT CHIKV or PBS (mock). At day 6 PV, NHPs were challenged subcutaneously with 10 5 PFU of the WT La Reunion strain of CHIKV. At day 4 PC, cells expressing NK cell markers were analyzed. Percent positive ( A ) or total number ( B ) of NK or NK T cells in PBMCs are shown. C Serum neutralizing activity against CHIKV 181/25 was measured by PRNT. D CHIKV E2 binding IgG responses at indicate time points by ELISA. E . ELISPOT quantification of peripheral T cell responses. PBMCs of macaques collected at day 4 PC were stimulated with CHIKV capsid, E3, E2 and E1 peptide pools for 24 h. SFCs were measured by IFN-γ ELISPOT. Data are shown as # of SFC per 10 6 cells. ** P < 0.01, or * P < 0.05 compared to mock. ## P < 0.01 compared to CHIKV 181/25.

Article Snippet: Millipore ELISPOT plates (Millipore Ltd, Darmstadt, Germany) were coated with CHIKV peptide pools (15 µg/ml), or CHIKV E2 recombinant protein (SinoBiological, USA, 15 ug/ml), 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).

Techniques: Expressing, Activity Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot