Journal: NPJ Vaccines

Article Title: Immunization-induced antigen archiving enhances local memory CD8+ T cell responses following an unrelated viral infection

doi: 10.1038/s41541-024-00856-6

Figure Lengend Snippet: a Experimental schematic for b – e . C57/BL6 mice were immunized subcutaneously in the footpad and/or flank with the indicated antigens and adjuvants. b Cells were stained with CD45, PDPN, CD31 and PD-L1. Cells were gated on CD45-PDPN + CD31- for FRC and CD45-PDPN + CD31+ for LECs. Shown are examples of LEC and FRC antigen-positive cells based on PD-L1 expression (floor, MARCO LEC) and ova-AF488+ from mice 2–3 weeks after immunization with ova conjugated to Alexa-Fluor 488 (AF488) and polyI:C and αCD40. c Quantification of the frequency of LEC, BEC, and FRC in the popliteal lymph node (pLN) that are positive for the indicated antigens administered with polyI:C and αCD40 at indicated time. d Same as ( b ) except for mice were immunized with SARS-CoV-2-RBD-AF488, polyI:C, and αCD40. e Same as in ( c ) except for SARS-CoV-2-RBD and CHIKV-E2 with polyI:C and αCD40. CHIKV-E2 was repeated for 9–14 days post-vaccine (~2 weeks). Statistical analysis was done using an unpaired t -test where the p -value between naïve and indicated antigen is <0.0001. In each experiment, at least n = 2–3 mice per group were evaluated and the experiment was repeated n = 2–5 times for c – e . Shown is the representative data from one of the experiments. Error bars are mean ± standard error of the mean. ns not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Ova (10 μg) was purchased from Sigma-Aldrich (Cat No. A5503) and Chikungunya virus envelope 2 protein (8 μg) (CHIKV-E2, strain SL-CK1) was purchased from Sino Biological (Cat. No. 40440-V08B).

Techniques: Staining, Expressing

Journal: Applied Microbiology and Biotechnology

Article Title: A broadly applicable split-luciferase biosensor approach for rapid antibody detection in emerging infectious diseases

doi: 10.1007/s00253-026-13712-5

Figure Lengend Snippet: Sensor pair selection for detection of SARS-CoV-2 and CHIKV antibodies. a Different sensor combinations were screened for their ability to reconstitute luciferase activity in the presence of anti-SARS-CoV-2 RBD antibodies. b Sensor pairs were similarly evaluated for luciferase complementation in the presence of anti-CHIKV E2 antibodies. Data represent means of duplicate tests; error bars indicate standard deviations

Article Snippet: The affinity-purified rabbit polyclonal antibodies against SARS-CoV-2 Spike RBD (K110843P, Solarbio, China) or against CHIKV E2 (40,440-T46; Sino Biological, China) (0.1 μL) were mixed with 40 ng of the LgBiT/C2 and SmBiT/C2 fusion sensors in dilution buffer (PBST containing 1% BSA); then, 100 μL of the mixture was added to plates and incubated for 30 min at 37 °C.

Techniques: Selection, Luciferase, Activity Assay

Journal: Applied Microbiology and Biotechnology

Article Title: A broadly applicable split-luciferase biosensor approach for rapid antibody detection in emerging infectious diseases

doi: 10.1007/s00253-026-13712-5

Figure Lengend Snippet: Analytical performance of the bioluminescent immunoassay for detection of anti-SARS-CoV-2 RBD (6.39 mg/mL) and anti-CHIKV E2 antibodies (1.0 mg/mL). Data represent means of duplicate tests; error bars indicate standard deviations

Article Snippet: The affinity-purified rabbit polyclonal antibodies against SARS-CoV-2 Spike RBD (K110843P, Solarbio, China) or against CHIKV E2 (40,440-T46; Sino Biological, China) (0.1 μL) were mixed with 40 ng of the LgBiT/C2 and SmBiT/C2 fusion sensors in dilution buffer (PBST containing 1% BSA); then, 100 μL of the mixture was added to plates and incubated for 30 min at 37 °C.

Techniques:

Journal: Molecular Therapy. Nucleic Acids

Article Title: A taRNA vaccine candidate induces a specific immune response that protects mice against Chikungunya virus infections

doi: 10.1016/j.omtn.2022.04.036

Figure Lengend Snippet: TR-RNAs are efficiently amplified by CHIKV replicase For the in vitro characterization of the taRNA vaccine combinations, HEK 293T cells were transfected with the replicase (nrRNA) or irrelevant RNA (TR-luc) together with the different TR-RNAs or infected with CHIKV (MOI 3). RNA levels were measured by qRT-PCR after 6 h and 16 h with specific primers directed against the E2 gene (A) or the capsid gene (B) and normalized to GAPDH. Numbers indicate the fold change in TR-RNA amount after 16 h mediated by the replicase expression. Black circles indicate nr-replicase-RNA-transfected cells and gray rectangles irrelevant RNA. Data are mean ± SEM of three independent experiments.

Article Snippet: After 24 h incubation at 37°C, cells were fixed, permeabilized with 0.5% Triton X-100 in PBS, and stained with primary antibodies directed against CHIKV E2 (Eurogentec, Cologne, Germany; custom made) or dsRNA (Scicons, Budapest, Hungary; no. 10010200).

Techniques: Amplification, In Vitro, Transfection, Infection, Quantitative RT-PCR, Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: A taRNA vaccine candidate induces a specific immune response that protects mice against Chikungunya virus infections

doi: 10.1016/j.omtn.2022.04.036

Figure Lengend Snippet: In vitro antigen expression of the taRNA constructs (A) E2 protein expression on the cell surface was determined 16 h after RNA transfection or CHIKV infection (MOI 3) by flow cytometric analysis. Data are mean ± SEM of three independent experiments. As a control, irrelevant RNA (TR-luc) instead of the replicase RNA (nrRNA) was transfected. The mean fluorescent intensity (MFI) indicates the amount of protein on the cell surface. (B) Expression of the indicated proteins was determined in cellular lysates of cells transfected with nr-replicase-RNA and TR-CS-RNA or CHIKV-infected cells (MOI 3) after 6 h and 24 h. (C) E2 and capsid protein expression after transfection of the nr-replicase-RNA with the TR-RNA combinations of either TR-CS, TR-S alone, or with both TR-S and TR-C was determined in cellular lysates or concentrated supernatants 48 h after transfection. The depicted western blots are representative of three independent experiments.

Article Snippet: After 24 h incubation at 37°C, cells were fixed, permeabilized with 0.5% Triton X-100 in PBS, and stained with primary antibodies directed against CHIKV E2 (Eurogentec, Cologne, Germany; custom made) or dsRNA (Scicons, Budapest, Hungary; no. 10010200).

Techniques: In Vitro, Expressing, Construct, Transfection, Infection, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Heterologous DNA Prime- Subunit Protein Boost with Chikungunya Virus E2 Induces Neutralizing Antibodies and Cellular-Mediated Immunity

doi: 10.3390/ijms241310517

Figure Lengend Snippet: Homologous DNA or E2* CHIKV protein prime-boost immunization induces robust humoral responses. C57BL/6 mice were either immunized intramuscularly twice with 100 μg of pVAX-E2 (non-targeted DNA vaccine), with scDEC-E2 (a DC-targeted DNA vaccine) followed by in vivo electroporation, or with 10 μg of E2* recombinant protein + poly (I:C) subcutaneously (immunization strategy displayed in ). The control group received empty pVAX vector and poly (I:C). Blood samples were collected 14 days after each immunization to evaluate the humoral immune response. ( a ) Total E2*-specific IgG titers. ( b ) E2*-Specific IgG subclasses after boost. ( c ) Antibody affinity of pooled mouse sera after incubation with increasing concentrations of ammonium thiocyanate. ( d ) For PRNT, pooled sera were incubated with 100 PFU of CHIKV and the NT 50 is displayed. Statistical analysis was performed by one-way ANOVA followed by Tukey post-hoc test. Data represent the mean ± SD and are representative from 3 independent experiments. (a,b) statistical significance when compared to the first dose. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: The E2 CHIKV ectodomain consensus protein sequence (aa 1 to 364) lacking the transmembrane region (aa 365 to 422) was generated after the alignment (ClustalW, MegAlign Pro V.17.4.2, DNAStar) of 79 sequences of the Brazilian virus isolates (Genbank Accession Numbers— ) and synthesized by GenScript (Piscataway, NJ, USA).

Techniques: In Vivo, Electroporation, Recombinant, Control, Plasmid Preparation, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Heterologous DNA Prime- Subunit Protein Boost with Chikungunya Virus E2 Induces Neutralizing Antibodies and Cellular-Mediated Immunity

doi: 10.3390/ijms241310517

Figure Lengend Snippet: Immunization with vaccines encoding E2 CHIKV induces robust humoral immune responses. C57BL/6 mice were either immunized intramuscularly twice with 100 μg of pVAX-E2 (non-targeted DNA vaccine), with 100 μg of a DC-targeted scDEC-E2 DNA vaccine followed by in vivo electroporation, or with 10 μg of E2* recombinant protein + poly (I:C) subcutaneously (immunization strategy displayed in ). For the heterologous DNA prime-protein boost, mice received one dose of a DNA vaccine (pVAX-E2 or scDEC-E2) followed by a boost with E2* recombinant protein + poly (I:C). Blood samples were collected 14 days after each immunization to evaluate the humoral immune response. ( a ) Total E2*-specific IgG titers. a- statistical analysis in comparison to the first dose. ( b ) E2*-specific IgG subclasses after the boost. ( c ) Antibody affinity of pooled mouse sera after incubation with increasing concentrations of ammonium thiocyanate. ( d ) For PRNT, pooled sera were incubated with 100 PFU of CHIKV and the NT 50 is displayed. Statistical analysis was performed with the one-way ANOVA followed by the Tukey post-hoc test. Data represent the mean ± SD and are representative of 2 independent experiments. ( a ) Statistical significance when compared to the first dose. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: The E2 CHIKV ectodomain consensus protein sequence (aa 1 to 364) lacking the transmembrane region (aa 365 to 422) was generated after the alignment (ClustalW, MegAlign Pro V.17.4.2, DNAStar) of 79 sequences of the Brazilian virus isolates (Genbank Accession Numbers— ) and synthesized by GenScript (Piscataway, NJ, USA).

Techniques: Vaccines, In Vivo, Electroporation, Recombinant, Comparison, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Heterologous DNA Prime- Subunit Protein Boost with Chikungunya Virus E2 Induces Neutralizing Antibodies and Cellular-Mediated Immunity

doi: 10.3390/ijms241310517

Figure Lengend Snippet: Immunization with vaccines encoding E2 CHIKV elicits robust T and B cell responses. C57BL/6 mice were immunized as described in (immunization strategy displayed in ). ( a ) Fifteen days after the boost, spleen cells were cultured in the presence of individual peptides from the E2* recombinant protein (10 μg/mL) to evaluate the number of IFN-γ-producing cells using the ELISpot assay. SFU: spot forming units. a, b, c, d represent statistical significance between the homologous and respective heterologous prime-boost strategies. #, €, &, $ represent the statistical significance between the pVAX-E2 and scDEC-E2 groups in the homologous immunization strategy. ( b ) Draining lymph node cells were cultured in the presence of E2* to evaluate the number of specific antibody-secreting cells (ASCs) by ELISpot. Statistical analysis was performed with the two-way ANOVA followed by Bonferroni’s post-hoc ( a ), or with the one-way ANOVA followed by the Tukey post-hoc test ( b ). Data represent the mean ± SD and are representative of 2 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: The E2 CHIKV ectodomain consensus protein sequence (aa 1 to 364) lacking the transmembrane region (aa 365 to 422) was generated after the alignment (ClustalW, MegAlign Pro V.17.4.2, DNAStar) of 79 sequences of the Brazilian virus isolates (Genbank Accession Numbers— ) and synthesized by GenScript (Piscataway, NJ, USA).

Techniques: Vaccines, Cell Culture, Recombinant, Enzyme-linked Immunospot

Journal: International Journal of Molecular Sciences

Article Title: Heterologous DNA Prime- Subunit Protein Boost with Chikungunya Virus E2 Induces Neutralizing Antibodies and Cellular-Mediated Immunity

doi: 10.3390/ijms241310517

Figure Lengend Snippet: Immunization in the presence of AS03 adjuvant elicits robust humoral responses with a strong neutralizing ability. C57BL/6 mice were immunized intramuscularly twice 15 days apart with 100 μg of the non-targeted pVAX-E2 DNA vaccine or the DC-targeted scDEC-E2 DNA vaccine followed by electroporation, or with 10 μg of E2* recombinant protein + AS03 subcutaneously (immunization strategy displayed in ). For the heterologous prime-boost, mice received one dose of a DNA vaccine (pVAX-E2 or scDEC-E2) followed by E2* recombinant protein + AS03. The control group received the empty pVAX vector and AS03. Blood samples were collected 14 days after each immunization to evaluate the antibody response. ( a ) Vero E6 cells were infected with CHIKV virus (MOI = 0.1) for 20 h, incubated with pooled sera, followed by donkey-anti mouse IgG-Alexa Fluor 488 and DAPI staining. ( b ) Total E2*-specific IgG titers. ( c ) E2*-specific IgG subclasses after the boost on a logarithm scale. ( d ) Antibody affinity from pooled sera after incubation with increasing concentrations of ammonium thiocyanate. ( e ) For PRNT, pooled sera were incubated with 100 PFU of CHIKV and the NT 50 is displayed. Statistical analysis was performed with the one-way ANOVA followed by the Tukey post-hoc test. Data represent the mean ± SD. a- statistical significance conducted when compared to the first dose. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: The E2 CHIKV ectodomain consensus protein sequence (aa 1 to 364) lacking the transmembrane region (aa 365 to 422) was generated after the alignment (ClustalW, MegAlign Pro V.17.4.2, DNAStar) of 79 sequences of the Brazilian virus isolates (Genbank Accession Numbers— ) and synthesized by GenScript (Piscataway, NJ, USA).

Techniques: Adjuvant, Electroporation, Recombinant, Control, Plasmid Preparation, Infection, Virus, Incubation, Staining

Journal: International Journal of Molecular Sciences

Article Title: Heterologous DNA Prime- Subunit Protein Boost with Chikungunya Virus E2 Induces Neutralizing Antibodies and Cellular-Mediated Immunity

doi: 10.3390/ijms241310517

Figure Lengend Snippet: Vaccines containing E2 CHIKV elicit cellular immune responses with a cytotoxic profile. C57BL/6 mice (n = 6) were immunized as described in (immunization strategy displayed in ). Fifteen days after the boost, mice were euthanized and spleen and draining lymph nodes were removed. ( a ) Specific IFN-γ production was examined with ELISpot against individual peptides. (a, b)) represents statistical significance between the homologous and heterologous strategies with the same vaccine used as a prime. #, €, & indicate the statistical significance between the pVAX-E2 and scDEC-E2 groups in the homologous regimen. ( b ) Draining lymph node cells were cultured in the presence of E2* to evaluate the number of specific antibody-secreting cells (ASCs) with ELISpot ( c ) In vivo cytotoxicity assay against target cells pulsed with the E2 355–364 peptide. Statistical analysis was performed with the one-way ANOVA followed by the Tukey post-hoc test. Data represent the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: The E2 CHIKV ectodomain consensus protein sequence (aa 1 to 364) lacking the transmembrane region (aa 365 to 422) was generated after the alignment (ClustalW, MegAlign Pro V.17.4.2, DNAStar) of 79 sequences of the Brazilian virus isolates (Genbank Accession Numbers— ) and synthesized by GenScript (Piscataway, NJ, USA).

Techniques: Vaccines, Enzyme-linked Immunospot, Cell Culture, In Vivo, Cytotoxicity Assay