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: Yonsei Medical Journal

Article Title: Expression and Evaluation of Chikungunya Virus E1 and E2 Envelope Proteins for Serodiagnosis of Chikungunya Virus Infection

doi: 10.3349/ymj.2008.49.5.828

Figure Lengend Snippet: The seroreactivity of the recombinant CHIKV E1 and E2 envelope proteins using indirect IgM ELISA. Forty anti-CHIKV positive serum samples were used to evaluate the seroreactivity of the recombinant CHIKV E1 (•) and E2 (•) envelope proteins. The absorbance was read at 450 nm. IgM capture ELISA data (○) were supplied from Laboratoire Marcel Merieux. CHIKV, chikungunya virus.

Article Snippet: The absorbance values of anti-CHIKV positive serum samples to CHIKV E1 and E2 envelope proteins appear to be higher than the absorbance values of anti-CHIKV positive serum samples from Laboratoire Marcel Merieux (Lyon, France), even though the direct comparison of these data is not possible because different ELISA methods were used.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Virus

Journal: Yonsei Medical Journal

Article Title: Expression and Evaluation of Chikungunya Virus E1 and E2 Envelope Proteins for Serodiagnosis of Chikungunya Virus Infection

doi: 10.3349/ymj.2008.49.5.828

Figure Lengend Snippet: Analysis of expression of CHIKV E1 (A) and E2 (B) envelope protein in Sf9 cells. Sf9 cell lysates were analysed by SDS-PAGE, and the gel was stained with Coomassie blue (lanes 1 to 3) or analysed by Western blot with pooled anti-CHIKV positive serum (lane 4). M; Prestained protein marker, lane 1; lysate of Sf9 cells infected with mock-vector, lane 2; lysate of Sf9 cells infected with recombinant virus containing CHIKV E1 (A) or E2 (B) envelope protein gene, lane 3; purified recombinant CHIKV E1 (A) or E2 (B) envelope protein, lane 4; Western blot analysis with pooled anti-CHIKV positive serum. CHIKV, chikungunya virus.

Article Snippet: The absorbance values of anti-CHIKV positive serum samples to CHIKV E1 and E2 envelope proteins appear to be higher than the absorbance values of anti-CHIKV positive serum samples from Laboratoire Marcel Merieux (Lyon, France), even though the direct comparison of these data is not possible because different ELISA methods were used.

Techniques: Expressing, SDS Page, Staining, Western Blot, Marker, Infection, Plasmid Preparation, Recombinant, Virus, Purification

Journal: Yonsei Medical Journal

Article Title: Expression and Evaluation of Chikungunya Virus E1 and E2 Envelope Proteins for Serodiagnosis of Chikungunya Virus Infection

doi: 10.3349/ymj.2008.49.5.828

Figure Lengend Snippet: Detection of anti-CHIKV IgM antibodies by recombinant CHIKV E1 (A) and E2 (B) envelope protein-based ELISA. Each well of a microtiter plate was coated with various amounts of recombinant CHIKV E1 and E2 envelope protein and evaluated for their reactivity with pooled anti-CHIKV positive or negative serum. Absorbance was read at 450 nm. The P/N ratio is A450 of positive serum/A450 of negative serum. CHIKV, chikungunya virus.

Article Snippet: The absorbance values of anti-CHIKV positive serum samples to CHIKV E1 and E2 envelope proteins appear to be higher than the absorbance values of anti-CHIKV positive serum samples from Laboratoire Marcel Merieux (Lyon, France), even though the direct comparison of these data is not possible because different ELISA methods were used.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Virus

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