Journal: Frontiers in Immunology

Article Title: Design and immunogenicity of a quadrivalent mRNA vaccine targeting HSV-2 with comparative evaluation of co-formulated and admixed formulations

doi: 10.3389/fimmu.2025.1712691

Figure Lengend Snippet: Characterization of the immunogenicity of gB2, gC2, gD2, and gE2 mRNAs. Mice were immunized twice intramuscularly with PBS or 10 μg of gB2, gC2, gD2, or gE2 mRNA. (A) Immunization groups and schedule. (B) Serum total IgG, IgG1, and IgG2a were measured using ELISA. (C) PRNT 50 neutralizing antibody titers were measured using PRNT. (D) An IL-4 ELISPOT assay was performed on splenocytes. (E, F) CD4 + T cells producing IFN-γ or TNF-α (E) and CD8 + T cells producing IFN-γ, TNF-α, or Granzyme B (F) in splenocytes were analyzed using ICS/flow cytometry. Splenocytes were stimulated with the peptide pools of gB2, gC2, gD2, or the gE2 peptides prior to the ELISPOT and ICS/flow cytometry assays. Experiments involved five mice per group (n = 5/group). P- values were calculated using the Kruskal-Wallis test with Dunn’s multiple comparisons test (B, C) and two-way ANOVA with Tukey’s multiple comparisons test (D–F) . * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Error bars represent 95% confidence intervals of the geometric means (B, C) and standard deviations of the means (D–F) . GMT, geometric mean titer; ELISA, enzyme-linked immunosorbent assay; PRNT, plaque reduction neutralization test; ELISPOT, enzyme-linked immunospot; ICS, intracellular cytokine staining.

Article Snippet: Colorimetric detection was performed by incubating for 1 h with horseradish peroxidase (HRP)-conjugated antibodies: anti-mouse IgG (Bethyl Laboratories, Montgomery, TX, USA) at a 1:3,000 dilution, IgG1 (Bethyl Laboratories) at a 1:3,000 dilution, or IgG2a (Bio-Rad, Hercules, CA, USA) at a 1:2,000 dilution, followed by incubation with 3,3′,5,5′-tetramethylbenzidine (TMB) substrates.

Techniques: Immunopeptidomics, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Flow Cytometry, Plaque Reduction Neutralization Test, Staining

Journal: Frontiers in Immunology

Article Title: Design and immunogenicity of a quadrivalent mRNA vaccine targeting HSV-2 with comparative evaluation of co-formulated and admixed formulations

doi: 10.3389/fimmu.2025.1712691

Figure Lengend Snippet: Comparison of IgG and neutralizing antibody responses between co-formulated and admixed quadrivalent mRNA vaccines. Mice were immunized twice intramuscularly with PBS, co-formulated quadrivalent mRNA vaccine, or admixed quadrivalent mRNA vaccine 4 weeks prior to HSV-2 challenge. 10 μg of mRNA per antigen was used for each group. (A) Schematic representation of the mRNA–LNP formulation. The quadrivalent vaccine, consisting of gB2, gC2, gD2, and gE2 mRNAs, was either co-formulated into a single LNP or formulated for each antigen and then admixed. (B) Immunization groups and schedule. (C, D) Serum IgG titers (C) and PRNT 50 neutralizing antibody titers (D) were measured using ELISA and PRNT, respectively, on weeks 1 and 2 post-second immunization. Sera for the PRNT were pooled and tested in triplicate. For the co-formulated group (1 wpi), data were analyzed in duplicate; one replicate was excluded due to edge-localized plaque formation. Experiments involved seven mice per group (n = 7/group). P- values were calculated using the Kruskal-Wallis test with Dunn’s multiple comparisons test. * p < 0.05, ** p < 0.01, ns, not significant. Error bars represent 95% confidence intervals of the geometric means. LNP, lipid nanoparticle; GMT, geometric mean titer; wpi, weeks post-second immunization.

Article Snippet: Colorimetric detection was performed by incubating for 1 h with horseradish peroxidase (HRP)-conjugated antibodies: anti-mouse IgG (Bethyl Laboratories, Montgomery, TX, USA) at a 1:3,000 dilution, IgG1 (Bethyl Laboratories) at a 1:3,000 dilution, or IgG2a (Bio-Rad, Hercules, CA, USA) at a 1:2,000 dilution, followed by incubation with 3,3′,5,5′-tetramethylbenzidine (TMB) substrates.

Techniques: Comparison, Vaccines, Formulation, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Design and immunogenicity of a quadrivalent mRNA vaccine targeting HSV-2 with comparative evaluation of co-formulated and admixed formulations

doi: 10.3389/fimmu.2025.1712691

Figure Lengend Snippet: Immunogenicity based on the dose of quadrivalent mRNA vaccine. Mice were immunized twice intramuscularly with PBS or 1 μg, 5 μg, or 10 μg of co-formulated quadrivalent mRNA vaccine consisting of gB2, gC2, gD2, and gE2 mRNA. The indicated dose refers to the amount of mRNA produced per antigen. (A) Immunization groups and schedule. (B, C) Serum IgG titers (B) and PRNT 50 neutralizing antibody titers (C) were measured using ELISA and PRNT, respectively. IgG titers: 1 μg dose vs . PBS ( p = 0.1263); 5 μg dose vs . PBS ( p = 0.0153); 10 μg dose vs . PBS ( p = 0.0023). PRNT 50 titers: 1 μg dose vs . PBS ( p > 0.9999); 5 μg dose vs . PBS ( p = 0.0574); 10 μg dose vs . PBS ( p = 0.0006). (D) An IFN-γ ELISPOT assay was performed on splenocytes. (E, F) CD4 + T cells producing IFN-γ or TNF-α (E) and CD8 + T cells producing IFN-γ, TNF-α, or Granzyme B (F) in splenocytes were analyzed using ICS/flow cytometry. Splenocytes were stimulated with the gD2 peptide pool or the gE2 peptides before the ELISPOT and ICS/flow cytometry assays. Experiments involved five mice per group (n = 5/group). P- values were calculated using the Kruskal-Wallis test with Dunn’s multiple comparisons test (B, C) and two-way ANOVA with Tukey’s multiple comparisons test (D–F) . * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, not significant. Error bars represent 95% confidence intervals of the geometric means (B, C) and standard deviations of the means (D–F) . GMT, geometric mean titer.

Article Snippet: Colorimetric detection was performed by incubating for 1 h with horseradish peroxidase (HRP)-conjugated antibodies: anti-mouse IgG (Bethyl Laboratories, Montgomery, TX, USA) at a 1:3,000 dilution, IgG1 (Bethyl Laboratories) at a 1:3,000 dilution, or IgG2a (Bio-Rad, Hercules, CA, USA) at a 1:2,000 dilution, followed by incubation with 3,3′,5,5′-tetramethylbenzidine (TMB) substrates.

Techniques: Immunopeptidomics, Produced, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Design and immunogenicity of a quadrivalent mRNA vaccine targeting HSV-2 with comparative evaluation of co-formulated and admixed formulations

doi: 10.3389/fimmu.2025.1712691

Figure Lengend Snippet: Evaluation of long-term immune responses and protective efficacy of quadrivalent mRNA vaccine. Mice were intravaginally challenged with 1×10 5 PFU of HSV-2 strain MS 16 weeks after two intramuscular immunizations with PBS or co-formulated quadrivalent mRNA vaccine (10 μg of each gB2, gC2, gD2, and gE2 mRNA). (A) Immunization groups and experimental design for the viral challenge. (B, C) Serum IgG titers (B) and PRNT 50 neutralizing antibody titers (C) at weeks 4 and 15 post-second immunization. (D) IFN-γ- or TNF-α-producing splenic CD4 + and CD8 + T cells were assessed after peptide stimulation at the study endpoint. (E–H) Survival (E) and weight loss (F) were monitored daily, and genital disease (G) and clinical signs (H) were scored every alternate day for 36 days. (I) HSV-2 titers on days 2 and 4 post-infection. (J, K) DRG and vaginal tissue were harvested at death or study endpoint. HSV-2 DNA copies in the DRG (J) and H&E-stained vaginal sections showing epithelial degeneration and immune cell infiltration (K) . Experiments involved six to seven mice per group (G1, n = 6; G2, n = 7). Unvaccinated and uninfected mice served as controls for baseline T cell levels and normal vaginal tissues (G3, n = 6). P- values were calculated using the two-tailed Mann-Whitney U test (B, C, I, J) , two-tailed Wilcoxon signed-rank test for within-group comparison (B, C) , two-way ANOVA with Sidak’s multiple comparisons test (D) , and one-way ANOVA with Tukey’s multiple comparisons test (K) . *** p < 0.001, **** p < 0.0001, ns, not significant. Error bars represent 95% confidence intervals of the geometric means (B, C, I, J) and standard deviations of the means (D, F–H, K) . GMT, geometric mean titer; wpi, weeks post-second immunization.

Article Snippet: Colorimetric detection was performed by incubating for 1 h with horseradish peroxidase (HRP)-conjugated antibodies: anti-mouse IgG (Bethyl Laboratories, Montgomery, TX, USA) at a 1:3,000 dilution, IgG1 (Bethyl Laboratories) at a 1:3,000 dilution, or IgG2a (Bio-Rad, Hercules, CA, USA) at a 1:2,000 dilution, followed by incubation with 3,3′,5,5′-tetramethylbenzidine (TMB) substrates.

Techniques: Infection, Staining, Two Tailed Test, MANN-WHITNEY, Comparison

Journal: The Veterinary Quarterly

Article Title: Evaluation of the safety and immunogenicity of a peptide vaccine against canine leishmaniosis: a double-blind, multicenter, controlled clinical trial in dogs

doi: 10.1080/01652176.2025.2591396

Figure Lengend Snippet: Serum IgG subtypes response to recombinant vaccine antigens in vaccinated dogs. (A) Shows IgG2a and (B) shows IgG1 titers in response to the recombinant vaccine proteins HisDTC and protein Q, as detected by ELISA at multiple time points post-vaccination. Data are presented as median ± interquartile range. Statistical significance is denoted by asterisks (** p < 0.01) compared with the His-PLGA group.

Article Snippet: HRP-conjugated anti-dog IgG2a (sheep) and IgG1 (goat) (Bethyl Laboratories, Montgomery, TX, USA), diluted 1:45000, were used for detection.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Infection and Immunity

Article Title: Rickettsia rickettsii inactivated whole cell antigen vaccine protects against Rocky Mountain spotted fever independent of the adjuvant used

doi: 10.1128/iai.00412-25

Figure Lengend Snippet: Development of antibodies by vaccinated dogs. Plasma samples collected from all dogs several days following vaccinations and infection challenges were assessed by ELISA for the presence of R. rickettsii -specific IgG ( A ), IgG1 ( B ), and IgG2 ( C ). All dogs receiving WCAV developed antigen-specific IgG responses following the primary vaccination, which increased following the booster vaccination. R. rickettsii -specific total IgG and IgG subclass; IgG1 and IgG2 in different groups of vaccinated dogs were compared by two-way ANOVA test followed by multiplicity-adjusted post hoc comparisons (Tukey). P -value < 0.05 was considered statistically significant, which was observed for total IgG and IgG 2 for Quil A compared to Montanide and Alhydrogel from day 37 onward with P -values of ≤0.0001. Vertical dotted blue lines refer to the days of booster vaccination.

Article Snippet: One hundred microliters of a 1:50,000 dilution of anti-canine total IgG (PA1-29738, ThermoFisher, USA), 1:10,000 anti IgG1 (AHP947P, Bio-Rad, Hercules, CA, USA), or 1:5,000 of anti IgG2 (AHP948P, Bio-Rad, Hercules, CA, USA) peroxidase-labeled antibodies was added to each well, and plates were incubated for 45 min at 37°C.

Techniques: Clinical Proteomics, Infection, Enzyme-linked Immunosorbent Assay