Journal: bioRxiv

Article Title: A modular mRNA–LNP vaccine platform enables integrated RNA, lipid and antigen design to protect against CCHFV

doi: 10.64898/2026.01.17.699915

Figure Lengend Snippet: a, Immunization and sampling scheme. Female C57BL/6J mice (8–10 weeks old; n = 5 per group) were immunized intramuscularly with 10 μg of ARCA-capped nucleocapsid (NP) mRNA–LNP on days 0 and 14 using three UTR configurations (CCHFV-UTR–NP, T-UTR–NP, or ACTA-1–UTR–NP). Negative-control animals received 1× PBS intramuscularly. On day 28, animals were euthanized and blood and spleens were collected for serology (ELISA) and cellular immunogenicity assays (ELISPOT and intracellular cytokine staining). b, Antigen-specific cytokine secretion by ELISPOT. Splenocytes harvested on day 28 were stimulated for 24 h with recombinant CCHFV NP (10 μg ml⁻¹) and analyzed for IFN-γ and IL-2 spot-forming cells (SFCs; normalized per 1.5 × 10⁶ input cells). Representative ELISPOT wells are shown adjacent to quantification. All NP mRNA–LNP vaccines induced significantly higher IL-2 secretion than stimulated negative controls. Constructs containing CCHFV and ACTA-1 UTRs elicited significantly greater IL-2 responses than the T-UTR construct and negative controls, whereas IFN-γ responses were also significantly increased relative to controls, with UTR-dependent differences indicated by the displayed P values. c, CD8⁺ T cell cytokine production. Day-28 splenocytes were stimulated for 5 h with recombinant NP (10 μg ml⁻¹) and analyzed by intracellular cytokine staining (ICS) to quantify frequencies of IFN-γ⁺, IL-2⁺, and TNF-α⁺ CD8⁺ T cells. The T-UTR construct increased CD8⁺ IFN-γ, IL-2, and TNF-α relative to negative controls, although the three vaccine candidates did not differ significantly from one another across these CD8⁺ readouts (as indicated by the annotated P values). d, CD4⁺ T cell cytokine polarization. Day-28 splenocytes were stimulated for 5 h with recombinant NP (10 μg ml⁻¹) and analyzed by ICS to quantify IFN-γ⁺, IL-2⁺, IL-4⁺, TNF-α⁺, and IL-17A⁺ CD4⁺ T cells. The ACTA-1–UTR construct induced markedly higher frequencies of all five cytokines compared with the CCHFV-UTR and T-UTR constructs and the negative-control group, indicating broadened helper polarization. CCHFV-UTR– and T-UTR–based constructs produced comparable but lower cytokine frequencies in CD4⁺ T cells. Bars represent group means with individual animals overlaid as points; error bars denote variability within groups. Statistical analyses were performed in GraphPad. Between-group comparisons used one-way ANOVA with Tukey’s multiple comparisons test; when normality assumptions were not met, the Kruskal–Wallis test was used. A significance threshold of P < 0.05 was applied unless otherwise indicated, and exact P values are shown in the figure.

Article Snippet: Plates were washed once with 0.1% Tween-20 in PBS and blocked with 200 μL ChonBlock ELISA Buffer (Chondrex, Cat #9068) for 2 hours at room temperature.

Techniques: Sampling, Negative Control, Enzyme-linked Immunosorbent Assay, Immunopeptidomics, Enzyme-linked Immunospot, Staining, Recombinant, Vaccines, Construct, Produced

Journal: bioRxiv

Article Title: A modular mRNA–LNP vaccine platform enables integrated RNA, lipid and antigen design to protect against CCHFV

doi: 10.64898/2026.01.17.699915

Figure Lengend Snippet: a, The immunization schedule and experimental workflow are shown for AG-capped mRNA–LNP formulations encoding nucleocapsid (NP), mutant nucleocapsid (NPmut), sGCs, and sGCs (GP38Δglyc). Female C57BL/6J mice (8–10 weeks old) were immunized intramuscularly with 10 µg of each mRNA–LNP vaccine on days 0 and 14. Blood, spleens, and inguinal lymph nodes were collected on day 28 for downstream immunological analyses, including ELISA, germinal center (GC) B and T follicular helper (Tfh) cell profiling, and ELISPOT assays. b, Splenocytes were stimulated for 24 hours with 10 µg/mL of recombinant nucleocapsid or GP38 proteins and analyzed by ELISPOT to quantify IFN-γ and IL-4 secretion. The NP and NPmut groups exhibited significantly higher IFN-γ–secreting cell frequencies compared with sGCs, sGCs (GP38Δglyc), and negative control groups, whereas sGCs and sGCs (GP38Δglyc) formulations elicited stronger IL-4 responses than NP, NPmut, and controls. c, GC B and Tfh cell populations were quantified in lymph nodes collected on day 28. The NPmut group displayed a significantly increased percentage of GC B and Tfh cells relative to negative controls, while the NP group showed a higher frequency of GC B cells compared with controls. Antigen-specific GC B cells were elevated across all vaccine groups relative to controls, with no significant differences among vaccine formulations. d, Antibody levels measured by in-house ELISA on day 28 showed that two out of eight mice in the NPmut group had detectable antigen-specific antibodies, while all samples in the sGCs and sGCs (GP38Δglyc) groups exhibited significantly higher antibody titers compared with negative controls. No significant differences were observed between sGCs and sGCs (GP38Δglyc) groups. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc multiple comparisons test. Significant differences are shown as p -values.

Article Snippet: Plates were washed once with 0.1% Tween-20 in PBS and blocked with 200 μL ChonBlock ELISA Buffer (Chondrex, Cat #9068) for 2 hours at room temperature.

Techniques: Mutagenesis, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Recombinant, Negative Control

Journal: bioRxiv

Article Title: A modular mRNA–LNP vaccine platform enables integrated RNA, lipid and antigen design to protect against CCHFV

doi: 10.64898/2026.01.17.699915

Figure Lengend Snippet: a, Female C57BL/6J mice were immunized intramuscularly with Cleancap M6 mRNA–LNP formulations encoding nucleocapsid (NP), mutant nucleocapsid (NPmut), sGCs, or sGCs (GP38Δglyc). Blood, spleens, and lymph nodes were collected on day 35 for downstream analyses, including ELISA, dendritic cell profiling, and ELISPOT assays. b, Splenocytes were stimulated for 24 hours with 10 µg/mL of nucleocapsid or GP38 proteins and analyzed by ELISPOT for IFN-γ and IL-4 secretion. NP and NPmut groups induced significantly higher IFN-γ responses compared with sGCs, sGCs (GP38Δglyc), and negative control, whereas sGCs and sGCs (GP38Δglyc) showed no significant IFN-γ response. None of the vaccine groups elicited significant IL-4 production. c, Splenocytes were stimulated ex vivo for 24 hours with 10 µg/mL of nucleocapsid or GP38 proteins and analyzed for intracellular cytokines IFN-γ, IL-2, and TNF-α in CD8⁺ and CD4⁺ T cells. All vaccine groups elicited robust CD4⁺ and CD8⁺ T cell responses compared with negative controls, except for sGCs (GP38Δglyc). NP vaccination induced significantly higher cytokine production in CD8⁺ T cells relative to control, whereas sGCs (GP38Δglyc) uniquely enhanced IL-2 and TNF-α in CD4⁺ T cells. No significant differences were observed among vaccine groups. d, Lymph nodes were analyzed for plasmacytoid dendritic cells (pDCs) and conventional CD11c⁺ DCs (cDC1). Only sGCs and NP vaccine groups demonstrated significantly increased pDC and cDC1 frequencies, respectively, relative to controls. e, Dots represent the distribution of individual serum samples at each dilution. Both immunogens elicited robust neutralizing activity at low serum dilutions (1:100–1:200). At higher dilutions (1:1600–1:6400), sera from mice immunized with MLD-GP38Δglyc exhibited significantly greater neutralization than the wild-type MLD-GP38 group, indicating that removal of GP38 glycans enhances the potency and/or durability of the neutralizing antibody response. Negative control sera (blue) showed minimal inhibition across all dilutions. f, ELISA revealed that 3 out of 6 mice in the NPmut group were positive, while no NP-vaccinated mice showed detectable antibody. Both sGCs and sGCs (GP38Δglyc) groups exhibited significantly higher antibody responses than negative controls, with no differences between these two groups. g and h, The immunization/grouping scheme in IS C57BL/6 mice. i, Weight, clinical score and survival of C57BL/6J mice vaccinated with CCHFV based mRNAs 35 days prior to challenge and provided a boost dose at 21 days prior to challenge (n = 10 per group). On the day of challenge, mice were transiently immunosuppressed with anti-mouse IFNAR-1 mAb IP and subsequently inoculated with CCHFV strain IbAr10200 SC. Weights depicted as percent change from baseline at D0. Clinical scores are shown as individual daily scores (0–10; increasing intensity of red indicates higher scores, and gray boxes denote end of monitoring due to euthanasia or reaching a lethal endpoint; top) or as mean scores for each experimental group (bottom).

Article Snippet: Plates were washed once with 0.1% Tween-20 in PBS and blocked with 200 μL ChonBlock ELISA Buffer (Chondrex, Cat #9068) for 2 hours at room temperature.

Techniques: Mutagenesis, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Negative Control, Ex Vivo, Control, Activity Assay, Neutralization, Inhibition