Journal: NPJ Vaccines
Article Title: Preserved efficacy of lyophilized SARS-CoV-2 mRNA vaccine incorporating novel ionizable lipids after one year at 25 °C
doi: 10.1038/s41541-025-01201-1
Figure Lengend Snippet: a Immunization scheme and sample collection schedule. Mice ( n = 5) were immunized intramuscularly at day 0 (prime) and 21(boost) with 1 µg of mRNA/animal. Blood samples were obtained at week 3 (prior to boost) and 6, and specific antibody levels were determined in serum. T-cell response was evaluated in splenocytes 3 weeks post-boost. b Reciprocal endpoint titers of antigen-specific IgG antibodies in serum samples determined by ELISA using RBD recombinant protein from wild-type variant. c Neutralization titers in serum samples were determined by neutralization assay using Spike-pseudotyped lentivirus and infection in HEK293T-ACE2-TMPRSS2 cells. NT50 titers refer to the dilution of a serum sample at which 50% of the pseudovirus infection is inhibited. d IFN-γ-secreting splenocytes quantification by ELISPOT after O.N. stimulation with SARS-CoV-2 Spike peptide pool. e IFN-γ quantification in supernatants of splenocytes after overnight stimulation with SARS-CoV-2 Spike peptide pool determined by ELISA. f T-lymphocyte CD4 and CD8 cell frequencies in spleen analyzed by flow cytometry. g Tfh and GC B cells analysis. Mice ( n = 5) were immunized intramuscularly with 5 µg of mRNA/animal. Inguinal lymph nodes were extracted on day 7. h Tfh (CD45R - , CD4 + , CD44hi, CXCR5 + , PD-1 + ) and GC B cell (CD19 + , CD95 + , GL7 + ) frequencies were determined by flow cytometry analysis. Graphs are represented as mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 as determined by two-way ANOVA ( a ) and one-way ANOVA ( c – h ) with Tukey post-test.
Article Snippet: Tfh cells were defined as CD45R - CD4 + , CD44 high , CXCR5 + , PD-1 + , and the following antibodies were used for surface staining: CD45R (B220)-APC-Vio770 (Miltenyi, 130-110-849), CD4-VioBright FITC (Miltenyi, 130-118-692), CD44-PerCP-Vio700 (Miltenyi, 130-128-625), CD185 (CXCR5)-APC (Miltenyi, 130-119-129), and CD279 (PD1)-PE (Miltenyi, 130-111-953).
Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Variant Assay, Neutralization, Infection, Enzyme-linked Immunospot, Flow Cytometry
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