Fig. 1: Enhanced durable humoral and cellular immune responses in MnARK-based subunit vaccine.
a Schematic diagram of immunization and sample collection. BALB/c mice were immunized with 10 μg RBD dimer with MnARK (52 μg Mn) or alum (Alhydrogel adjuvant 2%, Croda, 52 μg Al) on days 0, 21, and 42. Sera were collected for up to 360 days. b anti-RBD IgG titers were measured by ELISA. Data are shown as the means ± SD (n = 5), and individual data are presented. c Sera collected at 56 days and 360 days post the first immunization were tested for neutralizing live SARS-CoV-2 (prototype strain). The NT50 was calculated as the reciprocal of serum dilution required for 50% neutralization of viral infection (n = 5). The values are the geometric mean titer (GMT). d The germinal center (GC) B cell percentages (n = 5) on day 56. e Representative immunochemistry staining of GCs in lymph nodes from different vaccinated groups on day 90 post-immunization. Anti-B220 (yellow), anti-IgD (purple), anti-Ki67 (red), anti-CD3 (green) and DAPI (blue) are presented. Bar = 200 μm. f The percentage of memory B cells and central memory (TCM) CD4+ and CD8 T+ cells in spleens (n = 5) on day 90. g Ex vivo images of lymph nodes at 12 h and 24 h post-injection (n = 5). Cy5-labeled RBD dimer or vaccines was intramuscularly injected into the right leg of mice. IN inguinal nodes; AN axillary nodes. h Quantification of the total fluorescence intensity (n = 5). i Quantify the percentage of dimer positive cells in lymph node samples (n = 3). j Cellular distribution of dimer uptake (measured by flow cytometry) among alum and MnARK group (n = 3). k CyTOF analysis of immune responses in muscles and lymph nodes of mice after i.m. injection with MnARK. Single cells obtained from muscles and lymph nodes were stained by antibody cocktails and analyzed by mass cytometry. l The t-SNE plot visualization of CD45+ cells pooled from 10,000 cellular events in muscles. m Number of CD45+ cell subsets in samples of muscles. The cell number of each population was calculated by multiplying the total number of isolated immune cells by the percentage of the population. n Normalized expression of the major histocompatibility complex class II (MHC II) marker on the t-SNE map. o The t-SNE plot visualization of CD45+ cells pooled from 10,000 cellular events in lymph nodes. p, q Number of CD45+ cell subsets (p myeloid cells, q T and B cells) in the lymph nodes sample. All the data were analyzed with a two-way ANOVA test and are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001
