Extended Data Fig. 4: Immunogenicity of MICA/B α3 domain vaccine in non-human primates.
From: A vaccine targeting resistant tumours by dual T cell plus NK cell attack
a, Characterization of the MICA/B α3 immunogen used in the primate study. The α3 domains of rhesus macaque MICA and MICB were expressed as a fusion protein with ferritin to generate nanoparticles that displayed both α3 domains on the surface. Nanoparticles formed by this fusion protein were conjugated using click chemistry to CpG ODN 2935 as the adjuvant and characterized by HPLC gel filtration chromatography. Shown are HPLC traces of the protein following conjugation to the CpG oligonucleotide (red: 280 nm trace for detection of protein; blue: 350 nm trace for detection of bis-aryl hydrazone bond). b, SDS-PAGE analysis of purified macaque MICA/B α3 – ferritin protein under reducing (+) and non-reducing (−) conditions following CpG conjugation and final purification using a HPLC gel filtration column. c–e, Characterization of serum antibody responses to rhesus macaque MICA (left) and MICB (right) proteins (full-length extracellular domains without ferritin fusion partner) at different steps in the immunization process. Antibody responses were investigated in three animals (RBQ12, RVf10 and RQq15) at multiple timepoints (pre-immunization; three weeks following initial immunization and boosts 1-3, as illustrated in Fig. 1h) using a fluorescence-based ELISA (RFU, relative fluorescence units) at multiple serum dilutions (1:102 to 1:104). f–m, Binding of purified polyclonal serum IgG to cell surface MICA (left) and MICB (right) using HEK293T transfectants that displayed rhesus macaque MICA (Mamu-A*01) or MICB (Mamu-B*01) proteins. Pre-immune sera were used as a control (grey) for sera obtained following immunization (red). Representative histograms (left in f, g) and graphical summaries of flow cytometry data (right in f, g, h–m) are shown for the four immunized macaques (9312, RBQ12, RVf10 and RQq15). Representative data from two experiments (a–b). Data from a single macaque immunization experiment with technical replicates for each macaque analyzed (c–m). Two-way ANOVA with Tukey’s multiple comparison test (c-e). Data depict mean+/− SD.
