Immunogenicity and innate immunity to high-dose and repeated vaccination of modified mRNA versus unmodified mRNA

mRNA vaccines represent a new era with several novel constructs underway. We compared the responses of high doses and multiple repetitive immunizations of a nucleoside-modified mRNA construct to a sequence-codon-optimized unmodified mRNA construct encoding the identical model antigen (HIV-1 gag). Rhesus macaques were immunized five times at 2-week intervals, with a final boost 20 weeks later. At 24 h post-vaccination, both unmodified (160 μg) and modified (400 μg and 800 μg) mRNA constructs elicited clear but transient increase of plasmacytoid dendritic cells, intermediate CD14+ CD16+ monocytes, and neutrophils along with secretion of type I interferon (IFN)-related and inflammatory cytokines. Unmodified mRNA induced higher interleukin-7 (IL-7) and IFN-α levels, whereas modified mRNA induced higher IL-6 levels. Transcriptomic profiling showed significant upregulation of genes related to type I IFN signaling, antigen presentation, and innate immune activation induced by both mRNA constructs. The high-dose modified mRNA induced a higher number of differentially expressed genes at prime, which further increased after the fifth immunization. These differences in innate immune activation nonetheless led to similar levels and kinetics of gag-specific antibody and T cell responses. These findings offer insights into the immunogenic and reactogenic potential of different mRNA vaccine modalities, guiding future vaccine and therapy development.