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Abstract Adjuvants play a central role in enhancing the immunogenicity of otherwise poorly immunogenic vaccine antigens. Combining adjuvants has the potential to enhance vaccine immunogenicity compared with single adjuvants, although the cellular and molecular mechanisms of combination adjuvants are not well understood. Using the influenza virus hemagglutinin H5 antigen, we define the immunological landscape of combining CpG and MPLA (TLR-9 and TLR-4 agonists, respectively) with a squalene nanoemulsion (AddaVax) using immunologic and transcriptomic profiling. Mice immunized and boosted with recombinant H5 in AddaVax, CpG+MPLA, or AddaVax plus CpG+MPLA (IVAX-1) produced comparable levels of neutralizing antibodies and were equally well protected against the H5N1 challenge. However, after challenge with H5N1 virus, H5/IVAX-1–immunized mice had 100- to 300-fold lower virus lung titers than mice receiving H5 in AddaVax or CpG+MPLA separately. Consistent with enhanced viral clearance, unsupervised expression analysis of draining lymph node cells revealed the combination adjuvant IVAX-1 significantly downregulated immune homeostasis genes, and induced higher numbers of antibody-producing plasmablasts than either AddaVax or CpG+MPLA. IVAX-1 was also more effective after single-dose administration than either AddaVax or CpG+MPLA. These data reveal a novel molecular framework for understanding the mechanisms of combination adjuvants, such as IVAX-1, and highlight their potential for the development of more effective vaccines against respiratory viruses. 

Macrophages are essential for skeletal muscle homeostasis, but how their dysregulation contributes to the development of fibrosis in muscle disease remains unclear. Here, we used single-cell transcriptomics to determine the molecular attributes of dystrophic and healthy muscle macrophages. We identified six clusters and unexpectedly found that none corresponded to traditional definitions of M1 or M2 macrophages. Rather, the predominant macrophage signature in dystrophic muscle was characterized by high expression of fibrotic factors, galectin-3 (gal-3) and osteopontin (Spp1). Spatial transcriptomics, computational inferences of intercellular communication, and in vitro assays indicated that macrophage-derived Spp1 regulates stromal progenitor differentiation. Gal-3⁺macrophages were chronically activated in dystrophic muscle, and adoptive transfer assays showed that the gal-3⁺phenotype was the dominant molecular program induced within the dystrophic milieu. Gal-3⁺macrophages were also elevated in multiple human myopathies. These studies advance our understanding of macrophages in muscular dystrophy by defining their transcriptional programs and revealSpp1as a major regulator of macrophage and stromal progenitor interactions.