Cell painting with amphiphile-protein conjugate vaccines drives B cell activation and germinal center priming to enhance humoral immunity

Most licensed vaccines against infectious diseases mediate protection by priming B cells and humoral immune responses, where efficacy largely relies on activation of germinal centers (GCs) in lymphoid tissues that serve as the training ground for a high-affinity antibody response. While subunit vaccines offer safety advantages, they tend to be poorly immunogenic with weak and short-lived antibody responses, presenting a need for engineering strategies that enhance immunogenicity. Amphiphile-protein vaccines, consisting of protein antigens modified with an albumin-binding lipid tail via a polyethylene glycol (PEG) linker, hitchhike on albumin following subcutaneous injection to enhance lymphatic trafficking and antigen-specific immune activation. Amph-vaccines also demonstrate an ability to insert their lipid tail into cell membranes, effectively 'painting' cells with multivalent antigen. We hypothesized that cell painting may play a role in driving humoral immunity by sustaining antigen persistence in lymph nodes and generating multivalent antigen presentation for B cell activation. Here, we investigated how amph-vaccine molecular properties (antigen MW and PEG linker length) influenced conformational behaviors (micelle formation, albumin hitchhiking, and membrane insertion) along with the resulting humoral immune response. Using HIV env proteins as model antigens (eOD monomer and MD39 trimer), we investigated cell membrane insertion and B cell activation via calcium flux assay in vitro, followed by lymphatic trafficking and immunogenicity studies in vivo. Our results demonstrate that amphiphile conjugation is an effective strategy for enhancing humoral immunogenicity of both monomer and trimer protein antigens, where cell painting plays a significant role in driving B cell, GC, and humoral immune activation.