U35 - CD45-targeted antigens amplify humoral immunity against HIV-1 Env

Introduction: : mRNA vaccines have demonstrated remarkable success, yet their components, including the translated antigen, may be rapidly degraded. While rapid clearance can contribute to the safety profile of vaccines, it may also limit the duration of antigen exposure required to induce effective immune responses. Prolonging antigen availability within lymphoid tissues is a promising strategy to enhance and sustain vaccine immunogenicity, as extended antigen presence is associated with more robust germinal center (GC) reactions, higher antibody titers, and other correlates of protection against pathogens. Here, we present a novel approach that exploits CD45 – a pan-leukocyte surface marker expressed at extremely high levels (>100,000 copies per cell) with minimal receptor recycling, thereby extending antigen persistence in lymphoid tissues.

Materials and

Methods: : As proof of concept, we synthesized mRNA constructs encoding a stabilized HIV Env antigen fused to a CD45-targeting nanobody. A nanobody targeting Green Fluorescent Protein (GFP) was used as an irrelevant control. Synthesized mRNAs were electroporated in murine myoblast C2C12 cells and yield and folding of translated protein was evaluated in cell culture supernatant using ELISA. For in vivo studies, mRNA was encapsulated into Moderna-mimic lipid nanoparticles and injected intramuscularly in C57BL/6 mice. Surface expression of translated protein and germinal center B cell response were assessed in lymph nodes using flow cytometry. Antibody responses were measured in serum via ELISA.

Results, Conclusions, and Discussions:: Both CD45- and GFP-targeted constructs showed comparable protein expression and specific binding to their respective targets in vitro. Anchoring antigens to CD45 via nanobody fusion significantly delayed antigen clearance due to the receptor’s long surface residence time. CD45-anchored antigens, produced by electroporating the corresponding mRNA into murine C2C12 cells, were harvested from the supernatant and incubated with splenocytes. The antigens remained stably displayed on the surface of immune cells for over 24 hours in vitro, with more than 98% of splenocytes still presenting the construct. Twenty-four hr following intramuscular administration of lipid nanoparticle-encapsulated mRNA, CD45-targeted trimer was detected in lymph nodes by surface staining of immune cells, with 2-fold higher antigen levels across key immune subsets compared to GFP-targeted control antigen, including CD4⁺ T cells, plasmacytoid dendritic cells (DCs), and monocyte-derived DCs. Additionally, this targeting strategy enhanced myeloid cell activation, as evidenced by a 2-fold increase in CD86 expression on DCs, macrophages, and NK cells compared to control vaccinations. Further, this potent anchoring strategy elicited a 4-fold increase in total GC responses, 10-fold expansion of antigen-specific GC B cells, and 100-fold increase in serum antibody titers, compared to controls. Notably, a single dose of CD45-anchored antigen produced GC responses comparable to a complex “extended dosing” regimen designed to prolong antigen availability through seven doses over two weeks.

These results demonstrate that CD45-targeting significantly enhances the persistence and immunogenicity of mRNA vaccines, offering a powerful strategy to boost vaccine efficacy—particularly against challenging pathogens such as HIV.

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