Mechanisms of Enhancement of Antibody Responses by Escalating Dose Immunization in Mice

Abstract

Vaccination strategies targeting complex pathogens like HIV-1 require optimization to improve immune responses, particularly for subdominant epitopes. The HIV-1 fusion peptide (FP) epitope, a region of the HIV-1 envelope, is considered subdominant due to the much lower antibody titers it typically elicits in comparison to other dominant epitopes of the virus. It has been previously shown that slow-release immunization using nonmechanical osmotic pumps induces higher HIV-1 FP-specific antibody titers compared to conventional bolus immunization. A key question, therefore, is how slow-release immunization preferentially enhances antibody responses to subdominant epitopes like HIV-1 FP. While the effects of slow-release immunization on dendritic cells (DCs), T cells, and B cells have been described, the precise immunological mechanisms driving the induction of FP-specific antibodies - especially in terms of epitope specificity- remain poorly understood.This study elucidates the immunological mechanisms underlying the slow-release immunization method, escalating dose (ESD), where the gradual release of progressively higher vaccine doses enhances FP-specific antibody responses. I hypothesize that ESD enhances FP-specific antibody responses by skewing the immune response toward a Th2 phenotype, which promotes B cell activation and antibody production. To test this hypothesis, using a Helicobacter pylori ferritin nanoparticle-based HIV FP immunogen, I investigated how ESD modulates DC cell migration, dendritic cell subpopulations, Th1/Th2 polarization, and the expansion of FP-specific B cells in secondary lymphoid tissues. Additionally, I measured HIV-specific serum antibodies to assess the impact of ESD on early antibody responses and immunoglobulin subclass production.I found that ESD immunization significantly increased the frequency of migratory conventional DC (cDC) populations, including CCR7+ cDC1, cDC2, and plasmacytoid DCs (pDCs), which are essential for efficient antigen presentation and CD4+ T cell priming. These DC subsets promoted a Th2-biased immune response, characterized by elevated IL-4, IL-10, and GATA-3 expression in CD4+ T cells. Mouse strains expressing low levels of class II molecules (I-Ad and I-Ak), potentially through a Th2 pathway, elicit a stronger HIV FP antibody response, while strains with high class II expression (I-Ab), likely via a Th1 pathway, exhibit a weaker response. Notably, Th2-skewed environment was associated with a preferential expansion of HIV FP-specific B cells, particularly in the extrafollicular (EF) compartment, which is known for rapid, early antibody production. Crucially, ESD immunization preferentially enhanced antibody responses to the HIV FP epitope, with a marked increase in FP-specific antibody titers compared to conventional bolus immunization. This enhancement was selective for the subdominant FP epitope, as responses to the dominant ferritin nanoparticle epitope were similar between both regimens. The expansion of FP-specific B cells in the EF compartment suggests that ESD drives a shift toward early, robust antibody production targeting non-dominant epitopes without compromising responses to dominant epitopes. In conclusion, these findings provide mechanistic insights into how ESD immunization selectively enhances immune responses to subdominant epitopes like HIV FP. By modulating DC migration, T cell differentiation, and B cell activation, ESD promotes a Th2-biased environment that favors rapid antibody responses to non-dominant epitopes. These results highlight the potential of ESD as a strategy for improving vaccine efficacy by modulating epitope specificity and enhancing responses to subdominant epitopes in the development of next-generation HIV vaccines.