Role of Linker Length and Antigen Density in Nanoparticle Peptide Vaccine

[Image: see text] Multiple studies have been published emphasizing the significant role of nanoparticle (NP) carriers in antigenic peptide-based subunit vaccines for the induction of potent humoral and cellular responses. Various design parameters of nanoparticle subunit vaccines such as linker chemistry, the proximity of antigenic peptide to NPs, and the density of antigenic peptides on the surface of NPs play an important role in antigen presentation to dendritic cells (DCs) and in subsequent induction of CD8+ T cell response. In this current study, we evaluated the role of peptide antigen proximity and density on DC uptake, antigen cross-presentation, in vitro T cell proliferation, and in vivo induction of CD8+ T cells. To evaluate the role of antigen proximity, CSIINFEKL peptides were systematically conjugated to poly(ethylene glycol) (PEG) hydrogels through N-hydroxysuccinimide–PEG–maleimide linkers of varying molecular weights: 2k, 5k, and 10k. We observed that the peptides conjugated to NPs via the 2k and 5k PEG linkers resulted in higher uptake in bone marrow-derived DCs (BMDCs) and increased p-MHC-I formation on the surface of bone marrow-derived DCs (BMDCs) as compared to the 10k PEG linker formulation. However, no significant differences in vitro T cell proliferation and induction of in vivo CD8+ T cells were found among linker lengths. To study the effect of antigen density, CSIINFEKL peptides were conjugated to PEG hydrogels via 5k PEG linkers at various densities. We found that high antigen density NPs presented the highest p-MHC-I on the surface of BMDCs and induced higher proliferation of T cells, whereas NPs with low peptide density resulted in higher DC cell uptake and elevated frequency of IFN-γ producing CD8+ T cells in mice as compared to the medium- and high-density formulations. Altogether, findings for these experiments highlighted the importance of linker length and peptide antigen density on DC cell uptake, antigen presentation, and induction of in vivo CD8+ T cell response.