Deletion of antibody encoded tolerogenic signals to improve a dendritic cell targeted vaccine delivery platform system
Antibodies specific for DEC205, a dendritic cell (DC) endocytic receptor that traffics to the antigen presentation pathway, have been shown to be excellent tools for raising robust, sustained immune responses to co-delivered vaccine antigens; however, strong immune responses are only elicited with the aid of non-specific dendritic cell maturation factors, without which a tolerogenic immune response is induced. We hypothesize that regulatory T cell epitopes (Tregitopes) located in the αDEC205 sequence promotes tolerance, requiring the use of non-specific immuno-stimulators to promote pro-inflammatory immune responses. This hypothesis is based on previous research performed by De Groot et al. 2008 who characterized a set of natural regulatory T cell epitopes derived from human immunoglobulins (IgG) that were found to induce tolerance by stimulating regulatory T cells (CD4+CD25 +FoxP3+). ^ We believe that αDEC205 can be rendered less tolerogenic by modifying its regulatory T cell epitope content and improve its capacity to induce inflammatory responses without the aid of non-specific maturation factors to activate the immune system. ^ In this work, the αDEC205 sequence was computationally screened for putative HLA-Class II-restricted, regulatory T cell epitopes as targets for mutation. Mutations at key amino acid sites, relevant to peptide-HLA-DRB1*0401 binding, were carried out to reduce epitope binding affinity to the HLA-DRB1*0401 allele. Sequence modifications confirmed to disrupt peptide-HLA binding were incorporated into an array of αDEC205:OVA-ORG (original sequence) variants (mutant sequences) via site directed mutagenesis. Protein was produced on a large scale with CHO-S cellular transfection. Purified variant αDEC205:OVA-ORG sequences were utilized in DR4 in vivo immunizations and functional assays to observe T cell activation and proliferative immune responses. ^ The αDEC205:OVA variants (HC54-MOD1, VH77-MOD1 & VH77-MOD2) were shown to target and bind to dendritic cells as effectively as the non-modified αDEC205:OVA antibody. Splenocytes were re-stimulated with Ovalbumin in the T cell proliferation assay and with OVA Class I (257-264) and OVA Class II (323-339) peptides in the ELISpot assay to measure the memory responses. Both αDEC205:OVA-VH77-MOD1 and αDEC205:OVA-VH77-MOD2 showed statistically significant differences in CD8+ and CD4+ T cell proliferation in comparison to splenocytes previously immunized with the αDEC205:OVA-ORG antibody condition, respectively. However, a lack of statistically significant IFN-γ cytokine production was observed for all variant antibody immunization conditions. Further analyses are required to determine the true statistical significance observed from the αDEC205:OVA-VH77-MOD1 data set as evidenced by the possible outliers, which may be skewing the results. The αDEC205:OVA-HC54-MOD1 sequence failed to generate any kind of statistically significant data. ^ It was concluded that the αDEC205:OVA-VH77-MOD2 antibody did induce statistically significant pro-inflammatory responses in comparison to the baseline immune response levels of the αDEC205:OVA-ORG antibody. In regards to the other variant antibodies, further extensive modifications to the Tregitope sequences in the αDEC205:OVA antibody's overall sequence may be necessary to reduce tolerogenicity further and create a detolerized antibody capable of inducing a pro-inflammatory immune response without the aid of a non-specific immuno-stimulator.^
Biology, Molecular|Biology, Cell|Health Sciences, Immunology
"Deletion of antibody encoded tolerogenic signals to improve a dendritic cell targeted vaccine delivery platform system"
Dissertations and Master's Theses (Campus Access).