Date of Award


Degree Type


Degree Name

Master of Science in Biological and Environmental Sciences (MSBES)


Cell and Molecular Biology


Cell & Molecular Biology

First Advisor

Leonard Moise


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 elimination by mutation. Mutations affecting 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 by CHO-S cell transient transfection. Purified variant αDEC205:OVA-ORG recombinant proteins 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 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 elevated levels of T cell proliferation and IFN-γ secretion responses.

It was concluded that the αDEC205:OVA antibodies (VH77-MOD1 and VH77-MOD2) induced statistically significant elevated T cell proliferative responses in comparison to the baseline immune response levels of the nonmodified αDEC205:OVA-ORG antibody, suggesting that an improved vaccine delivery system is underway due to the epitope modifications at the VH77 Tregitope sequence, which potentially decreased the tolerogenicity of the αDEC-205:OVA antibody. In regards to the other variant antibodies, further modifications to the Tregitope sequences in the αDEC205:OVA antibody’s overall sequence may be necessary to reduce tolerogenicity further to begin to create a detolerized antibody capable of inducing a pro-inflammatory immune response without the aid of a non-specific immuno-stimulator.



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