The inherent weak immune response against carbohydrate antigens has directed several novel approaches towards increasing their immunogenicity for their use as vaccine components. We hypothesized that conjugation of an L-rhamnose (Rha) moiety to a carbohydrate antigen would increase the immune response against the antigen in mice possessing anti-Rha antibodies via an antibody-dependent antigen uptake mechanism. To explore this hypothesis we synthesized a single-molecule three-component vaccine containing the GalNAc- O-Thr (Tn) tumor specific antigen, a 20 amino acid helper T-cell epitope (YAF) derived from an outer membrane protein of Neisseria meningitides and a Rha moiety. Synthesis of the vaccine was achieved by automated Fmoc-based solid phase peptide synthesis and deacetylated by brief treatment with NaOMe. Groups of female BALB/c mice were immunized and boosted with Rha-ovalbumin (Rha-OVA) formulated with either TiterMax ® Gold or Sigma Adjuvant System® for a period of 35 days to generate optimum anti-Rha antibodies necessary for evaluating the vaccine. Anti-Rha antibody titers were >100 fold higher in groups of mice immunized with Rha-OVA than the control groups. Mice producing anti-Rha were challenged with Rha-YAF-Tn or YAF-Tn. Sera collected from the groups initially immunized with Rha-OVA and later challenged with Rha-YAF-Tn showed a two fold increase in anti-Tn titer at 1/100 serum dilution compared to mice not immunized with Rha-OVA. An in vitro T-cell proliferation study using cells primed with either Rha-YAF-Tn or YAF-Tn was performed to examine differences in antigen uptake and presentation on the MHC II in the presence of anti-Rha antibodies. In the presense of anti-Rha antibodies proliferation of T-cells showed a 10-fold decrease in the amount of antigen required. The result strongly suggests that T-cells present in the spleen were presented with higher concentrations of Rha-YAF-Tn as a result of the presence of the anti-Rha antibodies.
MUC1 variable number tandem repeats (VNTRs) conjugated to tumor-associated carbohydrate antigens (TACAs) have been shown to break self-tolerance in humanized MUC1 transgenic mice. Therefore, we hypothesize that a MUC1 VNTR TACA-conjugate can be successfully formulated into a liposome-based anti-cancer vaccine. The immunogenicity of the vaccine should be further augmented by incorporating surface displayed L-rhamnose (Rha) epitopes onto the liposomes to take advantage of a natural antibody-dependent antigen uptake mechanism. To validate our hypothesis we synthesized a 20-amino acid MUC1 glycopeptide containing a GalNAc- O-Thr (Tn) TACA by SPPS and conjugated it to a functionalized Toll-like receptor ligand (TLRL). An L-Rha-cholesterol conjugate was prepared using tetraethylene glycol (TEG) as a linker. The liposome-based anti-cancer vaccine was formulated by the extrusion method using TLRL-MUC1-Tn conjugate, Rha-TEG-cholesterol and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in a total lipid concentration of 30 mM. The stability, homogeneity and size characterization of the liposomes was evaluated by SEM and DLS measurements. The formulated liposomes demonstrated positive binding with both anti-Rha and mouse anti-human MUC1 antibodies. Groups of female BALB/c mice were immunized and boosted with a rhamnose-Ficoll (Rha-Ficoll) conjugate formulated with alum as adjuvant to generate the appropriate concentration of anti-Rha antibodies in the mice. Anti-Rha antibody titers were >25-fold higher in the groups of mice immunized with the Rha-Ficoll conjugate than the non-immunized control groups. The mice were then immunized with the TLRL-MUC1-Tn liposomal vaccine formulated either with or without the surface displaying Rha epitopes. Sera collected from the groups of mice initially immunized with Rha-Ficoll and later vaccinated with the Rha-displaying TLRL-MUC1-Tn liposomes showed a >8-fold increase in both anti-MUC1-Tn and anti-Tn antibody titers in comparison to the groups of mice that did not receive Rha-Ficoll. T-cells from BALB/c mice primed with a MUC1-Tn peptide demonstrated increased proliferation to the Rha-liposomal vaccine in the presence of antibodies isolated from Rha-Ficoll immunized mice compared to nonimmune mice, supporting the proposed effect on antigen presentation. The anti-MUC1-Tn antibodies in the vaccinated mice serum recognized MUC1 on human leukemia U266 cells.
|Advisor:||Sucheck, Steven J.|
|Commitee:||Sucheck, Steven J., Tillekeratne, L. M. Viranga, Wall, Katherine A., Yamamoto, Kana|
|School:||The University of Toledo|
|School Location:||United States -- Ohio|
|Source:||DAI-B 78/11(E), Dissertation Abstracts International|
|Subjects:||Chemistry, Biochemistry, Oncology|
|Keywords:||Anti-cancer vaccines, Anti-tumor vaccines|
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