Access Type

Open Access Dissertation

Date of Award

January 2014

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry

First Advisor

Zhongwu Guo

Abstract

The unique carbohydrates expressed on the surface of cancer, bacterial, viral and fungal cells are excellent target antigens for the design of therapeutic or preventive vaccines. However, as antigens carbohydrates have problems. First, carbohydrates usually have low immunogenicity. Second, even if immunogenic, carbohydrates typically elicit T cell-independent immune responses. To overcome these problems and design useful vaccines based on carbohydrate antigens, they are usually coupled with carrier proteins to form conjugates to enhance the immunogenicity of the antigens. However, there are still some issues existing in glycoprotein vaccines, such as poor reproducibility of the conjugates, difficulties in quality control and so on. To deal with these issues, our group explored a strategy to utilize synthetic carbohydrate antigens with well-defined structures for the construction of glycoprotein vaccines. In the meantime, our group has also developed new vaccine carriers, such as monophosphoryl lipid A (MPLA), to construct full-synthetic carbohydrate-based vaccines that have well-defined structures and improved immunological properties. The main aims of this dissertation are to study

and evaluate these semi- and full-synthetic glycoconjugates and develop carbohydrate-based vaccines against cancer and bacteria.

The first part of this dissertation (Chapters 2 and 3) is focused on antitumor vaccines targeting at tumor-associated carbohydrate antigens (TACAs). For TACAs, in addition to the problems associated with carbohydrate antigens mentioned above, there is another problem, namely immunotolerance, due to their structural similarity to normal carbohydrates on normal cells. To overcome the immunotolerance problem, our group developed a novel immunotherapeutic strategy based on glycoengineering of sialo-TACAs on cancer cells. An important requirement for this strategy to work is to engineer cancer cells to express unnatural sialo-TACAs. In Chapter 2, a convenient method was developed for the quantification of various sialic acids expressed by cells and used to analyze the efficiency of N-phenylacetyl-D-mannosamine (ManNPhAc) to metabolically glycoengineer SKMEL-28 cancer cell. In specific, after cancer cells were cultured with ManNPhAc, the cells were treated with 2M acetic acid to release sialic acids and then with 1,2-diamino-4,5-methylenedioxybenzene (DMB). Sialic acids could react with DMB to form the corresponding derivatives that had strong UV absorptions. The reaction mixture was then applied to HPLC-UV analysis to determine the amounts and the ratios of natural sialic acid and its unnatural analog. It was confirmed that after incubation with ManNPhAc, the SKMEL-28 cell was effectively glycoengineered to express a significant amount of unnatural sialic acid.

Another requirement for the new cancer immunotherapeutic strategy is to have effective vaccines made of TACAs that contain the correspondingly modified sialic acid. In Chapter 3, a new construct of carbohydrate-based cancer vaccines with MPLA as the

carrier molecule and a build-in adjuvant, which are full-synthetic and potentially possess strong and self-adjuvanting immunological activities, were investigated. For this purpose, four MPLA analogs were prepared and immunologically evaluated to identify the ideal vaccine carrier. It was confirmed MPLA conjugates of chemically modified sTn antigen induced robust immune responses, thus they can be used as effective vaccines for the new cancer immunotherapeutic strategy. Furthermore, the optimized MPLA was used to develop Globo H-based anti-breast cancer vaccine. The immunological results of Globo H-keyhole limpet hemocyanin (KLH) and Globo H-MPLA conjugates indicated that the Globo H-MPLA conjugate had better immunogenicity than that of Globo H-KLH, including the capability of stimulating a T cell-mediated immunity. Therefore, Globo H-MPLA had the potential for being further developed into clinically useful vaccines.

The second part of this dissertation (Chapter 4) is focused on the development of antibacterial vaccines based on their capsular polysaccharide antigens. Instead of using polysaccharides isolated from bacterial cells, oligomers of the polysaccharide antigen repeating units were synthesized and then conjugated with a carrier protein, such KLH, or a MPLA derivative to form semi- or full-synthetic vaccines. The resultant conjugates were evaluated in mice and their structure-activity relationships were analyzed to identify the proper repeating unit oligomers for vaccine development. In this dissertation, two types of bacteria Haemophilus Influenzae type b (Hib) and group C Neisseria meningitidis were studied. The target antigen for Hib was the repeating ribosylribitol phosphate (RRP) polysaccharide, and for group C N. meningitides, the target antigen was α[2,9]-linked polysialic acid. Immunological studies of these conjugates suggested that they all can stimulate strong T-cell mediated immune responses. Most importantly, it was concluded

that short oligomers of bacterial polysaccharide antigens can be highly immunogenic and induce immune responses that can recognize and bind the target pathogens.

In conclusion, in this dissertation, a new method was developed for quantitative analysis of cell surface sialic acids and analysis of the efficiency of sialic acid metabolic engineering. This method can be broadly useful for various cells and sialic acid analogs. In this dissertation, two different vaccine strategies, which could lead to semi- and fully synthetic vaccines, against cancer and bacteria have been investigated in great details. These vaccines showed promising properties and are worth further investigation. More importantly, the results of this dissertation have provided proof of principle for the new strategies, which may be widely applicable to other cancer and bacteria.

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