Previous studies by our group showed that vaccinia virus recombinants expressing Brucella abortus (BA) antigens heat shock protein GroEL, 18 kDa protein and Cu/Zn SOD, were unable to induce protective immune responses against Brucella challenge. This dissertation analyzes the possible reasons for this phenomenon, by using other genes/proteins from BA and Listeria monocytogenes (LM), various shuttle plasmids (pSC65, pSC11) and immune response modulators (CpG, IL-12, B7-1). As the first objective, a vaccinia virus recombinant (WRL7/L12), expressing the BA L7/L12 gene was generated. L7/L12 ribosomal protein was used as a T-cell reactive antigen, with protective potential to Brucella challenge. The WRL7/L12 was able to express the gene of interest and induce IgG2A type antibody response, but not a protective immune response against Brucella challenge. As a control, an antigen from LM proven to induce CTL and protective immune responses, was used to test the efficacy of vaccinia virus to induce protection. A portion of hly gene, encoding partial listeriolysin (pLLO), was inserted into the same vaccinia virus stain. This recombinant (WRpLLO) was able to induce protection against a Listeria challenge. Next another vaccinia virus recombinant expressing Brucella abortus Cu/Zn SOD was analyzed. Although a variety of approaches, including the enhancement of the protein expression by the pMCO2 synthetic promoter, booster immunization, addition of the oligomer CpG adjuvant (WRSODCpG) to enhance Th1 type response, were used, the SOD recombinant failed to protect mice against Brucella challenge. Lastly, vaccinia virus produces a family of proteins that bind cytokines, chemokines and interferons to evade the host defensive systems. Therefore, a vaccinia virus strain co-expressing murine IL-12, and cofactor B7-1, were used to generate the recombinant WRIL12L7/L12. In order to further boost the induction of Th 1 type response, the adjuvant CpG was used. A similar recombinant, WRIL12pLLO, was generated with partial hly gene to serve as a positive control for protection. Mice immune responses to these recombinants, with and without adjuvant CpG, were analyzed, and compared with the recombinants generated with vaccinia strain WR. Co-expression of IL12 and B7 abrogated the protective efficacy of the vaccinia/ pLLO recombinant. / Ph. D.

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This study was undertaken to examine the specificity of the cell-mediated immune response to vesicular stomatitis virus in mice, using the recombinant adenovirus vector AdG12. AdG12 contains the coding region for VSV glycoprotein (G) within the genome of adenovirus type 5. Ultimately, these studies attempted to provide a model for the use of adenovirus vectors to elicit specific CTL responses in mice against an inserted foreign protein. Cell-mediated immunity was examined using a standard ⁵¹Cr release assay. Splenocyte effectors from VSV or AdG12 primed mice were tested for their ability to lyse labelled infected target cells. A number of target cell lines were analyzed for productive infection by AdG12 and expression of VSV-G. Of the lines tested, B10.D2 (H-2ᵈ) and PAK (H-2ᵇ) lines were shown to be infectible with AdG12 and expressed VSV-G 36 hours post infection. Cell lines P815 (H-2ᵈ) and EL-4 (H-2ᵇ) did not appear to be AdG12 infectible. Responses were measured in mice intravenously infected with AdG12. Results demonstrated that peak cytotoxic activity from AdG12 primed mice occurred six days post-infection against syngeneic target cells infected with AdG12, Ad5 wt or VSV. However, these effectors also significantly lysed allotargets infected with VSV, implying that VSV infected targets were lysed in a non-MHC restricted manner. In subsequent experiments, it was discovered that VSV infected B10.D2 and PAK targets were markedly lysed by effectors from immunized and non-immunized Balbic, C57B1/6 and CBA/J mice. Thus, it appeared that these mouse strains contained an inherent or natural cytotoxic activity against VSV infected targets that was unlike classical CTL killing. Depletion experiments showed that this activity was not due to adherent or Thy1 bearing cells within spleen cell populations. To further characterize this activity, splenocyte effectors were tested for their ability to lyse NK-sensitive YAC-1 targets, but no significant lysis was demonstrated. However, despite these results, it appeared that this activity was that of an NK-like effector. The presence of NK-like cytotoxicity against VSV infected targets precluded efforts to define specific anti-VSV responses in these mice. / Thesis / Master of Science (MS)

Alginate is a well-known naturally-derived biomaterial that has been widely used in preparing microparticles for drug delivery and in preparing scaffolds for tissue engineering. Despite desirable properties, alginate has been shown to activate inflammatory cells in vivo. The mechanisms are still unclear. In this thesis, the mechanism by which alginate caused innate immune responses was investigated in vitro by using RAW264.7 cells, a macrophage-like cell line. The NF-(kappa)B pathway, an important signaling pathway in macrophages, has been tracked to identify cellular responses. The secretion of cytokines IL-1(beta), IL-6, IL-12(p40) and TNF-(alpha) was quantified to determine the activation outcomes. Also the interaction between alginate and serum was studied. Experimental results indicated that alginate induced the activation of RAW264.7 cells with a time and dose dependent behavior. Like lipopolysaccharide, a bacterial product and known activator of innate immunity, alginate induced macrophage activation through the NF-(kappa)B pathway and eventually led to detectable IL-1(beta), IL-6 and TNF-(alpha) cytokine secretion. Serum influenced alginate recognition by macrophages in an unknown mechanism. Also, alginate promoted cell survival in a nutrition starvation condition. These results revealed in vitro alginate stimulation, and provided much information for further research. / Thesis / Master of Applied Science (MASc)

The use of polymeric biomaterials in regenerative medicine and drug delivery is a continually growing practice. Alginic acid (alginate) is widely used in these fields because of its beneficial properties from an engineering and mechanical perspective. Still, alginate has not yet been fully investigated from a biological perspective. For disciplines that anticipate in vivo use of their devices, it is crucial to understand the biological interactions between the device and the host. In this project, the in vitro and in vivo immunological effects of alginate are examined in two model systems: one with a protein antigen and one with a xenogeneic cell antigen. The former system is used as a proof of principle study for alginate's immunological effect on simple protein-based systems, similar to those found in protein/drug deli very applications and certain types of vaccines. This model uses bovine serum albumin (BSA) as the protein antigen. The latter system is used to demonstrate alginate's effect on more complex antigens, such as whole cells. Thus, Chinese hamster ovary (CHO) cells are used as the as the cell antigen. This model represents a system that may be found in tissue engineering applications, where whole cells are delivered with a biomaterial scaffold. Antibody production from blood serum indicated that alginate solution has adjuvant abilities while alginate microspheres do not. Thus, alginate solution possesses great potential in the field of vaccines. In addition, in vivo alginate challenges were found to have effects on second-set responses of splenocytes to in vitro alginate and antigen challenges. Splenocytes from alginate-injected mice were overall equally or less responsive to in vitro challenges than splenocytes without previous alginate immunization. Therefore, alginate solution may also have immunosuppressive effects, although the results from this project merely speculate on this possibility. Still, this ability would be helpful in overcoming current transplantation problems as well as certain tissue engineering hurdles. / Thesis / Master of Science (MS)

Earlier studies have demonstrated that nitrosoureas such as 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) and chlorozotocin (CLZ) can cure almost 100% of C57BL/6 mice bearing syngeneic LSA tumor. In contrast, similiar or higher doses of streptozotocin (STZ) completely failed to cure LSA-bearing mice. Further studies revealed that the efficacy of nitrosoureas may depend on their immunomodulating properties. In the current study, therefore, attempts were made to investigate the effects of these nitrosoureas on the immune system of normal and LSA tumor-bearing mice. Treatment of normal C57BL/6 mice with 5 intraperitoneal injections of 20 mg/kg body weight of BCNU or CLZ caused an increase in the percentage of CD4⁻ CD8⁻ T cells and a decrease in the percentage of CD4⁺CD8⁺ T cells in the thymus. In addition, such treatment also caused an increase in the percentage of CD4⁺ T cells without significantly affecting the CD8⁺ T cells in the thymus. However, when total cellularity of the thymus was studied, BCNU and CLZ were found to decrease the total number of CD4⁺CD8⁺ T cells without significantly affecting the other subsets. In contrast, similiar or higher (100mg/kg body weight) doses of STZ had no significant effect on the total number and percentages of T ceil subsets in the thymus. Also, BCNU and CLZ but not STZ-treatment caused a 50% decrease in the total number of CD4⁺ and CD8⁺ T cells in the spleen. Interestingly in tumor-bearing mice, BCNU treatment was followed by a ten-fold increase in the percentage of CD4⁺ T cells found in the peritoneal cavity. The percentages of CD8⁺ cells increased also, but to a lesser degree. These changes were limited to the peritoneal cavity which is the site of tumor growth. When T cells in the spleens of nitrosourea-treated normal mice were functionally analyzed, it was observed that BCNU and CLZ caused a dramatic decrease in the T cell responsiveness to Con A, anti-CD3, and PMA + calcium ionophore stimulation. In contrast, STZ treatment failed to significantly inhibit the T cell responsiveness to these activation signals. Using the accessory cell-dependent and independent assays, BCNU and CLZ were found to suppress the functions of both T cells and macrophages in normal mice. BCNU and CLZ also suppressed the B cell responsiveness to lipopolysaccharide (LPS). Also, addition of growth factors such as IL-1, IL-2, IL-4 and IL-6 failed to reconstitute the defective responsiveness of BCNU and CLZ-treated T cells and macrophages. Together these data suggest that nitrosoureas have varying immunomodulating properties and this may in turn determine their efficacy in the treatment of cancer. / Master of Science