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Infectious diseases remain one of the leading causes of morbidity and mortality of humans and livestock worldwide. Diseases such as malaria, rabies, rinderpest, and foot-and-mouth disease still afflict millions each year, while newer pathogens like human immunodeficiency virus (HIV) are spreading in epidemic proportions. Although traditional vaccination strategies such as live attenuated or whole inactivated vaccines now exist for some of these diseases, in the majority of cases, their cost, relative lack of availability in developing countries, and safety concerns, restrict their widespread application. In other cases, no effective vaccines currently exist. It is imperative that new approaches to vaccine development continue to be explored so that new and improved vaccines can be used to control and eradicate such diseases. One such approach that has generated great excitement is DNA immunization, in which an antigen is synthesized in vivo after inoculation of its encoding sequence. DNA vaccines are attractive for various reasons: 1) they are easy and inexpensive to manufacture in large quantities, 2) they do not require special transportation or storage conditions since they are heat-stable, and 3) they are effective in inducing both humoral and cell-mediated immune responses. In fact, the best non-live attenuated vaccines in the simian immunodeficiency virus (SIV) model for AIDS are cytokine-adjuvanted DNA and prime-boost (DNA priming followed by poxvirus boost) vaccination strategies.<P>
This project aims to test and develop DNA vaccines in a vesicular stomatitis virus (VSV) / mouse model system, so that the resulting technology can be applied to develop new and improved vaccines for livestock diseases. Although immune responses elicited by DNA vaccines are quite pliant, they are not as potent as those induced by other vaccine strategies, possibly resulting from the lack of effective adjuvants and low antigen expression. There are many possible strategies to optimize immunity to DNA vaccines, including changing the method or location of immunization, adding intron sequences or multiple copies of immunostimulatory motifs within the plasmid backbone, altering the immunization regimen, or co-expressing genes for cytokines or co-stimulatory molecules.<P>
In this study, we will focus on two strategies. Our hypothesis is that we can modulate (enhance) the immune response to DNA immunization by adding adjuvant genes (immumomodulatory cytokines) and by selecting the proper form of antigen expression (e.g., secreted). <P>
Objective 1: To construct DNA vaccines that elicit potent Th1 and/or Th2 immune responses by incorporating immunomodulatory cytokines (IL-18 and IL-6), and expressing either a secreted or membrane-bound form of the antigen (VSV-G). <P>
Objective 2: To test the ability of the developed DNA vaccines to direct the immune response towards either a humoral or cytotoxic T-lymphocyte response.
NON-TECHNICAL SUMMARY: Although the immune responses elicited by DNA vaccination are quite pliant, they are not as potent as those induced by other vaccine strategies, possibly resulting from the lack of effective adjuvants as well as low antigen expression. There are many possible strategies to optimize immunity to DNA vaccines. In this study, we will focus on two strategies. Our hypothesis is that we can modulate (enhance) the immune response to DNA immunization by adding adjuvant genes (immumomodulatory cytokines) and by selecting the proper form of antigen expression (e.g., secreted).
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APPROACH: For the first objective, we will generate 4 types of DNA vaccines, incorporating within the plasmid backbone a specific T helper 1 (Th1) or Th2 type of cytokine (IL-18 or IL-6), in addition to two forms of the antigen (secreted or membrane-bound VSV-G), in an attempt to induce the desired humoral or cytotoxic T-cell immune response. Briefly, the two forms of the VSV-G gene will be cloned into plasmid pCI under the human cytomegalovirus major immediate-early gene enhancer/promoter region and the simian virus 40 late polyadenylation signal. The mouse IL-18 and IL-6 genes will be then the cloned into plasmid pRc/RSV under the Rous sarcoma virus enhancer/promoter region and the bovine growth hormone polyadenylation signal. These cassettes will be transferred to the pCI plasmid for co-expression of VSV-G and cytokines. Large-scale plasmid purification methods will be used to purify the DNA vaccines. For the second objective, using the murine VSV model, we will test the host's immune responses to DNA vaccination and resistance to virulent challenge. Briefly, mice (10/group) will be given each DNA vaccine intramuscularly (including controls with plasmids encoding only VSV-G, cytokines, or nothing). Animals will then be challenged intranasally 4 weeks later with 10 50%-lethal dose (LD50) of the New Jersey strain of VSV. IL-18 (a Th1 cytokine) is expected to enhance cell-mediated responses by inducing the production of interferon-g, favoring a Th1 response, and enhancing NK cell cytotoxicity. IL-6 (a Th2 cytokine) promotes B cell proliferation and differentiation and is expected to enhance humoral (especially mucosal) responses. Also, we expect the secreted form of the VSV-G antigen to induce better humoral responses than the membrane-bound form, which we expect to elicit better cytotoxic T-cell responses.
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PROGRESS: 2003/10 TO 2008/09 <BR>
OUTPUTS: Although the immune responses elicited by DNA vaccination are quite pliant, they are not as potent as those induced by other vaccine strategies, possibly resulting from the lack of effective adjuvants as well as low antigen expression. There are many possible strategies to optimize immunity to DNA vaccines. These include changing the method or location of immunization, adding intron sequences or multiple copies of immunostimulatory motifs within the plasmid backbone, altering the immunization regimen, or co-expressing genes for cytokines or co-stimulatory molecules. In this study, we will focus on two strategies. Our hypothesis is that we can modulate (enhance) the immune response to DNA immunization by adding adjuvant genes (immumomodulatory cytokines) and by selecting the proper form of antigen expression (e.g., secreted). Objective 1: To construct DNA vaccines that elicit potent Th1 and/or Th2 immune responses by incorporating immunomodulatory cytokines (IL-18 and IL-6), and expressing either a secreted or membrane-bound form of the antigen (VSV-G). Objective 2: To test the ability of the developed DNA vaccines to direct the immune response towards either a humoral or cytotoxic T-lymphocyte response. PARTICIPANTS: Dr. Tilahun Yilma, Principal investigator Dr. Paulo Verardi, Researcher Dr. Fatema Aziz, Graduate Student TARGET AUDIENCES: Individuals and organizations interested in new vaccines for livestock diseases.
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IMPACT: 2003/10 TO 2008/09<BR>
Although the immune responses elicited by DNA vaccination are quite pliant, they are not as potent as those induced by other vaccine strategies, possibly resulting from the lack of effective adjuvants as well as low antigen expression. There are many possible strategies to optimize immunity to DNA vaccines. In this study, we will focus on two strategies. Our hypothesis is that we can modulate (enhance) the immune response to DNA immunization by adding adjuvant genes (immumomodulatory cytokines) and by selecting the proper form of antigen expression (e.g., secreted). We have constructed DNA vaccines that incorporate the immunomodulatory cytokines IL-18 or IL-6, and express either a secreted or membrane-bound form of the antigen, the surface glycoprotein of vesicular stomatitis virus (VSV-G). We have incorporated IL-18, cloned through RT-PCR using murine spleen template cDNA, into a DNA vaccine. We have confirmed IL-18 expression in vitro using western blotting. We have also constructed an IL-6-expressing plasmid that is expected to induce a Th2 response. These vaccines will be tested in animals when funding becomes available.