Novel Influenza Virus Vaccines Bearing Human Specific Immonomodulators

NON-TECHNICAL SUMMARY: Avian influenza represents a significant threat to both humans and the poultry industry. Vaccines that protect both poultry flocks and humans from avian influenza virus are urgently needed. This purpose of this project is to significantly improve influenza vaccines that provide protection against avian influenza virus.

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APPROACH: In objective 1, we will specifically fuse immune stimmulatory molecules such as human cytokines or chemokines directly to a portion of the viral glycoprotein, hemagglutinin. This fusion anchors the immune stimmulatory molecule to the surface of cells when expressed. Upon viral infection, newly synthesized virus particles will incorporate these fusion proteins as the virus leaves the infected cell. Virus will subsequently be harvested from the media of infected cell cultures and inactivated and purified using standard techniques. Objective 1 will be complete after we verify that the inactivated viruses still possess a bioactive form of the incorporated immune stimmulatory molecule. This will be accomplished by standard bioassays. In objective 2, we will test these inactivated viruses bearing immune stimulatory molecules using a mouse model of influenza virus infection. This will be accomplished by vaccinating mice by different routes of administration and by challenging the mice with highly infectious mouse-adapted influenza virus strains at different times following vaccination. We will test the ability of these vaccines to generate protective antibody responses in the mice as well as determine the type of protective responses that were achieved. Finally, we will compare protective responses generated in both young and aged mice. A successful vaccine should be effective in both the young and the aged animal.
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PROGRESS: 2006/10 TO 2007/09<BR>
OUTPUTS: The goal of this study was to make membrane-bound, human-specific, immunomodulators that could effectively be incorporated into influenza virus particles. It is expected that these immunomodulatory proteins when presented in context with the inactivated virus particles will serve as potent adjuvants enhancing the establishment of anti-viral immune responses. We have established important interactive collaborations with Wake Forest University, to explore potential use of the vaccine platforms for the aging as well as to explore new options for vaccine development against other animal pathogens. Some of this work is being presented at the Dean's Forum on Health, Food and Nutrition, Skelton Conference Center at Virginia Tech, Blacksburg, VA, November 5, 2007. Further events include a seminar entitled "Membrane-bound Immunomodulators as Adjuvants for Viral Vaccines. Virginia-Maryland Regional College of Veterinary Medicine, Maryland campus, College Park, MD November 14, 2007.<BR> PARTICIPANTS: Andrew S. Herbert and Tila Khan are both graduate students working on vaccine development at the College of Veterinary Medicine at Virginia Tech. Dr. Chris Roberts, Associate Professor served as P.I. on this Project. Drs. Gogal and Switonsky served as collaborators directing the immunological aspects of the project. <BR> TARGET AUDIENCES: Target audiences were graduate programs at the Maryland Campus of the Virginia-Maryland College of Veterinary Medicine and professors working at that campus.
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IMPACT: 2006/10 TO 2007/09<BR>
We have already begun construction of the fusion constructs that are required for membrane-bound insertion. Specifically, we have acquired (commercially) the coding sequences of human IL-2, IL-12 and IL-15. Using PCR amplification, we have amplified the region coding for the mature protein and modified it such that the necessary restriction sites have been incorporated at both the 5' and 3' ends. All of these constructs will be inserted as a fusion into our plasmid pcDNA3-HA1521, which encodes for the HA transmembrane and cytoplasmic tail regions of the viral hemagglutinin. By using specific restriction sites, insertion of the cytokines results in an inframe insertion, such that the immunomodulatory region of the cytokine is oriented outward with the transmembrane region of the HA protein serving as an anchor in the membrane. Once these constructs have been verified by DNA sequencing, we will proceed to the next milestone, which is to make stable cell lines expressing the bioactive, membrane-bound form of our fusion constructs at the cell surface of MDCK cells, our virus producing cell line