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United States Department of Agriculture

Agricultural Research Service

Title: Phylogeny of bluetongue virus isolates by sequence analysis of the VP5 coding gene

item Wu, Shixuan
item Letchworth, Geoffrey
item Mecham, James

Submitted to: Annual Conference on Vaccine Research
Publication Type: Abstract Only
Publication Acceptance Date: 3/9/2007
Publication Date: 5/2/2007
Citation: Wu, S., Letchworth, G., Mecham, J.O. 2007. Phylogeny of bluetongue virus isolates by sequence analysis of the VP5 coding gene. Tenth Annual Conference on Vaccine Research. Baltimore, MD. April 30-May 2, 2007.

Interpretive Summary: West Nile virus (WNV) is transmitted by mosquitoes to birds, horses, humans and a number of other animals; and is of interest to both agriculture and public health. From an agricultural point of view, the equine industry in the U.S. represents a multibillion-dollar enterprise. The affects of WNV are costly not only in terms of mortality and morbidity to valuable animals, but also in terms of its affect on international livestock trade and movement. Since West Nile virus is transmitted by mosquitoes, it causes disease in animals by circulating in the blood, crossing the blood-brain barrier, and damaging the central nervous system. Antibodies that neutralize virus in the blood, and therefore prevent spread into the central nervous system, afford protection against disease. Vaccination with either killed virus or a genetically engineered virus engenders the production of protective (neutralizing) antibodies. However, engineered vaccines are very expensive and killed vaccine preparations have the potential of carrying incompletely inactivated virulent virus; thereby themselves causing disease. Vaccination with subunits (parts of the virus) avoids the risks associated with whole virus preparations; however, these subunit vaccines may engender suboptimal immunity due to poor immunogenic properties. Chimeric proteins, consisting of an antigen and complement component C3d, have engendered antibody responses at approximately 10,000-fold lower doses than the same antigen without C3d. The reported study sought to enhance the neutralizing immune response to West Nile virus (WNV) truncated envelope (Et) protein by linking it with murine C3d. Mice were immunized with the chimeric protein expressed in Cos1 cells, the cDNA chimeric construct, or with a combination of both the expressed protein and cDNA. Serum samples were collected from the mice and tested for neutralizing antibody to infectious WNV. Results showed that C3d enhanced antibody formation but these antibodies were not neutralizing and therefore not likely to protect against WNV.

Technical Abstract: Chimeric proteins consisting of complement component C3d and several antigens have been shown to engender antibody responses at approximately 10,000-fold lower doses than the same antigens without C3d. We sought to enhance the neutralizing immune response to West Nile virus (WNV) envelope (E) protein by chimerizing it with murine C3d. We inserted into the pcDNA3 expression plasmid sequences encoding the extra-membrane portion of WNV E protein (Et) followed by either none, one, or three copies of sequences encoding murine C3d. Six histidine codons were included to facilitate protein purification. We transfected plasmids into Cos1 cells, purified and quantified the recombinant proteins, and vaccinated mice twice at a 21 day interval. Other mice were vaccinated with purified full-length WNV E protein, pCBWN DNA expressing the full-length WNV E protein (Davis et al., 2001, J. Virol. 75:4040-4047), or pcDNA3 expressing Et followed by purified Et. Blood samples were taken 15 days after the second immunization and tested for WNV neutralization and reaction in a binding ELISA with either purified Et protein or Et-3xC3d. As previously reported (Davis et al., 2001), full-length E, either as a DNA vaccine or as a protein vaccine, engendered neutralizing antibody. WNV Et DNA followed by Et protein engendered neutralizing antibody; however, Et chimerized with murine C3d DNA did not. We surmise that murine C3d altered the conformation of chimeric Et so that it was unable to engender neutralizing antibody. On the other hand, Et chimerized with C3d protein produced more ELISA binding antibody than the Et protein alone, suggesting that C3d enhanced the antibody response. No DNA vaccine produced detectable ELISA binding antibody, whether it produced neutralizaing antibody or not.

Last Modified: 06/26/2017
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