Lauren Syndergaard and Brian Poole, Micro and Molecular Biology
Introduction
Dengue virus is a mosquitoes-borne virus that causes dengue fever. It is found primarily in tropical areas with over a third of the world’s population living in at-risk areas (WHO). The number of infections has grown rapidly in recent years with as many as 50 to 100 million infections occur each year (WHO). Though not widely present in the United States (small outbreaks occur with some regularity in Texas from the Mexican border; Hawaii and Florida relatively recently had cases), many travelers endemic areas contract the virus. Though several are being studied, as yet, there are no vaccines available for dengue. Treatment is for symptoms and for maintenance of vascular volume if dengue hemorrhagic fever occurs. This semester, I have worked primarily on harvesting anti-dengue antibodies produced by memory B cells.
In addition to working on harvesting anti-Dengue antibodies, I have also been working on various other projects. These include writing a review about Epitope Spreading and EBI2 experiments.
Methodology
To begin my work, I harvested blood from those previously infected with dengue virus. BYU provided an ideal location for subjects, with return missionaries from South America and Southeast Asia. I found many volunteers who were willing and collected a total of 9 blood samples. Upon harvesting, I isolated the B cells using lymphocyte separation medium using blood sample processing protocols. I then transfected the B cells with EBV. I also added cyclosporine to suppress T cells found in the lymphocyte population. Transfection with EBV allowed the cells lines to be immortalized.
Cell lines were frozen down at this step for future use. B cells were separated into 4 cell lines and stimulated with pokeweed mitogen. The supernatant containing antibodies and cell lines were frozen down as well.
Results
Overall, all steps were successful. We were able to harvest B cells from those previously infected with Dengue. Information regarding location and year of infection was also collected to allow for future research into the particular serotypes of Dengue. Using the protocols, we were able to collect B cell lines and the antibodies produced by them. These samples are frozen down and may used in the future, particularly in the testing of them with ELISA.
Discussion
There are four serotypes of dengue virus, and infection with one does not provide protection from subsequent infection with the remaining serotypes (except for a few months of crossprotection). In fact, a second infection is at higher risk for a more severe outcome. Though this phenomenon is not entirely understood, but if we were to isolate anti-dengue antibodies this could provide the basis for research into the genes and makeup responsible. The next step in the process will be to test the antibodies against Dengue virus using an ELISA protocol to see if they are anti-dengue antibodies. From there, we can study the mechanisms of inhibition and look at any cross-reactivity.
In addition to the Dengue project, I have been working on projects and papers. This past November, I was published in a review regarding Epitope Spreading. I took part in gathering the literature and information regarding epitope spreading and wrote about the intramolecular epitope spreading, namely the T and B cell interaction. I also studied diseases in which epitope spreading is known to occur. I have also been working on EBI2 experiments. I have finished a paper that addresses the effects of BRF1 on EBI2. This involved RNA extraction, PCR, and Transfection.
Conclusion
The study into Dengue antibodies can provide new information regarding the mechanism of inhibition and Antibody-Dependent Enhancement. Future directions include commercializing Abs as prophylactic treatment for those traveling to at-risk areas, and investigating the genes of anti-dengue antibodies responsible for this antibody specificity.