Creation of stable transfectants expressing HLA Alleles thought to influence Dengue Disease severity.

Dengue fever/dengue hemorrhagic fever, considered by the World Health Organization as the most important resurgent tropical infectious disease, constitutes one of the leading causes of hospitalization of children in Southeast Asia. Several immunopathological mechanisms are thought to influence disease severity, including complex interactions between HLA alleles and the infecting virus serotype (DEN-1,-2,-3, or-4). In this project, stable transfectants expressing HLA alleles thought to influence dengue disease severity were created. To do this, HLA alleles HLA-A*0207 and HLA-B*0705/6 were amplified using PCR from a B lymphoblastoid cell line that was generated from a dengue immune Thai subject. These alleles were transformed into the pBluescriptII bacterial vector and subsequently subcloned into the RSV.5 neo mammalian expression vector. The latter vector was then used to transfect adherent mammalian cell lines BHK-21 and COS-7, and stable transfectants were selected using the drug G418. The resulting cells will be useful for studies of the expression of dengue virus antigens to T lymphocytes in the context of specific HLA alleles found in the Thai population