We have isolated a novel HTLV-1 like virus from a baboon (designated BTLV). The primary objectives of this investigation are to further characterize this BTLV isolate and to develop a recombinant envelope protein or envelope gene DNA construct for use as a vaccine which can be evaluated in rhesus macaques for protection against mucosal exposure to STLV. The envelope gene of BTLV was cloned in an eukaryotic expression vector and was found to direct the expression of a precursor protein of approximately 62 kda. We also expressed a soluble form of the envelope protein and this construct was used for DNA immunization studies in mice. Intramuscular and intradermal routes of DNA immunization were compared. For intramuscular immunization, groups of mice (containing 5 in each group) were injected with 200 fg of DNA either alone or in combination with Lipofectin (Gibco BRL). The animals were subsequently boosted twice at four week intervals. Intradermal immunizations were accomplished through the use of a gene gun. For gene gun mediated DNA delivery, plasmid DNA was precipitated onto gold particles in the presence of calcium chloride and spermidine. DNA was targeted to the abdominal epithelial cells of mice using the Accell particle delivery system (Auragen, Inc, WI). The animals were similarly boosted three times at 4 week intervals and bleeding was performed as described previously. Blood was collected from all mice prior to each immunization and following the final boost, and the sera were used for evaluation of the immune response. An antibody-capture ELISA kit (Virotech International Inc.) was used to evaluate the immune response in immunized mice. The ELISA results failed to show the presence of significant levels of antibody in the sera subsequent to DNA immunization. The same sera were further used to immunoprecipitate envelope proteins synthesized in transfected cells. For that purpose, HeLa T4 cells were infected with vaccinia virus expressing T7 RNA polymerase and then transfected with plasmid DNA encoding the envelope protein. The cells were radiolabeled and the expressed proteins were immunoprecipitated with antibodies raised in mice. The proteins were resolved by SDS-PAGE and visualized using autoradiography. The results indicated that mice immunized with the DNA plasmid have developed antibodies which precipitated the labeled glycoprotein of 62 kda. Similar results were observed when the antisera were used to detect the labeled soluble form of the envelope protein. A syncytium inhibition assay was used to assess the neutralization effect of the sera. Briefly, coculture of XC cells (transformed cell lines permissible to HTLV-1 induced cell fusion), with 1621 cells (developed from the baboon and expressing the viral proteins) will result in the formation of syncytia (Vincent et al., 1996, in press). We observed a concentration-dependent inhibition in syncytium formation when 1621 cells were incubated with dilutions of heat-inactivated sera prior to coculture with XC cells. These results show that intradermal DNA immunization has elicited an immune r esponse against the envelope protein of BTLV in mice. Our initial attempt at intramuscular DNA immunization failed to elicit detectable antibody titers as judged by immunoprecipitation and syncytia inhibition assays. We are now attempting to develop a challenge system by which we can consistently infect rhesus macaques by intravenous, intravaginal, or oral inoculation with STLV-infected cells. In our future studies we hope to investigate whether a similar env-gene based DNA vaccine will protect rhesus macaques from experimental STLV infection.
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