Identification and characterization of protective antigens for control of Ixodes scapularis infestations

The objective of this research was to identify and characterize antigens protective against infestations of the black legged tick, Ixodes scapularis , that could serve as candidate antigens for development of a vaccine for control of tick infestations. Thus far, identification of protective antigens has been a limiting factor in the development of tick vaccines. In this research, expression library immunization (ELI) was used in a tick/mouse model to screen an I. scapularis cDNA library for selection of protective antigens. Mice were immunized twice with 50 mug of plasmid DNA and challenge-exposed with I. scapularis larvae. After several screenings, cDNAs that resulted in immunity against larval tick infestations were considered for further screening. Genes encoding for three protective antigens, 4E6, 4D8, and 4F8, were selected for characterization. While 4F8 was to be nucleotidase, 4D8 and 4E6 were of unknown function. The three proteins were expressed in E. coli and used to produce rabbit-antiserum for immunohistochemistry (IHC) studies. All three proteins were localized by IHC on cultured tick cells, and 4E6 and 4D8 were found on gut cells in tick cross sections. Expression of 4E6, 4D8 and 4F8 genes was confirmed by RT-PCR in eggs, larvae, and nymphs, and in guts and salivary glands from adult I. scapularis. Subsequently, expression of these three genes was confirmed by RT-PCR in guts and salivary glands of other tick species. Gene 4F8 was expressed in I. ricinus, gene 4E6 was expressed in I. ricinus, I. pacificus and Amblyomma americanum, and the 4D8 gene was expressed in I. pacificus, I. ricinus, A. americanum, D. variabilis, Rhipicephalus sanguineus, and R. (Boophilus) microplus. Based on immunization trials against larval and nymphal ticks in rabbits and by artificial feeding experiments of adults, these recombinant antigens, especially 4D8, appear to be good candidates for development of a vaccine for control of I. scapularis and several other medically important species of ticks.