Evidence to date suggests that in animals chronically infected with F. hepatica a Th2 response is dominant with IgG1 being the dominant antibody isotype in chronically infected cattle [1], sheep [2, 3], and rats [4]. By contrast, in animals that develop resistance against infection, high levels of IgG2 and IFN-delta have been observed [5]. This indicates that while Th2 response is associated to susceptibility of infection, the Th1-response could be associated to resistance. The identification of parasite antigens which are involved in inducing the Th1 response during infection is crucial for the development of a vaccine against parasite. We have recently reported a novel member of the Fasciola saposin-like / NK-lysin protein family, termed FhSAP-2, which is highly immunogenic in mice and rabbits. It is a Fasciola / Schistosoma cross-reactive antigen that is expressed at an early stage of F. hepatica infection. In addition, FhSAP-2 exhibit a potent lytic activity on human erythrocytes and peripheral blood mononuclear cells. A detailed structural analysis of FhSAP-2 has shown that it contains 6 conserved cysteine residues arranged within 5 amphipathic alpha-helix domains and seven hydrophobic residues in strictly conserved positions. Additionally, computer algorithms applied to primary structure of FhSAP-2 have predicted the existence of several MHC class II complex-binding regions as well as of regions that might form T-cell epitope [ 16, 17]. Since FhSAP-2 is a F. hepatica / S. mansoni cross-reactive antigen it is obvious that some of these T-cell epitopes could be F. hepatica / S. mansoni cross-reactive epitopes. The first long term objective of this proposal is to identify and characterize epitopes from FhSAP-2 which are restricted to the Th1 or Th2 phenotype and then to identify those that are Fasciola / Schistosoma cross-reactive epitopes. For this, a peptide libray from the entire sequence of FhSAP-2 will be synthetized with overlapping in the last ten amino acids. Spleen and inguinal lymph node lymphocytes from BALB/c (H-2d) and C57BL/6 (H-2b) mice primed with FhSAP-2 and boosted with pools of peptides will be estimulated with single peptides in cell-proliferation assays. The magnitude of the immune response generated in the animals by vaccination with peptides containing T-cell epitopes will be studied in terms of isotype of antibodies and Th cytokine profile. The second long term objective of this proposal is to asses the level of protection conferred by pools of responders peptides and two lineal MAP constructs in mouse and rabbit as experimental model and to establish the correlation between vaccine-induced immune responses and post-challenge parasite burden in the animals vaccinated. For this, mice and rabbits will be prime with pools of responders peptides or MAPs before a Fasciola hepatica challenge infection. Protection will be measured by reduction of parasite burden, reduction of liver damage, and/or reduction of parasite egg excretion. The results will be useful to identify FhSAP-2 epitopes that could form part of a multi-component vaccine against fascioliasis or schistosomiasis.
NIH ExPorter
NIH ExPorter