Leishmania major-Phlebotomus duboscqi interactions: inhibition of anti-LPG antibodies and characterisation of two proteins with shared epitopes
Tonui, W.K.; Ngumbi, P.M.; Mpoke, S.S.; Orago, A.S.; Mbati, P.A.; Turco, S.J.; Mkoji, G.M.
East African Medical Journal 81(2): 97-103
2004
ISSN/ISBN: 0012-835X PMID: 15125094 Document Number: 580042
Objectives: To assess the effect of monoclonal antibodies (MABs) raised against L. major-derived lipophosphoglycan (LPG) on L. major development in vitro and in its natural vector P. duboscqi, and to determine whether the LPG molecule and the sand fly gut lysates have shared epitopes. Setting: Colony bred P. duboscqi sand flies and all other experiments were done under laboratory conditions. Methods: Laboratory-reared sand flies were allowed to feed beneath a blood filled membrane feeder containing 1x106 amastigotes in 20 micro l mixed with 0.5 ml of defibrinated rabbit blood with a 1:100 dilution of anti-LPG MABs. Control blood contained a similar number of amastigotes but without MABs. At least five previously-fed female sand flies were later dissected on days two, four, and six post-feeding, and midgut samples were examined for promastigotes and parasite loads. In vitro, the same number of amastigotes in 100 micro l complete Schneider's Drosophila medium was mixed in a 96 well plate with either 100 micro l of 1:100 anti-LPG MABs, 1:1000 anti-LPG MABs, or undiluted sera from L. major-infected mice. The control well contained a similar number of amastigotes, but with no antibodies added. Following an overnight incubation in a CO2 incubator at 37 degrees C, and growth at 26 degrees C, parasites were assessed at 3-, 6- and 24-hour intervals for changes in their developmental forms. Results: The 1:100 dilution of anti-LPG MABs when mixed with amastigotes was effective in reducing L. major development at the early log phase or procyclic stage both in vitro and within the sand fly (p<0.05). The control cultures or sand flies that fed on amastigotes alone and without MABS supported full parasite development up to the metacyclic stage. Sand flies that fed on MABs showed low parasitaemia levels of 2+, compared to a high density of 4+ for their controls (p<0.5). Conclusions: These findings showed that anti-LPG MABs were effective in reducing sand fly infections. This study also showed that P. duboscqi midgut lysates and the proteins present in L. major-derived LPG share two common proteins of molecular weights 105 kDa and 106 kDa. Further analysis of these individual proteins from the midgut should be studied to determine their vaccine potential.