CHARACTERIZATION OF NATIVE PATHOGENIC ANTIGENS OF ONCHOCERCA-VOLVULUS- IDENTIFICATION OF HIGH-MOLECULAR-MASS PROTEIN ANTIGENS ELICITING INTERSTITIAL KERATITIS IN A GUINEA-PIG MODEL

Sclerosing keratitis is the predominant cause of blindness due to onch ocerciasis which is a major human parasitic disease caused by the fila rial parasite Onchocerca volvulus. In the present investigation, nativ e pathogenic antigens of O. volvulus which are particularly potent in causing interstitial keratitis were characterized utilizing a guinea p ig model, Following demonstration of the protein nature of these antig ens using pronase digestion, the crude O. volvulus antigen extract was subjected to stepwise procedures of protein purification. At each sta ge of purification, pooled antigen fractions were injected into one co rnea of presensitized guinea pigs followed by clinical evaluation of s tromal inflammation and vascularization at different intervals of lime after intrastromal challenge. Initial purification of the pathogenic antigens was carried out in the following order: molecular sieve chrom atography on Bio-gel A-5m, anion exchange chromatography on Mono Q fol lowed by DEAE-Sepharose CL-GB and cation exchange chromatography on Mo no S. Two out of six different pools from the Mono S column (pool a el uted unbound at 10 mM-NaCl and pool e eluted between 130 mM and 475 mM -NaCl) were found to be most pathogenic, Further purification of Mono S pool a and pool e separately by gel filtration chromatography using Superose 12 demonstrated that the fractions which were most potent in inducing interstitial keratitis contained proteins with approximate mo lecular masses between 100 and 200 kDa. These results show that minor subfractions of total crude antigens of O. volvulus are largely respon sible for induction of experimental interstitial keratitis. We have de monstrated the presence of these antigens in O. volvulus microfilariae by their cross-reactivities with anti-microfilarial antibodies, and h ence the relevance of the purified antigens to ocular onchocerciasis i n man since sclerosing keratitis is associated with invasion of the co rnea by O. volvulus microfilariae. Isolation of these two pathogenic a ntigen pools represents the practical limits of purification and subse quent animal experiments possible with the available amounts of native parasite material obtained from infected human individuals in the abs ence of a suitable non-human host or of an in vitro culture system for O. volvulus.