Scand. J. Immunol.
3 6 , S u p p l . I I , 57 6 1 . W 9 2
Cell-Mediated and Monoclonal AntibodyDependent Killing oi Onchocerca volvulus Microfilariae* V. p. K. TITANJI. P. N. NDE & W. F. MBACHAM The Biotechnology Center and Department of Biochemistry. Faculty of Science. Yaounde University, Yaounde. Cameroon
Titanji VPK, Ndc PN, Mbactiam Wl". Cell-MediHti.*d Hud Monoclonal Antibody-Dependent Killing oXOmhovcrta volvulus Microfilariae. Scand J Immunol l992:.^6(Suppl.l l):57 61 To idenlily the target antigens implicated in the adhfrcncc and killing of mifrolilahaL" (mr) by leucocytes. Me mcubated nodular mTund teuctes in ihe presence otanti-vuticukirmontvlonal aniibodie!i(M()Vs)and Tresh scrum. Leucocvledonors were patlenls with a mean age of .^7 years (withO I mr snip), who had lived in thcendemicvillagcstudied rural least IOyear« AHer 16 20h of incubation, up to 74".. of the mf could be seen wiih 10 or more cells adhering to them. By .16 4 0 h up lo 54",I of the mf had been killed hy Ihc icuoKyies in the pa-scncc ofa cocktail of moniKlonal antibtnlies termed MOV GI V. The degree of killing in tonirol exficnments with Ihe monoclonal antibody MOV I remained lower (A'\ und/or onchocercal anti);en in the urine [6].
57
58
111 lino Killing ol O. volvulus mf (comprising MOV 25, 29, 30 and M), up to 74% of ihe mf could be seen wilh 10 or more cells adhering to them. Some of ihc mf were covered with large ckistcts of" cells, while others were strclchcd. sluggish or itiimobili
MOVl
Ul
^
mtm 1
MOV GIV
NMS
MOVl
MOV GIV
FKJ. 2. CcHular adhcrcriee and killing of O. vohulm microfilariae. O. Cyioadherence of > 10 cells; • . dead mf (sireiched and immotile): MOV, monoclonal antibodies raised lo culicular anli(!cns of O. vohulus females; NMS. normal mouse scrum
59
60
In Vitro Killing ofO. volvulus mf Effect of complement on cellular adherence and killing of Otv:hocerca votvufus mrcrofilanae 100
MOV-1 25
29
30
GIV
MOV-1 25
29
30
32
GIV
FIG. 3. Eflect of complement on cellular adherence and killing of O. volvulux microlilariae. O. Cytoadherence of > 10 cells (40 44 h). • . dead mf (40 44 h); MOV. monoclonal antibodies lo culicular antigens of O. volculiu females; Comp.. complement.
Stages by immune cells have been demonstrated previously in the presence of polycloiia! anlibody (I. 2. 11. 13] and mont>clonal antibixlies [12] but the anligens involved in these reactions were not identified. This investigation identifies some of the onchocercal antigens ihat mediate mf clearance by Immune cells. Earlier we had postulated that cuticular antigens would play an important role in host-parasilc intcraetions [8]. Further experiments to determine the protective role of the identified antigens are required.
ACKNOWLEDGMENTS This investigation received fmyncial support from the United States Agency for International Development, the International Science Program, Uppsala. Sweden and Yaounde University. Cameroon. W.F.M. is indebted to the TDR/ WHO lor a studentship (No. TIM81/M8/M99). We ihank Drs O. Zelterqvisl. K. Rubin and R. Holmdahl. Department of Medical and Physiological Chemistry. Uppsala University. Sweden, for
p. K. Titanji er al.
61
REKERENCES IDENTIFICXnON OF T A H Q E T
ANTIQEN8 BY
25 29 30 32 FIG. 4. Identification of target antigens of MOVs. Ref.. reference proteins (Pharmacia. Uppsala. Sweden); N. normal mouse serum; F. front; MOVs {1. 25. 29.30 and 321, monoclonal antibodies to cucicular antigens of O. volvulu.s females.
facilities lo prepare monoclonal antibodies. Drs R. Tata. F. Soh and J. Ticha for examining patients and performing nodulcctomics, and Mr T. Pogonka for adviee on cell isolation.
1 Brattig NW, Tischcndorf FW, Strote G. Mcdina-de la Garza CE. Eosinophil-larval interaction in onchocerciasis: Heterogeneity of in vitro adherence of eosinophils lo mleciive third and fourth stage larvae and microliluriac of Onchocerva volvulus. Parasil Immunol I99I;I3:I3 22. 2 Greene BM. Taylor HR. Aikawa M. Cellular killing of microfilariac of Ondmcerca tolrulu.\ Eosinophils and neulrophils mediated serum-dependent destruciion. J Immunol I98I;I27;I611-18. 3 Bultner DW. Racz P. Macro- and microfilariae in nodules from onchocerciasis palicnts in ihe Yemen Arab Republic. Tropenmed Parasil 19S3;34:113 21. 4 Holmdahl R. Moran T, Anderson M. A rapid and etlieient immunization protocol for ihe production of monoclonal aniibiHlies reactive wiih autoantibodies. J Immunol Meih 1985:83:379 84. 5 Mbacham WF. Rubin K. Holmdahl R. Tilanji VPK. Thumberg L. Laurent T. Characterisalion of Ihe cuticle of Omiuxena tolvultts wilh monoclonal antibodies. Excerpt a Medica I988;81O:61. 6 Mbacham WF. Surface properties of the developmental stages of OmhnciTca VOIVUIU.K. Thesis. Biochemistry E>eparlmcnl. University of Yaounde. 1989, 7 Tilanji VPK. Mbaeham WF. Leke GRF. Lectinbinding studies of Onchocena volvulus development stages. Ann Univ Sc Same I987;4:396-4OI. 8 Titanji VPK. Mbaeham WF. Sakwe A. Idcntifieation of different radiolahelled antigens of the developmental stages of Onchoi-ena volvulus. Acta Trop I99O;47;307-2L 9 Trager W. Cultivation of erythroeytic stages of malaria. WHO,TDR Series I98O;3;3 14. 10 HsuS-M. RaineL. FangerHj. Useofavidin biotin complex (ABC) in immunopcroxidase techniques: A comparison between ABC and unlabelled antibody PAP procedures. J Histochem Cytoehcm 1981; 29:577-84. 11 Leke RGF. Boto W. Lando G. Ngu JL. Immunity to O. volvulus: Scrum mediated leukocyte adherence to infective larvae in vitro. Trop Med Parasit 1989:40:39 41. 12 Tan MAJA. Mak JW. Yong HS. In iifw activity of some monoclonal antibodies against Brugia malayi microfilariae and Infective larvae. Trop Med Parasit 1989;40:3I7-2l. 13 Williams JF. Ghalib H W. Mackenzie CD. Elkhalifa MY. Ayuya JM. Kron MA. Cell adherence lo microfilariae of
3 6 , S u p p l . I I , 57 6 1 . W 9 2
Cell-Mediated and Monoclonal AntibodyDependent Killing oi Onchocerca volvulus Microfilariae* V. p. K. TITANJI. P. N. NDE & W. F. MBACHAM The Biotechnology Center and Department of Biochemistry. Faculty of Science. Yaounde University, Yaounde. Cameroon
Titanji VPK, Ndc PN, Mbactiam Wl". Cell-MediHti.*d Hud Monoclonal Antibody-Dependent Killing oXOmhovcrta volvulus Microfilariae. Scand J Immunol l992:.^6(Suppl.l l):57 61 To idenlily the target antigens implicated in the adhfrcncc and killing of mifrolilahaL" (mr) by leucocytes. Me mcubated nodular mTund teuctes in ihe presence otanti-vuticukirmontvlonal aniibodie!i(M()Vs)and Tresh scrum. Leucocvledonors were patlenls with a mean age of .^7 years (withO I mr snip), who had lived in thcendemicvillagcstudied rural least IOyear« AHer 16 20h of incubation, up to 74".. of the mf could be seen wiih 10 or more cells adhering to them. By .16 4 0 h up lo 54",I of the mf had been killed hy Ihc icuoKyies in the pa-scncc ofa cocktail of moniKlonal antibtnlies termed MOV GI V. The degree of killing in tonirol exficnments with Ihe monoclonal antibody MOV I remained lower (A'\ und/or onchocercal anti);en in the urine [6].
57
58
111 lino Killing ol O. volvulus mf (comprising MOV 25, 29, 30 and M), up to 74% of ihe mf could be seen wilh 10 or more cells adhering to them. Some of ihc mf were covered with large ckistcts of" cells, while others were strclchcd. sluggish or itiimobili
MOVl
Ul
^
mtm 1
MOV GIV
NMS
MOVl
MOV GIV
FKJ. 2. CcHular adhcrcriee and killing of O. vohulm microfilariae. O. Cyioadherence of > 10 cells; • . dead mf (sireiched and immotile): MOV, monoclonal antibodies raised lo culicular anli(!cns of O. vohulus females; NMS. normal mouse scrum
59
60
In Vitro Killing ofO. volvulus mf Effect of complement on cellular adherence and killing of Otv:hocerca votvufus mrcrofilanae 100
MOV-1 25
29
30
GIV
MOV-1 25
29
30
32
GIV
FIG. 3. Eflect of complement on cellular adherence and killing of O. volvulux microlilariae. O. Cytoadherence of > 10 cells (40 44 h). • . dead mf (40 44 h); MOV. monoclonal antibodies lo culicular antigens of O. volculiu females; Comp.. complement.
Stages by immune cells have been demonstrated previously in the presence of polycloiia! anlibody (I. 2. 11. 13] and mont>clonal antibixlies [12] but the anligens involved in these reactions were not identified. This investigation identifies some of the onchocercal antigens ihat mediate mf clearance by Immune cells. Earlier we had postulated that cuticular antigens would play an important role in host-parasilc intcraetions [8]. Further experiments to determine the protective role of the identified antigens are required.
ACKNOWLEDGMENTS This investigation received fmyncial support from the United States Agency for International Development, the International Science Program, Uppsala. Sweden and Yaounde University. Cameroon. W.F.M. is indebted to the TDR/ WHO lor a studentship (No. TIM81/M8/M99). We ihank Drs O. Zelterqvisl. K. Rubin and R. Holmdahl. Department of Medical and Physiological Chemistry. Uppsala University. Sweden, for
p. K. Titanji er al.
61
REKERENCES IDENTIFICXnON OF T A H Q E T
ANTIQEN8 BY
25 29 30 32 FIG. 4. Identification of target antigens of MOVs. Ref.. reference proteins (Pharmacia. Uppsala. Sweden); N. normal mouse serum; F. front; MOVs {1. 25. 29.30 and 321, monoclonal antibodies to cucicular antigens of O. volvulu.s females.
facilities lo prepare monoclonal antibodies. Drs R. Tata. F. Soh and J. Ticha for examining patients and performing nodulcctomics, and Mr T. Pogonka for adviee on cell isolation.
1 Brattig NW, Tischcndorf FW, Strote G. Mcdina-de la Garza CE. Eosinophil-larval interaction in onchocerciasis: Heterogeneity of in vitro adherence of eosinophils lo mleciive third and fourth stage larvae and microliluriac of Onchocerva volvulus. Parasil Immunol I99I;I3:I3 22. 2 Greene BM. Taylor HR. Aikawa M. Cellular killing of microfilariac of Ondmcerca tolrulu.\ Eosinophils and neulrophils mediated serum-dependent destruciion. J Immunol I98I;I27;I611-18. 3 Bultner DW. Racz P. Macro- and microfilariae in nodules from onchocerciasis palicnts in ihe Yemen Arab Republic. Tropenmed Parasil 19S3;34:113 21. 4 Holmdahl R. Moran T, Anderson M. A rapid and etlieient immunization protocol for ihe production of monoclonal aniibiHlies reactive wiih autoantibodies. J Immunol Meih 1985:83:379 84. 5 Mbacham WF. Rubin K. Holmdahl R. Tilanji VPK. Thumberg L. Laurent T. Characterisalion of Ihe cuticle of Omiuxena tolvultts wilh monoclonal antibodies. Excerpt a Medica I988;81O:61. 6 Mbacham WF. Surface properties of the developmental stages of OmhnciTca VOIVUIU.K. Thesis. Biochemistry E>eparlmcnl. University of Yaounde. 1989, 7 Tilanji VPK. Mbaeham WF. Leke GRF. Lectinbinding studies of Onchocena volvulus development stages. Ann Univ Sc Same I987;4:396-4OI. 8 Titanji VPK. Mbaeham WF. Sakwe A. Idcntifieation of different radiolahelled antigens of the developmental stages of Onchoi-ena volvulus. Acta Trop I99O;47;307-2L 9 Trager W. Cultivation of erythroeytic stages of malaria. WHO,TDR Series I98O;3;3 14. 10 HsuS-M. RaineL. FangerHj. Useofavidin biotin complex (ABC) in immunopcroxidase techniques: A comparison between ABC and unlabelled antibody PAP procedures. J Histochem Cytoehcm 1981; 29:577-84. 11 Leke RGF. Boto W. Lando G. Ngu JL. Immunity to O. volvulus: Scrum mediated leukocyte adherence to infective larvae in vitro. Trop Med Parasit 1989:40:39 41. 12 Tan MAJA. Mak JW. Yong HS. In iifw activity of some monoclonal antibodies against Brugia malayi microfilariae and Infective larvae. Trop Med Parasit 1989;40:3I7-2l. 13 Williams JF. Ghalib H W. Mackenzie CD. Elkhalifa MY. Ayuya JM. Kron MA. Cell adherence lo microfilariae of
