Use of recombinant Onchocerca volvulus antigens for diagnosis and surveillance of human onchocerciasis

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1 ~ VOLUML 1 NO. 5 PP OCTOBER 1996 Use of recombinant Onchocerca volvulus antigens for diagnosis and surveillance of human onchocerciasis R. Chandrashekar', A. F. Ogunrinade' and G. J. Weil' ' Departments of Medicine and Molecular Microbiology, Washington University School of Medicine arid The Icioisb Hospital of St Louis, St Louis, Missouri , USA Department of Veterinary Mzcrobiology and Parasatology, University of Ibadan, Ihadan, Nigeria Summary The diagnostic value of ELISAs based on recombinant Onchocerca volvulus antigens OC 3.6 and OC 9.3 was evaluated with sera from endemic areas in West Africa, Guatemala and Ecuador. IgG assays were slightly more sensitive than those that detected IgG,, and the antigen combination was significantly more sensitive than either antigen alone (OC 3.h, 93%; OC 9.3, 847'0, combined 98%). These assays were also evaluated with sera from 2 villages in the Onchocerciasis Control Programme area of West Africa including one village (Pendie) with recent recrudescence of infection and one (Niarba) where transmission had been interrupted for 15 years by vector control. The OC 3.6 IgG antibody assay was sensitive for new infections and exposure in Pendie; 24/24 (roo%) of people with positive skin snips and 15/74 (20%) of sera from MF negative people had IgG antibodies to this antigen. In addition, antibodies to OC 3.6 often preceded the onset of skin snip positivity in Pendie. In contrast, IgG antibodies to OC 3.6 and OC 9.3 were rarely seen in children born during the 15 years since transmission was interrupted by vector control in Niarba. These encouraging results suggest that antibody assays based on OC 3.6 and OC 9.3 may be valuable tools for surveillance of onchocerciasis and also for monitoring the efficacy of control programmes. keywords recombinant antigen, onchocerciasis, Onchocerca, diagnosis, surveillance, ELISA correspondence G. J. Weil, Infectious Diseases Division, Barnes-Jewish Hospital at Washington University Medical Center, 216 S. Kingshighway, St Louis, Missouri 63 I I o, USA Introduction Onchocerciasis, a disease caused by the filarial nematode Onchocerca volvulus, is an important cause of blindness and severe dermatitis in sub-saharan Africa and in Latin America (WHO 1976). At present, diagnosis of human onchocerciasis depends primarily on the demonstration of microfilariae in skin snips (WHO 1976). However, skin snip examination is not sensitive for detection of early infections or for diagnosis of individuals with low microfilaria densities in the skin (Prost 1980; Taylor et nl. r989). Further, the skin snip procedure is inconvenient, the instruments used to obtain snips are expensive and, as they are not disposable, special care must be taken to ensure that blood-borne viruses are not transmitted in field surveys. Recently, a sensitive method has been developed for detecting 0. volvulus DNA: parasite DNA in skin snips is amplified by the polymerase chain reaction, and the product is detected by ELISA (Nutman et al. I 994; Zimmerman et nl. 1994). Leaving aside practical issues such as field applicability and cost, this approach is not likely to gain wide acceptance for Blackwell Science Ltd

2 ~ ~ ~ VOLUME I NO. 5 PI OCTOB~K ~- -. R Chandrashekar et al. Recombinant 0. volvulus antigens for surveillance of onchocerciasis primary surveillance, because the method still requires collection of skin snips. Antibody-based diagnostic tests for onchocerciasis are potentially useful as tools for identifying early infections, for use in epidemiological surveys, and also for monitoring the efficacy of control programmes (Greene 1992; Ramachandran 1993; Egwang et al. 1994). Preliminary reports from our laboratory showed that antibody assays based on antigens encoded by cdna clones OC 3.6 and OC 9.3 are sensitive and specific for diagnosis of onchocerciasis in humans (Chandrashekar et a/. 1991; Ogunrinade et al. 1993). OC 3.6 encodes an 0. volvulus antigen that is similar to the protein product of Ov33 which is believed to be a pepsin inhibitor (Chandrashekar et al. 1991; Willenbucher et al. 1993; Tawill et al. 1995). OC 9.3 encodes a I 5 kda native antigen; it is closely related to OV7 which encodes an 0. volvulus cystatin (Lustigman et al. 1991). Prior studies have shown that these proteins are present in developing larvae and adult worms of 0. volvu/us and that antibodies to these antigens can develop long before the onset of microfilarial patency (Lucius et al. 1988; Lustigman et al. 1991; Chandrashekar et al. 1995). The goals of the present study were to attempt to confirm the diagnostic value of antibody assays based on these antigens, singly and combined, with sera from several endemic areas in Latin America and Africa, and to demonstrate the value of antibody serology as a means of detecting onchocerciasis transmission (persistence or recrudescence) in areas subject to vector control. Materials and methods Human sera Nigerian sera were collected from people who reside in villages near New Bussa that are mesoendemic for savannah-type onchocerciasis (Chandrashekar et al. 1990; 1993 ). Guatemalan sera collected from residents of coffee plantations in the Lake Atitlan endemic area were kindly provided by Dr R. Lujan. Ecuadoran sera collected from an endemic area of Esmeraldas Province were provided by Dr T. Nutman (Elson et al. 1994). The diagnosis of onchocerciasis was based on clinical examination for subcutaneous nodules or onchocercal dermatitis and examination of at least four skin snips for the presence of microfilariae as previously described (Chandrashekar et al. 1990; 1993). Sera from z villages in the Onchocerciasis Control Programme (OCP) area in West Africa were provided by the WHO filariasis serum bank (Dr N. Weiss, Basel). These included I I I sera collected in 1989 from Pendie, a village in West Africa in the OCP with documented reinvasion or recrudescence of onchocerciasis, and 87 sera from Niarba, a village in Burkina Faso where vector control by OCP had interrupted transmission of 0. volvulus for I 5 years. Lymphatic filariasis sera for specificity testing were collected in South India. Recombinant antigens and antibody assay Recombinant 0. volvulus antigens OC 3.6 and OC 9.3 expressed as glutathione-s-transferase (GST) fusion proteins and purified by affinity chromatography (Ogunrinade et al j were used in the present study. An indirect ELISA, essentially as described by Ogunrinade et al. (1993), was used to measure IgG and IgG, antibodies to OC 3.6 and OC 9.3-GST fusion proteins in sera from patients. Briefly, recombinant antigens OC 3.6, OC 9.3, or both OC 3.6 and OC 9.3 and the control antigen GST (100 Fl/well; 1.0 pg/ml in 0.06 M carbonate buffer, ph 9.6) were incubated in polyvinyl microtitre plates (Dynatech Laboratories, Alexandria, Virginia, USA) overnight at 37T. Plates were washed 3 times with PBS containing o.o5%0 Tween 20 (Sigma Chemical Co., St Louis, Missouri, USA) (PBS/T) and blocked with PBS/T containing 5% fetal calf serum (PBST/FCS) for I hour at 37T. Sera diluted 1:300 in PBS/T/FCS were added to duplicate wells and incubated for 2 hours at 37T. Plates were washed 3 times with PBS/T. IgG antibody binding was detected with peroxidase-conjugated goat antihuman IgG antibody (Organon Teknika-Cappel, Malvern, Pennsylvania, USA) in PBS/T/FCS. After I hour incubation at 37X, the plates were washed and substrate was added (0-phenylenediamine (Eastman Kodak, Rochester, New York, USA) with H,O,). The enzyme reaction was stopped after 10 minutes at room temperature with 4 M H,SO,. Optical density (OD) was read versus a PBS blank at 490 nm Blackwell Science Lrd

3 ~~~~~~~~~ -~ ~~~~ ~~~ - VOLUMt 1 NO 5 1'1' OLIOBlR 1996 ~ R. Chandrashekar et ol. Recombinant 0. volvulus antigens for surveillance of onchocerciasis Table I Comparative sensitivities of IgG and IgG, ELISAs for antibodies to recornbinant antigens in sera from onchocerciasis patients Serum source Nigerian children Nigerian adults Guatemalan adults Ecuadoran adults Total no. positivelno. tested (Yo positive) "Difference between IgG and IgG, significant by xl Table 2 IgC and IgG, antibody reactivities to recombinant 0. volvulus antigens in sera from onchocerciasis patients from Pendie, a West African village with recent recrudescence of infection No. of qerurn specimens po\itive/no. tested (% positive) oc 3.6 oc 9.3 Serum source ~~~ IgG WJ, IgG IgG, ' These subjects had negative skin snips when sera were collected in 1989 and became skin snip positive either in May 1990 or November with an EL 3 I ze ELISA reader (Bio-Tek Instruments, Winooski, Vermont, USA). Background OD obtained with GST was subtracted to determine net OD, a measure of specific antibody reactivity. As quality controls, positive and negative control sera were tested on each plate. Serum samples were coded and tested blindly. Sera which produced net OD values greater than the mean +3 SD of values obtained with a panel of 20 serum specimens from adult residents of St Louis, Missouri, USA, were considered positive. IgG, antibodies to recombinant 0. volvulus antigens were detected as described above, except the serum dilution tested was I:IOO and IgC, antibody reactivity was detected with horse-radish peroxidase- conjugated monoclonal antibody HP6023, which binds to the Fc portion of human IgC, (Reimer ct ul. 1984). Results Sensitivity of IgG and IgG, ELISAs for antibodies to recombinant antigens in sera from onchocerciasis patients A total of 201 serum specimens from patients with onchocerciask were tested for IgG and IgG, aiitibody reactivity to OC 3.6, OC 9.3 and to combined antigens (OC 3.6+0C 9.3) by enzyme imniunoassay (Table I). Similar results were obtained with sera Blackwell Science Ltd

4 ~ ~ R Chandrashekar et of Recombinant 0. volvulus antigens for surveillance of onchocerciasis VOIUME I NO. 5 I P 57~-58o OCIORIR iyqh ~ ~ ~ _ rca) I , I I O L a Age Figure I lgg antibodies (net OD) to recornbinant antigens a, OC: 3.6 and h, OC: y.3 are shown by age for residents of Niarha, a West African village where Omhocercu volvulus transmission had lxen interrupted for I j years by 0C:P vector control. Cut-off values for a positive antibody test (mean +3 SD obtained with a panel of sera from a non-endemic area) arc shown with horizontal broken lines. Points to the left of the vertical broken line represent net 01) values obtained with sera from children under TS years of age. a patient with brugian filariasis) had borderline positive IgG reactivity with the antigen. Antibodies to recombinant antigens in sera from onchocerciasis patients from vector controlled areas OC 3.6 and OC 9.3 were evaluated with sera from z villages in areas subject to vector control by OCP. One of these villages (Pendie) was known to have experienced a recrudescence of onchocerciasis in 1989 when the sera were collected. All 24 sera from subjects in Pendie with positive skin snips had positive OC 3.6 IgG antibody assays (Table 2, Group 111). Nine of 73 subjects (69%) with negative skin snips in 1989 who became skin-snip positive in iyyo had antibodies to OC 3.6 in 1989 (Table 2, Group 11). Thus, the OC 3.6 IgG assay was sensitive for detecting prepatent, existing and new infections in an area with recrudescent onchocerciasis. We also evaluated these antibody assays with sera from a second OCP village (Niarba) where onchocerciasis transmission was documented to have been interrupted by vector control for I 5 years prior to collection of sera. Antibodies to OC 3.6 and OC 9.3 were still present in many people who lived in the village before transmission was interrupted by OCP. However, antibodies to these antigens were almost entirely absent from children horn during the 15 years of vector control in Niarba (Figure 1 ). from Nigeria, Guatemala and Ecuador. IgG antibody assays were somewhat more sensitive than those which detected IgG,. This difference was most obvious with sera from Nigerian children (8-14 years of age) (Table I). Prior studies have shown that OC 3.6 and OC 9.3 are specific for onchocerciasis; sera from patients with lymphatic filariasis, loiasis and dracunculiasis and schistosomiasis are not reactive with these antigens (Chandrashekar et al. 1997; Ogunrinade et al. I 993). Additional specificity tests were performed with the combined OC 3.d9.3 antigen in this study. Excellent specificity was again observed. None of 20 sera from Indian patients with lymphatic filariasis ( I o with Wuchereria bancrofti and TO with Brugia malayi) had IgC, antibodies to the antigen combination. One of 20 lymphatic filariasis sera (from Discussion Early attempts to develop antibody diagnostic tests for onchocerciasis relied upon crude or partially purified native antigens which are cross-reactive with antigens from other nematode species (Ambroise- Thomas 1980). Several groups have reported that the specificity of such antibody tests can be improved by using a low molecular weight antigen fraction (Cabrera & Parkhouse r987; Weiss tcc Karam 1989) or by detecting antibodies of isotypes IgG, or IgE (Weiss et al. 1982; Weil et al. 1990; Egwang et al. 1994). Despite these refinements, none of the assays based on native antigens has satisfactory sensitivity and specificity. Prior studies from our laboratory have shown that antibody assays based on recombinant antigens OC 3.6 and OC y.3 are sensitive and specific for diagnosis of onchocerciasis 578 c 1996 Blackwell Science Lcd

5 ~~ ~~ VOIUMt 1 NO OLrOBFK 1996 R. Chandrashekar et ol Recombinant 0. volvulus antigens for surveillance of onchocerciasis in humans (Chandrashekar et al. 1991; Ogunrinade et al. 1993). In the present study, we evaluated these antigens with a large panel of human onchocerciasis sera from several endemic areas and from villages within the Onchocerciasis Control Programme of the World Health Organization. OC 3.6 and OC 9.3 were tested singly and in combination with sera from different endemic areas. The best results were obtained by detecting IgG antibodies to the antigen combination, and this was true for sera from all of the endemic areas tested. Bradley et al. (1993) reported similar results with a cocktail of 3 recombinant antigens (OVMBP 10, IT, 12). We and others have previously reported that measurement of IgG, antibodies to 0. volvulus antigens resulted in improved diagnostic sensitivity and specificity for onchocerciasis (Weil et al. 1990; Ogunrinade et al. 1993; Egwang et al. 1994). However, in the present study, IgG assays were more sensitive than IgG, assays. This difference was most obvious with sera from Nigerian children. IgG, antibodies are believed to be produced in response to chronic antigenic stimuli (Hamilton 1987). However, IgG4 anti bodies develop relatively slowly in children; adult levels of IgG, are not achieved in North American children until they reach 9-11 years of age (Schur et al. 1979). This may explain the relatively poor sensitivity of OC IgG, antibody assays with sera from Nigerian children. Results obtained with sera from villages in the OCP suggest that antibody assays based on OC 3.6 and OC 9.3 may be very useful for monitoring the effect of vector control programmes on onchocerciasis transmission. The results showed that the OC 3.6 IgG assay was sensitive for the detection of existing and new infections in an African village with documented recent recrudescence of onchocerciasis. Interestingly, most skin snip converters had IgG antibodies to OC months before microfilariae were detected in skin snips (69%). It is not surprising that some prepatent infections were not detected. Early prepatent infections were probably missed, since antibodies to OC 3.6 are first detected 4-6 months after infection in chimpanzees (Ogunrinade et al. 1993; Chandrashekar et al. 1995). IgG antibodies to OC 3.6 and OC 9.3 were almost uniformly absent from children born in an OCP village after 0. volvulus transmission was interrupted by vector control. However, several children below 15 years of age had positive IgG antibody tests with each of the recornbinant antigens. Antibodies in these children may be due to suhclinical infections which occurred even though transmission was believed to have been completely interrupted in the village by vector control. Alternatively, these children may have been exposed or infected in a different area with ongoing transmission during some part of their childhood. Finally, we cannot exclude the possibility that these were false positive results. Additional follow-up studies were not performed on these children. In conclusion, serodiagnostic assays based on OC 3.6 and OC 9.3 appear to be quite useful for diagnosis of onchocerciasis infections in different endemic zones. In addition, our results suggest that antibody serology may be a useful tool for detecting reinvasion or recrudescence of onchocerciasis in previously controlled areas. Acknowledgements This work was supported by National Institutes of Health grant A and the Onchocerciasis Control Programme in West Africa-UNDPAX/orld Bank/ WHO Special Program for Research and Training in Tropical Diseases, Macrofil Chemotherapy Project. Dr A. F. Ogunrinade was supported in part by a Biotechnology Research Fellowship from the Rockefeller Foundation. We wish to thank Drs Thomas B. Nutman and Ricardo Lujan for providing onchocerciasis sera collected from Ecuador and Guatemala, respectively. We also would like to thank the OCP and the TDR Filariasis Serum Bank (Dr N. Weiss, curator) for providing sera from Niarba and Pendie villages. Excellent technical assistance was provided by Mrs Fanya Liftis and Kurt Curtis. References Ambroise-Thomas P (I 980) Filariasis. In Ininircizolo~ic.cil lnvestigations of Tropical Parasitic Diseases (cd V Houba), Churchill-Livingstone, Edinburgh, pp Bradley JE, Trenholme KR, Gillespie AJ et rrl (I 993) A sensitive serodiagnostic test for onchocerciasis using a cocktail of recombinant antigens. Amrricaii /ournu1 of Tropical Medicine and Hygiene 48, I Blackwell Science Ltd

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