Effect of ivermection prophylaxis on antibody responses to Onchocerca volvulus recombinant antigens in experimentally infected chimpanzees

0020-7519(95)OW11-9

R. CHANDRASHEKAR,*Q

B. VAN SWINDEREN,* and G. J. WEIL*t

H. R. TAYLOR4

Departments of *Internal Medicine and tA4olecular Microbiology, Washington University School of Medicine, St Louis, MS63110, U.S.A. EjDepartment of Ophthalmology, The Royal Victorian Eye and Ear Hospital, University of Melbourne, Victoria 3002, Australia (Received

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25 November

1994; accepted

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Key words:

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. b&motiouol Jm*nal of Parusit&gy c-hlekerea

31 January

Onchocerca

of the later outcome

of the infecth.

volvulus; ivermectin; infective larvae; recombinant antigens; ELISA; chimpanzee.

1987). However, skin snip examination is not sensitive for detection of early infections or for diagnosis of individuals with low micr&laria Latin America (World Health Organization, 1987). densitiesin the skin (Prost, 1980;Taylor, Munoz, At present. diagnosis of human onchocerciasis Keyvan-Larijani & Greene, 1989).Further, the skin dependsprimarily on the demonstration of micro- snip procedureis inconvenient, the instrumentsused filariae in skin snips (World Health Organization, to obtain snipsare expensiveand, as they are not disposable,specialcaremust be taken to ensurethat $To whom correspondeuce. should be addressed at: blood-borne viruses are not transmitted in field surveys. Recently, sensitive methods have been Infectious Diseases Division, Jewish Hospital, Washington developed for detecting 0. v&ulus microfilrriae and University Medical Center, 216 S. Kingshighway, St Louis, DNA by PCR (Nutman, Zimmerman, Kubofcik & MO 63110, U.S.A. INTRODUCTION

Onchocerciasis is an important cause of blindness and severe dermatitis in sub-Saharan Africa and in

983

984

R. Chandrashekar et al

Kostyu, 1994).Leaving asidepractical issuessuchas field applicability and cost, this approachis not likely to gain wide acceptance,becausethe method still requiresthe collection of skin snips.A practical and sensitiveantigen test would improve understanding of the epidemiologyof onchocerciasis, and would be potentially useful for monitoring the successof vector control efforts (Duke, 1990;Taylor, Pacque & Greene, 1990).Unfortunately, sucha test is not yet available. Antibody-based diagnostictestsfor onchocerciasis can be usefulastools for identifying early infections, for use in epidemiologicalsurveys, and also for monitoring efficacy of chemotherapeutic drugs (Greene, 1992; Ramachandran, 1993). However, antibody assaysfor onchocerciasis basedon mixtures of native antigens are hampered by scarcity of antigen, and they suffer from poor specificity (Ambroise-Thomas, 1980). In recent years several recombinant0. volvulusantigenshave beendescribed that appear to be more specific for antibody diagnosis of onchocerciasisthan native antigens (Garate, Conrath, Harnett, Buttner & Parkhouse, 1990; Lobos, Weiss, Karam, Taylor, Ottesen & Nutman, 1991;Bradley, Helm, Lahaise& Maizels, 1991; Chandrashekar, Masood, Alvarez, Ogunrinade, Lujan, Richards & Weil, 1991; Lustigman, Brotman, Huima & Prince, 1991; Ramachandran, 1993).Prior studiesfrom our laboratory have shown that antibody assays based on the recombinant antigensOC 3.6 and OC 9.3 are sensitiveand specific for diagnosisof onchocerciasisin humansand that theseantigensare alsoimmunogenicin chimpanzees (Chandrashekaret al., 1991;Ogunrinade, Chandrashekar,Eberhard& Weil, 1993).OC 3.6 is a 24 kDa antigen that is similar to the previously described 0~33 which codes for a major pepsin inhibitor (Chandrashekaret al., 1991;Willenbucher, Hofle & Lucius, 1993). OC 9.3 codes for a 15 kDa native antigen; it is closely related to OV7 which has been recently identified asan 0. vo/vuluscystatin (Lustigman et al., 1991). Important questionsremain regarding the interpretation of antibody serologyresultsobtained with theseantigens.For example,can suchtestsbe usedto specifically identify people with active, mature 0. volvulus infection and distinguishthem from people who have been exposed to the parasite without establishmentof mature infection? We also would like to know whether antibody testscan be usedto accurately monitor the successof preventive measures (drugs, vaccines, or vector control) and/or therapiesfor onchocerciasis.It is logistically difficult to answer these questionswith sera from humans who have been naturally exposedto the parasitein

uncontrolledconditions. Studieswith serafrom drug trials performedwith animalsmay allow usto answer someof thesequestions,and we were fortunate to have accessto an extensiveserumcollection from a prior study of ivermectinprophylaxis in chimpanzees (Taylor, Trpis, Cupp, Brotman, Newland, Soboslay & Greene,1988)for systematicevaluationof the effect of ivermectin prophylaxis on antibody responsesto OC 3.6 and OC 9.3. MATERIALS of chimpanzees.

AND

METHODS

The chimpanzees (Pan troglodytes) (age 4-7 years) used in the present study were all raised at the Liberian Institute for Biomedical Research, Liberia, West Africa. Each animal was inoculated S.C.with 250 third-stage infective larvae (Ls) of 0. volvulus (Cupp, Bernado, Kiszewski, Trpis & Taylor, 1988; Taylor et al., Infection

1988). Treatment

with ivermectin. Eighteen chimpanzees were divided into 3 groups of 6 animals each (Taylor et al., 1988). Group I was used to test the efficacy of ivermectin (Merck, Sharp & Dohme, U.S.A.) on Ls. These animals were treated with ivermectin by stomach tube (200 ug/kg body weight) on the same day that they were infected with 0. volvuZus. Group II was used to test efficacy of ivermectin on fourthstage larvae (Lb) and juvenile adult 0. volvulus. These animals were inoculated with living Ls on day 0 and treated with a single dose of ivermectin at 200 ug/kg 28 days postinfection. Control chimpanzees in group III were infected but not treated with ivermectin. Parasitological examination. Skin snips were obtained monthly as previously described (Taylor et al., 1988) starting 2 months before infection and continuing for 25 months. When skin snips were taken, blood was also collected, and sera were separated and stored at - 20°C. Antibody assay. An indirect ELISA essentially as described by Ogunrinade et al. (1993) was used to measure IgG antibodies to OC 3.6 and OC 9.3 glutathione-4 transferase (GST) fusion proteins in sera from infected chimpanzees. Briefly, recombinant antigens OC 3.6, OC 9.3, and the control antigen GST (100 pg/well; 1.0 pg/ml in 0.06M carbonate buffer, pH 9.6) were incubated in polyvinyl microtiter plates (Dynatech Laboratories, Alexandria, VA) overnight at 37°C. Plates were washed 3 times with PBS containing 0.05% Tween 20 (Sigma) (PBS/T) and blocked with PBS/T containing 5% fetal calf serum (PBS/T/FCS) for 1 h at 37°C. Sera diluted 1: 100 in PBS/T/FCS were added to duplicate wells and incubated for 2 h at 37°C. Plates were washed 3 times with PBS/T. IgG antibody binding was detected with peroxidase-conjugated goat antihuman IgG antibody (Organon Teknika-Capped, Malvern, PA) in PBS/T/FCS. After 1 h incubation at 37°C the plates were washed and substrate was added [o-phenylene-diamine (Eastman Kodak, Rochester, NY) with HzO& The enzyme reaction was stopped after 10 min at room temperature with 4 M HzS04. Optical density (O.D.) was read versus a PBS blank at 490 nm with a EL 312e Bio-Kinetics reader (BioTek Instruments, Winooski, WI). Background OD obtained with GST was subtracted to give net O.D., a measure of

0. volvulus infection in chimpanzees

45

speciIic antibody reactivity. As quality controls, positive and negative control sera were tested on each plate. Sera which produced net O.D. values greater than the mean+2 SD. of values obtained with pre-infection sera were considered positive for antibodies (0.35 cut off for OC 3.5; 0.15 for

oc 9.3). Statistical analysis. Data analysis was performed on a microcomputer with ABstat@ 7.04 software (Anderson-Bell,

Arvada,CO).The nonparametric Kruskal-Wallistestwas usedto determinethe significance of differences between groups. OOL-f

RESULTS Parasitological

observations

1

3

7 MONTHS

Nine of 18 chimpanzeesdeveloped patent infections (Taylor et al., 1988). The prepatent period varied from 12 to 28 months post-infection (p.i.). Only 1 of 6 animalsbecamemicrofiladexmiapositive when ivermectin was given on the day of infection (group I). Four of 6 animalstreated with ivermectin 28 days p.i. (group II), and 4 of 6 untreated animals (group III) developedpatent infections.Theseresults suggestthat ivermectin may have a partial prophylactic effect againstthe L3 of 0. voIvulusbut no effect

11

15

1 19

-i 23

POST-INFECTION

Fig. 2. Comparison of IgC; antibody reactivity to recomhinant 0. volvulu~ antigen 06 3.6 in chimpanzees with patent and non-patent infectitons. Each point represents

meanrtS.E. of O.D. values obtainedwith sera from chimpanzees in groups II and III. Mean net O.D. values were significantly higher in animals that developed patent infection. The difference was statistically significant at iY months p.i. (P = 0.027).

zees.Antibody levelsincreasedwith microfilarial (mf) patency, althoughin thoseanimalsthat neverbecame patent, meanantibody levelsremainedrelatively low throughout the period of observation (Fig. 2). Antibody levelsto OC 3.6 19monthsp.i. were higher Antibody responses to OC 3.6 and OC 9.3 in infected in animalsthat developedpatent infections(Fig. 2). chimpanzees Only 1 of 6 animals in group I had a strong Significant variability was observed in antibody responseswithin each group of animals. However, antibody responseto OC 9.3. Ironically, this animal analysisof results by group revealed a number of neverdevelopedmf patency.Three other animalshad interestingfindings. Antibodies to OC 3.6 developed borderline antibody responsesto this antigen during the prepatent period in all three groups (Table 1). In contrast, all 12 animalsin groups II (Fig. 1) and were detectedin all infected chimpan- and III producedantibodiesto OC 9.3. Antibodies to OC 9.3 tendedto developlater than antibodiesto OC 3.6, and they were usually first detectedaround the against 1988).

later stages of the parasite

(Taylor

et al.,

time of onset of mf patency

8 8 0.8 5

(Fig. 3 and Table

1).

Interestingly, several animals in groups II and III that never developedmf patency had early antibody responses to OC 9.3 which peaked7-15 months p.i. and then declined (Fig. 4). This pattern was quite different from that observedin animalswith patent infections (Fig. 4).

0.6

MSC’USSION As previously reported, ivermectin may have 0.2 prophylactic activity against0. volvulusin chimpanzees, but only if the drug is given on the day of 0.0 23 -1 3 7 11 15 19 infection. This suggeststhat ivermectin is active in MONTHS POST-INFECTION vivo againstL3 but not Ld of 0. volvulus(Taylor et al., 1988).The presentstudy examinedthe effectsof 1. IgG antibody reactivity to recombinant 0. volvulus Fig antigen OC 3.6 in sera from infected chimpanzees. Each ivermectin prophylaxis on humoral responses to the point represents mean+ S.E.of O.D. valuesobtainedwith recombinant0. volvulusantigensOC 3.6 and OC 9.3 serafrom 6 chimpanzees in each group. Group 1 (treated with ivermectin on day 0); groupII (treatedwith ivermectin in infected chimpanzeesOur prior studiesshowed that chimpanzeesdevelop antibodiesto OC 3.6 4--Ei on day 28 p.i.); group III (untreated controls). 0.4

R. Chandrashekar et al.

986

Table I-Timing

of onset of patency and of first antibody responses to recombinant Onchocerca and OC 9.3 in infected chimpanzees

Animal no.

Time of patency*

Antibody to OC 3.6

volvulus antigens OC 3.6

Antibody to OC 9.3

First detected?

Maximum net O.D.

First detected?

Maximum net O.D.

Group I 195 205 213 223 268 293

NPS NP NP NP NP 26

7 3 I I 11 I

0.715 1.556 0.559 0.685 1.308 1.220

NIL 23 19 17 7 NIL

0.119 0.281 0.245 0.226 1.695 0.028

Group II 177 181 222 227 267 294

14 17 17 19 NP NP

I 7 19 I 3

1.588 1.929 1.598 0.660 0.649 0.713

15 15 15 I 7 I

0.960 2.336 1.158 2.064 0.459 0.519

Group III 161 174 175 187 240 251

21 12 NP NP 28 17

7 7 3 I I 7

1.746 2.102 0.583 1.315 I.066 1.459

15 19 I 3 15 23

0.849 1.039 0.907 2.251 0.334 0.407

1

*Month during which the animals became patent for mf. tTiming (months post-infection) of first antibody response to recombinant antigen. $Never-patent. months after infection while antibodies to OC 9.3 usually develop around the time of onset of microfilarial patency (Ogunrinade et al., 1993). Results from the present study suggest that antibody reactivity of OC 3.6 in chimpanzees indicates early infection, irrespective of the later outcome of the

infection. Thus, OC 3.6 may not he useful for monitoring trials of prophylactic drugs or vaccines. The timing of antibody responses to OC 3.6 and OC 9.3 was different. In general, antibodies to OC 9.3 first appeared around the time of onset of microfilarial patency and increased uniformly after

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0.6

.

0.4

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0.2

0.2

0.0

0.0

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7

MONTHS

11

15

19

-

23

POST-INFECTION

Fig. 3. Antibodies to OC 9.3 in 0. volvulus-infected chimpanzees. Each point represents meanf S.E. of O.D. values obtained with sera from 6 chimpanzees in each group. Group I (treated with ivermectin on day 0); group II (treated with ivermectin on day 28 p.i.); group III (untreated controls). The net O.D. values (antibody levels) were significantly higher in groups II and III animals than those in Group I, 19 and 23 months p.i. (PcO.05).

MONTHS

POST-INFECTION

Fig. 4. Comparison of IgG antibody reactivity to recombinant 0. volvulus antigen Oc 9.3 in chimpanzees that did or did not develop patent infections. Each point represents meanfS.E. of O.D. values obtained with se.ra from chimpanzees from groups II and III. Antibody levels were significantly higher in never-patent animals 7 and 11 months after infection (JVO.05). Differences at other time points were not statistically significant.

0.

volvulus

infection in chimpanzees

patency, as observed in a prior study with sera from a different set of animals (Ogunrinade et al., 1993). This result is similar to that of Soboslay, Weiss, Dreweck, Taylor, Brotman, Shultz-Key & Greene (1992) who showed that serological reactivity of chimpanzeesto low-molecular weight 0. volvulus antigensincreased13 months p.i. and continued to rise with patency. In the presentstudy, only 1 of 6 animalsin group I (treated with ivermectin on the day of infection) had a strong antibody responseto OC 9.3. This result is consistentwith the conclusion that ivermectin has significant prophylactic activity againstthe LJ stageof 0. volvuIusin chimpanzees.In contrast, all 6 animals in group II produced antibodiesto OC 9.3. Again, this result is consistent with parasitological results, which suggestedthat ivermectin hasno prophylactic effect on Ld or early L5 stagesof the parasite.Theseresultssuggestthat, unlike OC 3.6, OC 9.3 may be of value in monitoring the efficacy of drugs or vaccinesfor prevention of onchocerciasis. Another interesting observation in the present study wasthe early antibody responseto OC 9.3 in 4 chimpanzeesin groups II and III that did not develop patent infection (Fig. 4). The samephenomenon was reported by Lustigman et al. (1991) with recombinantantigen OV7, which isclosely relatedto OC 9.3 and is present in all stagesof the parasite. One explanation for the early antibody responses to OC 9.3 in never-patentchimpanzeesmay be parasite death with premature exposureof internal antigens that are not normally exposeduntil patency. These animalsmay have clearedtheir infection late in the prepatent period. A second explanation for this observation is that a subset of animals develop persistent,non-patent0. volvulus infection and that a strongearly antibody responseto OC 9.3 is somehow related to this phenomenon. The present study has evaluated the effect of ivermectin prophylaxis and early treatment on antibody responses to OC 3.6 and OC 9.3 in 0. volvulusinfected chimpanzeeswith the hope that the results might also be applicable to humans. Additional studiesare neededto determinethe utility of these antigens for monitoring the efficacy of preventive measuresand therapiesfor onchocerciasisin animals and humans.

BetsyBrotmanand her staff at the LiberianInstituteof BiomedicalResearch, Robertsfred,Liberia,who handted the chimpanzees throughout this experiment.

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Immunabg~cal

of Tropical D&eases. (Edited by ffouba V.), pp. 84-103. Churchill-Living&one, Edinburgh. Bradley J. E., Helm R., Lahaise M. t M&&s R. M. 1991. cDNA clones of Onchocercu volvul~s low molecular weight antigens provide immunologically specific diagnostic probes. Molecular Biochemical Parasitology: 46: 219-228. Chandrashekar R., Masood K., Alvarez R. M., Ogunrinade A. F., Lujan R., Richards F. 0. & Weil G. J. 1991. Investigations

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and Hygiene

38: 596-600.

Duke B. 0. L. 1990. Onchocerciasis (river blindness)----can it be eradicated? Parasitology Today 6: 82-84. Garate T., Conrath F. J., Hamett W., Buttner DI W. Br Parkhouse R. M. E. 1990. Cloning of specific diagnostic antigens in onchocerciasis. Tropical Medicine and Farasirdogy

41: 245-250.

Greene B. M. 1992. Modern medicine versus an ancient scourge: progress toward control of onchocerciasis. Journal of” Infectious Diseases 166: 15-21. Lobos E. N., WeissN., KaramM., Taylor H. R., Ottesen E. A. & Nutman 1’. B. 1991. An immunogenic Onchocerca volvulus antigen: a specific and early marker of infection. Science 231: 1603-1605. Lust&man S., Brotman B..,Huima T. & Prince A. M. 1991. Characterization of an Onchocerca volvuius cDNA clone encoding a genus-specific antigen present in infective larvae and adult wonns. Molecular and Biochemical Parasitology

45: 65-16.

Nutman T. B., Zimmermau P. A. Kubofcik & Kostyu D. D. 1994. A universally applicable diagnostic approach to filarial and other infections. Parasitology Today IO: 239-243. Ogunrinade A. F., Chandrashekar R., Eberhard M. 1,. Br Weil G. J. 1993. Preliminary evaluation of recombinant Onchocerca volvulus antigens for serodiagnosis of onchocerciasis. Journal’ qf Clinical Microbiolo.gy 31: 1741-1745. Prost A. 1980. Latence parasitize dam l’onchocercose. Bulletin of the World Health Organization s& 923-925. Ramachandran C. P. 1993. Improved immunodiagnostic tests to monitor onchocerciasis control programmes-- -a Acknowledgements-This work wassupported by National multicenter effort. Parasitology Today 9: 76-79. Institutesof HealthgrantAI 22488andthe OnchocerciasisSoboslay P. T., Weiss N., Dreweck C. M., Taylor H. R., Control Programin West Africa-UNDP/World Bank/ Brotman B., Sbultz-Key H. & Greene B. M. 1992. WHO SpecialProgramfor Researchand Training in Experimental onchocerciasis in chimpanzees: antibody TropicalD&eases Macrofit. Chemotherapy Project. Excelresponse and antigen recognition after primary infection lent technical assistance was provided by Dr Fanya Liftis. with Onchocerca volvulus. Experimental Parasitology 74: We would also like to acknowledge the contribution of 367- 380.

R. Chandrashekar et al.

988

Taylor H. R., Trpis M., Cupp E. W., Brotman B., Newland H. N., Soboslay P. T. & Greene B. M. 1988. Ivermectin prophylaxis against experimental Onchocerca volvulus infection in chimpanzees. American Journal of Tropical Medicine

and Hygiene

39: 8&90.

Taylor H. R., Munoz B., Keyvan-Larijani E. & Greene B. M. 1989. Reliability of detection of microfilariae in skin snips in the diagnosis of onchocerciasis. American Journal of Tropical Medicine and Hygiene 41: 467747 1.

Taylor M. B., Pacque B. & Greene B. M. 1990. Impact of mass treatment of onchocerciasis with ivermectin on transmission of infection. Science 250: 116-l 18. Willenbucher J., Hofle W. & Lucius R. 1993. The filarial antigens Av33/0v33-3 show striking similarities to the major pepsin inhibitor from Ascaris suum. Molecular and Biochemical Parasitology 57: 349-35 1. World Health Organization. 1987. WHO Expert Committee on onchocerciasis: 3rd report: World Health Organization Technical Report Series 152: 1-167. WHO, Geneva.