The effect of ivermectin treatment on the antibody response to antigens of Onchocerca volvulus

456 TRANSACTIONS OF THE ROYAL Socvxr

UF TROPNXL MEDICINE AND HTGJENE (1994) 88, 456-460

The effect of ivermectin Onchocerca volvulus

treatment

on the antibody

response

to antigens

of

A. J. Gillespie’, S. Lust&an 2, A. R. Rivas-Alcala3 and J. E. Bradley ’ ‘Department of Biology, Imperial College of Science, Technology and Medicine, Prince Consort Road, London, S W7 ZBB, UK; 2Laboratory of Virologly and Parasitology, The Lindsey F. Kimball Research Institute of the New York Blood Center, New York, NY, USA; 3Deceased; formerly of CIES, Carretera Panamericanay Periferico Sur SIN, San Christobal de Las Casas, Chiapas, Mexico Abstract The effect of the ~~ro~laricid~ drug ivermectin on the antibody response to a detergent extract of adult Onchocerca volvulus (OvAg) and a number of specific recombinant peptides was examined. Three of the peptides were combined in a serodiagnostic ‘cocktail’ and the effect of ivermectin on the diagnostic performance of this assaywas assessed.Immunoglobulin (Ig) Gl serum levels in responseto OvAg significantly decreased following ivermectin treatment. The antibody response to only one recombinant peptide (OvMBP29) was significantly affected, with IgG levels decreasingfollowing treatment. Levels of total IgE increased following treatment. No correlation was observed between initial antibody level (or change in antibody level) and any adverse reaction to treatment. The serodiagnostic ‘cocktail’ was 100% sensitive before and after the use of ivermectin. A serodiagnostic assayusing specific recombinant peptides can be used to evaluate infection in the absenceof dermal microfilariae in areaswhere ivermectin is used. The use of ivermectin has revolu~on~ed the treatment of onchocerciasis. Originally developed as an anti-parasitic drug for use with livestock, the microfilaricidal activity of ivermectin against Onchocerca volvulus infection in humans was first reported by AZIZ et al. (1982a, 1982b). Following extensive human trials, the French Government first approved the use of ivermectin in 1987 and it is now the drug of choice for onchocerciasis treatment world-wide. Diagnosis of 0. volvulus currently involves examination of skin snips for microfilariae (mf), a technique rendered ineffective in areaswhere ~~ro~laricid~ ivermectin is used; furthermore, skin snip diagnosis does not detect prepatent infections and is insensitive in low level patent infections (BUCK, 1974). As an alternative approach, a serodiagnostic test was developed employing recombinant peptides specifically recognized by sera from persons infected with 0. volvulus (see BRADLEYet al., 1991, 1993a). Two of these peptides are being considered by the World Health Organization (WHO) for use in a diagnostic assaywhich could replace, or provide an additional tool to, the use of skin smp diagnosis within the Onchocerciasis Control Programme (OCP) (RA~CHANDRAN, 1993). As the OCP has recently introduced ivermectin trea~ent as a complementary control measure to vectoricidal spraying, it is important to assess any change in the performance of the diagnostic assay that may result from the use of the drug. Adverse reactions to ivermectin treatment range from pruritis to more serious conditions such as hypotension, although severe reactions such as the latter are rare (DE SOLEet al., 1989: WHITWORTHet al., 1992). As is true for most ofthe pathology associatedwith O.‘volvulus infection, the adverse reactions to treatment with the previous drug of choice, diethylcarbamazine, and probably those to ivermectin treatment, are mediated by inappropriate immune responsesof the host (OTTESEN,1985). It is therefore ~port~t to assessany influence ivermectin may have on host immune responses.STEELet al. (1991) reported changes in antibody responsesto an extract of adult 0. volvulus following ivermectin treatment. A more complete picture may be provided by the examination of antibody responsesto stage- and species-specificrecombinant peptides of 0. volvulu.~. The primary aim of the present study was to examine the effect of ivermectin treatment on the diagnostic performance of a ‘cocktail’ of recombinant peptides, previously shown to exhibit high levels of sensitivity and specificity (BRADLEY et al., 1993a). The study design Address for correspondence: Dr Janette E. Bradley, Department of Biology, Imperial College of Science, Technofogy and Medicine, Prince Consort Road, London, SW7 ZBB, UK.

also enabled the ~~lestigation of any association between changes in antibody levels and the development of adverse reaction to treatment. Materials and Methods Study population

Eighty randomly selected males from the 0. voZvulusendemic region of southern Chiapas in Mexico participated in a single ‘blind’ placebo-controlled trial of ivermectin. All patients were aged between 12 and 60 years and had the presence of dermal mf confirmed by skin snip examination. Doses of ivermectin (ISO-22Ovg) or placebo were given 3 times at intervals of 6 months. Patients were clinically examined each time and serum samples were collected before, and 6 months after, the initial treatment and 6 months after the second treatment. Control sera

Non-endemic control serawere obtained from 48 individuals living in an area of Venezuela known to be free from 0. volvulus and Wuchereria bancrofti transmission. Antigens 0. volvulus detergent extract. iidult female 0. volvuL~

were dissected from nodules obtained from patients resident in the study area. A detergent extract (OvAg) was prepared as described previously (TRENHOLME et al., 1994). Recombinant proteins. Three recombinant antigens,

OvMBPilO, OvMBPill and OvMBPI29, had been previously selected for their specificity and sensitivity as diagnostic probes (BRADLEY er al., 1991, 1993a). They were over-expressed and purified as described by BRAD1,EYet al., (1993a), and are referred to as the diagnostic ‘cocktail’ when used in combination. A fourth recombinant peptide, 0~103, not included in the diagnostic assay, was over-expressed and purified in the pGEX-1N plasmid vector, producing a fusion polypeptide with the carboxy terminus of the Sch~s~osoma japonicum glutathione S-transferase protein (GST) (SMITH et al., 1987; LUSTIGMANet al., 1992b). OvMBP/lO (corresponding to 0~7; LUSTIGMANet al., 1991) is found in the L3, L4 adult stages of the worm and bv103 is found in the adult femaik and mf stages (LUSTIGMAN et al.. 199213).Localization studies for &MBP/ll and OvMBPI29 are incomplete, although both are known to be present in adult female worms (BRADLEYet al., 1991). Statistical ~aZ~a~~on

The frequency distributions of isotype responses to

some recombinant antigens did not permit the use of par-

457

ametric analyses (CONOVER,1980); therefore non-parametric procedures were used. A ranked one-way analysis of variance, utilizing van der Waerden scores, was used to assessdifferences in isotype responses between the examination times in each treatment group. Enzyme-linked immunosorbentassays

The enzyme-linked immunosorbent assay (ELISA) for IgG and IgG subclassesassayhas been described in detail elsewhere (BRADLEY et al., 1993b). The recombinant antigens OvMBPilO, OvMBPill and OvMBPI29 were fused to the Escherichia coli maltose binding protein (MBP) and 0~103 was fused to GST. As it was expected that antibodies reactive to MBP and GST might be found in human sera, the anti-MBP and anti-GST responsesof all sera used in the study were evaluated by performing an anti-MBP and anti-GST assayin parallel. Absorbance values (optical density, O.D.) were corrected to replicates of a high titre anti-O. volvulus sera pool present on each individual plate, and the anti-MBP and anti-GST values subtracted before any further analysis. The cut-off values for the diagnostic ‘cocktail’ assaywere determined as the mean O.D. plus 3 standard deviations of the 48 Venezualan control sera. A specific IgE ELBA was performed as follows. Sera were preabsorbed before use with protein G sepharose (Sigma) overnight at 4”C, in order to remove IgG antibodies and so limit competitive inhibition of IgE bindin (HUSSAIN& OTTESEN, 1986). Nunc-Immuno Maxisorp”% plates were coated overnight at 4°C with OvAg in 0.05 M carbonate buffer! pH9.6, at 50 yg/mL. The recombinant peptides and fusion proteins were coated at the following concentrations: MBP, 5 PgimL; OvMBPilO, 2.5 PgimL; OvMBPIll, 5 yg/mL; OvMBPI29, 2.5 kg/mL; GST, 5 PgimL; and 0~103, 5 yg/mL. The plates were then rinsed with 0.05 M NaCl containing 0.01% Tween 20@ and blocked for one hour at 37°C in phosphate-buffered saline (PBS) containing 5% fat-free milk powder and 5% foetal calf serum (FCS). Following incubation with the sera for 2h, plates were washed and incubated with rabbit anti-human IgE (Dako A094) for 3h, also at room temperature. Plates were then incubated with peroxidase-conjugatedswine anti-rabbit antibodies overnight at 4”C, and antibody binding was visualized by o-phenylenediamine (OPD). Plates were washed 6 times between each incubation.

powder and 5% FCS. A titration curve for each individual serum sample was constructed and the plates were incubated for 2 h, followed by incubation with a peroxidase-conjugated rabbit anti-IgE antibody (Dako P295) for a further 3 h. Antibody binding was visualized by OPD. A standard curve was constructed on each plate from dilutions of pooled reference sera, against which all individual patient values were compared. Results Sample size of treatment groups

Complete serum sets were available for 28 patients who received ivermectin and 16 who received the placebo. Clinical reactions to treatment

Reactionsto treatment reported included pruritis, headache, tender and enlarged lymph nodes, muscle and joint pain, and 3 casesof hypotension. The incidence of each reaction was significantly greater in the ivermectin group than in the placebo group (P
The effect of ivermectin treatment on the dermal mf loads of patients, as measured by skin snip, is shown in Fig. 1. There was a significant decline in mf loads (P
Total serumIgE measurement

Total IgE levels were measured by an antibody class capture assay. Nunc-Immuno Maxisorp@ microtitre plates were coated overnight at 4°C with a rabbit anti-IgE antibody (Dako A094) in 0.05 M carbonate buffer at a concentration of 2 yg/mL. Plates were then washed and blocked for one hour at 37°C in PBS containing 5% milk 120

Pie

1st

2nd

Treatment

El

Pre

1 St

2nd

Treatment

Fig.1. The effect of ivermectintreatmenton dermalmicrofilarialoads (Dmfimg). Eachsquarerepresentsone treatedindividual, with the median for eachgroup shownby the black diamonds.The blocksshowthe medianlevelsin theplacebogroup.

Treatment Fig.2. IgG (a) andIgGl (b) responses to the detergent-extract 0. vo2uulus antigenOvAg. Eachsquarerepresentsoneindividual receivingivermectin, with the medianfor eachgroupshownby the black diamonds.The blocksshowthemedianlevelsin theplacebogroup.

458

loads remained stable throughout the course of the trial with no significant change. Six months after the initial treatment, total IgG responsesto OvAg were significantly lower than pre-treatment levels (P
1.2

% u8

1.00.8’

I

o

Pf@

1st

2nd

Treatment Fig.4. IgG responses to recombinant 0. volv~l~s peptide OvMBPR9. Each square represents one individual receiving ivermectin, with the median for each group shown by the black diamonds. The blocks show the median leveis in the placebo group.

level (Fig.5). This 100% sensitivity was not affected by ivermectin treatment even though a significant (P
0.60.40*20.01 0

.

I 20

*

I 40

.

I . =i 60 80 Dmf/mg

%

(b) lv2j

I . 100 120

1

%

1.0

.

% 0.8

Pie

1st

2nd

Treatment

0

20

40

60 80 100 120 Dmf/mg Fig.3. Correlation of IgG (a) and IgG4 (b) responsesto recombinant 0. ~ol~Z~ peptide 0~103 before treatment and the dermal microfilaria ioad (Dmf~mg).

Antibody responsesto the recombinant peptides

IgG and IgG4 responsesto 0~103 showed a weak but significant negative correlation with pre-treatment mf loads (F&.3). No correlation between IgGl responses and mf loads was observed. The effect of ivermectin treatment on IgG, IgGl and IgG4 antibody responses to Ov~BP~29 is shown in Fig.4. There was a significant decline (P
of the diagnostic ‘cocktail

Before treatment, every patient in both the ivermectin and placebo groups exhibited responsesabove the cut-off

Fig.S. The effect of ivermectin treatment on the performance of the diagnostic ‘cocktail’ for onchocerciasis (recombinant peptides OvMBl’ilO, 11 and 29). The hatched basal region represents absorbancevalues below the cut-offlevel.

Total IgE levels

Following ivermectin trea~ent there was a significant (J’
The inadequacies of skin snip diagnosis have long been recognized (AMBROISE-THOMAS, 1974; NGU et al., 1981; CABRERA & PARKHOUSE, 1987) and are perhaps magnified when using the techni ue to monitor control programmes such as the OCP. Ta e microfilaricidal action of ivermectin has rendered the use of skin snip diagnosis inappropriate in areaswhere the drug is used. The use of ivermectin in conjunction with vectoricidal spraying in the OCP has thus intensified the need for a new diagnostic method to detect any new foci of infection. In this study, it was found that ivermectin treatment affected only total IgG responsesto one of the recombinant peptides studied, OvMBM29, the least diagnostically sensitive of the pe tides composing the diagnostic ‘cocktail’ (BRADLEY et aT:., 1993a) (Fig.4). Before the initial treatment, all individuals in both the ivermectin and

459

or amount of dead mf antigen, following treatment was sufficient to induce maximal levels of antibody responses to this peptide. In this study, before treatment, we found that total IgG and IgG4 responses showed a weak but significant negative correlation with mf burden (Fig.3). Together wirh the observation that antibodv directed against 0~103 can mediate mf killina and thk fact that the vevtide is more frequently recoinized by individuals wiih low level patent infections (~~sTI~~A~ et al., 1992b), this negative correlation may indicate a role in limiting the mf burden in v&o for antibodies directed against ov103.

Pre

1st

2nd

Treatment

Fig.& The effect of ivermectintreatmenton thelevelof non-specifictotal IyE ~internationa1 unitsi~l).

placebo groups exhibited antibody responsesto the diagnostic ‘cocktail’ above the cut-off level. Although a significant decreasein responsesto the diagnostic cocktail was seen following the initial ivermectin treatment, the absolute sensitivity of the test was retained. Responsesto the individual DeDtides, OvMBPilO and OvMBPill, were not affected by ivermectin treatment; these peptides are currently being assessedby WHO for inclusion in a serodiagnostic test to be employed in the OCP. The microfilaricidal action of ivermectin has been demonstrated bv many authors (AZIZ et al., 1982% 1982b; CUPP, 1992; W~IT~OR~H,. 1992), and was con: firmed in the present study (Fig.1). By lowering the available micro~laria1 ‘pool’, long-term use of ivermectin may lead to a decrease in exposure to infective larvae, which has already been shown to affect antibody responsesto the peptides composing the diagnostic ‘cocktail’ (BRADLEYet al.. 199313).Whether the sensitivitv of this diagnostic test will be unaffected by long term usk of ivermectin remains to be determined. The observed effects of ivermectin treatment on IgG and IgG subclass responsesto OvAg agreewith the findings of STEEL et al. (1991), with both studies showing a general decrease in levels following treatment (Fig.2). This decline probably reflects a decreasein the stimulatory mf antigen load. A decreasein the level of antibody responsesto OvAg may indicate the existence of a number of proteins shared between adults and mf, as illustrated by localization studies on 0~103 (LUSTIGMAN et al., 1992b). Although OvAg is an extract of adult worms, mf and eggs in the uterus of the female are present and may represent a major antigenic component of the extract to which responsesoccur. The decline in IgG and IgG subclass responses to OvAg is also reflected by the decline of these responses to OvMBIPI29 (Fig.3). IgGl and IgG4 responsesdeclined following treatment (although not signi~cantly), but the summation of the subclass responses probably explains the significant decline observed in the IgG isotype response. Localization studies are as yet incomplete for OvMBIV29, but the decline in antibody responses observed may indicate the presenceof the native protein in the mf stage. The presence of OvMBPilO in the thirdstagelarvae and adult worms (LUSTIGMANet al., 1992a), but not the mf, may explain why no significant change in antibody responses to this peptide was observed following treatment. The position of 0~103 on the mf surface (LUSTIGMAN et al., 1992b) suggested that the dramatic decreasein mf load following trea~ent would lead to a change in levels of antibody d&ected against this clone; however, no such change was observed. As no single person completely cleared the mf load, it may be that the residual mf load,

It is important to assessfully the effect of ivermectin treatment on host immune responses and to see if these responses,or changesin them, relate to any adverse reaction to treatment or change in pathology. Several adverse reactions to treatment were reported by patients receiving ivermectin, including pruritis, muscle and joint pain and, in 3 cases,hypotension. Many of the mild reactions were also reported in the placebo group, although the incidence of all these reactions was signi~c~tly higher in the ivermectin group. There was no correlation between initial antibody level (or change in antibody level) and adverse reactions to treatment, which suggests that these responses are not involved in the generation of post-treatment reactions. However, adverse reactions to ivermectin treatment usually develop within 48 h (WHITWORTH et al., 1992) and. following: ivermectin treatment of Bancroftian filariasis, IgG4 leiels rise steadily for approximately 30 d before declining to below pre-treatment levels (WAMAE et al., 1992). It may therefore be more appropriate to look for such correlations much closer to the time of treatment, as the initial increase in antibody levels may be far more ~formative than the long term decline. In this study, a significant increase in total IgE levels was observed followine treatment iFia.6) contrasting with the decreasefound by STEELet,l.-( 1991). The increasedescribed here may be the result of the increase in interleukin 4 levels, and decrease in CD23 levels, that follow ivermectin treatment (DELESPESSE et al., 1991; SOBOSLAY et al., 1992). As the levels of specific IgE against 0vA.g and the recombinant peptides remain stable, the in&ease in non-specific IgE-level has the effect of decreasing the svecificinon-sDecific IeE ratio. This ratio decrea&d sign~~~antly in the caseof&ibody to the recomb~ant peptides but not with that to OvAg, indicating that titres of specific IgE to these peptides decreaseas a proportion of the total IgE pool. This study has shown that the short term use of ivermectin does not diminish the sensitivity of our serodiagnostic ‘cocktail’, even though antibody responsesto one of the antigens is significantly lowered. The diagnostic test described previously by BRADLEY et al. (1993a) can therefore be used as an additional tool to monitor control programmes, such as the OCP, where ivermectin has been, or is being, administered. Acknowledgements

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1993;