Biochemical and immunochemical characterization of surface and excretory-secretory antigens of Loa loa microfilariae

Molecular and Biochemical Parasitology, 31 (1988) 251-262 251 Elsevier

MBP 01045

Biochemical and immunochemical characterization of surface and excretory-secretory antigens of Loa loa microfilariae

T h o m a s G . E g w a n g , A l a i n D u p o n t , J e a n - P a u l A k u 6 a n d M a r g a r e t P i n d e r

International Centre for Medical Research (CIRMF), Franceville, Gabon

(Received 28 April 1988: accepted 27 June 1988)

Detergent solubilized extracts of ~ZSlodogen surface-labelled Loa loa microfilariae revealed a relatively simple profile of two strongly labelled molecules of 23 and 67 kDa for blood microfilariae and several strongly labelled molecules of 23, 40, 42-67 kDa for in vitro born microfilariae. In addition, there were other weakly labelled molecules which were resolved after prolonged autoradiographic exposure. Surface molecules of 28, 29, and 33 kDa were unique to blood microfilariae, a 14.4 kDa molecule was unique to in vitro born microfilariae and molecules of 23, 4(1, and 75-84 kDa were common to both forms of microfilariae. The profile of excretory-secretory products consisted of molecules of 14.4-198 kDa. H u m a n albumin was a predominant component of surface molecules and excre tory- secretory products from blood microfilariae. Immunoprecipi ta t ion with occult and microfilaremic Ioaiasis sera demons t ra ted that the 23 kDa surface molecule and excretory-secretory products of 14.4 and 33 kDa were only recognised by occult Ioaiasis sera whereas surface molecules of 4(/and 75-84 kDa and excretory-secretory products of 28 and 67 kDa were recognised by both sera. Studies with heterologous sera demonst ra ted that with the exception of the 75-84 kDa antigens, all the L. loa microfilarial surface antigens contained epitopes which were restricted to filarial parasites. Further studies revealed that the 23 kDa antigen was a protein which contained neither asparagine-N-linked oligosaccharides nor interchain disulfide-linkages.

Key words: Loa loa: Microfilaria: Surface antigen; Immuni ty

Introduction

Loaiasis afflicts some 3 million people in West- ern Equatorial Africa where the transmission is maintained by the tabanid flies Chrvsops dimi- diata and C. silacea [1]. Within the endemic zone, 20--40% of the population is microfilaremic [1,2]; there is also a similar proportion of subjects with occult loaiasis who are amicrofilaremic but carry adult worms as manifested by the passage of adult worms under the skin or beneath the conjunctiva

Correspondence address: T.G. Egwang, CIRMF, B.P. 769, Franceville, Gabon.

Abbreviations: A D C C , ant ibody-dependent cellular-mediated cytotoxicity; DOC, sodium deoxycholate: endo H, cndo-[3-N-acetylglucosaminidase H; ES, concentrated excretory-secretory products: IFA, indirect immunofluorescent as- say; PBS, phosphate-buffered saline: PMSF, phenylmethylsulfonyl fluoride; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis: TCA, trichloroacetic acid.

of the eye (Dupont, Zue-Ndong and Pinder, in preparation). The immune mechanisms control- ling microfilaremia in loaiasis have not been studied in depth and immunological studies of loaiasis generally have been scanty [3-7]. The major con- straints to detailed immunological and biochemical studies remain the lack of a suitable laboratory animal model and the inability to propagate the complex life cycle of Loa loa in the laboratory. Previous workers in this laboratory have perfected a technique for the purification of L. loa microfilariae from the blood of highly microfilaremic subjects [8]. This has made it possible to ob- tain microfilariae in numbers sufficient for limited biochemical and immunochemical studies. Since virtually no information is available on L. loa microfilarial antigens, we radioiodinated living microfilariae and excretory-secretory (ES) products and proceeded to identify antigens of potential importance in the immunobiology of loaiasis using sera from parasitologically and din-

0166-6851/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

ically defined subjects from a loaiasis endemic zone. In this paper, we describe our findings and the partial characterization of an immunodomi- nant surface antigen of L. loa microfilariae.

Materials and Methods

Parasites and parasite preparations. L. loa microfilariae were purified from the blood of heavily microfilaremic subjects as already described [8], cryopreserved, and thawed when needed according to Cesbron et al. [9]. The microfilariae were

3 o incubated overnight at ,7 C in RPMI-1640 medium, pH 7.4, supplemented with glutamine, penicillin and streptomycin, to allow the parasites to release cryopreservatives. The following day, 5-10 × 10 3 microfilariae were washed in phosphate-buffered saline (PBS), pH 7.4, labelled with l:SI using the Iodogen technique and extracted in PBS containing protease inhibitors, 1% Triton X-100 and 1% deoxycholate (DOC) as described [10]. After centrifugation at 10000 × g for 30 rain at 4°C, the supernatant was re- trieved: the pellet was re-extracted in the same extraction buffer containing, in addition, 5% [3- mercaptoethanol. The Triton X-100/DOC soluble extract was used for all studies: the Triton X- 100/DOC/mercaptoethanol extract was used only to compare the degree of detergent solubilisation of the surface molecules in the absence of [3-mercaptoethanol. In vitro born microfilariae were obtained by culturing an adult female L. loa (re- moved surgically under local anaesthesia from the eye of a patient for ophthalmic reasons) in serum- free RPMI-1640 medium supplemented with gentamicin and glutamine for 72-96 h at 37°C in a humidifed COe incubator. Every day, the medium was checked for in vitro born microfilariae and replenished. The in vitro born microfilariae were recovered by centrifugation, washed 3 times in PBS, surface-iodinated and extracted as described. Concentrated ES products of microfilariae were prepared as follows: 5-10 × 10 ~ microfilariae were cultured in serum-free supplemented RPMI-1640 medium for 48-72 h at 37°C in a humidified CO2 incubator. The parasites were separated from the medium by centrifugation and the medium was concentrated 10-20 fold by ul- trafiltration using a filter with a molecular weight

cut-off point of 10 kDa. Concentrated ES products were iodinated by the Iodogen technique and fractions containing peak trichloroacetic acid (TCA) precipitable radioactivity were stored in aliquots at -70°C until used. All the reagents including lodogen, detergents and protease inhibitors were from Sigma Chemical Co., St. Louis, MO. Nal2SI (specific activity: 14 Ci mg -1) was from Amersham International, Amersham, U.K.

Sera. Individual or serum pools from occult and microfilaremic loaiasis Gabonese subjects were employed in immunoprecipitations to identify microfilarial surface antigens, Apart from the difference in their parasitological status, both groups of patients manifested similar clinical symptoms including passage of adult worms under the conjunctiva. A serum sample from a European who had not been exposed to loaiasis was used as a control in all immunoprecipitation studies with human sera. Sera from BALB/c female mice repeatedly immunized with living L. loa microfilariae and a mandrill infected with L. loa infective larvae 377 days previously were also used to identify antigenic surface components of microfilariae.

To determine the distribution of L. loa antigens among filarial and non-filarial nematodes, serum pools from patients or animals with heterologous infection sera were employed: microfilaremic onchocerciasis individuals from Guate- mala and microfilaremic Wuchereria bancrofti infected individuals from the Philippines (both from the W H O Filariasis Serum Bank); a cat pat- ently infected with Brugia malayi (obtained from Dr. J. McCall, University of Georgia, Athens, GA); hamsters infected with Dipetalonema viteae (provided by Dr. N. Weiss, Swiss Tropical Insti- tute, Basel, Switzerland); rats infected 3 times either with Nippostrongylus brasiliensis or Trichi- nella spiralis (provided by Dr. A.D. Befus, De- partment of Microbiology and Infectious Dis- eases, University of Calgary, Calgary, Canada).

lrnmunoprecipitation. Microfilarial extracts or ES products were pre-cleared, incubated overnight with various sera, and the immune complexes im- mobilised on protein A-Sepharose and prepared for sodium dodecyl sulfate-polyacrylamide gel

electrophoresis (SDS-PAGE) as already described [10].

Physico-chemical and biochemical characterization. To provide an insight into the protein or carbohydrate nature of the labelled molecules, similar amounts of TCA precipitable radioactivity of the microfilarial extract were incubated with various concentrations of pronase E (Sigma P- 5147) or trypsin (Sigma T-8642) at 37°C for 15 rain. The enzyme activity was stopped by boiling for 5 min. The digested extracts were then prepared for SDS-PAGE. The contribution of proteins and carbohydrate moieties to the antigenicity was assessed by variously treating microfilarial extracts before incubation with a highly reactive occult loaiasis serum. The extracts were (1) heated at 90°C for 15 min, with the control held at room temperature; (2) incubated in 0.05 M sodium metaperiodate (Merck, Darm- stadt, F .R .G. ) in 0.05 M sodium acetate buffer, pH 5.5 in the dark at 4°C for 24 h, with the control in sodium acetate buffer only; and (3) incubated at 37°C for 18 h with 50 milliunits of endo- [3-N-acetylglucosaminidase H (endo H) (Sigma E 6878) in 0.05 M phosphate buffer pH 7.0 containing 2 mM phenylmethylsulfonyl fluoride (PMSF), 90 mM E D T A and 50 txg ml 1 gentamicin, with the control in phosphate buffer alone. The re- suiting immune complexes were prepared for SDS-PAGE as described.

Concanavalin A-Sepharose fractionation. A column of concanavalin A-Sepharose (Pharmacia Fine Chemicals, Uppsala, Sweden), in a 5-ml sy- ringe was equilibrated with 10 mM Tris-HCl, pH 8.0, 150 mM NaCI and 2% Nonidet P-40. An extract of surface-labelled microfilariae was then added to the column and allowed to equilibrate for 1 h at room temperature. The column was washed with equilibration buffer until no further radioactivity was detected in the wash through. The elution of specifically bound glycoproteins was carried out using 200 mM C~-D-methyl man- nopyranoside in equilibration buffer. Fractions of the wash through and the eluate containing peak radioactivity were separately pooled, concentrated, and prepared for SDS-PAGE.

253

SDS-PAGE, autoradiography and densitometric scanning. Samples were boiled for 5 min with an equal volume of SDS-PAGE buffer and run on 11-12% acrylamide gels according to Laemmli [11]. In experiments for assessing the subunit structure, extracts were run on SDS-PAGE with and without [3-mercaptoethanol. The gels were run overnight at 10 mA or for 4 h at 45 mA per gel and prepared for autoradiography as described [10]. All SDS-PAGE chemicals, molecular weight standards (14.4-200 kDa range), and apparatus were from Bio-Rad Laboratories, Richmond, CA. In one experiment, scanning of the autoradiographic signals was carried out using an LKB 2202 UltroScan laser densitometer (LKB Produkter AB, Bromma, Sweden).

Resul t s

Profiles of detergent-soluble surface molecules and ES products Of L. loa microfilariae. The profiles

A B C ,== 1=.. ic=

zoo - 200--

200- - 66.2-- ~ 84

31--

21.5-- t

14.4--

40 !

23 t

14.4--

t O 1 2 MF

60.2-

1 2 MF

198 150

110 72 67

5e 52

46

Fig. 1. The profiles of detergent-soluble ~:51-surface-labelled molecules of L. loa microfilariae and ]>l-labelled ES products of blood microfilariae. (A) Blood microfilariae: short exposure (lane 1), longer exposure of the same lane (lane 2). (B) Profile of in vitro born microfilariae (lane 1) run on the same gel with an extracl of blood microlilariae (lane 2) for comparison. (C) Profile of ES (lane 2) run alongside that of an extract of blood microfilariae (lane 1) for comparison. Panels A and B were from lick acrylamide gels and panel C from a 101~ acrylamide gel; in the latter, a band of 14.4 kDa ran off the gel. The numbers refer to molecular weight s tandards and

calculated molecular weights (× l(J~).

4 2.2-

28

1 2 HF ES

254

of detergent-soluble surface molecules of both blood and in vitro born microfilariae, and of ES products of blood microfilariae are presented in Fig. 1. Soluble surface molecules of blood microfilariae consistently comprised of two strongly labelled bands of 23 and 67 kDa and a weakly labelled band at 75-84 kDa (Fig. 1A, lane 1). Longer exposures revealed the presence of other weakly labelled bands at 58, 40, 33~ 29 and 28 kDa (Fig. 1A, lane 2). When the detergent-insoluble pellet was re-extracted in buffer containing 5% [3- mercaptoethanol , there was further release of the 23, 40, and 75-84 kDa bands; however, no additional bands were solubilised (data not presented). When surface-labelled microfilariae were exposed to 100 p~g ml-] trypsin for 1 h at 37°C before extraction, the intensity of the labelled bands was considerably reduced, particularly that of the 23 kDa molecule (data not presented). There was no difference between the profiles of cryopreserved and freshly prepared blood microfilariae. The profile of detergent-soluble surface molecules of in vitro born microfilariae comprised of strongly labelled bands at 23 and 40 kDa,

a smear at 42-67 kDa and weakly labelled bands at 14.4, 75, and 84 kDa (Fig. 1B, lane 1); for comparison purposes, a profile of surface-labelled blood microfilariae was run alongside that of in vitro born microfilariae on the same gel (Fig. 1B, lane 2). Extraction of the detergent-insoluble pellet of in vitro born microfilariae resulted in further release of only the 14.4 and 23 kDa bands (data not presented). The profile of l~SI-labelled ES products of L. loa microfilariae comprised of molecules of 23, 28, 33, 46, 52, 58, 67, 72, 110, 150 and 198 kDa (Fig. 1C, lane 2). In all preparations, the 67 kDa band dominated the profile of the ES products.

Antigenicity of surface molecules and ES products of L. loa microfilariae. Infection sera from human loaiasis subjects immunoprecipi tated several molecules from detergent-soluble extracts of surface-labelled microfilariae and labelled ES products of microfilariae (Fig. 2). The only strongly immunoprecipitated band from extracts of blood microfilariae was at 23 kDa and this was only by the occult loaiasis serum pool (Fig. 2A, lane 4);

A

Mr 200-

66.2-

42.7-

21.5-

B C Mr M r

! ! i i ~ 2 0 O-

I

66.2-

42.7-

1 2 3 4 1 2 3 4 1 2 3

BLOOD MF IN VITRO MF ES

14A

Fig. 2. The antigenicity of L. loa microfilarial surface molecules and ES products. Immunoprecipitation of extracts of blood (A) and in vitro born microfilariae (B) and of ES products (C). For A and B, total profile (lane 1), immunoprecipitation by normal European serum (lane 2), microfilaremic loaiasis serum (lane 3) and occult loaiasis serum (lane 4). For C, immunoprecipitation

by individual microfilaremic sera (lanes 1 and 2) and occult loaiasis sera (lanes 3 and 4).

255

certain other bands were visible at 75-84 and 40 kDa on the autoradiograph after prolonged exposure but were too weak to photograph. Similar results were obtained when individual sera rather than serum pools were employed in immunoprecipitations: 7 out of 8 occult sera immunoprecipitated, whereas 10/10 microfilaremic sera did not immunoprecipi ta te the 23 kDa surface antigen of blood microfilariae (Pinder, Dupont and Eg- wang, in preparat ion). Both microfilaremic and occult loaiasis serum pools immunoprecipi ta ted a band of 40 kDa (Fig. 2B, lanes 3 and 4) whereas, again, only the occult loaiasis serum pool immunoprecipitated the 23 kDa band f rom extracts of in vitro born microfilariae (Fig. 2B, lane 4, ar- rowed). In one preparat ion of blood microfilariae, the 67 kDa band was immunoprecipi ta ted by homologous and heterologous filarial infection sera (see Fig. 4) which suggested that a component of this band was parasite-derived. Normal European serum did not immunoprecipi ta te any of the bands (Fig. 2A and B, lane 2).

When labelled ES products were immunoprecipitated, both loaiasis serum categories recognised two bands of 67 and 28 kDa whereas only the occult loaiasis sera recognised two additional bands of 14.4 and 33 kDa (Fig. 2C, lanes 3 and 4). The prominent 46 kDa band immunoprecipitated by both categories of serum was also immunoprecipi ta ted by normal European serum (data not presented). Interestingly, the 23 kDa ES molecule was not immunoprecipi ta ted by any sera, suggesting that the ES and surface molecules of 23 kDa were not the same. Thus several ES and surface antigens of L. loa microfilariae were identified and some of these were only recognised by sera f rom occult loaiasis, i.e. amicrofilaremic subjects.

Demonstration of human albumin on the surface of L, loa microfilariae. In some preparat ions of surface-labelled blood microfilariae, the 67 kDa band was the most strongly labelled in comparison with the 23 and 75-84 kDa bands. Since host proteins including human albumin have been reported on the surface of microfilariae of other species, immunoprecipi ta t ions with anti-human albumin, anti-human serum, and anti-human immunoglobulins were carried out. Both anti-hu-

man albumin and anti-human serum consistently immunoprecipi tated only the 67 kDa band; anti- human immunoglobulins did not precipitate any bands (data not presented). The 67 kDa band thus appeared to contain predominantly human albumin which was consistent with the observation that microfilariae born in vitro in the absence of serum did not demonstrate this band. The prom- inence of the 67 kDa band suggested that albumin was a major surface component which might be of immunological significance when raising antibodies in laboratory animals. This was indeed confirmed by raising anti-microfilarial sera in mice. Sera from BALB/c mice repeatedly inocu- lated with living microfilariae immunoprecipitated the 67 kDa band from extracts of surface- labelled microfilariae (Fig. 3A, lanes 3-5); nor-

A B

M ~ ,,,,-,- L""~ c",,,~ I ~ r z : E : E : E ~ ~: I z ~

6 6.2- i ~

21.5-

~ q i i l

A 1 2 3 4 5 6 1 2

MF ES Fig. 3. The demonstration of human albumin on the surface and in ES products of L. loa microfilariae. (~,) Extracts of blood microfilariae. Total profile of surface-labelled molecules (lane 1); immunoprecipitation of an extract of surface- labelled molecules by normal mouse serum (lane 2), sera of. individual immunized mice (lanes .3--5), and rabbit anti-human albumin (lane 6). (B) ES products of blood microfilariae. Total profile (lane 1): immunoprecipitation by normal rabbit serum (NRS) (lane 2) and rabbit anti-human albumin

(lane 3).

256

mal sera from mice never exposed to microfilariae did not recognise the 67 kDa band (lane 2). A positive control rabbit IgG anti-human albumin also immunoprecipitated the band (lane 6). The rabbit anti-human albumin also immunoprecipitated a 67 kDa band from iodinated ES products (Fig. 3B, lane 3) whereas normal rabbit serum did not (lane 2). These data demonstrated that human albumin was a major component of the surface and ES products of L. loa blood-derived microfilariae.

Species distribution of L. loa microfilarial surface antigens. To study the species distribution of L. loa microfilarial surface antigens or epitopes in other nematode parasites, sera from 4 microfilaremic heterologous filarial and 2 non-filarial nematode infections were employed (Fig. 4). The 23 kDa antigen was immunoprecipitated only by the serum pool from O. volvulus subjects (lane 7) but not by sera from D. viteae, B. malayi, or W.

M r 2 0 0 -

66.2-

42.7-

31-

21.5 -+

14.4-

+ + + + +++ I r a + .

.......... + + O w l ~ g

O

1 2 3 4 5 6 7 8 9 10 11 1213

Fig. 4. The recognition of L. loa microfilarial surface antigens by sera from heterologous filarial and non-filarial nematode infections. Total profile of surface-labelled molecules (lane 1); immunoprecipi ta t ion by normal hamster serum (lane 2), D. viteae 12, 31) and 50 week post-infection sera (lanes 3-5), B. malayi serum (lane 6), O. volvulus serum pool (lane 7), W. bancrofti serum pool (lane 8), N. brasiliensis serum (lane 9), T. spiralis serum (lane 10), microfilaremic loaiasis serum (lane 11). occult Ioaiasis serum (lane 12), and microfilaremic man-

drill loaiasis serum (lane 13).

bancrofti infections (lanes 3-6, 8). As controls, homologous loaiasis sera from infected human subjects and a mandrill immunoprecipitated the antigen (lanes 11-13), with the strongest reaction being shown by the occult loaiasis serum (lane 12). The non-filarial N. brasiliensis and T. spiralis sera did not immunoprecipitate the 23 kDa antigen (lanes 9, 10). The 40 kDa antigen was immunoprecipitated by the sera from most filarial infections (lanes 3-8+ 12-13) but not from non-filarial infections (lanes 9, 10). A 67 kDa band was strongly immunoprecipitated by the B. rnalayi infection serum (lane 6) and less strongly by the microfilaremic human and mandrill loaiasis sera (lanes 12 and 13). Interestingly, weakly labelled antigens in the 75-84 kDa range were broadly cross-reactive with both filarial and non-filarial infection sera. Thus epitopes of the 23, 40 and 67 kDa antigens appeared to be restricted to the filarial nematodes.

Subunit structure of the 23 kDa antigen. It was of interest to study the 23 kDa molecule further since it was a prominent surface molecule and was dif- ferentially recognised by occult versus microfilar-

Mr #-M E 4" -

200 - -

14.4--

L

1 2 Fig, 5. The subunit structure of the 23 kDa surface antigen. Migration on SDS-PAGE of an extract of surface-labelled microfilariae under reducing (lane 1) and non-reducing condi-

tions (lane 2).

emic loaiasis sera. In order to gain some insights into its subunit structure, an extract of surface-labelled blood microfilariae was resolved on SDS- P A G E in the presence and absence of [3-mercaptoethanol, a reducing agent of disulfide bonds (Fig. 5). The omission of the reducing agent resulted in no major alteration in migration of the 23 kDa band, suggesting that this antigen does not consist of polypeptide chains linked by disulfide bonds (Fig. 5, lane 2).

Further biochemical and physico-chemical characterization. In order to determine whether the surface antigens were proteinaceous in nature, their susceptibility to proteases was studied (Fig. 6). The 23 kDa and all other minor L. loa microfilarial surface antigens were susceptible to de-

257

gradation by pronase E (Fig. 6, lanes 2-6) and trypsin (lanes 8-11). Enzyme concentrations of 10 mg ml-1 resulted in complete digestion of the 23 kDa antigen (lanes 2 and 8) whereas concentrations of 100 ixg ml - ' or less resulted in incom- plete digestion with the generation of discrete fragments (lanes 3-6, 9-11).

It was also of interest to determine whether protein or carbohydrate residues played a signif- icant role in the antigenicity of the 23 kDa antigen. Microfilarial extracts were therefore sub- jected to various physico-chemical treatments as detailed in the Materials and Methods before immunoprecipitation with a highly.reactive occult loaiasis serum (Fig. 7). Neither endo H digestion nor heat treatment affected the antigenicity as assessed by the cpm immunoprecipitated and the

M r

200-

66.2-

423-

31-

l - nO

= - = ~ = -" Z -

~ ,,,,, , .b

21.5-

14.4-

1 2 3 4 5 6 7 8 9 10 11

PRONASE TRYPSIN Fig. 6. The susceptibility of the 23 kDa surface antigen t6 proteolysis. An extract of surface-labelled microfilariae was incubated with various concentrat ions (mg ml ~) of pronase E (lanes 2-6) or trypsin (lanes 8-11) and resolved on SDS- PAGE. Control (no enzyme) (lane 1), 10 (lane 2), 1 (lane 3). (I.1 (lane 4), 0.01 (lane 5), 0.001 (lane 6), 10 (lane 8), 0.1 (lane 9), 0.01 (lane 10), 0.1 plus 0.1 mg ml- ~ soybean trypsin inhibitor

(lane 11).

200-

ENDOH I 04+ HEAT

66.2-

42.7-

31-

21.5-R

1 2 3 4 5 6 Fig. 7. The effect of various physico-chemical t reatments on the antigenicity and migration on SDS-PAGE of the 23 kDa antigen. Phosphate buffer alone (lane 1): endo H in phosphate buffer (lane 2): sodium acetate buffer alone (lane 3); sodium metaperiodate in sodium acetate buffer (lane 4): room

temperature (lane 5): heat (lane 6).

258

migration of the antigen on S DS -P AGE (lanes 1 and 2, 5 and 6). Periodate t reatment did, however, exert a slight effect on antigenicity in that in 3 experiments, there was a mean reduction of 30% in the cpm immunoprecipi ta ted from periodate-treated extracts by comparison with that immunoprecipi ta ted from controls (data not presented). This resulted in a reduction in the thick- ness of the 23 kDa band (lanes 3 and 4). Scan- ning of the bands in lanes 3 and 4 using a laser densi tometer indicated that the autoradiographic signal was reduced by 23%. Some non-specific degradation appears to have occurred as evident by the smearing but there was no shift in M r of the 23 kDa antigen (lanes 1-3, 5, 6). The effect of possible protease contamination in the endo H preparat ion was ruled out by inclusion of the protease inhibitors PMSF and E D T A in the buffer. In toto, these results suggested that carbohydrate moieties played a limited, if any, role in the antibody binding of the 23 kDa antigen. The absence of oligosaccharides containing mannose or glucose residues was further confirmed by the lack of binding of the 23 kDa antigen to concanavalin A-Sepharose (data not presented).

Discussion

In this study, we concentrated our efforts on the identification of the molecules present on the surface of living microfilariae of L. loa and the characterization of those which might play a role in stimulating protective human humoral responses during loaiasis. Living microfilariae were surface- labelled with 125Iodine by the Iodogen method. This technique was employed because there is evidence that it specifically iodinates surface structures of microfilariae of the related B. pahangi [12,13] as well as the more distant D. viteae [14]. In our hands, the high viability and vigorous motility of microfilariae after iodination coupled with the fact that surface-labelled molecules on living microfilariae were susceptible to trypsin digestion, suggested that the labelling of L. loa microfilariae was restricted to the surface. Con- centrated ES products of cultured microfilariae were also iodinated using the Iodogen technique to identify important ES antigens.

The profiles of detergent-soluble surface-la-

belled molecules of blood and in vitro microfilariae were comparable to a limited extent, with several differences. Firstly, on blood microfilariae only two bands were strongly labelled. These were the 23 kDa band of parasite origin and the 67 kDa band which was mainly human albumin. By contrast, on in vitro born microfilariae, several bands of M r 23, 40 and 42-67 kDa were strongly labelled whereas the 67 kDa band was conspicuously weaker. Secondly, some molecules were expressed by only one form of microfilariae. For example, bands of 28, 29 and 33 kDa were observed only in prof les of blood microfilariae whereas a 14.4 kDa band was unique to in vitro born microfilariae. The differences observed between in vitro born and blood microfilariae may be explained, in part, by age-related differences between in vitro born microfilariae obtained 0-24 h after release and in vivo born microfilariae which may be several months old [15]. Develop- mental changes in the expression of microfilarial surface molecules have been described in Brugia species [16-18]. The profile of ES products comprised of molecules in the 14-198 kDa range and some of 23, 28, 83 and 58 kDa co-migrated with surface-labelled molecules. However , the 23 kDa band which predominated in the profiles of surface-labelled microfilariae was considerably weaker in ES products and was not immunoprecipitated by any sera. This observation suggests that the 23 kDa surface molecule is not released in vitro to a great extent. Host albumin dominated the profile of ES products, and in some preparat ions it was the only strongly labelled molecule.

The presence of host albumin on the surface of microfilariae of various filarial species has been reported [19-21]. In this study we documented that human albumin was a predominant component of the microfilarial surface and ES products. The significance of human albumin in the immunobiology of loaiasis remains unclear, We spec- ulate that the more restricted profile of iodinated surface molecules of blood microfilariae may be attributable, in part, to the presence of host albumin, as has been previously reported for skin and blood microfilariae of O. gibsoni and W. bancrofti respectively [19,21],

The surface molecules of 23 kDa and ES tool-

ecules of 14.4 and 33 kDa were immunoprecipitated by the occult loaiasis sera only, whereas the surface molecules of 40, 67, 75-84 kDa and ES molecules of 28 and 67 kDa were immunoprecipitated by both occult and microfilaremic serum pools. We have recently presented evidence that occult sera contained antibodies which were reactive with the surface of living L. Ioa microfilariae in IFA and antibody-dependent cellular cytotoxicity (ADCC), while the microfilaremic sera did not contain surface reactive antibodies and were negative in both IFA and ADCC (Pinder, Dupont and Egwang, in preparation). The pauc- ity of certain antibodies in microfilaremic sera may explain the lack of reactivity of these sera with the 23 kDa surface and with 2 of the ES antigens. This might be due to the absorption of such antibodies by the heavy microfilarial load. However, this possibility is unlikely since immunoprecipitation with anti-human immunoglobulins of extracts of surface-labelled microfilariae or IFA on living microfilariae, purified from the blood of heavily microfilaremic subjects, have not demonstrated immunoglobulins on the surface of microfilariae. The alternative explanation might be that there is a specific immunoregulatory mechanism in microfilaremic individuals which results in a relative deficit of antibodies to certain antigens. The ex- istence of such mechanisms in microfilaremic subjects with lymphatic filariasis and an immunosuppressive factor from B. malayi microfilariae has been reported [22-26]. It remains to be de- termined whether such mechanisms operate during loaiasis.

Immunologic cross-reactivity between different filarial parasites has been extensively reported [27-29]. We used sera from animal and human subjects with heterologous nematode infections to investigate possible cross-reactions between L. loa microfilariae and other nematodes. Antigens in the 75-84 kDa range were broadly cross-reactive with both heterologous filarial and non-filarial sera. The 67 kDa band was strongly immunoprecipitated by B. malayi infection serum, while the 40 kDa band was immunoprecipitated by most filarial but not non-filarial sera. The predominant 23 kDa band was strongly immunoprecipitated by the O. volvulus sera only, while W. bancrofti, B. malayi or D. viteae infection sera were not reac-

259

tive. W. bancrofti and O. volvulus sera have been reported to cross-react with a 22 kDa surface antigen of B. malayi [10] and O. gibsoni microfilariae [27]. The poor reactivity of most of the heterologous filarial sera with the 23 kDa antigen might be explained by the fact that these sera were from microfilaremic infections. The lack of reactivity of sera from N. brasiliensis or T. spiralis infections with the 23, 40 and 67 kDa antigens suggested that epitopes of these L. loa microfilarial antigens are restricted to filarial nematodes.

Further characterization of the 23 kDa antigen revealed that it was a protein, which did not contain disulfide-linked polypeptide chains, and was not released in vitro as an ES antigen. The lack of binding to concanavalin A-Sepharose and in- sensitivity to endo H suggested that the 23 kDa antigen did not contain high mannose asparagine-N-linked oligosaccharides. The antigenicity of this molecule was only slightly affected by periodate oxidation which indicated that periodate- sensitive amino acids such as tryptophan or ty- rosine [30] and carbohydrate moieties [31] played only a limited role in antibody binding. The limited availability of ES products and the abun- dance of human albumin in these did not permit a detailed characterization of ES antigens.

The immunological control of microfilaremia in lymphatic filariasis is strongly suggested by the fact that antibodies reacting with the surface of microfilariae are found almost exclusively in amicrofilaremic individuals [32-34]. However , the target antigens of these antibodies have remained virtually unknown. Recently, a 25 kDa antigen of B. malayi microfilariae, which was protective against an intravenous microfilarial challenge in mice, has been described; in addition, amicrofilaremic W. bancrofti patients had higher antibody levels to this antigen than microfilaremic patients [35]. In this work, we have identified and partially characterized a 23 kDa surface antigen of L. loa microfilariae which is immunoprecipitated only by serum antibodies from amicrofilaremic but not microfilaremic loaiasis subjects. After molecular cloning and expression of this antigen in E. coli by recombinant D N A tech- niques, its role in the immunobiology of loaiasis will be studied further in both human infections and a non-human primate model of loaiasis.

260

Acknowledgements

W e w o u l d l ike to t h a n k D r . J. Z u e - N d o n g ,

C h e f d e S e r v i c e d ' O p h t h a l m o l o g i e , H 6 p i t a l de

F r a n c e v i l l e , for his a s s i s t ance a n d c o o p e r a t i o n , L.

T r o t t e i n a n d N. O k i a s f o r t y p i n g t h e m a n u s c r i p t ,

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Documents

Biochemical and immunochemical characterization of surface and excretory-secretory antigens of Loa loa microfilariae