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Studies on a 14-kilodalton surface protein of Onchocerca microfilariae
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Molecular and Biochemical Parasitology, 46 (1991) 103-112 © 1991 Elsevier Science Publishers B.V. / 0166-6851/91/$03.50 ADONIS 016668519100044H MOLBIO 01514
Studies on a 14-kilodalton surface protein of Onchocerca microfilariae Franz J. Conraths 1"' , Michael J. W o r m s 1, Graham Preece 2, William Harnett ~and R. Michael E. Parkhouse 2 Divisions of Iparasitology and 2Immunology, National Institute for Medical Research, London, U.K. (Received 9 August 1990; accepted 22 November 1990)
A 14-kDa antigen present on the surface of uterine microfilariae of Onchocerca spp. has been identified using monoclonal antibodies. The antigen was also found in skin microfilariae, but in a masked or cryptic form. A complementary DNA clone encoding the epitope recognised by one of the monoclonal antibodies was identified in a ~gtl 1 library. Nucleotide sequencing revealed that the 233-bp cDNA fragment codes for the carboxy-terminus of the antigen. The deduced amino acid sequence consists of three hydrophobic domains with high potential for [3-sheetformation. The amino-terminalhydrophobic domain is followed by 4 positively charged residues (positions 22-25) which contribute to the rather basic character of the protein. Another interesting feature of the polypeptide is its richness in phenylalanine (12.7%). From the sequence information, a synthetic peptide was synthesised which was recognised by one of the monoclonal antibodies directed against the 14-kDa antigen and a small number of sera from patients with onchocerciasis. The relevance of this to vaccination is discussed. Key words: Onchocerca volvulus; Microfilariae; Surface antigen; Monoclonal antibody; Deduced partial amino acid sequence
Introduction
Human onchocerciasis is a tropical disease occurring in many parts of Africa, Central America and some regions of South America. The disease is associated with severe dermal lesions and blindness [1]. Many of the observed pathological Correspondence address: R.M.E. Parkhouse, Division of Immunology,National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, U.K. *Present address: Institut fur Parasitologie, Rudolf-Buchheim-Str. 2, D-6300 Giessen, F.R.G. Note: Nucleotide sequence data reported in this paper have been submitted to the GenBankTM data base with the accession number M 58412. Abbreviations: BSA, bovine serum albumin; cDNA, complementary DNA; ELISA, enzyme-linkedimmunosorbentassay; PBS, phosphate-buffered saline; PMSF, phenylmethylsulphonyl fluoride; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; TLCK,N-ct-tosyl-L-lysine chloromethyl ketone; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone.
changes are thought to arise as a consequence of immune responses of the host to microfilarial antigens [2]. Antibodies to microfilarial antigens are easily demonstrated in filarial infections in general. At the same time, the humoral response to surface components is difficult to detect and targetted to a few molecules [3-5]. The response in onchocerciasis appears to be typical of this, as Taylor and co-workers [6] have reported that many sera from human patients do not immunoprecipitate radioactively labelled surface antigens of Onchocerca volvulus microfilariae. In spite of the potential role of microfilarial surface antigens as targets for protective or pathological immune responses, information on their structure is very limited. One reason is the restricted availability of O. volvulus microfilariae. It is known, however, that surface proteins of O. volvulus and the related cattle parasites Onchocerca lienalis and Onchocerca gibsoni cross-react [3,7,25]. This cross-reactivity offers an alternative source of material for study. The scarcity of O. volvulus material can also be overcome by expressing cloned genes of the para-
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site [8]. Such expressed recombinant antigen, together with hybridoma antibodies may allow immunological and biochemical studies of individual parasite components. In this study, we have employed such techniques to characterise a 14-kDa surface antigen of Onchocerca microfilariae. Materials and Methods
Parasites.
Living O. lienalis microfilariae and adult worms were isolated from naturally infected British cattle. Cryopreserved O. gibsoni microfilariae and nodules containing O. gibsoni worms were provided by B. Copeman, Townsville, Australia. Uterine microfilariae were obtained by dissection of adult female worms. Adult males of O. volvulus isolated by collagenase digestion from onchocercomata of Venezuelan patients were provided by Z. Cabrera, NIMR. Human patient sera were obtained from the WHO filariasis serum bank.
Monoclonal antibodies.
BALB/c mice received an injection of 20 000 microfilariae suspended in Freund's complete adjuvant into each of the rear footpads. Three days later, a similar dose was given in incomplete Freund's adjuvant. At 3-4-day intervals, 4 further injections of similar numbers of microfilariae were given in PBS. One day after the last administration, the draining lymph nodes were removed, lymphocytes isolated and fused with JKAg4/1 cells. Hybridoma cell lines were selected according to standard procedures [9]. Tissue culture supernatants were screened for antibodies to microfilarial antigens in indirect immunofluorescent assays, radioimmune precipitations and Western blot experiments. Positive hybridoma cultures were cloned by limiting dilution.
Indirect immunofluorescent assays.
2 000 living microfilariae were reacted with 100 ~l hybridoma supernatant for 30 min on ice. After 3 washes with ice-cold RPMI-1640 medium, the microfilariae were incubated with anti-mouse IgG-FITC conjugate (Seralab) for 30 min on ice. Three further washes with ice-cold PBS were followed by a 30min incubation with 10% (w/v) formaldehyde in PBS at room temperature. After 3 washes with PBS at room temperature, the microfilariae were mounted on slides and microscopically examined.
Radioimmunoprecipitation.
Approximately 105 living microfilariae were radio-iodinated using the Iodogen method according to the instructions of the manufacturer (Pierce Chemical Co., Rockford, IL). Tyrosine was added in excess to bind unreacted radioactive iodine. Radiolabelled parasites were washed twice in saline and once in 100 mM Tris, pH 7.5. The parasites were suspended in 20 mM Tris, pH 8.3, containing 1.5 mM N-~-tosyl-L-lysine chloromethyl ketone (TLCK, Sigma); 1.5 mM Ntosyl-L-phenylalanine chloromethyl ketone (TPCK, Sigma); 1 mM phenylmethylsulphonyl fluoride (PMSF, Sigma); sodium deoxycholate (BDH) was then added to a final concentration of 2% (w/v). After 4 min sonication at 0°C the parasite lysate was centrifuged at 10 000 x g for 30 min. Approximately 5 ~tl of the supernatant of this crude antigen preparation (20 000 cpm of radioactivity) were reacted with 100 ~tl hybridoma supernatant or 5 ~tl mouse serum, respectively. Antigen-antibody complexes were precipitated and analysed by SDSPAGE as described [ 10].
Western blotting. Parasites were incubated in 100 mM Tris, pH 7.5/2% (w/v) sodium deoxycholate/ 1.5 mM TLCK/1.5 mM TPCK/1 mM PMSF for 15 min at 0°C, sonicated for 4 min and centrifuged (30 min, 10 000 x g). The supernatant of this preparation was denatured under reducing conditions and electrophoresed on 5-25% (w/v) gradient SDS-polyacrylamide gels as described [11 ]. Separated proteins were transferred to nitrocellulose membranes (Millipore Corp., 45/xm pore size). Membrane strips were blocked with 2% (w/v) bovine serum albumin and 0.05% (v/v) Tween 20 (Sigma) in 50 mM Tris, pH 8.0/150 mM sodium chloride/2 mM MgC12 (TBS). Monoclonal antibodies or sera, respectively, were appropriately diluted in blocking buffer and reacted with the nitrocellulose membranes overnight at room temperature. Bound antibodies were detected with anti-mouse IgG (H+L chain) alkaline phosphatase conjugate (Promega, Madison, Wisconsin, USA) and nitroblue tetrazolium in combination with 5bromo-4-chloro-3-indolyl phosphate (Sigma) as substrate. Isolation ofcDNA clones. A )~gtl 1 cDNA library derived from female O. volvulus worms isolated in
105
Kumba, Cameroons, was provided by J. Donelson, University of Iowa, Iowa City, IA, U.S.A. [8]. Fusion proteins encoded by recombinant phage were identified using monoclonal antibodies to O. gibsoni or O. lienalis microfilariae essentially as described [ 12]. DNA was isolated from plaque-purified phage and the inserts subcloned into the unique EcoRI site of the plasmid vector pUC8 [13] using standard techniques [ 14].
DNA sequencing.
Single-stranded DNA was prepared from recombinant plasmids by alkali treatment [15] followed by chain termination sequencing using BRL reagents and equipment [16]. Computer analyses of DNA and amino acid sequences were done using the the software package of the University of Wisconsin Genetics Computer Group [ 17].
Synthetic peptide.
A peptide with the sequence GIQKQCVLLKYMFPKKNKALF was manufactured by the Laboratory of Peptide Chemistry, NIMR, London on LKB Bioxlynx 4170 equipment.
Dot blot and ELISA.
10 ~g of the synthetic peptide or parasite antigen in 2 ktl volume were spotted onto nitrocellulose filters. The air-dried filters were processed as described for Western blots. Alternatively, 96-well polystyrene plates were coated with 10 ~tg peptide in 0.1 M sodium borate buffer, pH 9.0, at 4°C overnight. Remaining binding sites were blocked with 3% (w/v) BSA in the same buffer for 2 h at room temperature. Sera, appropriately diluted in PBS with 0.05% Tween 20, were reacted with the antigen for 1 h at room temperature. Bound antibodies were detected by incubation with appropriate anti-immunoglobulin alkaline phosphatase
B
C
Fig. 1. Immunofluorescencewith monoclonal antibodies to surfaceantigens of uterine O. gibsoni microfilariae. Monoclonalantibody 2A5B9 or normal mouse serum were incubated with uterine microfilariae, embryosand eggs derived from frozen nodules. Bound antibodies were visualisedusing a FITC-labelledanti-mouse IgG conjugate. (A) 2A5B9, immunofluorescence.(B) Normal mouse serum, immunofluorescence.(C) 2A5B9,bright field. (D) Normal mouse serum, bright field.
106
conjugates (1 h, room temperature). After washing, substrate (p-nitrophenyl phosphate; Sigma) was added and the reaction stopped after 30 min by adding NaOH to a final concentration of 1 M. Adsorption at 405 nm was measured in an automatic ELISA reader (Titertek Inc.).
LL.
o~
o')
0,~
~1
'<
Results
Monoclonal antibodies to a 14-kDa surface antigen. Hybridoma supernatants were employed in immunofluorescent assays using uterine and skin O. lienalis (live) and O. gibsoni (cryopreserved) microfilariae. None of the supernatants reacted with skin microfilariae of either species, but two (2A4E9 and 2A5B9) showed evenly distributed surface fluorescence on approximately 90% of the uterine microfilariae. The antigen(s) recognised were also present in the egg-shell and on the surface of embryos from the late morula stage onwards, but not on earlier stages (Table I, Fig. 1). In immunoprecipitation experiments, these antibodies reacted with radio-iodinated molecules of 14-kDa and 60 kDa from lysates of surface-labelled O. lienalis skin microfilariae (Fig. 2). The former antigen was also recognised when the monoclonal antibodies were employed to probe Western blots ofmicrofilaTABLE I Characteristics of monoclonal antibodies to surface antigens of Onchocerca microfilariae Monoclonal antibodies 2A4E9
2A5B9
Immunofluorescence, uterine microfilariae O. gibsoni O. lienalis
+ +
+ +
60 14
60 14
14 14
14 92
Immunofluorescence, skin microfilariae O. gibsoni O. lienalis
Radioimmune precipitation t25I-labelled O. lienalis microfilariae, (kDa) Western blot O. lienalis microfilariae (kDa) O. volvulus adult, male (kDa)
-
200kD
-
92.5
-
69
-
46
-
21.5
-
14.3
Fig. 2. Immunoprecipitations of radiolabelled surface antigens of O. lienalis microfilariae. Living O. lienalis skin microfilariae were radiolabelled with ~25I(Iodogen method) and solubilised. Antigen preparations were reacted with mouse sera or monoclonal antibodies. Antigen/antibody complexes were precipitated using goat anti-mouse IgG, analysed in SDS-polyacrylamide gels and autoradiographed. FI 17, mouse hyperimmune serum to O. gibsoni mf; 2A4E9, monoclonal antibody; 2A5B9, monoclonal antibody; NMS, normal mouse serum; AG, antigen preparation.
rial antigen, whereas the 60-kDa antigen is not detected under these conditions (Fig. 3A). In adult worm extracts, antibody 2A5B9 does not recognise the 14-kDa protein, but binds to a 92-kDa protein (Fig. 3B; Table I). cDNA cloning. Since the monoclonal antibody 2A5B9 reacted with a 92-kDa antigen present in adult worms in addition to the 14-kDa surface protein of microfilariae, we focussed on the antibody 2A4E9 as a probe for the 14-kDa antigen. The antibody was employed to screen a ~,gt 11 cDNA library derived from female worms from Kumba, Cameroons. It has previously been shown that this gene bank contains clones of microfilarial origin [8]. A recombinant phage designated ~,-2A4.1, which was selectively recognised by the antibody probe, was plaque-purified and further analysed.
107
LL
i ~i~i i i i i[~!i
i~iiiiiiii~%
i~ii!~i~iii~
<~
200kD
-200
kD
iiii! ,i
[~i[i ~i~ii~i)
92.5
92.5
69
69
i
~i.... ~!i!i ~ ~ii~!i~i[i i [~ii ~i!i!
~i!~i~i~~(!/!~[i ~,~i!....i~i~ ~i~iJ'i~?!~
~i!iii~i!
~!iiii!
-
46
-
30
-
21.5
46
iii~iiiiii!!~!i~iill
i ~i?i i i~i!i~i !!ili
30
20 -
14.3
A
.....
1 4,3
B
Fig. 3. Western blot analyses. O. gibsoniskin microfilariae (A) or adultmale O. volvulus(B) were solubilised, electrophoresed and transferred to nitrocellulose. The membranes were cut into strips which were individually incubated with monoclonal antibodies or sera. Bound antibodies were visualised with alkaline phosphataselabelled anti-mouse IgG and nitro blue tetrazoliumand 5-bromo4-chloro-3-indolyl phosphateas substrate.2A4E9, monoclonal antibody;2A5B9, monoclonal antibody;F 117, mouse hyperimmune serum; NMS, normalmouse serum.
Nucleotide and deduced amino acid sequence of~L2A4.1. The insert of the phage ~L-2A4.1 was subcloned into pUC 8 and sequenced (Fig. 4). It is 233 nucleotides long and apparently represents approximately half of the complete message. An amino-terminal start codon could not be found and the theoretical molecular weight of the deduced polypeptide remains well below the expected value of 14 kDa. A stop codon beginning at position +196
may indicate that ~,-2A4.1 encodes the carboxyterminus of the protein. The sequence following nucleotide position +217 could possibly serve as a polyadenylation signal, although there is a mismatch in position +220 (A:T) with respect to the reported consensus motif [18]. The deduced amino acid sequence of ~-2A4.1 is dominated by 3 stretches of mainly hydrophobic amino acids (positions 1-20; 33-42; 5 7 - 6 3 ) with high potential for
108 1 1
GAA
TTC
CAA Q
GTA V
ATT I
AGC S
AGG R
CGT R
TTT F
GGG G
ATA I
CAA AAA Q K
CAG Q
TGC C
GTT V
49 15
TTA L
CTG L
AAG K
TAC Y
ATG M
TTT F
CCA AAA P K
AAA K
AAT N
AAG K
GCA A
TTA L
TTT F
TCA S
CTG L
97 31
GAC D
ATC I
TTA L
CCG P
TTC F
GTT V
AGA R
GTG V
TAC Y
TAT Y
GGC ACA G T
TTT F
TCC S
AAC N
GAA E
145 47
ATT I
TTG L
TCT S
GCA A
TTA L
TTC F
CAG AAG Q K
TGC C
GGA G
ATG M
TGC C
TTT F
TTT F
ATT I
GCA A
193 63
TAT Y
TGA *
TTC
TGA
GAA
TTT
AGA
CAT AAA
AAA
AGG
AAT
TC
CTG
Fig. 4. cDNA and deduced amino acid sequence of k-2A4.1. Numbering corresponds to relative positions in a partial cDNA lacking the 3' end of the gene.
1
2
3
4
5
A B 6
C Fig. 5. Mapping of the epitope recognised by the monoclonal antibody 2A4E9. O. gibsoni antigen (1), PBS (2), pep2A4.1, comprising amino acids 8-29 of the sequence translated from the insert of )~-2A4.1 (3), BSA (4), k-2A4.1 fusion protein (5) and an irrelevant recombinant O. volvulus antigen (6) were spotted onto a nitrocellulose membrane and probed with the monoclonal antibodies 2A4E9 (A), 2A5B9 (B), and an irrelevant monoclonal antibody directed against an irrelevant O. volvulus antigen (C).
[3-sheet formation according to secondary structure predictions (data not shown) [19]. The amino-terminal hydrophobic domain is followed by 4 positively charged residues which contribute to a region of coil in secondary structure prediction. Likewise, the second and third hydrophobic B-sheet domains are separated by a stretch of coil and a short sequence of c~-helix forming amino acids, respectively, but charged residues are lacking in these regions. In general, the amino acid sequence encoded by )~-2A4.1 is rich in hydrophobic amino acids, in particular phenylalanine (12.7%). It also contains a
number of positively charged residues which make the protein rather basic. Three cysteine residues may influence the tertiary structure or interact with other molecules. There is no indication of any Nglycosylation sites in the deduced amino acid sequence. Determination of the epitope detected by monoclonal antibody 2A4E9. Potential epitopes on this molecule were predicted using the algorithm described by Modrow and Wolf [20]. Two regions ranging from amino acid positions 18-26 and 4045 showed an antigenic index above 1.0. A peptide comprising amino acids 8-29 (pep2A4.1) was synthesised. Dot blot assays revealed that pep2A4.1 is specifically recognised by the monoclonal antibody 2A4E9, but not by the monoclonal antibody 2A5B9 or a irrelevant monoclonal antibody directed against a different O. volvulus antigen (Fig. 5). A small number of human onchocerciasis sera (N=9) were examined for their reactivity with pep2A4.1. We found that only 2 of these sera recognised the peptide, with titres in the range of 1/160 1/1280 (Table II). Discussion
In this publication, we report the characterisation and partial predicted sequence of a markedly hydrophobic surface antigen of Onchocerca microfilariae. Complementary DNA clones presumably coding for this antigen, were selected from an existing expression library, which has previously yielded nucleotide sequences corresponding to important 'house-keeping' genes and antigens of diagnostic
109 TABLE II Reactivity of human filariasis patient sera with pep2A4.1 Serum No.
Origin
Clinical Manifestation
Titre
86 89 91 92 162 165 185 Pool NHS
Ivory Coast Ivory Coast Ivory Coast Ivory Coast Yemen Yemen Yemen Liberia United Kingdom
O O O O O,S O,S O,S O negative
negative negative 1/640 negative negative 1/1280 negative negative negative
Peptide pep2A4.1 was attached to polystyrene plates (10/zg/well) and reacted with serial dilutions of human onchocerciasis sera from patients with generalised onchocerciasis (O) or 'sowdah' (O,S), or normal human serum (NHS). Bound antibodies were detected with rabbit anti-humanIgG alkaline phosphatase conjugates and end-point titres determined.
relevance of O. volvulus [9,21,22]. Specifically, a cDNA clone (~,-2A4.1) was selected by a monoclonal antibody (2A4E9) reactive with a 14-kDa microfilarial surface antigen. The amino acid sequence of this clone apparently represents the carboxy-terminus of the surface polypeptide and displays 3 predominantly hydrophobic domains with a high potential for 13-sheet formation. These features are of interest as they would certainly allow the molecule to interact with other hydrophobic components of the microfilarial epicuticle. Whether they are organised in the living microfilarial surface in this manner, however, remains a challenge for future investigations. One possible arrangement which must also be considered is an association with other proteins, since the 14-kDa component does in fact interact with a 60-kDa molecule (for results of immunoprecipitations, see below). The precise location of the epitope recognised by the monoclonal antibody 2A4E9 is suggested but not proved by calculations of the antigen index according to the algorithm of Modrow and Wolf [20]. This analysis indicates that a peptide spanning the amino acid positions 20-26 is the most likely target of an immune response (data not shown). In line with this hypothesis, a peptide comprising amino acids 8-29 was synthesised and shown to be recognised by the original monoclonal antibody 2A4E9. The same peptide was also recognised by 2 out of 9 sera of infected humans, and it will be of considerable interest to survey a larger number of sera to determine if this low level of reactivity persists. Surface epitopes which are of poor immunogenicity
during natural infection may merit investigation as potential vaccines. It is possible that the epitope present in the peptide may fall into this category. The epitopes of the 14-kDa polypeptide recognised by the monoclonal antibodies 2A4E9 and 2A5B9 become masked or cryptic upon release of the microfilariae from the uterus of female worms in both O. lienalis and O. gibsoni. In this respect, our findings confirm results obtained by other investigators with polyvalent sera [6,23]. Forsyth and co-workers [23] suggested that host albumin may contribute to covering parasite molecules which are potential targets of the immune response. Although our own investigations confirmed the presence of bovine albumin on the surface of O. lienalis skin microfilariae (unpublished observations), it has yet to be established, whether albumin masks the epitopes recognised by the monoclonal antibodies 2A4E9 and 2A5B9. The results indicating the presence of masked or cryptic epitopes may reflect a maturation process which could have consequences for the recognition by antibody in vivo. It may be the case that microfilariae will only bind antibody for short period following release from the uterus. Surface components in the range of 14-15 kDa have been found in microfilariae of O. volvulus [6] and O. cervicalis [24]. Our own results show the presence of similar molecules in O. lienalis and O. gibsoni microfilariae. Our finding that these low molecular weight components contain cross-reactive epitopes is consistent with observations of Edwards and colleagues [24]. The immunoprecipitation analysis of surface-labelled O. lienalis
110
microfilariae may suggest, that the 14-kDa polypeptide recognised by the monoclonal antibodies forms a non-covalently associating heterodimer with a 60-kDa molecule which is also present on the surface of microfilariae. Although the monoclonal antibodies described in this publication do not react with epitopes present on the 60-kDa component, its purification is perfectly feasible using monoclonal antibody 2A4E9 and detergent lysate of microfilariae. More detailed information on this molecule is required in order to assess its relation to surface components with molecular weights similar to those of other Onchocerca species [23,24]. An important question is whether the microfilarial 14kDa antigen is endogenously synthesised or, on the other hand, exogenously acquired from the adult female. This question may be addressed by probing mRNA derived from different stages with the cDNA clone of the 14-kDa protein - given sufficient microfilariae. In summary, a 14-kDa surface antigen of Onchocerca microfilariae has been identified using monoclonal antibodies, partially cloned, and sequenced. The structure revealed is particularly interesting, being mainly hydrophobic and unusually rich in phenylalanine. At the same time, there is an obvious antigenic determinant, which, when synthesised as an oligopeptide, was recognised by the original monoclonal antibody and a restricted number of onchocerciasis patient sera. The possibility that this sequence is protective is currently under investigation.
Acknowledgements This work received financial support from the Edna McConnell Clark Foundation. FJC was supported by a fellowship grant of the German Academic Exchange Service.
References 1 Mackenzie, C.D., Williams, J.F., Guderian, R.H. and O'Day, J. (1987) Clinical responses in human onchocerciasis: parasitological and immunological implications. In: Filariasis: Ciba Foundation Symposium 127, pp. 46-67. Wiley, Chichester. 2 Philipp, M., Davis, T.B., Storey, N. and Carlow, C.K.S. (1988) Immunity in filariasis: perspectives for vaccine development. Annu. Rev. Microbiol. 42,685-716.
3 Forsyth, K.P., Copeman, D.B., Anders, R.F. and Mitchell, G.F. (l 981) The major radioiodinated cuticular antigens of Onchocerca gibsoni microfilariae are neither species nor onchocerca specific. Acta Trop. 38,343-352. 4 Maizels, R.M., Selkirk, M.E., Sutanto, 1. and Partono, F. (1987) Antibody responses in human lymphatic filarial parasites. In: Filariasis: Ciba Foundation Symposium 127, pp. 189-200, Wiley, Chichester. 5 Egwang, T.G., Dupont, A., Aku6, J.-P. and Pinder, M. (1988) Biochemical and immunological characterization of surface and excretory-secretory antigens ofLoa loa microfilariae. Mol. Biochem. Parasitol. 31,251-262. 6 Taylor, D.W., Goddard, J.M. and McMahon, J.E. (1986) Surface components of Onchocerca volvulus. Mol. Biochem. Parasitol. 18,283-300. 7 Garate, T., Cabrera, Z., Copeman, D.B., Harnett, W., McLaren, D.J., Pattersen, M. and Parkhouse, R.M.E. (In Press) Surface antigens of male worms and microfilariae of Onchocerca gibsoni. Int. J. Parasitol. 8 Donelson, J.E., Duke, B.O.L., Moser, D., Zeng, W., Erondu, N.E., Lucius, R., Renz, A., Karam, M. and Zea Flores, G. (1988) Construction of Onchocerca volvulus cDNA libraries and partial characterization of the cDNA for a major antigen. Mol. Biochem. Parasitol. 31,241-250. 9 K/Jhler, G. and Milstein C. (1976) Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion. Eur. J. Immunol. 6, 511-519. 10 Hamett, W., Meghji, M., Worms, M.J. and Parkhouse, R.M.E. (1986) Quantitative and qualitative changes in production of excretions/secretions by Litosomoides carinii during development in the jird (Meriones unguiculatus). Parasitology 93, 317-331. 11 Parkhouse, R.M.E., Philipp, M. and Ogilvie, B.M. (1981) Characterisation of surface antigens of Trichinella spiralis infective larvae. Parasite Immunol. 3,339-352. 12 Huynh, T.V., Young, R.A. and Davis, R.W. (1985) Constructing and screening cDNA libraries in lambda gt I 0 and lambda gtl 1. In: DNA Cloning. A Practical Approach, Vol. I (Glover, D.M., ed.), pp.49-78, IRL Press, Oxford. 13 Messing, J. and Vieira, J. (1982) A new pair ofM 13 vectors for selecting either DNA strand of double digest restriction fragments. Gene 19,269-272. 14 Maniatis, T., Fritsch, E.E. and Sambrook, F. (1982) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 15 Hattori, M. and Sakaki, Y. (1986) Dideoxy sequencing method using denatured plasmid templates. Anal. Biochem. 152,232-238. 16 Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463-5467. 17 Devereux, J., Haeberli, P. and Smith, O. (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12, 387-395. 18 Fitzgerald, M. and Shenk, T. (1981) The sequence 5'AAUAAA-3' forms part of the recognition site for polyadenylation of late SV40 mRNAs. Cell 24, 251-260. 19 Chou, P.Y. and Fasman, G.D. (1978) Prediction of the secondary structure of proteins from their amino acid se-
Ill quence. Adv. Enzymol. Rel. Areas Mol. Biol. 47, 45-148. 20 Modrow, S. and Wolf, H. (1986) Characterization of two related Epstein-Barr virus-encoded membrane proteins that are differentially expressed in Burkitt lymphoma and in vitro-transformed cell lines. Proc. Natl. Acad. Sci. USA 83, 5703-5707. 21 Lucius, R., Erondu, N., Kern, A. and Donelson, J.E. (1988) Molecular cloning of an immunodominant antigen of Onchocerca volvulus. J. Exp. Med. 168, 1199-1204. 22 Garate, T., Conraths, F.J., Harnett, W., Btittner, D.W. and Parkhouse, R.M.E. (1990) Cloning of specific diagnostic antigens of Onchocerca volvulus. Trop. Med. Parasitol. 41, 245-250.
23 Forsyth, K.P., Copeman, D.B., Mitchell, G.F. (1984) Differences in the surface of iodinated skin and uterine microfilariae of Onchocerca gibsoni. Mol. Biochem. Parasitol. 10, 217-229. 24 Edwards, M.K., Busto, P., James, E.R., Carlow, C.K.S. and Philipp, M. (1990) Antigenic and dynamic properties of the surface of Onchocerca microfilariae. Trop. Med. Parasitol. 41,174-180. 25 Edwards, M.K., James, E.R. and Philipp, M. (1987) Biochemical and immunological properties of the surface of Onchocerca lienalis microfilariae. J. Cell. Biochem. (Suppl.) 11A, 166.
Molecular and Biochemical Parasitology, 46 (1991) 103-112 © 1991 Elsevier Science Publishers B.V. / 0166-6851/91/$03.50 ADONIS 016668519100044H MOLBIO 01514
Studies on a 14-kilodalton surface protein of Onchocerca microfilariae Franz J. Conraths 1"' , Michael J. W o r m s 1, Graham Preece 2, William Harnett ~and R. Michael E. Parkhouse 2 Divisions of Iparasitology and 2Immunology, National Institute for Medical Research, London, U.K. (Received 9 August 1990; accepted 22 November 1990)
A 14-kDa antigen present on the surface of uterine microfilariae of Onchocerca spp. has been identified using monoclonal antibodies. The antigen was also found in skin microfilariae, but in a masked or cryptic form. A complementary DNA clone encoding the epitope recognised by one of the monoclonal antibodies was identified in a ~gtl 1 library. Nucleotide sequencing revealed that the 233-bp cDNA fragment codes for the carboxy-terminus of the antigen. The deduced amino acid sequence consists of three hydrophobic domains with high potential for [3-sheetformation. The amino-terminalhydrophobic domain is followed by 4 positively charged residues (positions 22-25) which contribute to the rather basic character of the protein. Another interesting feature of the polypeptide is its richness in phenylalanine (12.7%). From the sequence information, a synthetic peptide was synthesised which was recognised by one of the monoclonal antibodies directed against the 14-kDa antigen and a small number of sera from patients with onchocerciasis. The relevance of this to vaccination is discussed. Key words: Onchocerca volvulus; Microfilariae; Surface antigen; Monoclonal antibody; Deduced partial amino acid sequence
Introduction
Human onchocerciasis is a tropical disease occurring in many parts of Africa, Central America and some regions of South America. The disease is associated with severe dermal lesions and blindness [1]. Many of the observed pathological Correspondence address: R.M.E. Parkhouse, Division of Immunology,National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, U.K. *Present address: Institut fur Parasitologie, Rudolf-Buchheim-Str. 2, D-6300 Giessen, F.R.G. Note: Nucleotide sequence data reported in this paper have been submitted to the GenBankTM data base with the accession number M 58412. Abbreviations: BSA, bovine serum albumin; cDNA, complementary DNA; ELISA, enzyme-linkedimmunosorbentassay; PBS, phosphate-buffered saline; PMSF, phenylmethylsulphonyl fluoride; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; TLCK,N-ct-tosyl-L-lysine chloromethyl ketone; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone.
changes are thought to arise as a consequence of immune responses of the host to microfilarial antigens [2]. Antibodies to microfilarial antigens are easily demonstrated in filarial infections in general. At the same time, the humoral response to surface components is difficult to detect and targetted to a few molecules [3-5]. The response in onchocerciasis appears to be typical of this, as Taylor and co-workers [6] have reported that many sera from human patients do not immunoprecipitate radioactively labelled surface antigens of Onchocerca volvulus microfilariae. In spite of the potential role of microfilarial surface antigens as targets for protective or pathological immune responses, information on their structure is very limited. One reason is the restricted availability of O. volvulus microfilariae. It is known, however, that surface proteins of O. volvulus and the related cattle parasites Onchocerca lienalis and Onchocerca gibsoni cross-react [3,7,25]. This cross-reactivity offers an alternative source of material for study. The scarcity of O. volvulus material can also be overcome by expressing cloned genes of the para-
104
site [8]. Such expressed recombinant antigen, together with hybridoma antibodies may allow immunological and biochemical studies of individual parasite components. In this study, we have employed such techniques to characterise a 14-kDa surface antigen of Onchocerca microfilariae. Materials and Methods
Parasites.
Living O. lienalis microfilariae and adult worms were isolated from naturally infected British cattle. Cryopreserved O. gibsoni microfilariae and nodules containing O. gibsoni worms were provided by B. Copeman, Townsville, Australia. Uterine microfilariae were obtained by dissection of adult female worms. Adult males of O. volvulus isolated by collagenase digestion from onchocercomata of Venezuelan patients were provided by Z. Cabrera, NIMR. Human patient sera were obtained from the WHO filariasis serum bank.
Monoclonal antibodies.
BALB/c mice received an injection of 20 000 microfilariae suspended in Freund's complete adjuvant into each of the rear footpads. Three days later, a similar dose was given in incomplete Freund's adjuvant. At 3-4-day intervals, 4 further injections of similar numbers of microfilariae were given in PBS. One day after the last administration, the draining lymph nodes were removed, lymphocytes isolated and fused with JKAg4/1 cells. Hybridoma cell lines were selected according to standard procedures [9]. Tissue culture supernatants were screened for antibodies to microfilarial antigens in indirect immunofluorescent assays, radioimmune precipitations and Western blot experiments. Positive hybridoma cultures were cloned by limiting dilution.
Indirect immunofluorescent assays.
2 000 living microfilariae were reacted with 100 ~l hybridoma supernatant for 30 min on ice. After 3 washes with ice-cold RPMI-1640 medium, the microfilariae were incubated with anti-mouse IgG-FITC conjugate (Seralab) for 30 min on ice. Three further washes with ice-cold PBS were followed by a 30min incubation with 10% (w/v) formaldehyde in PBS at room temperature. After 3 washes with PBS at room temperature, the microfilariae were mounted on slides and microscopically examined.
Radioimmunoprecipitation.
Approximately 105 living microfilariae were radio-iodinated using the Iodogen method according to the instructions of the manufacturer (Pierce Chemical Co., Rockford, IL). Tyrosine was added in excess to bind unreacted radioactive iodine. Radiolabelled parasites were washed twice in saline and once in 100 mM Tris, pH 7.5. The parasites were suspended in 20 mM Tris, pH 8.3, containing 1.5 mM N-~-tosyl-L-lysine chloromethyl ketone (TLCK, Sigma); 1.5 mM Ntosyl-L-phenylalanine chloromethyl ketone (TPCK, Sigma); 1 mM phenylmethylsulphonyl fluoride (PMSF, Sigma); sodium deoxycholate (BDH) was then added to a final concentration of 2% (w/v). After 4 min sonication at 0°C the parasite lysate was centrifuged at 10 000 x g for 30 min. Approximately 5 ~tl of the supernatant of this crude antigen preparation (20 000 cpm of radioactivity) were reacted with 100 ~tl hybridoma supernatant or 5 ~tl mouse serum, respectively. Antigen-antibody complexes were precipitated and analysed by SDSPAGE as described [ 10].
Western blotting. Parasites were incubated in 100 mM Tris, pH 7.5/2% (w/v) sodium deoxycholate/ 1.5 mM TLCK/1.5 mM TPCK/1 mM PMSF for 15 min at 0°C, sonicated for 4 min and centrifuged (30 min, 10 000 x g). The supernatant of this preparation was denatured under reducing conditions and electrophoresed on 5-25% (w/v) gradient SDS-polyacrylamide gels as described [11 ]. Separated proteins were transferred to nitrocellulose membranes (Millipore Corp., 45/xm pore size). Membrane strips were blocked with 2% (w/v) bovine serum albumin and 0.05% (v/v) Tween 20 (Sigma) in 50 mM Tris, pH 8.0/150 mM sodium chloride/2 mM MgC12 (TBS). Monoclonal antibodies or sera, respectively, were appropriately diluted in blocking buffer and reacted with the nitrocellulose membranes overnight at room temperature. Bound antibodies were detected with anti-mouse IgG (H+L chain) alkaline phosphatase conjugate (Promega, Madison, Wisconsin, USA) and nitroblue tetrazolium in combination with 5bromo-4-chloro-3-indolyl phosphate (Sigma) as substrate. Isolation ofcDNA clones. A )~gtl 1 cDNA library derived from female O. volvulus worms isolated in
105
Kumba, Cameroons, was provided by J. Donelson, University of Iowa, Iowa City, IA, U.S.A. [8]. Fusion proteins encoded by recombinant phage were identified using monoclonal antibodies to O. gibsoni or O. lienalis microfilariae essentially as described [ 12]. DNA was isolated from plaque-purified phage and the inserts subcloned into the unique EcoRI site of the plasmid vector pUC8 [13] using standard techniques [ 14].
DNA sequencing.
Single-stranded DNA was prepared from recombinant plasmids by alkali treatment [15] followed by chain termination sequencing using BRL reagents and equipment [16]. Computer analyses of DNA and amino acid sequences were done using the the software package of the University of Wisconsin Genetics Computer Group [ 17].
Synthetic peptide.
A peptide with the sequence GIQKQCVLLKYMFPKKNKALF was manufactured by the Laboratory of Peptide Chemistry, NIMR, London on LKB Bioxlynx 4170 equipment.
Dot blot and ELISA.
10 ~g of the synthetic peptide or parasite antigen in 2 ktl volume were spotted onto nitrocellulose filters. The air-dried filters were processed as described for Western blots. Alternatively, 96-well polystyrene plates were coated with 10 ~tg peptide in 0.1 M sodium borate buffer, pH 9.0, at 4°C overnight. Remaining binding sites were blocked with 3% (w/v) BSA in the same buffer for 2 h at room temperature. Sera, appropriately diluted in PBS with 0.05% Tween 20, were reacted with the antigen for 1 h at room temperature. Bound antibodies were detected by incubation with appropriate anti-immunoglobulin alkaline phosphatase
B
C
Fig. 1. Immunofluorescencewith monoclonal antibodies to surfaceantigens of uterine O. gibsoni microfilariae. Monoclonalantibody 2A5B9 or normal mouse serum were incubated with uterine microfilariae, embryosand eggs derived from frozen nodules. Bound antibodies were visualisedusing a FITC-labelledanti-mouse IgG conjugate. (A) 2A5B9, immunofluorescence.(B) Normal mouse serum, immunofluorescence.(C) 2A5B9,bright field. (D) Normal mouse serum, bright field.
106
conjugates (1 h, room temperature). After washing, substrate (p-nitrophenyl phosphate; Sigma) was added and the reaction stopped after 30 min by adding NaOH to a final concentration of 1 M. Adsorption at 405 nm was measured in an automatic ELISA reader (Titertek Inc.).
LL.
o~
o')
0,~
~1
'<
Results
Monoclonal antibodies to a 14-kDa surface antigen. Hybridoma supernatants were employed in immunofluorescent assays using uterine and skin O. lienalis (live) and O. gibsoni (cryopreserved) microfilariae. None of the supernatants reacted with skin microfilariae of either species, but two (2A4E9 and 2A5B9) showed evenly distributed surface fluorescence on approximately 90% of the uterine microfilariae. The antigen(s) recognised were also present in the egg-shell and on the surface of embryos from the late morula stage onwards, but not on earlier stages (Table I, Fig. 1). In immunoprecipitation experiments, these antibodies reacted with radio-iodinated molecules of 14-kDa and 60 kDa from lysates of surface-labelled O. lienalis skin microfilariae (Fig. 2). The former antigen was also recognised when the monoclonal antibodies were employed to probe Western blots ofmicrofilaTABLE I Characteristics of monoclonal antibodies to surface antigens of Onchocerca microfilariae Monoclonal antibodies 2A4E9
2A5B9
Immunofluorescence, uterine microfilariae O. gibsoni O. lienalis
+ +
+ +
60 14
60 14
14 14
14 92
Immunofluorescence, skin microfilariae O. gibsoni O. lienalis
Radioimmune precipitation t25I-labelled O. lienalis microfilariae, (kDa) Western blot O. lienalis microfilariae (kDa) O. volvulus adult, male (kDa)
-
200kD
-
92.5
-
69
-
46
-
21.5
-
14.3
Fig. 2. Immunoprecipitations of radiolabelled surface antigens of O. lienalis microfilariae. Living O. lienalis skin microfilariae were radiolabelled with ~25I(Iodogen method) and solubilised. Antigen preparations were reacted with mouse sera or monoclonal antibodies. Antigen/antibody complexes were precipitated using goat anti-mouse IgG, analysed in SDS-polyacrylamide gels and autoradiographed. FI 17, mouse hyperimmune serum to O. gibsoni mf; 2A4E9, monoclonal antibody; 2A5B9, monoclonal antibody; NMS, normal mouse serum; AG, antigen preparation.
rial antigen, whereas the 60-kDa antigen is not detected under these conditions (Fig. 3A). In adult worm extracts, antibody 2A5B9 does not recognise the 14-kDa protein, but binds to a 92-kDa protein (Fig. 3B; Table I). cDNA cloning. Since the monoclonal antibody 2A5B9 reacted with a 92-kDa antigen present in adult worms in addition to the 14-kDa surface protein of microfilariae, we focussed on the antibody 2A4E9 as a probe for the 14-kDa antigen. The antibody was employed to screen a ~,gt 11 cDNA library derived from female worms from Kumba, Cameroons. It has previously been shown that this gene bank contains clones of microfilarial origin [8]. A recombinant phage designated ~,-2A4.1, which was selectively recognised by the antibody probe, was plaque-purified and further analysed.
107
LL
i ~i~i i i i i[~!i
i~iiiiiiii~%
i~ii!~i~iii~
<~
200kD
-200
kD
iiii! ,i
[~i[i ~i~ii~i)
92.5
92.5
69
69
i
~i.... ~!i!i ~ ~ii~!i~i[i i [~ii ~i!i!
~i!~i~i~~(!/!~[i ~,~i!....i~i~ ~i~iJ'i~?!~
~i!iii~i!
~!iiii!
-
46
-
30
-
21.5
46
iii~iiiiii!!~!i~iill
i ~i?i i i~i!i~i !!ili
30
20 -
14.3
A
.....
1 4,3
B
Fig. 3. Western blot analyses. O. gibsoniskin microfilariae (A) or adultmale O. volvulus(B) were solubilised, electrophoresed and transferred to nitrocellulose. The membranes were cut into strips which were individually incubated with monoclonal antibodies or sera. Bound antibodies were visualised with alkaline phosphataselabelled anti-mouse IgG and nitro blue tetrazoliumand 5-bromo4-chloro-3-indolyl phosphateas substrate.2A4E9, monoclonal antibody;2A5B9, monoclonal antibody;F 117, mouse hyperimmune serum; NMS, normalmouse serum.
Nucleotide and deduced amino acid sequence of~L2A4.1. The insert of the phage ~L-2A4.1 was subcloned into pUC 8 and sequenced (Fig. 4). It is 233 nucleotides long and apparently represents approximately half of the complete message. An amino-terminal start codon could not be found and the theoretical molecular weight of the deduced polypeptide remains well below the expected value of 14 kDa. A stop codon beginning at position +196
may indicate that ~,-2A4.1 encodes the carboxyterminus of the protein. The sequence following nucleotide position +217 could possibly serve as a polyadenylation signal, although there is a mismatch in position +220 (A:T) with respect to the reported consensus motif [18]. The deduced amino acid sequence of ~-2A4.1 is dominated by 3 stretches of mainly hydrophobic amino acids (positions 1-20; 33-42; 5 7 - 6 3 ) with high potential for
108 1 1
GAA
TTC
CAA Q
GTA V
ATT I
AGC S
AGG R
CGT R
TTT F
GGG G
ATA I
CAA AAA Q K
CAG Q
TGC C
GTT V
49 15
TTA L
CTG L
AAG K
TAC Y
ATG M
TTT F
CCA AAA P K
AAA K
AAT N
AAG K
GCA A
TTA L
TTT F
TCA S
CTG L
97 31
GAC D
ATC I
TTA L
CCG P
TTC F
GTT V
AGA R
GTG V
TAC Y
TAT Y
GGC ACA G T
TTT F
TCC S
AAC N
GAA E
145 47
ATT I
TTG L
TCT S
GCA A
TTA L
TTC F
CAG AAG Q K
TGC C
GGA G
ATG M
TGC C
TTT F
TTT F
ATT I
GCA A
193 63
TAT Y
TGA *
TTC
TGA
GAA
TTT
AGA
CAT AAA
AAA
AGG
AAT
TC
CTG
Fig. 4. cDNA and deduced amino acid sequence of k-2A4.1. Numbering corresponds to relative positions in a partial cDNA lacking the 3' end of the gene.
1
2
3
4
5
A B 6
C Fig. 5. Mapping of the epitope recognised by the monoclonal antibody 2A4E9. O. gibsoni antigen (1), PBS (2), pep2A4.1, comprising amino acids 8-29 of the sequence translated from the insert of )~-2A4.1 (3), BSA (4), k-2A4.1 fusion protein (5) and an irrelevant recombinant O. volvulus antigen (6) were spotted onto a nitrocellulose membrane and probed with the monoclonal antibodies 2A4E9 (A), 2A5B9 (B), and an irrelevant monoclonal antibody directed against an irrelevant O. volvulus antigen (C).
[3-sheet formation according to secondary structure predictions (data not shown) [19]. The amino-terminal hydrophobic domain is followed by 4 positively charged residues which contribute to a region of coil in secondary structure prediction. Likewise, the second and third hydrophobic B-sheet domains are separated by a stretch of coil and a short sequence of c~-helix forming amino acids, respectively, but charged residues are lacking in these regions. In general, the amino acid sequence encoded by )~-2A4.1 is rich in hydrophobic amino acids, in particular phenylalanine (12.7%). It also contains a
number of positively charged residues which make the protein rather basic. Three cysteine residues may influence the tertiary structure or interact with other molecules. There is no indication of any Nglycosylation sites in the deduced amino acid sequence. Determination of the epitope detected by monoclonal antibody 2A4E9. Potential epitopes on this molecule were predicted using the algorithm described by Modrow and Wolf [20]. Two regions ranging from amino acid positions 18-26 and 4045 showed an antigenic index above 1.0. A peptide comprising amino acids 8-29 (pep2A4.1) was synthesised. Dot blot assays revealed that pep2A4.1 is specifically recognised by the monoclonal antibody 2A4E9, but not by the monoclonal antibody 2A5B9 or a irrelevant monoclonal antibody directed against a different O. volvulus antigen (Fig. 5). A small number of human onchocerciasis sera (N=9) were examined for their reactivity with pep2A4.1. We found that only 2 of these sera recognised the peptide, with titres in the range of 1/160 1/1280 (Table II). Discussion
In this publication, we report the characterisation and partial predicted sequence of a markedly hydrophobic surface antigen of Onchocerca microfilariae. Complementary DNA clones presumably coding for this antigen, were selected from an existing expression library, which has previously yielded nucleotide sequences corresponding to important 'house-keeping' genes and antigens of diagnostic
109 TABLE II Reactivity of human filariasis patient sera with pep2A4.1 Serum No.
Origin
Clinical Manifestation
Titre
86 89 91 92 162 165 185 Pool NHS
Ivory Coast Ivory Coast Ivory Coast Ivory Coast Yemen Yemen Yemen Liberia United Kingdom
O O O O O,S O,S O,S O negative
negative negative 1/640 negative negative 1/1280 negative negative negative
Peptide pep2A4.1 was attached to polystyrene plates (10/zg/well) and reacted with serial dilutions of human onchocerciasis sera from patients with generalised onchocerciasis (O) or 'sowdah' (O,S), or normal human serum (NHS). Bound antibodies were detected with rabbit anti-humanIgG alkaline phosphatase conjugates and end-point titres determined.
relevance of O. volvulus [9,21,22]. Specifically, a cDNA clone (~,-2A4.1) was selected by a monoclonal antibody (2A4E9) reactive with a 14-kDa microfilarial surface antigen. The amino acid sequence of this clone apparently represents the carboxy-terminus of the surface polypeptide and displays 3 predominantly hydrophobic domains with a high potential for 13-sheet formation. These features are of interest as they would certainly allow the molecule to interact with other hydrophobic components of the microfilarial epicuticle. Whether they are organised in the living microfilarial surface in this manner, however, remains a challenge for future investigations. One possible arrangement which must also be considered is an association with other proteins, since the 14-kDa component does in fact interact with a 60-kDa molecule (for results of immunoprecipitations, see below). The precise location of the epitope recognised by the monoclonal antibody 2A4E9 is suggested but not proved by calculations of the antigen index according to the algorithm of Modrow and Wolf [20]. This analysis indicates that a peptide spanning the amino acid positions 20-26 is the most likely target of an immune response (data not shown). In line with this hypothesis, a peptide comprising amino acids 8-29 was synthesised and shown to be recognised by the original monoclonal antibody 2A4E9. The same peptide was also recognised by 2 out of 9 sera of infected humans, and it will be of considerable interest to survey a larger number of sera to determine if this low level of reactivity persists. Surface epitopes which are of poor immunogenicity
during natural infection may merit investigation as potential vaccines. It is possible that the epitope present in the peptide may fall into this category. The epitopes of the 14-kDa polypeptide recognised by the monoclonal antibodies 2A4E9 and 2A5B9 become masked or cryptic upon release of the microfilariae from the uterus of female worms in both O. lienalis and O. gibsoni. In this respect, our findings confirm results obtained by other investigators with polyvalent sera [6,23]. Forsyth and co-workers [23] suggested that host albumin may contribute to covering parasite molecules which are potential targets of the immune response. Although our own investigations confirmed the presence of bovine albumin on the surface of O. lienalis skin microfilariae (unpublished observations), it has yet to be established, whether albumin masks the epitopes recognised by the monoclonal antibodies 2A4E9 and 2A5B9. The results indicating the presence of masked or cryptic epitopes may reflect a maturation process which could have consequences for the recognition by antibody in vivo. It may be the case that microfilariae will only bind antibody for short period following release from the uterus. Surface components in the range of 14-15 kDa have been found in microfilariae of O. volvulus [6] and O. cervicalis [24]. Our own results show the presence of similar molecules in O. lienalis and O. gibsoni microfilariae. Our finding that these low molecular weight components contain cross-reactive epitopes is consistent with observations of Edwards and colleagues [24]. The immunoprecipitation analysis of surface-labelled O. lienalis
110
microfilariae may suggest, that the 14-kDa polypeptide recognised by the monoclonal antibodies forms a non-covalently associating heterodimer with a 60-kDa molecule which is also present on the surface of microfilariae. Although the monoclonal antibodies described in this publication do not react with epitopes present on the 60-kDa component, its purification is perfectly feasible using monoclonal antibody 2A4E9 and detergent lysate of microfilariae. More detailed information on this molecule is required in order to assess its relation to surface components with molecular weights similar to those of other Onchocerca species [23,24]. An important question is whether the microfilarial 14kDa antigen is endogenously synthesised or, on the other hand, exogenously acquired from the adult female. This question may be addressed by probing mRNA derived from different stages with the cDNA clone of the 14-kDa protein - given sufficient microfilariae. In summary, a 14-kDa surface antigen of Onchocerca microfilariae has been identified using monoclonal antibodies, partially cloned, and sequenced. The structure revealed is particularly interesting, being mainly hydrophobic and unusually rich in phenylalanine. At the same time, there is an obvious antigenic determinant, which, when synthesised as an oligopeptide, was recognised by the original monoclonal antibody and a restricted number of onchocerciasis patient sera. The possibility that this sequence is protective is currently under investigation.
Acknowledgements This work received financial support from the Edna McConnell Clark Foundation. FJC was supported by a fellowship grant of the German Academic Exchange Service.
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3 Forsyth, K.P., Copeman, D.B., Anders, R.F. and Mitchell, G.F. (l 981) The major radioiodinated cuticular antigens of Onchocerca gibsoni microfilariae are neither species nor onchocerca specific. Acta Trop. 38,343-352. 4 Maizels, R.M., Selkirk, M.E., Sutanto, 1. and Partono, F. (1987) Antibody responses in human lymphatic filarial parasites. In: Filariasis: Ciba Foundation Symposium 127, pp. 189-200, Wiley, Chichester. 5 Egwang, T.G., Dupont, A., Aku6, J.-P. and Pinder, M. (1988) Biochemical and immunological characterization of surface and excretory-secretory antigens ofLoa loa microfilariae. Mol. Biochem. Parasitol. 31,251-262. 6 Taylor, D.W., Goddard, J.M. and McMahon, J.E. (1986) Surface components of Onchocerca volvulus. Mol. Biochem. Parasitol. 18,283-300. 7 Garate, T., Cabrera, Z., Copeman, D.B., Harnett, W., McLaren, D.J., Pattersen, M. and Parkhouse, R.M.E. (In Press) Surface antigens of male worms and microfilariae of Onchocerca gibsoni. Int. J. Parasitol. 8 Donelson, J.E., Duke, B.O.L., Moser, D., Zeng, W., Erondu, N.E., Lucius, R., Renz, A., Karam, M. and Zea Flores, G. (1988) Construction of Onchocerca volvulus cDNA libraries and partial characterization of the cDNA for a major antigen. Mol. Biochem. Parasitol. 31,241-250. 9 K/Jhler, G. and Milstein C. (1976) Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion. Eur. J. Immunol. 6, 511-519. 10 Hamett, W., Meghji, M., Worms, M.J. and Parkhouse, R.M.E. (1986) Quantitative and qualitative changes in production of excretions/secretions by Litosomoides carinii during development in the jird (Meriones unguiculatus). Parasitology 93, 317-331. 11 Parkhouse, R.M.E., Philipp, M. and Ogilvie, B.M. (1981) Characterisation of surface antigens of Trichinella spiralis infective larvae. Parasite Immunol. 3,339-352. 12 Huynh, T.V., Young, R.A. and Davis, R.W. (1985) Constructing and screening cDNA libraries in lambda gt I 0 and lambda gtl 1. In: DNA Cloning. A Practical Approach, Vol. I (Glover, D.M., ed.), pp.49-78, IRL Press, Oxford. 13 Messing, J. and Vieira, J. (1982) A new pair ofM 13 vectors for selecting either DNA strand of double digest restriction fragments. Gene 19,269-272. 14 Maniatis, T., Fritsch, E.E. and Sambrook, F. (1982) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 15 Hattori, M. and Sakaki, Y. (1986) Dideoxy sequencing method using denatured plasmid templates. Anal. Biochem. 152,232-238. 16 Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463-5467. 17 Devereux, J., Haeberli, P. and Smith, O. (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12, 387-395. 18 Fitzgerald, M. and Shenk, T. (1981) The sequence 5'AAUAAA-3' forms part of the recognition site for polyadenylation of late SV40 mRNAs. Cell 24, 251-260. 19 Chou, P.Y. and Fasman, G.D. (1978) Prediction of the secondary structure of proteins from their amino acid se-
Ill quence. Adv. Enzymol. Rel. Areas Mol. Biol. 47, 45-148. 20 Modrow, S. and Wolf, H. (1986) Characterization of two related Epstein-Barr virus-encoded membrane proteins that are differentially expressed in Burkitt lymphoma and in vitro-transformed cell lines. Proc. Natl. Acad. Sci. USA 83, 5703-5707. 21 Lucius, R., Erondu, N., Kern, A. and Donelson, J.E. (1988) Molecular cloning of an immunodominant antigen of Onchocerca volvulus. J. Exp. Med. 168, 1199-1204. 22 Garate, T., Conraths, F.J., Harnett, W., Btittner, D.W. and Parkhouse, R.M.E. (1990) Cloning of specific diagnostic antigens of Onchocerca volvulus. Trop. Med. Parasitol. 41, 245-250.
23 Forsyth, K.P., Copeman, D.B., Mitchell, G.F. (1984) Differences in the surface of iodinated skin and uterine microfilariae of Onchocerca gibsoni. Mol. Biochem. Parasitol. 10, 217-229. 24 Edwards, M.K., Busto, P., James, E.R., Carlow, C.K.S. and Philipp, M. (1990) Antigenic and dynamic properties of the surface of Onchocerca microfilariae. Trop. Med. Parasitol. 41,174-180. 25 Edwards, M.K., James, E.R. and Philipp, M. (1987) Biochemical and immunological properties of the surface of Onchocerca lienalis microfilariae. J. Cell. Biochem. (Suppl.) 11A, 166.