EXPERIMENTALPARASITOLOGY75, 379-389 (1992)
Characterization
of a Muscle-Associated Wuchereria
N. RAGHAVAN,*
Antigen
from
bancrofti’
C. V. MAINA,? P. C. FITZGERALD,~ R. S. TUAN,§ B. E. SLATKO,~ E. A. OTTESEN,* AND T. B. NUTMAN*
*Laboratory of Parasitic Diseases, Building 4lRoom 126, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, U.S.A.; tNew England Biolabs, 32 Tozer Road, Beverly, Massachusetts 01915, U.S.A.; $Division of Computer Research and Technology, Building IZAIRoom 2051 National Institutes of Health, Bethesda, Maryland 20892, U.S.A.; and $Department of Orrhopedic Research, 1015 Walnut Street, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, U.S.A.
RAGHAVAN, N., MAINA, C. V., FITZGERALD,P. C., TUAN, R. S., SLATKO, B. E., OTTESEN, E. A., AND NUTMAN, T. B. 1992. Characterization of a muscle-associated antigen from Wuchereria bancrofti. Experimental Parasitology 75, 37%389. A recombinant clone, WbNl, isolated from a genomic expression library of Wuchereria bancrofti and showing restricted specificity at the DNA level (Southern and PCR analyses) for Wuchereria bancrofti and Brugia malayi has been previously described. Sequence analysis of WbNl indicated that it had notable similarity to myosin. Further characterization using in situ hybridization has localized the mRNA in the muscle of the adult parasite and in the microfilariae. Rabbit polyclonal antiserum, raised against the recombinant WbNl fused to the maltosebinding protein, recognized a 200-kDa polypeptide in immunoblots containing B. malayi antigen extracts. The same antibody also recognized myosin extracted from Brugia pahangi, Onchocerca volvulus, and Caenorhabditis elegans. Localization using the rabbit antiserum revealed the presence of the antigen in the adult muscle tissue and in the microfilariae; the same antibody inhibited the binding of a monoclonal antibody 28.2 (directed toward MHC B of C. elegans myosin) to the recombinant WbNl antigen and also to purified C. elegans myosin. Based on homology data, structural location, competitive ELISA, and immunoblot we conclude that WbNl is related to myosin or a similar myofibrillar protein. 0 19!32Academic Press, Inc. INDEX DESCRIPTORS AND ABBREVIATIONS:Nemata; Filarioidea; Wuchereria bancrofti; Brugia malayi; Brugia pahangi; Onchocerca volvulus; Lymphatic tilariasis; Myosin-like antigen; Base pair (bp); Phosphate-buffered saline (PBS); Maltose-binding protein (MBP); Open reading frame (ORF); Enzyme-linked immuno-sorbent assay (ELISA); Isopropyl p-othiogalactopyranoside (IPTG); 5-bromo-4-chloro-3-indolyl phosphate (BCIP); Nitroblue tetrazolium (NBT); Diethylcarbamazine (DEC); Polymerase chain reaction (PCR); Myosin heavy chain (MHC); Monoclonal antibody (Mab); Normal rabbit serum (NRS); Chronic lymphatic obstruction (CP); Asymptomatic microtilaremia (MF); Tropical pulmonary eosinophilia (TPE).
The isolation and characterization of the recombinant clone WbNl (2.5 kb) has been previously described (Raghavan ef al. 1991). Although derived from a genomic library, WbNl has an ORF of t 380 bp coding ‘ Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession No. M38213.
for a protein of 460 amino acids with a molecular weight of 51 kDa. The sequence of WbNl showed similarity to myosin of several species (Raghavan et al. 1991). To characterize this antigen further, the 2.5kb WbNl insert was subcloned in the plasmid expression vector pPR682 to produce a fusion product with the maltose binding protein of the Ma1 E gene. Antiserum raised in rabbits against this fusion product enabled 379 0014.4894/92$5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
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detailed characterization of the recombinant antigen expressed by WbNl. Such characterization shows that WbNl codes for a muscle-associated myosin-like product.
ETAL.
nol until used. The fixed parasites were embedded in paraffin, and 0.3~pm thick paraffin sections of the parasite were sequentially deparafllnized in 108% xylene (twice), 100% ethanol (twice), 90, 80, and 70% ethanol (once each) followed by single washes with distilled water and PBS for 2 to 3 min each. The deparaffinized sections were blocked with 3% BSA for 30 min at MATERIALSANDMETHODS room temperature and incubated overnight at 4°C with a 1:250 dilution of the primary antibody. The sections Parasites. Microfilariae of Wuchereria bancrofti were obtained from the blood of patients between 10 were then washed three times with PBS containing 0.05% Triton X-100 for 10min each and incubated with PM and midnight and purified as described earlier the second antibody conjugated to alkaline phos(Raghavan et al. 1991). Adult parasites and microfilarphatase for 1 hr at room temperature. The sections iae of Brugia malayi were obtained from the peritoneal cavity of jirds. Adult Brugia pahangi were obtained were washed, and the color reaction was developed using BCIP/NBT as the substrate/chromogenic indicafrom TRS Laboratories (Athens, GA). Caenorhabditis elegans N2 were cultured in the laboratory on Esche- tor. The sections were then dehydrated sequentially in richia coli OPSO for 10 days prior to harvesting the 70,80, and 90% ethanol once, 100%ethanol twice, and adult organisms (Wood 1988). Adult female On- xylene twice, for 1 to 3 min each. The stained sections chocerca volvulus were isolated from nodules by col- were then air dried and mounted using a solution of 50% Permount (Fisher Scientific Company, Fair lagenase digestion (Schulz-Key et al. 1977). Lawn, NJ). Production of recombinant protein and antibodies against the MBP-WbNl fusion product. The 2.5kb In situ hybridization analysis. Live B. malayi adult insert from WbNl was subcloned into the malE ex- female parasites were fixed, processed, and hybridpression vector pPR682 (Guan et al. 1988;Maina et al. ized either with the biotinylated WbNl DNA isolated 1988) as previously described (Raghavan et al. 1991). from a pUC19 subclone (pUC19-WbNl) or pUC19 Rabbits were immunized subcutaneously with the re- DNA used as the control (Tuan et al. 1988; Lo 1986). combinant MBP-WbNl fusion product with 500 pg of The labeled sections were examined with an Olympus BH-2 microscope using Nomarski optics and photoantigen in RIBI (RIB1 Immunochem Research, Inc., Hamilton, MT) adjuvant. The rabbits were boosted graphed using Kodak Ektachrome-160 film. Histological staining. Histological staining of paraevery 2 weeks over a 6-week period. Sera were colsite tissue sections were done with Gill’s hematoxylin lected a week after the final boost. Zmmunoblots. The recombinant MBP-WbNl fusion (No. 2) and counterstained with eosin (Kieman 1981). product or the myosins extracted (Frederiksen and Antifilarial antibody measurement. Antililarial antiCunningham 1982)from B. malayi, B. pahangi, C. el- bodies were measured by ELISA using either B. maegans, and 0. volvulus were solubilized in sample layi antigen extracts (Nutman et al. 1987) or the rebuffer (63 m&f Tris-HCl, pH 6.8, 2% w/v SDS, 5% combinant MBP-WbNl fusion protein. 2-mercaptoethanol, and 10% v/v glycerol), boiled for 3 Competitive ELISA. Competitive ELISA was performed by coating a 96-well microtiter plate with 100 to 5 min, separated on 4-20% SDS-polyacrylamide kg/ml of recombinant antigen in carbonate/ gels (Laemmli 1970), and transferred to nitrocellulose paper (Towbin et al. 1979). The filters were blocked bicarbonate buffer. After washes with PBS containing with a 3% solution of skim milk powder followed by 0.05% Tween-20, rabbit anti-WbNl antibodies, normal incubation with the primary antibody of interest and rabbit serum (NRS), or rabbit anti-Ag43 (directed togoat anti-rabbit IgG conjugated to alkaline phos- ward an unrelated filarial antigen), were serially diphatase (Jackson Immunoresearch Labs, Inc., West luted and added to the wells. Mabs 5-6 and 28.2 (Miller Grove, PA). The filters were washed four times, at et al. 1983;Ardizzi and Epstein 1987)directed toward IO-mm intervals between each incubation, in a buffer different epitopes of C. elegans myosin were also containing 0.25 M Tris, pH 8.0, 0.2 M glycine, 0.3% added at dilutions normalized by their reactivity to the recombinant antigen. After an overnight incubation, Tween-20, and 0.05% Triton X-100. After the final wash, the filters were developed with BCIP as the sub- goat anti-mouse IgG conjugated to alkaline phosstrate and NBT as the chromogenic indicator (Kirkephatase was added, incubated for 2 hr, washed, and gaard and Perry, Gaithersburg, MD). The reactions developed as decribed earlier (Nutman et al. 1987). Competitive immunoblots. Myosin extracted from were stopped by washing the filter extensively with C. elegans was solubilized in sample buffer, separated distilled water. Zmmunostaining. Live B. malayiadult parasites were on a 10% SDS-polyacrylamide preparative gel, and washed extensively in PBS, and fixed for 2 to 3 hr in a immunoblotted as described earlier. Four-millimeter strips were cut from the imrnunoblots and incubated solution of Bouin-Hollande (Lillie 1954). Following this, the fixed parasites were suspended in 70% etha- with C. elegans Mab 28.2 alone (1:50,000), or in com-
MYOSIN-LIKE ANTIGEN OF w. bUnCrOfti bination with varying dilutions of rabbit anti-WbNl antibodies (1:20, l:lOO, 1:500, and 1:2500) or rabbit anti-Ag43 antibodies (1:20) (antibody raised against an unrelated 43-kDa antigen isolated from the infective larvae of B. maluyQ or normal rabbit serum (1:20). The strips were washed and incubated with goat antimouse IgG conjugated with alkaline phosphatase and developed.
RESULTS
Expression of recombinant antigen and production ofrabbit antiserum. The 2.5-kb insert of WbNl was subcloned in the BamHZ site of the Mu1 E expression vector pPR682 (Raghavan et al. 1991). Expression of the recombinant antigen, induced with IO mM IPTG, resulted in the production of a fusion protein composed of the recombinant antigen (51 kDa) and the maltosebinding protein (39 kDa; Fig. 1, lane 2). This 90-kDa fusion product was readily purified using an amylose affinity column and eluted with free maltose as shown (Fig. 1,
MWl
2
200 kDa 97.4 kDa 69
kDa-
46
kDa-
30 kDa-21.5 kDa 14.3 kDa -
FIG. 1. Lmmunoblot analysis of recombinant WbNl. An immunoblot containing MBP-WbNl fusion protein (lane I) and MBP (lane 2) were probed with rabbit anti-MBP antiserum. Arrow indicates the position of the 90-kDa MBP-WbNl fusion protein.
381
lane 1). The smaller proteins present in the eluted fraction are presumably degradation products of the 90-kDa recombinant fusion protein or possibly truncated products of translation. Antiserum was raised in rabbits against this MBP-WbN 1 90-kDa fusion protein for further biochemical and histological analyses. Zmmunobtot unalysis and identification of the native WbNl antigen. In order to identify the native product encoded by WbNl, antigen extracts from B. malayi adults were electroblotted and reacted with either immune rabbit serum (Fig. 2, lane l), anti-MBP (Fig. 2, lane 2), or normal preimmune rabbit serum (Fig. 2, lane 3). The antiserum against the 90-kDa MBP-WbNl fusion recognized a 200-kDa parasite protein, and neither the normal rabbit nor the anti-MBP antisera recognized this 200-kDa protein. This indicated that the reactivity of the antibody to the high molecular weight 200-kDa protein was specifically derived from the WbNl region and not the MBP portion of the recombinant fusion protein. Zmmunolocalization of the native WbNl antigen. The anti-WbN1 antiserum was next used to localize the antigen in tissue sections of B. maluyi adult parasite (Fig. 3). Specific staining was observed in the muscle of the adult female and, to a lesser degree, in the microfilariae. No staining was observed when either the anti-MBP antibodies or the preimmune rabbit serum were used. Similar results were obtained with male parasites (data not shown). In situ hybridization. Localization of the transcript encoded by WbNl was studied by in situ DNA-mRNA hybridization. B. malayi adult female tissue sections were probed with either a biotinylated pUCl!% WbNl DNA (Fig. 4B) or pUC19 DNA (Fig. 4A) as a control. Positive hybridization signals were detected as purple/brown coloration in the tissue sections. As predicted by the antibody localization described above, a strong positive signal was observed in the
382
RAGHAVAN
12 MW Markers 200 kDa -
92.5 kDa 69 kDa 46 kDa -
3
ET AL.
elegans MHC family (MHC A, myo3; MHC B, uric-54; MHC C, myo-2 and MHC D, myo-1; Karn et al. 1983; Dibb et al. 1989) are shown in Fig. 5. As noted previously (Raghavan et al. 1991), there is notable similarity of WbNl to the C. elegans sequence. Further, based on the secondary structure predictions, there is a structural similarity between WbNl and the a-helical coiled-coil proteins such as myosin. Both the Chou-Fasman (Chou and Fasman 1978) and Garnier-Osguthorpe-Robson (Garnier et al. 1978) predictions for WbN1 show a predominance of a-helical conformations. Immunoblot analyses of purified myosins. To clarify further the antigenic nature
of WbNl, myosin was partially purified from B. malayi, B. pahangi, C. elegans, and 0. volvulus (Frederiksen and Cunning21.5 kDa ham 1982). The partially purified preparations of myosin were subjected to SDS14.3 kDa PAGE, immunoblotted, and reacted with the anti-WbNl antiserum (Fig. 6). As seen, anti-WbN 1 antiserum strongly recognized a a-MBP a-MBP NM specific 200-kDa peptide antigen from the + WbNl myosin prepared from B. malayi and, to a FIG. 2. Identification of the native WbNl antigen. lesser degree, from B. pahangi, 0. volvuAn immunoblot containing 15 kg/lane of B. malayi tolus, or C. elegans. The reactivity seen with tal antigen extract was probed with immune rabbit sesome of the lower molecular weight bands rum (lane l), anti-MBP antiserum (lane 2), or normal rabbit serum (lane 3). Arrow indicates the position of in the myosin preparations is likely due to the native antigen (200 kDa) recognized by the immune recognition of either some breakdown rabbit serum. product of myosin or the tropomyosins, which copurify during the preparation of muscle layer of the adult female and in the crude myosin and which may exhibit antigenie cross-reactivity with myosin. microfilariae. There was no hybridization Competitive ELISA. In order to deterwith the control pUC19 DNA. mine more precisely whether WbNl is reThus, the immunoblotting, immunostainlated to myosin, the ability of anti-WbNl ing, and in situ hybridization analyses indiantibodies to inhibit competitively the bindcate that WbNl encodes a high molecular weight (200 kDa) protein localized predom- ing of two monoclonal antibodies that each inantly in the adult body wall and to a lesser preferentially recognizes one of two myosin isoforms-monoclonal antibody 5-6 diextent in the microfilariae. Amino acid sequence and homology of rected toward MHC A and monoclonal anWbNl. The deduced amino acid sequence tibody 28.2 directed toward myosin B of WbNl and its alignment with the C. ele- (Mdler et al. 1983; Ardizzi and Epstein determined in a gans uric-54 (Karn ef al. 1983) sequence 1987) of C. elegans-was and a consensus sequence derived by align- competitive ELISA (Fig. 7). While both ing sequences from the rod region of the C. monoclonal antibodies 5-6 and 28.2 recog30 kDa -
MYOSIN-LIKE
ANTIGEN
OF
W. bUnCrofti
383
FIG. 3. Immunolocalization of the WbNl-antigen in adult parasite sections of B. malayi. Hematoxylin-eosin-stained section (A) and immunostained sections: anti-MBP antiserum (B), normal rabbit serum (C), and immune rabbit serum (D) were photographed under Normasky optics (magnification 365~). Intense labeling is seen in the muscle and, to a lesser degree, in the microtilariae.
nized the recombinant antigen WbNl (data not shown), anti-WbNl antibody inhibited by 68% the binding of Mab 28.2 and by only 18% the binding of Mab 5-6, thus suggesting that WbNl is related to nematode myosin and more so to MHC B than MHC A. In addition, neither the NRS nor an antibody to an unrelated filarial antigen (a Ag43) were able to inhibit the binding of Mab 28.2 to WbNl . This indicates the specificity of the recognition by these two antibodies of similar antigenic sites. To determine whether the inhibition of binding of Mab 28.2 to WbNl by antiWbNl antibodies (based on the ELISA results) was due to strict competition (and not to low-affinity binding of the C. elegans Mab 28.2), competitive immunoblotting was performed using C. elegans myosin ex-
tracts. Partially purified C. elegans myosin extracts were separated by preparative SDS-PAGE, immunoblotted, and analyzed individually with either Mab 28.2 alone or with Mab 28.2 in combination either with varying dilutions of rabbit anti-WbNl antibodies or an antibody to an unrelated filarial antigen (anti-Ag 43) or NRS (Fig. 8). As seen, anti-WbNl at a dilution of 1:20 effectively inhibited most of the recognition of C. elegans myosin by Mab 28.2, and this effect sequentially decreased at higher dilutions of the anti-WbNl antibody. In contrast, neither the anti-Ag 43 antibodies nor NRS even at a 1:20 dilution had any effect on the recognition of C. elegans myosin by Mab 28.2, indicating that Mab 28.2 and WbNl were directed to the same antigenic site(s) in the rod region of C. elegans myosin.
384
RAGHAVAN
ET AL.
FIG. 4. In siru localization of WbNl transcript. B. mnluyi adult female parasite tissue sections were probed with either the biotinylated pUC19 DNA (A) or pUC19-WbNI DNA (B) and photographed under Normasky optics (magnification 340x). Hybridization was observed in the adult muscle and in the microflariae with WbNl DNA and not with pUC19 DNA.
Immune recognition and specific reactivity of WbNI. It has been demonstrated previously (Ottesen et al. 1985) that patients with various clinical manifestations of lymphatic tilariasis showed variable IgG and IgG4 reactivities to total B. malayi antigen extracts. To assess the level of reactivity to WbNl , sera from a small group of normal individuals (n = 9) and those with chronic lymphatic obstruction (CP; IZ = lo), asymptomatic microfilaremia (MF; IZ = lo), and tropical pulmonary eosinophilia (TPE; n = 10) were tested in an ELISAbased assay against the recombinant WbNl fusion protein. As seen in Fig. 9, neither the normal individuals (9/9) nor the CP patients (9/10) recognized WbNl. In marked contrast, both the MF (9/10) and TPE (lo/lo) patients recognized WbNl , which indicates that a WbNl epitope is presented to the host immune system and that this antigen is immunogenic in infected individuals. Further, the recognition of WbNl by the MF patients markedly increased after treatment with the microtilaricidal drug DEC (data not shown), which suggests that microfdarial myosins released after therapy may boost the host immune response to WbNl. DISCUSSION
The responses to human lymphatic filar-
ial infection are diverse and include a broad spectrum of clinical manifestations including elephantiasis (or chronic lymphatic obstruction), TPE, and asymptomatic microfilaremia (where individuals are asymptomatic but have persistent circulating microfilariae). There are varied cellular (Ottesen 1984) and humoral (Ottesen et al. 1982) responses in the different clinical groups. It has been proposed that this diversity is related to the magnitude of the immune response or perhaps to the nature of parasite-derived antigens (Ottesen et al. 1985). The identification of such antigens is important to the understanding of the hostparasite interaction. In order to define such antigens, a recombinant antigen WbNl was derived from a genomic Xgtll expression library. It had been identified initially by its strong reactivity to a pool of sera derived from patients (Raghavan et al. 1991). The genomic characterization of WbNl (2.5 kb) indicated that it was a partial clone derived from a low copy gene, which hybridized to an 8-kb mRNA transcript (from B. mafayi adult RNA) and expressed a 51-kDa protein. Sequence analyses indicated that the 2.5-kb insert had an ORF of 1380 bp, corresponding to a predicted protein sequence of 460 amino acids. The protein sequence was
MYOSIN-LIKE WbNl Cons Mwkw
ANTIGEN
OF
W. buncrojli
385
. . . . . . . . ..NS qk-k-e-Ecs-1 AKKKIEAEVEAL
FLNKYITSQDLL
1136 .............. EQGGATAAQVEVN
1234
1284
1334
1384 WbNl Cons Mwkw
[email protected] KT FFYL ---ete---gl---eEleeaKkkqaq--q AR@GE.. .GLLKTIDELEDAKRRQAQKIN&&ii&:~~
WbNl Cons Mwkw
LLI 1431
1481
FIG. 5. Amino acid sequence and homology of WbNl . Alignment of the deduced 460 amino acid sequence of WbNl (WbNl) with the consensus sequence (Cons) derived from the sarcomeric myosin rod sequences of the C. elegans MHC family (Kam et al. 1983; Ardizzi and Epstein 1987; Dibb et al. 1989) and the uric-54 sequence of C. efegans (Mwkw). Alignments were calculated by the Genetics Computer Group (Deveraux et al. 1985) program PILEUP, using a gap weight of 3.0 and a gap increment weight of 0.2. In the consensus sequence (Cons) uppercase letters indicate invariant amino acids, and lowercase letters indicate amino acids conserved in three or more of the aligned sequences from which the consensus was derived. Hyphens represent positions not characterized by a clear consensus and gaps are represented by dots. Dark boxes with reverse fonts indicate identical amino acids at given positions in all three sequences compared and grey shaded areas indicate conservative amino acid changes. The numbering scheme used is that of the uric-54 (Mwkw) sequence of C. elegans and indicates the position of the right most amino acid.
shown to have similarity to the rod region of myosin (Raghavan er al. 1991). Since myosin-like coiled-coil proteins are highly immunogenic molecules (Werner et al. 1989; Donelson et al. 1988; Erondu and Donelson 1990; Newport et al. 1987), it was of interest to characterize this myosin-like
antigen and to study the immune response elicited by this molecule. Because of the low degree of identity at the sequence level of WbNl to myosin, one of the major objectives was to define the relationship of WbN 1 to myosin. Rabbit antiserum raised against the MBP-WbNl re-
386
RAGHAVAN
combinant fusion protein recognized a 200kDa antigen from B. malayi antigen extracts. Furthermore, the same antibodies recognized a 200-kDa protein from myosin preparations isolated from B. malayi, B. pahangi, 0. volvulus, and C. elegans. That the recombinant WbNl antigen is immunologically related to myosin was further established by competitive ELISA (Fig. 7) where anti-WbN1 antibody alone was able to inhibit the binding of monoclonal 28.2 directed toward body wall myosin (MHC B) of C. elegans (Miller et al. 1983; Ardizzi and Epstein 1987; Dibb ef al. 1989). Further, the differential inhibition of antiWbNl antibody of the two Mabs of myosin indicated that WbN 1 is immunologically most closely related to MHC B. Finally, competitive immunoblots (Fig. 8) confirmed that WbNl and the rod region of C.
3021.5 14.3 -
FIG. 6. Immunoblot analysis of purified nematode myosins. Fifty micrograms each of purified myosins from B. malayi (Bm), B. pahangi (BP), 0. volvulus (Ov), and C. elegans (Ce) were immunoblotted and probed with immune rabbit serum. Arrows indicate position of MHC.
ET AL.
E i=
60
G 5 z
40
FIG. 7. Competition of myosin-specific Mabs by varying dilutions of anti-WbNl antibody. The data are expressed as the percentage inhibition (Y-axis) of binding of the Mabs 54 (open circles) and 28.2 (closed circles) NRS (cross) and a-Ag43 (diamond) to the recombinant antigen WbNl at a given dilution of antiWbNl antibody (X-axis).
elegans (MHC B) myosin shared antigeni-
tally similar regions. Localization of WbNl within the parasite was demonstrated using B. malayi adult tissue sections. In situ hybridization of the 2.5-kb WbNl insert to these tissue sections showed that the mRNA was localized in the adult body-wall muscle tissue and to a lesser extent in the microfilariae. Localization of WbNl using antiserum raised against the recombinant fusion protein also showed the antigen to be present extensively in the body-wall muscle and to a lesser degree in the microfilariae. Taken together, the low, but significant homology to myosin, the hybridization to an 8-kb mRNA transcript (Raghavan et al. 1991), a native parasite protein of 200 kDa, antigenic competition of myosin specific Mabs, and localization of both the mRNA and the antigens in the body wall of the adult and microfilariae indicate the close relationship of WbNl to myosin. The low degree of sequence identity between myosins and WbNl may be related to the high variability commonly seen in the helical rod region of myosin (Karn ef al. 1983; Dibb et al. 1989). In our earlier report (Raghavan et al. 1991), it was shown that WbNl had the
MYOSIN-LIKE
ANTIGEN
387
OF W. bancrcfti
et al. 1988; Werner et al. 1989) is responsible for lowering the homology between the various nematode species at the nucleic acid level, significant immunological similarity exists at the protein level. A similar observation has already been reported in the cloning of a myosin-like antigen of B. malayi (Werner et al. 1989). The immunogenicity of myosin and paramyosin and their ability to evoke humoral responses in both mice and humans has already been shown in other helminth infections (Newport et al. 1987). The mode of presentation to the immune system of myosin or myosin-like proteins, both of which are usually constituents of muscle fibers (and therefore compartmentalized internally), is intriguing. One possible mechanism to explain presentation of the antigen is that it is released following the death of the parasite or during normal physiologic turnover. When the immune recognition of the myosin-like WbNl was examined using various patient groups, it was clearly seen that WbNl was recognized to the highest
NW
69 -
46 -
*1 1
1
Mab 28.2 FIG. 8. Competition of Mab 28.2 by anti-WbNl antibody to C. elegans myosin. A preparative immunoblot of C. elegans myosin was cut into 4-mm strips and incubated with either Mab 28.2 alone (0) or in conjunction with varying dilutions of anti-WbNl antibodies (+ l/20, + l/100, + l/500 or + l/2.500), anti-Ag43 antibodies (+ l/20) or normal rabbit serum (+ l/20). The blots were then incubated with goat anti-mouse IgG and developed. The arrow indicates the position of MHC B with a molecular size of 200 kDa.
highest degree of identity (26%), even at the protein level with B. malayi myosin (Werner and Rajan, 1992) sequence. However, purified myosins from B. pahangi, C. elegans, and 0. volvulus were clearly recognized by the antiserum against WbNl, indicating that even if codon bias (Rothstein
9. f G l5 3 2
NL
CP
MF
TPE
FIG. 9. Recognition of WbNl by human sera. ELISA analysis of IgG reactivity to WbNl in sera of normal (NL) individuals (n = 9) or patients with chronic lymphatic obstruction (CP; n = lo), asymptomatic microfilaremia (MF; n = lo), and tropical pulmonary eosinophilia (TPE; n = 10). The shaded area represents values which are S3 standard deviations above the mean value calculated for the normal individuals. The A OD WbNlIMBP-MBP represents the relative OD units obtained from the difference between the OD values for the WbNl-MBP reactivity and the MBP reactivity.
388
RAGHAVAN
extent by patients with TPE, followed by the microfilaremics (Fig. 9). The presumed rapid clearance of microfilariae in patients with TPE may explain the higher antibody response to WbNl seen in these patients. Similarly, sera from patients with microfilaremia showed an increased recognition of WbN1 following treatment with DEC (data not shown). These data indicate that the exposure and presentation of WbN1 may play an important role in its recognition by the various patient groups. Likewise, it is of interest to note that when subjects with schistosomiasis are treated, levels of antischistosome myosin antibodies rise following chemotherapy (Warren et al. 1977). The relevance of such a myosin-like molecule in the pathogenesisof diseaseis yet to be understood; however, characterization of this antigen should contribute to the understanding of the host-parasite interaction. Such studies could identify parasitespecific epitopes suitable for targeting in vaccine strategies. ACKNOWLEDGMENTS
The authors thank Dr. C. Carlow and Dr. D. Hough for their assistance in purifying and raising antisera against WbNl, Dr. T. Burglin and Dr. B. Wightman for providing C. elegans and protocols for maintaining it in the laboratory, Dr. H. F. Epstein and Mr. D. Casey for providing Mabs. 5-6 and 28.2 and for their very valuable discussions on the biology of C. elegans myosin, Dr. T. V. Rajan for sharing with us the B. malayi myosin sequence for comparative analysis, and the LPD editorial staff for their help in preparing this manuscript. REFERENCES
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40, 213-224. FREDERIKSEN, D. W., AND CUNNINGHAM, L. W. 1982. Structural and contractile proteins. Part B. The contractile appartus and the cytoskelton. In “Methods in Enzymology” (D. W. Frederiksen and L. W. Cunningham, Eds.), Vol. 85, pp. 55-71. Academic Press, New York. GARNIER, J., OSGUTHORPE,D. J., AND ROBOSN,B. 1978. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. Journal of Molecular Biology 120, 97-120.
GUAN, C-D., LI, P., AND NOUYE, H. 1988. Vectors that simplify the expression and puritication of foreign peptides in Escherichia coli by fusion to maltose binding protein. Gene 67, 21-30. KARN, J., BRENNER,S., AND BARNETT, L. 1983. Protein structural domains in the Caenorhabditis elegans uric-54 myosin heavy chain are not separated by introns. Proceedings of the National Academy of Sciences of the United States of America 80, 42534257.
KIERNAN, J. 1981. “Histological and Histochemical Methods, Theory and Practice.” Pergamon Press, Oxford. LAEMMLI, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. LILLIE, R. D. 1954. “Histopathologic Technic and Practical Histochemistry,” pp. 29-48. McGrawHill, New York. Lo, C. 1986. Localization of low abundance DNA sequences in tissue sections by in situ hybridization. Journal of Cell Science 81, 142162. MAINA, C. V., RIGGS, P. O., GRANDEA, A. B., SLATKO, B. E., MORAN, L. S., AGLIAMONTE, J. A., MCREYNOLDS,L. A., AND GUAN, CD. 1988. An Escherichia coli vector to express and purify foreign proteins by fusion to and separation from maltose-binding protein. Gene 14, 365-373. MILLER, D. M., ORTIZ, I., BERLINER,G. C., AND EP-
MYOSIN-LIKE
ANTIGEN
STEIN, H. F. 1983. Differential localization of two myosins within nematode thick filaments. Cell 34, 471-490. NEWPORT, G. R., HARRISON, R. A., MCKERROW, J., TARR, P., KALLESTAD, J., AND AGABIAN, N. 1987. Molecular cloning of Schistosoma mansoni myosin. Molecular and Biochemical Parasitology 26, 29-38. NUTMAN, T. B., KUMARASWAMI, V., AND OTTESEN, E. A. 1987. Parasite specific anergy in human tilariasis. Journal of Clinical Investigation 19, 15161523. OTTESEN, E. A. 1984. Immunological aspects of lymphatic filariasis and onchocerciasis in man. Transactions of the Royal Society of Tropical Medicine nnd Hygiene 78, (Suppl.), 9-18. OTTESEN, E. A., SKVARIL, F., TRIPATHY, S. M., POINDEXTER, R. W., AND HUSSAIN, R. 1985. Prominence of IgG4 in the IgG antibody response to human tilariasis. Journal of Immunology 134, 27072112. OTTESEN, E. A., WELLER, P. F., LUNDE, M. N., AND HUSSAIN, R. A. 1982. Endemic filariasis on a Pacific island. American Journal of Tropical Medicine and Hygiene 31, 953-961. RAGHAVAN, N., MCREYNOLDS, L. A., MAINA, C. V., FEINSTONE, S. M., JAYARAMAN, K., OTTESEN, E. A., AND NUTMAN, T. B. 1991. A recombinant clone of Wuchereria bancrofti with DNA specificity for lymphatic fdarial parasites. Molecular and Biochemical Parasitology 47, 63-72. ROTHSTEIN, N., STOLLER, T. J., AND RA.JAN, T. V. 1988. DNA base composition of tilarial nematodes. Parasitology 97, 75-79.
OF
W. bUnCrOffi
389
SCHULZ-KEY, H., ALBIEZ, E. J., AND BUETTNER, D. V. 1977. Isolation of living Onchocerca volvulus from nodules. Tropical Medical Parasitology 28, 428-430. TOWBIN, M., STAEHELIN, T., AND GORDON, J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets, procedure and some applications. Proceedings of the National Academy of Sciences of the United States of America l&4350-4354. TUAN, R. S., LAMB, B. T., AND JENSINKEY, C. B. 1988. Mouse placental 57 kD calcium-binding protein. Differentiation 37, 198-204. WARREN, K. S., PELLEY, R. P., AND MAHMOUD, A. A. F. 1977. Immunity and immunopathology following reinfection of mice cured of chronic Schistosomiasis mansoni. American fournal of Tropical Medicine and Hygiene 26, 957-962. WERNER, C., HIGASHI, G. I., YATES, J. A., AND RAJAN, T. V. 1989. Differential recognition of two cloned Brugia malayi antigens by antibody class. Molecular and Biochemical Parasitology 35, 209218. WERNER, C., AND RAIAN, T. V. 1992. Characterization of a myosin heavy chain gene form Brugia malayi. Molecular and Biochemical Parasitology 50, 261-268. WOOD, W. B. 1988. “The Nematode-Cnenorhabditis elegans,” pp. 587-590. Cold Spring Harbor Press, Cold Spring Harbor, NY. Received 17 February August 1992
1992; accepted with revision
24
Characterization
of a Muscle-Associated Wuchereria
N. RAGHAVAN,*
Antigen
from
bancrofti’
C. V. MAINA,? P. C. FITZGERALD,~ R. S. TUAN,§ B. E. SLATKO,~ E. A. OTTESEN,* AND T. B. NUTMAN*
*Laboratory of Parasitic Diseases, Building 4lRoom 126, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, U.S.A.; tNew England Biolabs, 32 Tozer Road, Beverly, Massachusetts 01915, U.S.A.; $Division of Computer Research and Technology, Building IZAIRoom 2051 National Institutes of Health, Bethesda, Maryland 20892, U.S.A.; and $Department of Orrhopedic Research, 1015 Walnut Street, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, U.S.A.
RAGHAVAN, N., MAINA, C. V., FITZGERALD,P. C., TUAN, R. S., SLATKO, B. E., OTTESEN, E. A., AND NUTMAN, T. B. 1992. Characterization of a muscle-associated antigen from Wuchereria bancrofti. Experimental Parasitology 75, 37%389. A recombinant clone, WbNl, isolated from a genomic expression library of Wuchereria bancrofti and showing restricted specificity at the DNA level (Southern and PCR analyses) for Wuchereria bancrofti and Brugia malayi has been previously described. Sequence analysis of WbNl indicated that it had notable similarity to myosin. Further characterization using in situ hybridization has localized the mRNA in the muscle of the adult parasite and in the microfilariae. Rabbit polyclonal antiserum, raised against the recombinant WbNl fused to the maltosebinding protein, recognized a 200-kDa polypeptide in immunoblots containing B. malayi antigen extracts. The same antibody also recognized myosin extracted from Brugia pahangi, Onchocerca volvulus, and Caenorhabditis elegans. Localization using the rabbit antiserum revealed the presence of the antigen in the adult muscle tissue and in the microfilariae; the same antibody inhibited the binding of a monoclonal antibody 28.2 (directed toward MHC B of C. elegans myosin) to the recombinant WbNl antigen and also to purified C. elegans myosin. Based on homology data, structural location, competitive ELISA, and immunoblot we conclude that WbNl is related to myosin or a similar myofibrillar protein. 0 19!32Academic Press, Inc. INDEX DESCRIPTORS AND ABBREVIATIONS:Nemata; Filarioidea; Wuchereria bancrofti; Brugia malayi; Brugia pahangi; Onchocerca volvulus; Lymphatic tilariasis; Myosin-like antigen; Base pair (bp); Phosphate-buffered saline (PBS); Maltose-binding protein (MBP); Open reading frame (ORF); Enzyme-linked immuno-sorbent assay (ELISA); Isopropyl p-othiogalactopyranoside (IPTG); 5-bromo-4-chloro-3-indolyl phosphate (BCIP); Nitroblue tetrazolium (NBT); Diethylcarbamazine (DEC); Polymerase chain reaction (PCR); Myosin heavy chain (MHC); Monoclonal antibody (Mab); Normal rabbit serum (NRS); Chronic lymphatic obstruction (CP); Asymptomatic microtilaremia (MF); Tropical pulmonary eosinophilia (TPE).
The isolation and characterization of the recombinant clone WbNl (2.5 kb) has been previously described (Raghavan ef al. 1991). Although derived from a genomic library, WbNl has an ORF of t 380 bp coding ‘ Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession No. M38213.
for a protein of 460 amino acids with a molecular weight of 51 kDa. The sequence of WbNl showed similarity to myosin of several species (Raghavan et al. 1991). To characterize this antigen further, the 2.5kb WbNl insert was subcloned in the plasmid expression vector pPR682 to produce a fusion product with the maltose binding protein of the Ma1 E gene. Antiserum raised in rabbits against this fusion product enabled 379 0014.4894/92$5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
380
RAGHAVAN
detailed characterization of the recombinant antigen expressed by WbNl. Such characterization shows that WbNl codes for a muscle-associated myosin-like product.
ETAL.
nol until used. The fixed parasites were embedded in paraffin, and 0.3~pm thick paraffin sections of the parasite were sequentially deparafllnized in 108% xylene (twice), 100% ethanol (twice), 90, 80, and 70% ethanol (once each) followed by single washes with distilled water and PBS for 2 to 3 min each. The deparaffinized sections were blocked with 3% BSA for 30 min at MATERIALSANDMETHODS room temperature and incubated overnight at 4°C with a 1:250 dilution of the primary antibody. The sections Parasites. Microfilariae of Wuchereria bancrofti were obtained from the blood of patients between 10 were then washed three times with PBS containing 0.05% Triton X-100 for 10min each and incubated with PM and midnight and purified as described earlier the second antibody conjugated to alkaline phos(Raghavan et al. 1991). Adult parasites and microfilarphatase for 1 hr at room temperature. The sections iae of Brugia malayi were obtained from the peritoneal cavity of jirds. Adult Brugia pahangi were obtained were washed, and the color reaction was developed using BCIP/NBT as the substrate/chromogenic indicafrom TRS Laboratories (Athens, GA). Caenorhabditis elegans N2 were cultured in the laboratory on Esche- tor. The sections were then dehydrated sequentially in richia coli OPSO for 10 days prior to harvesting the 70,80, and 90% ethanol once, 100%ethanol twice, and adult organisms (Wood 1988). Adult female On- xylene twice, for 1 to 3 min each. The stained sections chocerca volvulus were isolated from nodules by col- were then air dried and mounted using a solution of 50% Permount (Fisher Scientific Company, Fair lagenase digestion (Schulz-Key et al. 1977). Lawn, NJ). Production of recombinant protein and antibodies against the MBP-WbNl fusion product. The 2.5kb In situ hybridization analysis. Live B. malayi adult insert from WbNl was subcloned into the malE ex- female parasites were fixed, processed, and hybridpression vector pPR682 (Guan et al. 1988;Maina et al. ized either with the biotinylated WbNl DNA isolated 1988) as previously described (Raghavan et al. 1991). from a pUC19 subclone (pUC19-WbNl) or pUC19 Rabbits were immunized subcutaneously with the re- DNA used as the control (Tuan et al. 1988; Lo 1986). combinant MBP-WbNl fusion product with 500 pg of The labeled sections were examined with an Olympus BH-2 microscope using Nomarski optics and photoantigen in RIBI (RIB1 Immunochem Research, Inc., Hamilton, MT) adjuvant. The rabbits were boosted graphed using Kodak Ektachrome-160 film. Histological staining. Histological staining of paraevery 2 weeks over a 6-week period. Sera were colsite tissue sections were done with Gill’s hematoxylin lected a week after the final boost. Zmmunoblots. The recombinant MBP-WbNl fusion (No. 2) and counterstained with eosin (Kieman 1981). product or the myosins extracted (Frederiksen and Antifilarial antibody measurement. Antililarial antiCunningham 1982)from B. malayi, B. pahangi, C. el- bodies were measured by ELISA using either B. maegans, and 0. volvulus were solubilized in sample layi antigen extracts (Nutman et al. 1987) or the rebuffer (63 m&f Tris-HCl, pH 6.8, 2% w/v SDS, 5% combinant MBP-WbNl fusion protein. 2-mercaptoethanol, and 10% v/v glycerol), boiled for 3 Competitive ELISA. Competitive ELISA was performed by coating a 96-well microtiter plate with 100 to 5 min, separated on 4-20% SDS-polyacrylamide kg/ml of recombinant antigen in carbonate/ gels (Laemmli 1970), and transferred to nitrocellulose paper (Towbin et al. 1979). The filters were blocked bicarbonate buffer. After washes with PBS containing with a 3% solution of skim milk powder followed by 0.05% Tween-20, rabbit anti-WbNl antibodies, normal incubation with the primary antibody of interest and rabbit serum (NRS), or rabbit anti-Ag43 (directed togoat anti-rabbit IgG conjugated to alkaline phos- ward an unrelated filarial antigen), were serially diphatase (Jackson Immunoresearch Labs, Inc., West luted and added to the wells. Mabs 5-6 and 28.2 (Miller Grove, PA). The filters were washed four times, at et al. 1983;Ardizzi and Epstein 1987)directed toward IO-mm intervals between each incubation, in a buffer different epitopes of C. elegans myosin were also containing 0.25 M Tris, pH 8.0, 0.2 M glycine, 0.3% added at dilutions normalized by their reactivity to the recombinant antigen. After an overnight incubation, Tween-20, and 0.05% Triton X-100. After the final wash, the filters were developed with BCIP as the sub- goat anti-mouse IgG conjugated to alkaline phosstrate and NBT as the chromogenic indicator (Kirkephatase was added, incubated for 2 hr, washed, and gaard and Perry, Gaithersburg, MD). The reactions developed as decribed earlier (Nutman et al. 1987). Competitive immunoblots. Myosin extracted from were stopped by washing the filter extensively with C. elegans was solubilized in sample buffer, separated distilled water. Zmmunostaining. Live B. malayiadult parasites were on a 10% SDS-polyacrylamide preparative gel, and washed extensively in PBS, and fixed for 2 to 3 hr in a immunoblotted as described earlier. Four-millimeter strips were cut from the imrnunoblots and incubated solution of Bouin-Hollande (Lillie 1954). Following this, the fixed parasites were suspended in 70% etha- with C. elegans Mab 28.2 alone (1:50,000), or in com-
MYOSIN-LIKE ANTIGEN OF w. bUnCrOfti bination with varying dilutions of rabbit anti-WbNl antibodies (1:20, l:lOO, 1:500, and 1:2500) or rabbit anti-Ag43 antibodies (1:20) (antibody raised against an unrelated 43-kDa antigen isolated from the infective larvae of B. maluyQ or normal rabbit serum (1:20). The strips were washed and incubated with goat antimouse IgG conjugated with alkaline phosphatase and developed.
RESULTS
Expression of recombinant antigen and production ofrabbit antiserum. The 2.5-kb insert of WbNl was subcloned in the BamHZ site of the Mu1 E expression vector pPR682 (Raghavan et al. 1991). Expression of the recombinant antigen, induced with IO mM IPTG, resulted in the production of a fusion protein composed of the recombinant antigen (51 kDa) and the maltosebinding protein (39 kDa; Fig. 1, lane 2). This 90-kDa fusion product was readily purified using an amylose affinity column and eluted with free maltose as shown (Fig. 1,
MWl
2
200 kDa 97.4 kDa 69
kDa-
46
kDa-
30 kDa-21.5 kDa 14.3 kDa -
FIG. 1. Lmmunoblot analysis of recombinant WbNl. An immunoblot containing MBP-WbNl fusion protein (lane I) and MBP (lane 2) were probed with rabbit anti-MBP antiserum. Arrow indicates the position of the 90-kDa MBP-WbNl fusion protein.
381
lane 1). The smaller proteins present in the eluted fraction are presumably degradation products of the 90-kDa recombinant fusion protein or possibly truncated products of translation. Antiserum was raised in rabbits against this MBP-WbN 1 90-kDa fusion protein for further biochemical and histological analyses. Zmmunobtot unalysis and identification of the native WbNl antigen. In order to identify the native product encoded by WbNl, antigen extracts from B. malayi adults were electroblotted and reacted with either immune rabbit serum (Fig. 2, lane l), anti-MBP (Fig. 2, lane 2), or normal preimmune rabbit serum (Fig. 2, lane 3). The antiserum against the 90-kDa MBP-WbNl fusion recognized a 200-kDa parasite protein, and neither the normal rabbit nor the anti-MBP antisera recognized this 200-kDa protein. This indicated that the reactivity of the antibody to the high molecular weight 200-kDa protein was specifically derived from the WbNl region and not the MBP portion of the recombinant fusion protein. Zmmunolocalization of the native WbNl antigen. The anti-WbN1 antiserum was next used to localize the antigen in tissue sections of B. maluyi adult parasite (Fig. 3). Specific staining was observed in the muscle of the adult female and, to a lesser degree, in the microfilariae. No staining was observed when either the anti-MBP antibodies or the preimmune rabbit serum were used. Similar results were obtained with male parasites (data not shown). In situ hybridization. Localization of the transcript encoded by WbNl was studied by in situ DNA-mRNA hybridization. B. malayi adult female tissue sections were probed with either a biotinylated pUCl!% WbNl DNA (Fig. 4B) or pUC19 DNA (Fig. 4A) as a control. Positive hybridization signals were detected as purple/brown coloration in the tissue sections. As predicted by the antibody localization described above, a strong positive signal was observed in the
382
RAGHAVAN
12 MW Markers 200 kDa -
92.5 kDa 69 kDa 46 kDa -
3
ET AL.
elegans MHC family (MHC A, myo3; MHC B, uric-54; MHC C, myo-2 and MHC D, myo-1; Karn et al. 1983; Dibb et al. 1989) are shown in Fig. 5. As noted previously (Raghavan et al. 1991), there is notable similarity of WbNl to the C. elegans sequence. Further, based on the secondary structure predictions, there is a structural similarity between WbNl and the a-helical coiled-coil proteins such as myosin. Both the Chou-Fasman (Chou and Fasman 1978) and Garnier-Osguthorpe-Robson (Garnier et al. 1978) predictions for WbN1 show a predominance of a-helical conformations. Immunoblot analyses of purified myosins. To clarify further the antigenic nature
of WbNl, myosin was partially purified from B. malayi, B. pahangi, C. elegans, and 0. volvulus (Frederiksen and Cunning21.5 kDa ham 1982). The partially purified preparations of myosin were subjected to SDS14.3 kDa PAGE, immunoblotted, and reacted with the anti-WbNl antiserum (Fig. 6). As seen, anti-WbN 1 antiserum strongly recognized a a-MBP a-MBP NM specific 200-kDa peptide antigen from the + WbNl myosin prepared from B. malayi and, to a FIG. 2. Identification of the native WbNl antigen. lesser degree, from B. pahangi, 0. volvuAn immunoblot containing 15 kg/lane of B. malayi tolus, or C. elegans. The reactivity seen with tal antigen extract was probed with immune rabbit sesome of the lower molecular weight bands rum (lane l), anti-MBP antiserum (lane 2), or normal rabbit serum (lane 3). Arrow indicates the position of in the myosin preparations is likely due to the native antigen (200 kDa) recognized by the immune recognition of either some breakdown rabbit serum. product of myosin or the tropomyosins, which copurify during the preparation of muscle layer of the adult female and in the crude myosin and which may exhibit antigenie cross-reactivity with myosin. microfilariae. There was no hybridization Competitive ELISA. In order to deterwith the control pUC19 DNA. mine more precisely whether WbNl is reThus, the immunoblotting, immunostainlated to myosin, the ability of anti-WbNl ing, and in situ hybridization analyses indiantibodies to inhibit competitively the bindcate that WbNl encodes a high molecular weight (200 kDa) protein localized predom- ing of two monoclonal antibodies that each inantly in the adult body wall and to a lesser preferentially recognizes one of two myosin isoforms-monoclonal antibody 5-6 diextent in the microfilariae. Amino acid sequence and homology of rected toward MHC A and monoclonal anWbNl. The deduced amino acid sequence tibody 28.2 directed toward myosin B of WbNl and its alignment with the C. ele- (Mdler et al. 1983; Ardizzi and Epstein determined in a gans uric-54 (Karn ef al. 1983) sequence 1987) of C. elegans-was and a consensus sequence derived by align- competitive ELISA (Fig. 7). While both ing sequences from the rod region of the C. monoclonal antibodies 5-6 and 28.2 recog30 kDa -
MYOSIN-LIKE
ANTIGEN
OF
W. bUnCrofti
383
FIG. 3. Immunolocalization of the WbNl-antigen in adult parasite sections of B. malayi. Hematoxylin-eosin-stained section (A) and immunostained sections: anti-MBP antiserum (B), normal rabbit serum (C), and immune rabbit serum (D) were photographed under Normasky optics (magnification 365~). Intense labeling is seen in the muscle and, to a lesser degree, in the microtilariae.
nized the recombinant antigen WbNl (data not shown), anti-WbNl antibody inhibited by 68% the binding of Mab 28.2 and by only 18% the binding of Mab 5-6, thus suggesting that WbNl is related to nematode myosin and more so to MHC B than MHC A. In addition, neither the NRS nor an antibody to an unrelated filarial antigen (a Ag43) were able to inhibit the binding of Mab 28.2 to WbNl . This indicates the specificity of the recognition by these two antibodies of similar antigenic sites. To determine whether the inhibition of binding of Mab 28.2 to WbNl by antiWbNl antibodies (based on the ELISA results) was due to strict competition (and not to low-affinity binding of the C. elegans Mab 28.2), competitive immunoblotting was performed using C. elegans myosin ex-
tracts. Partially purified C. elegans myosin extracts were separated by preparative SDS-PAGE, immunoblotted, and analyzed individually with either Mab 28.2 alone or with Mab 28.2 in combination either with varying dilutions of rabbit anti-WbNl antibodies or an antibody to an unrelated filarial antigen (anti-Ag 43) or NRS (Fig. 8). As seen, anti-WbNl at a dilution of 1:20 effectively inhibited most of the recognition of C. elegans myosin by Mab 28.2, and this effect sequentially decreased at higher dilutions of the anti-WbNl antibody. In contrast, neither the anti-Ag 43 antibodies nor NRS even at a 1:20 dilution had any effect on the recognition of C. elegans myosin by Mab 28.2, indicating that Mab 28.2 and WbNl were directed to the same antigenic site(s) in the rod region of C. elegans myosin.
384
RAGHAVAN
ET AL.
FIG. 4. In siru localization of WbNl transcript. B. mnluyi adult female parasite tissue sections were probed with either the biotinylated pUC19 DNA (A) or pUC19-WbNI DNA (B) and photographed under Normasky optics (magnification 340x). Hybridization was observed in the adult muscle and in the microflariae with WbNl DNA and not with pUC19 DNA.
Immune recognition and specific reactivity of WbNI. It has been demonstrated previously (Ottesen et al. 1985) that patients with various clinical manifestations of lymphatic tilariasis showed variable IgG and IgG4 reactivities to total B. malayi antigen extracts. To assess the level of reactivity to WbNl , sera from a small group of normal individuals (n = 9) and those with chronic lymphatic obstruction (CP; IZ = lo), asymptomatic microfilaremia (MF; IZ = lo), and tropical pulmonary eosinophilia (TPE; n = 10) were tested in an ELISAbased assay against the recombinant WbNl fusion protein. As seen in Fig. 9, neither the normal individuals (9/9) nor the CP patients (9/10) recognized WbNl. In marked contrast, both the MF (9/10) and TPE (lo/lo) patients recognized WbNl , which indicates that a WbNl epitope is presented to the host immune system and that this antigen is immunogenic in infected individuals. Further, the recognition of WbNl by the MF patients markedly increased after treatment with the microtilaricidal drug DEC (data not shown), which suggests that microfdarial myosins released after therapy may boost the host immune response to WbNl. DISCUSSION
The responses to human lymphatic filar-
ial infection are diverse and include a broad spectrum of clinical manifestations including elephantiasis (or chronic lymphatic obstruction), TPE, and asymptomatic microfilaremia (where individuals are asymptomatic but have persistent circulating microfilariae). There are varied cellular (Ottesen 1984) and humoral (Ottesen et al. 1982) responses in the different clinical groups. It has been proposed that this diversity is related to the magnitude of the immune response or perhaps to the nature of parasite-derived antigens (Ottesen et al. 1985). The identification of such antigens is important to the understanding of the hostparasite interaction. In order to define such antigens, a recombinant antigen WbNl was derived from a genomic Xgtll expression library. It had been identified initially by its strong reactivity to a pool of sera derived from patients (Raghavan et al. 1991). The genomic characterization of WbNl (2.5 kb) indicated that it was a partial clone derived from a low copy gene, which hybridized to an 8-kb mRNA transcript (from B. mafayi adult RNA) and expressed a 51-kDa protein. Sequence analyses indicated that the 2.5-kb insert had an ORF of 1380 bp, corresponding to a predicted protein sequence of 460 amino acids. The protein sequence was
MYOSIN-LIKE WbNl Cons Mwkw
ANTIGEN
OF
W. buncrojli
385
. . . . . . . . ..NS qk-k-e-Ecs-1 AKKKIEAEVEAL
FLNKYITSQDLL
1136 .............. EQGGATAAQVEVN
1234
1284
1334
1384 WbNl Cons Mwkw
[email protected] KT FFYL ---ete---gl---eEleeaKkkqaq--q AR@GE.. .GLLKTIDELEDAKRRQAQKIN&&ii&:~~
WbNl Cons Mwkw
LLI 1431
1481
FIG. 5. Amino acid sequence and homology of WbNl . Alignment of the deduced 460 amino acid sequence of WbNl (WbNl) with the consensus sequence (Cons) derived from the sarcomeric myosin rod sequences of the C. elegans MHC family (Kam et al. 1983; Ardizzi and Epstein 1987; Dibb et al. 1989) and the uric-54 sequence of C. efegans (Mwkw). Alignments were calculated by the Genetics Computer Group (Deveraux et al. 1985) program PILEUP, using a gap weight of 3.0 and a gap increment weight of 0.2. In the consensus sequence (Cons) uppercase letters indicate invariant amino acids, and lowercase letters indicate amino acids conserved in three or more of the aligned sequences from which the consensus was derived. Hyphens represent positions not characterized by a clear consensus and gaps are represented by dots. Dark boxes with reverse fonts indicate identical amino acids at given positions in all three sequences compared and grey shaded areas indicate conservative amino acid changes. The numbering scheme used is that of the uric-54 (Mwkw) sequence of C. elegans and indicates the position of the right most amino acid.
shown to have similarity to the rod region of myosin (Raghavan er al. 1991). Since myosin-like coiled-coil proteins are highly immunogenic molecules (Werner et al. 1989; Donelson et al. 1988; Erondu and Donelson 1990; Newport et al. 1987), it was of interest to characterize this myosin-like
antigen and to study the immune response elicited by this molecule. Because of the low degree of identity at the sequence level of WbNl to myosin, one of the major objectives was to define the relationship of WbN 1 to myosin. Rabbit antiserum raised against the MBP-WbNl re-
386
RAGHAVAN
combinant fusion protein recognized a 200kDa antigen from B. malayi antigen extracts. Furthermore, the same antibodies recognized a 200-kDa protein from myosin preparations isolated from B. malayi, B. pahangi, 0. volvulus, and C. elegans. That the recombinant WbNl antigen is immunologically related to myosin was further established by competitive ELISA (Fig. 7) where anti-WbN1 antibody alone was able to inhibit the binding of monoclonal 28.2 directed toward body wall myosin (MHC B) of C. elegans (Miller et al. 1983; Ardizzi and Epstein 1987; Dibb ef al. 1989). Further, the differential inhibition of antiWbNl antibody of the two Mabs of myosin indicated that WbN 1 is immunologically most closely related to MHC B. Finally, competitive immunoblots (Fig. 8) confirmed that WbNl and the rod region of C.
3021.5 14.3 -
FIG. 6. Immunoblot analysis of purified nematode myosins. Fifty micrograms each of purified myosins from B. malayi (Bm), B. pahangi (BP), 0. volvulus (Ov), and C. elegans (Ce) were immunoblotted and probed with immune rabbit serum. Arrows indicate position of MHC.
ET AL.
E i=
60
G 5 z
40
FIG. 7. Competition of myosin-specific Mabs by varying dilutions of anti-WbNl antibody. The data are expressed as the percentage inhibition (Y-axis) of binding of the Mabs 54 (open circles) and 28.2 (closed circles) NRS (cross) and a-Ag43 (diamond) to the recombinant antigen WbNl at a given dilution of antiWbNl antibody (X-axis).
elegans (MHC B) myosin shared antigeni-
tally similar regions. Localization of WbNl within the parasite was demonstrated using B. malayi adult tissue sections. In situ hybridization of the 2.5-kb WbNl insert to these tissue sections showed that the mRNA was localized in the adult body-wall muscle tissue and to a lesser extent in the microfilariae. Localization of WbNl using antiserum raised against the recombinant fusion protein also showed the antigen to be present extensively in the body-wall muscle and to a lesser degree in the microfilariae. Taken together, the low, but significant homology to myosin, the hybridization to an 8-kb mRNA transcript (Raghavan et al. 1991), a native parasite protein of 200 kDa, antigenic competition of myosin specific Mabs, and localization of both the mRNA and the antigens in the body wall of the adult and microfilariae indicate the close relationship of WbNl to myosin. The low degree of sequence identity between myosins and WbNl may be related to the high variability commonly seen in the helical rod region of myosin (Karn ef al. 1983; Dibb et al. 1989). In our earlier report (Raghavan et al. 1991), it was shown that WbNl had the
MYOSIN-LIKE
ANTIGEN
387
OF W. bancrcfti
et al. 1988; Werner et al. 1989) is responsible for lowering the homology between the various nematode species at the nucleic acid level, significant immunological similarity exists at the protein level. A similar observation has already been reported in the cloning of a myosin-like antigen of B. malayi (Werner et al. 1989). The immunogenicity of myosin and paramyosin and their ability to evoke humoral responses in both mice and humans has already been shown in other helminth infections (Newport et al. 1987). The mode of presentation to the immune system of myosin or myosin-like proteins, both of which are usually constituents of muscle fibers (and therefore compartmentalized internally), is intriguing. One possible mechanism to explain presentation of the antigen is that it is released following the death of the parasite or during normal physiologic turnover. When the immune recognition of the myosin-like WbNl was examined using various patient groups, it was clearly seen that WbNl was recognized to the highest
NW
69 -
46 -
*1 1
1
Mab 28.2 FIG. 8. Competition of Mab 28.2 by anti-WbNl antibody to C. elegans myosin. A preparative immunoblot of C. elegans myosin was cut into 4-mm strips and incubated with either Mab 28.2 alone (0) or in conjunction with varying dilutions of anti-WbNl antibodies (+ l/20, + l/100, + l/500 or + l/2.500), anti-Ag43 antibodies (+ l/20) or normal rabbit serum (+ l/20). The blots were then incubated with goat anti-mouse IgG and developed. The arrow indicates the position of MHC B with a molecular size of 200 kDa.
highest degree of identity (26%), even at the protein level with B. malayi myosin (Werner and Rajan, 1992) sequence. However, purified myosins from B. pahangi, C. elegans, and 0. volvulus were clearly recognized by the antiserum against WbNl, indicating that even if codon bias (Rothstein
9. f G l5 3 2
NL
CP
MF
TPE
FIG. 9. Recognition of WbNl by human sera. ELISA analysis of IgG reactivity to WbNl in sera of normal (NL) individuals (n = 9) or patients with chronic lymphatic obstruction (CP; n = lo), asymptomatic microfilaremia (MF; n = lo), and tropical pulmonary eosinophilia (TPE; n = 10). The shaded area represents values which are S3 standard deviations above the mean value calculated for the normal individuals. The A OD WbNlIMBP-MBP represents the relative OD units obtained from the difference between the OD values for the WbNl-MBP reactivity and the MBP reactivity.
388
RAGHAVAN
extent by patients with TPE, followed by the microfilaremics (Fig. 9). The presumed rapid clearance of microfilariae in patients with TPE may explain the higher antibody response to WbNl seen in these patients. Similarly, sera from patients with microfilaremia showed an increased recognition of WbN1 following treatment with DEC (data not shown). These data indicate that the exposure and presentation of WbN1 may play an important role in its recognition by the various patient groups. Likewise, it is of interest to note that when subjects with schistosomiasis are treated, levels of antischistosome myosin antibodies rise following chemotherapy (Warren et al. 1977). The relevance of such a myosin-like molecule in the pathogenesisof diseaseis yet to be understood; however, characterization of this antigen should contribute to the understanding of the host-parasite interaction. Such studies could identify parasitespecific epitopes suitable for targeting in vaccine strategies. ACKNOWLEDGMENTS
The authors thank Dr. C. Carlow and Dr. D. Hough for their assistance in purifying and raising antisera against WbNl, Dr. T. Burglin and Dr. B. Wightman for providing C. elegans and protocols for maintaining it in the laboratory, Dr. H. F. Epstein and Mr. D. Casey for providing Mabs. 5-6 and 28.2 and for their very valuable discussions on the biology of C. elegans myosin, Dr. T. V. Rajan for sharing with us the B. malayi myosin sequence for comparative analysis, and the LPD editorial staff for their help in preparing this manuscript. REFERENCES
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1992; accepted with revision
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