Molecular cloning of Brugia malayi antigens for diagnosis of lymphatic filariasis

MOLECULAR AND

ELSEVIER

Molecular and Biochemical Parasitology 64 (1994) 261-271

BIOCHEMICAL PARASITOLOGY

Molecular cloning of Brugia malayi antigens for diagnosis of lymphatic filariasis Ramaswamy Chandrashekar a'b'*, Kurt C. Curtis a, Reda M. Ramzy c, Fanya Liftis a, Ben-Wen Li a, Gary J. Weil a'b Departments of" "Medicine and hMolecular Microbiology, Washington University School of Medicine and The Jewish Hospital of St. Louis, St. Louis, MO 63110, USA; and "Center for Research and Training on Vectors of Diseases, Ain Shams University. Cairo, Egypt

Received 9 September 1993; accepted 17 January 1994

Abstract Immunological crossreactivity among nematodes has hampered development of specific serodiagnostic assays for lymphatic filariasis. In the present study, we report the molecular cloning and characterization of two filaria-specific recombinant clones (BmM5 and BmM 14) with immunodiagnostic potential. BmM5 is a 505-bp cDNA which codes for a protein of 130 residues that ends with an endoplasmic reticulum targeting sequence. BmM14 is closely related to a recently reported clone (SXP-I), and it has 62% homology (deduced amino acid sequence) with a previously described Onchocerca volvulus clone, 2RAL-2. Glutathione S-transferase fusion proteins of BmM5 and BmM 14 were tested in various ELISA formats. The best results were obtained by measuring IgG4 antibodies to the fusion proteins. ELISA studies showed that approximately 90% of 111 sera from Indian and Egyptian patients with brugian and bancroftian filariasis were reactive with both antigens. Nonendemic sera as well as sera from patients with schistosomiasis or intestinal helminths were uniformly nonreactive. Assays based on BmM5 and BmM14 may be useful for large scale screening as an alternative to microfilaria or filarial antigen detection as a means of obtaining a rough index of filariasis endemicity in previously unstudied areas. Key words. Nematode; Antibody; IgG4; Serology; Immunodiagnosis

~ponding author. Infectious Diseases Division, The Jewish Hospital at Washington University Medical Center, 216 S. Kingshighway,St. Louis, MO 63110, USA. Tel.: 314-454-7782, Fax: 314-454-5293 Abbreviations: BCIP/NBT, 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium; BmA, Brugia malayi adult extract; GST, glutathione S-transferase; IPTG, isopropyl-//-D-thiogalactoside; MF, microfilaria; ORF, open reading frame; PBS/T/ FCS, phosphate buffered saline with 0.05% Tween 20 and 5% fetal calf serum: PCR, polymerase chain reaction.

0166-6851/94/$7.00 (C~ 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 6 - 6 8 5 1 ( 9 4 ) 0 0 0 3 5 - L

1. Introduction A n estimated 80 million people are infected with parasites responsible for lymphatic filariasis, namely, W u c h e r e r i a bancrofti, Brugia m a l a y i a n d

Note: Nucleotide sequence data reported in this paper have been submitted to GenBankT M database with the accession numbers M95550 (BmM5) and M95546 (BmM14).

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R. Chandrashekar et al./ Molecular and Biochemical Parasitology 64 (1994) 261 271

B. timori [1[. Improved methods for diagnosis of filarial infections are needed to facilitate surveillance activities, to monitor control efforts, and to evaluate new drugs. Clinical examination is a relatively simple diagnostic method, but it is insensitive, because many who are infected do not have symptoms or signs of disease [1]. The most widely used method for diagnosis of filarial infections is examination of blood for microfilariae (MF). However, there are practical and biological limitations to this approach. First, the sensitivity of such tests depends on the volume of blood examined. The most sensitive method, membrane filtration of venous blood, is expensive and impractical in many areas. Second, microfilarial tests are inconvenient for survey teams and their target populations because in most areas nocturnal periodicity of microfilaremia .necessitates night blood collections. Finally, microfilaria detection is insensitive for diagnosis of patients with suspected clinical filariasis; all patients with filarial puhnonary eosinophilia and most with elephantiasis or hydrocele are amicrofilaremic [24]. Serological tests based on detection of parasite excretion products ('antigen tests') have been developed for bancroftian filariasis [5-9]. However, these tests are often negative in amicrofilaremic patients with clinical symptoms [10 12], and they are not useful for diagnosis of Brugia infections in humans. Attempts to develop antibody serology tests for diagnosis of lymphatic filariasis have been hampered by poor specificity caused by immunological crossreactivity between filariae and other nematodes. Much of this crossreactivity is due to nonprotein determinants (e.g., carbohydrates and phosphorylcholine) which are present in many components of crude antigen extracts prepared from adult worms [8,13]. Several groups have described non-crossreactive recombinant parasite antigens that are useful for antibody diagnosis of onchocerciasis [14-19], and recent reports have described recombinant antigens that may be useful for diagnosis of lymphatic filariasis [20,21]. This article describes studies we have performed to develop and evaluate recombinant antigen-based antibody tests for lymphatic filariasis.

2. Materials and methods

Human sera. Bancroftian filariasis sera were collected from patients with microfilaremia and/or evidence of lymphatic obstruction in endemic areas in India and in Egypt. Sera from patients with brugian filariasis were obtained from individuals residing in Chetikade (Kerala, India). Clinical and parasitological methods employed have been previously described [10,12]. Nonendemic control sera were obtained from healthy residents of St. Louis (MO, USA), and from adults residing in Cairo who had no history of residence in areas endemic for filariasis. Other control sera were obtained from patients infected with Schistosoma mansoni, Loa loa, Mansonella perstans, Ascaris lumbricoides, Dracunculus medinensis, Strongyloides stercoralis, Ancylostoma duodenale, Hymenolepis n a n a , Enterobius vermicularis or Onchocerca volvulus. A bancroftian filariasis serum pool was prepared by combining equal volumes of sera from ten patients with microfilaremia. A broadly reactive anti-helminth serum pool was prepared with sera from patients infected with S. mansoni, L. loa, M. perstans, A. lumbricoides, D. medinensis, S. stercoralis, A. duodenale and H. nana. Parasite antigens and anti-parasite antibodies. Soluble antigens of Brugia malayi adult worms (BmA) were extracted in 0.01 M phosphate buffered saline (PBS), pH 7.4, and antibodies to BmA were produced in rabbits as previously described [22]. Antibodies to glutathione S-transferase fusion proteins of clones BmM5 and BmMI4 (BmM5-GST; BmM14-GST) were produced in 6-week-old female Balb/c mice by foot-pad injection of 10/~g of purified recombinant GST-fusion proteins (see below) in FCA followed by a second injection of antigen in IFA 4 weeks later. Sera were collected 1 week after the booster immunization. Screening of a gene expression library and selection of recombinant clones. A B. malayi adult worm cDNA library in 2gtll prepared in our laboratory [23] was used for immunoscreening. The library was screened to identify filaria-specific

R. Chandrashekaret al./Molecular and Biochemical Parasitology64 (1994) 261 271 clones essentially as previously described [14]. Clones that were reactive with the bancroftian filariasis serum pool but not with the broad antinematode serum pool were selected and purified by repeated cycles of immunoselection. The reactivity of serum pools to fusion proteins expressed by purified recombinant phage was studied by plaque-dot immunoblot analysis as previously described [14]. PCR was employed to amplify the cDNA inserts of selected recombinant 2gtll clones with the GeneAmp D N A amplification kit (Perkin Elmer-Cetus, Norwalk, CT) as previously described [24]. D N A dot hybridization was performed using peroxidase-labeled DNA fragments [25] to study the relationship among the selected clones. Affinity purification of human antibodies reactive with recombinant clones and immunoblot analysis. Antibodies to /~-galactosidase fusion proteins expressed by BmM5 and BmM14 were affinity purified according to the method of Ozaki et al. [26]. Affinity purified antibodies were used to probe immunoblots of B. malayi adult antigens to identify the native antigens that correspond to recombinant fusion proteins [14,27,28]. Alternatively, mouse antibodies raised to recombinant proteins were used to probe immunoblots to identify native antigens that correspond to recombinant proteins. Southern blot analysis and DNA sequencing. B. malayi genomic D N A (2/~g) was cut with selected restriction endonuclease enzymes. Digestion products were electrophoresed into a 1% agarose gel and transferred to H y b o n d - N + nylon transfer membrane (Amersham) by standard techniques and blots were probed with labeled cDNA inserts BmM5 and BmMI4 [29]. The dideoxynucleotide chain termination method [30] was used for double stranded DNA sequencing using the TaqTrack Sequencing System (Promega Corporation, Madison, WI) with T3 and T7 pBluescript primers and with synthetic oligonucleotide primers synthesized by the Washington University Protein Chemistry Laboratory. The P C / G E N E D N A Sequence Analysis Software (Intelligenetics, Inc., Mountain View, CA) was

263

used to analyze nucleotide and deduced amino acid sequences and to determine sequence homologies with previously reported sequences in the GenBank T M data base and the Protein Sequence Data Base of the Protein Identification Resource of the National Biomedical Research Foundation (Washington, DC). Expression of recombinant proteins in pGEX2A. cDNA inserts were subcloned into the plasmid expression vector pGEX-2A, which was generously provided by Dr. Richard Lucius. Recombinant antigens were purified by affinity chromatography with glutathione-agarose (Sigma Chemical Co., St. Louis, MO) essentially as described by Smith and Johnson [31]. Enzyme immunoassay.for detection of antibodies to recombinant antigens. Preliminary studies were carried out to determine the optimal concentration of recombinant antigens to use for the lgG and IgG4 antibody assays. As similar results were obtained when 0.5, 1.0 or 2.0/tg m l - 1 of antigen were used to coat the plates, 0.5 #g ml J was chosen for later studies to conserve antigen. Recombinant antigens, BmM5-GST, BmMI4GST and GST control antigen (100 #l/well; 0.5 #g ml i in 0.06 M carbonate buffer, pH 9.6) were incubated in polyvinyl microtiter plates (Dynatech Laboratories, Alexandria, VA) overnight at 37°C. Plates were blocked with 0.01 M PBS (pH 7.4) with 0.05% Tween 20 (Sigma) and 5% fetal calf serum (PBS/T/FCS) for 1 h at 37°C. Sera diluted 1/100 in PBS/T/FCS were incubated in duplicate wells for 2 h at 37°C. Plates were washed 3 times with PBS/T. IgG antibody binding was detected with a peroxidase-conjugated goat anti-human IgG antibody (Organon Teknika-Cappel, Malvern, PA) in PBS/T/FCS. After 1 h incubation, the plates were washed and substrate was added (o-phenylenediamine, Eastman Kodak, Rochester, NY) with H202. The enzyme reaction was stopped after 10 min at room temperature with 4 M H2SO4. Optical density (OD) was read versus a PBS blank at 490 nm with a Bio-Kinetics Reader EL 312e (Bio-Tek Instruments, Inc., Winooski, VT). Sera were tested for antibody reactivity to GST

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R. Chandrashekar et al./ Molecular and Biochemical Parasitology 64 (1994) 261 271

and to recombinant antigens. OD values obtained with GST were subtracted from OD values obtained with recombinant antigens to give net OD. Sera with net OD greater than the mean + 3 S.D. of results obtained with a panel of US nonendemic sera (n = 20) were considered positive. If duplicate wells produced disparate results, the assay was repeated. Positive and negative control sera were tested on each plate for quality control. lgG4 antibodies to recombinant B. malayi antigens were detected as described above except the serum dilution tested was 1/50 and IgG4 antibody binding was detected with peroxidase-conjugated monoclonal antibody HP 6023 which binds to the Fc portion of human IgG4 [32]. In other studies, subclass-specific antibodies to recombinant antigens were detected with biotinconjugated isotype-specific mouse monoclonal antibodies (Hybridoma Reagent Laboratories, Baltimore, MD) followed by avidin-peroxidase (Organon Teknika).

tive B. malayi antigens by immunoblot (Fig. 1). Antibodies to BmM14 bound to a 13-kDa parasite antigen in BmA. Control antibodies purified with fl-galactosidase did not bind to the 13-kDa antigen. Antibodies purified with BmM5 did not recognize any band in BmA (data not shown). Molecular biological characterization of recombinant clones BmM5 and BmM14. The cDNA insert of the recombinant clone BmM5 contains 505 bp with an open reading frame (ORF) of 390 bp in frame with the fl-galactosidase gene. Interestingly,

123 kDa

200-

-

3. Results

Selection and preliminary characterization o[" recombinant B. malavi cDNA clones. Approximately 200 000 phage plaques from a B. malayi c D N A expression library were screened to select 33 clones that were reactive with the W. bancro[?i serum pool but not with the pool of sera from patients with other helminthic infections. These clones were rescreened with separate serum pools from patients infected with each of the parasites represented in the anti-helminthic serum pool. Twenty filaria-specific clones were tested by plaque-dot immunoblot for reactivity with sera from filariasis patients and controls. The two best clones (BmM5 and BmM14) were selected for further study. DNA dot hybridization studies showed that the clones did not cross-hybridize to one another under high stringency conditions (data not shown). The BmM5 and BmM14 fi-galactosidase fusion proteins (130 and 132 kDa, respectively, vs. 116 kDa for unfused figalactosidase) were used to affinity purify antibodies from the human bancroftian filariasis serum pool to identify immunologically crossreactive ha-

93

-

66-

45-

31-

14-

; ~:ii

i

Fig. I. Identification of native B. malayi antigens that are immunologically crossreactive with B m M I 4 fusion protein, lmmunoblot of B. malayi adult extract was developed with: lane 1, antibody to BmM14 affinity purified from h u m a n bancroftian filariasis sera; lane 2, unfractionated filariasis sera; lane 3, control for lane I with antibody affinity purified from h u m a n filariasis sera with fi-galactosidase produced by wild-type )~gt 11.

R. Chandrashekar et al./Molecular and Biochemical Parasitology 64 (1994) 261 271

B14v15 BZ4M14 SXP-I RAL2

EFRS ............ VF .............. GEEQQFMIPPFLLGAPES ............ IPFF-FLSIGLIVAASAQREAQLPQPEIPPFLSGAPSH EFRVTSSLNLTKMKYFIFLSIGLIAAASAQREAQLPQPEIPPFLSGVPSH ............................. QRD .... EREIPPFLEGAPPS ***** *. *

B~145 BZ4MI 4

AVREMEE LFQKYANKPD VVKQFFDLLKADE SKTD VVKQFFDLLKADE SKTD V IDE FYNLLKTDENKTD ..... * .... *.*

24 37 50 17

S QLEAAVEEWVN GKGG~4I KEKY SQFKANMQLMH PQTEAD IEAFIRRLGGDYQTRFEQFKQE IKIKEK PQTEAD IEAF IRRLGGDY QTRFE QFKQE IKKE K QQTEADVEAF INRL GGS Y KVRFTQFMEE VKKAR * * * . . * .... ** .... ** ......

74 87 i00 67

B~a45 BMM14 SXP-I RAL2

E KTALLRKAMAENL S PEAKKADVD L SN IGKDKTL SE QQKRE KFKE Y LRNL AQ YE KFHQRALLKF S PAAREADAKMSA IAD S TQLTNHQKTE Q IKA IMD S L A Q Y E K I H Q A A L L K F S P A A R E A D A K I 4 S A I A D S T Q L T N H Q K T E Q I K A II~3 S L ADYERI HQQAVARFSPAAFdDADARMSAIAD SPHLTTRQKSQQ IQAIMD SL . . . . . . . . * * . * o .**. .*.*... *...** ....... *

124

BI4M5 BI4MI 4 SXP-I RAL2

SPVVRNEL ........ SEAVRKE ILEGFNSQSEAVP.KE I LEGFNSQ SE SVRRE I INAL SPQE * **.*.

SXP-I RAL2

265

137 150 117

132 152 165 133

Fig. 2. Comparison of the amino acid sequences of BmM5, BmMI4, SXP-1 and the O. volvulus c D N A clone, ,~RAL-2, using the multiple (Clustal) alignment that gives the highest number of identical residues. Identical residues are marked with an asterisk (') and conserved residues are marked with a dot ( • ).

the C-terminal end of the protein sequence contains the endoplasmic reticulum (ER) targeting sequence Arg-Asn-Glu-Leu (RNEL), a variant of the classical K D E L signal sequence [33]. This feature suggests that the BmM5 protein is an ER luminal protein. Computer searches failed to reveal significant homology between the D N A sequence of BmM5 with previously reported nucleotide sequences in GenBank TM. However, a comparison of the deduced amino acid sequence of BmM5 with previously reported sequences from filarial parasites in our own database revealed that the protein encoded by BmM5 has 37% identity with a previously described O. volvulus cDNA clone, )~RAL-2 (Fig. 2) [34]. The sequence of BmMI4 contains a single translational O R F that encodes 151 amino acids and 106 bp of untranslated DNA at the 3' end. The deduced protein of B m M I 4 contains a short hydrophobic region at the N-terminal end that is adjacent to a highly hydrophilic region. BmM 14 is closely related to a B. malayi clone, SXP-1, that was recently described by Dissanayake et al. [20]. A computer search of deduced protein sequences in our database revealed that BmM14 has 62% homology with O. volvulus clone, 2RAL-2. The results of a multiple sequence alignment [35] of the proteins encoded by BmM5, BmM14, SXP-1

and 2RAL-2 are shown in Fig. 2. This alignment shows 20% identity and 34% similarity among these clones. In order to identify the genomic DNA fragments carrying the gene(s) corresponding to BmM5 and BmM14, B. malayi DNA was digested with EcoRI, HindIII and HaeIII, and

B

1 2 kb

A

i i :i i~i ~i i i i i!~i~ iiiii~iii~i~;~i

i!iiiiiiiiii:i!iiiiiiiiiiiiiiiiiii.i!iii !

?4

1 2 -

8.0

-

6.0

- -

3.5

~iiiii!ii!!iiiiii~iii~iiii~i!!ii~i~i!~!iii --

1.9

Fig. 3. Southern blot of genomic D N A of B. malayi probed with labeled D N A from clones BmM5 and BmM 14. Genomic D N A was digested with EcoR1 (lanes I, 3 in panel A), with Hindlll (lanes 2, 4 in panel A) or with Haelll (lanes 1, 2 in panel B), electrophoresed on a 1% agarose gel, and transferred to nylon membrane. The membrane was probed with peroxidase-labeled c D N A inserts of clones BmM5 (panel A, lanes 1, 2; panel B, lane I) and BmM14 (panel A, lanes 3, 4: panel B, lane 2).

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R. Chandrashekar et al./ Molecular and Biochemical Parasitology 64 (1994) 261 271

Southern blot analysis was performed as described in Methods. As shown in Fig. 3A, the inserts from BmM5 and BmM14 hybridized to similarly sized, single bands in B. malayi genomic D N A digested with either EcoRI or HindIII. However, with HaeIII the binding pattern was different showing that BmM5 and BmM14 are separate gene products (Fig. 3B). No hybridization signals were detected with 2DNA cut with the same restriction enzymes (data not shown). Expression of B m M 5 and B m M I 4 in pGEX-2A plasmid vector. The expression and purification of BmM5 and BmM14 are shown in Fig. 4. Fusion proteins with apparent molecular weights of 41 kDa for BmM5 (Fig. 4A, lane 2) and 43 kDa for BmM14 (Fig. 4B, lane 2) were evident by SDSP A G E and immunoblot. Both GST-fusion proteins were purified from cell lysates by affinity chromatography (lane 3). Typical yields from 1 1 cultures were 0.8 1.0 mg of BmM5-GST and 0.50.7 mg for BmM14-GST.

A I

2

Immunoblot analysis of mouse antibodies to recombinant antigens. Sera from mice immunized with recombinant GST-fusion proteins were tested for antibodies to fl-galactosidase fusion proteins of BmM5 and BmM14. Interestingly, antibodies raised to either recombinant protein bound to flgalactosidase fusion proteins of both BmM5 and BmM14 (data not shown). The reactivity of these sera with BmA by immunoblot is shown in Fig. 5. Antibodies raised to BmM5-GST bound to a 50kDa parasite antigen that was not recognized by mouse anti-GST antibodies. Mouse antibodies to BmM14-GST bound to a 13-kDa antigen in BmA. This result was consistent with the result obtained with antibodies affinity purified with BmM 14 (see above).

12545 kDa 200

-

B :.5

I

2

!i!~ill

3

116

-

9 3 -

i!!!!~ii:i;/i

kDo 66

116 92-

-

66-

66 4 5 -

45-

'4-5

31

31-

-

~i!!iiJ!!i

22-

31

.!

Fig. 4. S D S - P A G E and i m m u n o b l o t analysis of expression and purification of B m M 5 - G S T (A) and B m M I 4 - G S T (B). 10% S D S - P A G E was loaded with E. coli extract without IPTG induction (lane 1), after IPTG induction (lane 2), and with purified fusion protein eluted from glutathione-agarose beads (lane 3), The immunoblots were developed with h u m a n filariasis serum pool (1:500 dilution), enzyme-labeled anti-hum a n lgG second antibody, and substrate.

22

-

14

-

iil;i,il if!!!

i:iiiiiiiii-~---

Fig. 5. lmmunoblot of B. malayi adult worm extract developed with: lane 1, normal mouse serum: lane 2, mouse antibody to B m M 5 - G S T f u s i o n protein: lane 3, m o u s e a n t i b o d y to BmM 14-GST fusion protein: lane 4, mouse antibody to GST; lane 5, rabbit antibodies to B. malayi adult worm.

267

R. Chandrashekar et al./ Molecular and Biochemical Parasitology 64 (1994) 261 271

Comparison of IgG and I g G 4 ELISA for antibodies to recombinant B. malayi antigens. Preliminary studies were carried out with sera from patients infected with W. bancrofti to compare IgG and IgG4 reactivity to BmM5 and BmMI4. Although 13 of 14 sera were positive by IgG ELISA for BmM14-GST, optical densities obtained with the sera in the IgG4 assay were much higher (Fig. 6B). The difference between the IgG and IgG4 assays was even more dramatic for BmM5-GST (Fig. 6A). The sensitivity of the BmM5-GST IgG4 assay for this serum set was 100% compared to 71% for IgG. In general, human sera had little or no background reactivity with GST in the IgG4 assay, but IgG reactivity with GST was significant and highly variable. Thus, the best signal-tonoise ratios were obtained by measuring IgG4 antibodies to the fusion proteins. Sensitivity and specificity of I g G 4 ELISA for antibodies to recombinant B. malayi antigens. Sensi-

tivity data are shown in Table 1. Both antigens were equally sensitive when tested with sera from Indian and Egyptian patients with bancroftian filariasis and Indians with brugian filariasis. Nonendemic control sera from Egypt and the USA were uniformly nonreactive in the assay. Control sera from patients with various other nematode infections were also tested in the IgG4 assay for antibodies to BmM5 and BmM14 (Table 1). The only control sera that were reactive with these antigens were from patients with onchocerciasis.

IgG subclass antibody reactivity to recombinant antigens. IgG subclass antibody reactivity was determined for 30 human filariasis sera by ELISA with recombinant antigens BmM5 and BmM14. Specific antibody reactivity to both antigens was limited to the IgG4 subclass except for one serum which also had IgGi antibody reactivity with BmM5 (data not shown).

4. Discussion _~3.0; *loe .

The goals of this study were to clone, characterize, and overexpress B. malayi recombinant antigens with immunodiagnostic potential. Recombi-

z.o' Z 1.0

Q

•

A

-~.-210 ;

g,l g,

A ->3.0

Table 1 Sensitivity and specificity of IgG4 ELISA for human antibodies to recombinant B. malayi antigens Serum source

g

->3. l0 20 I0 --t-= ol

B

,~o

2'°

_>3B.0

IgG (OD49o)

Fig. 6. Comparison of results obtained in enzyme immunoassays for IgG and IgG4 antibodies to recombinant antigens BmM5 (A) and BmM14 (B) in a preliminary study with 14 bancroftian filariasis sera. Net OD cut off values (mean + 3 S.D. obtained with a panel of nonendemic control sera) are s h o w n with b r o k e n lines (OD49¢) cut o f f values BmM5 lgG-0.300; BmM5 I g G 4 - 0.110; BmMI4 lgG 0.095; BmMI4 IgG4 - 0.060).

Bancroftian filariasis (India) Bancroftian filariasis (Egypt) Brugian filariasis (India) Nonendemic controls (USA) Nonendemic controls (Egypt) Ascaris lumbricoides Schistosoma mansoni Itymenolepis nana Enterobius vermicularis Onchocerca volvulus

Number of sera tested

30 49 32 20 28 10 15 4 9 8

Number of sera reactive a with BmM5

BmMI4

29 ND 28 0 0 0 0 0 0 3

28 42 27 0 0 0 0 0 0 3

aSera were considered to have positive antibody tests if they had net OD values greater than mean + 3 S.D. obtained with a panel of nonendemic sera. ND, not done.

268

R. Chandrashekar et al./ Molecular and Biochemical Parasitology 64 (1994) 261-271

nant clones that expressed filaria-specific antigens were identified by several cycles of differential immunoscreening, and the most immunoreactive and specific clones (BmM5 and BmM14) were selected for more detailed studies. These clones code for the most promising recombinant diagnostic antigens for lymphatic filariasis that have been identified to date. BmM5 codes for a novel protein that appears to correspond to a 50-kDa native B. malayi antigen. The protein encoded by BmM5 contains an endoplasmic reticulum (ER) targeting sequence (RNEL) at its C-terminal end. Proteins that permanently reside in the lumen of the ER seem to be distinguished from newly synthesized secretory proteins by the presence of the C-terminal sequence Lys-Asp-Glu-Leu (KDEL) [36], It is believed that proteins bearing this signal are not simply held in the ER; they are selectively retrieved from a post-ER compartment and returned to the ER lumen [36]. Thus, the protein encoded by BmM5 joins a select group of ER luminal proteins described so far [3740]. BmM14 codes for a protein that appears to correspond to a 13-kDa native B. malayi antigen. This clone is very closely related to a recently described B. malayi clone, SXP-1. SXP-I was selected from a B. malayi adult male worm cDNA library with sera from patients with bancroftian filariasis. Our results confirm the findings of Dissanayake et al. [20] and extend them by providing information on the native antigen encoded by the clone and homology of these clones with O. volvulus clone 2RAL-2. Dissanayake et al. [20] reported the paradoxical finding that the B. malavi recombinant SXP-1 was reactive with sera from patients infected with W. bancrofti but not reactive with 16 sera from patients infected with B. malavi. In contrast, we found that sera from patients with brugian and bancroftian filariasis were almost equally reactive with BmM 14. Both BmM5 and BmM14 have very interesting sequence homologies at the amino acid level to a previously reported O. volvulus clone, 2RAL-2 [34]. BmM14 is more closely related to 2RAL-2 with an amino acid identity of 62% compared to 37% for B m M 5 . 2 R A L - 2 was isolated from an O. volvulus cDNA library using rabbit antibodies to

O. volvulus infective larvae [41]. BmM5 and BmM14 are immunologically crossreactive with each other, and they are likely to be crossreactive with 2RAL-2 as well. All three antigens are recognized by antibodies in sera from onchocerciasis patients. This finding is not surprising in view of the sequence homology observed for these proteins. Southern blot analysis was performed to obtain information on localization of genes that correspond to BmM5 and BmM14 in B. malayi genomic DNA. The fact that both inserts hybridized to a single band after digestion with two different restriction enzymes (EcoRI and HindIII) suggests that there is only one copy of the genes that encode BmM5 and BmMl4. Both probes hybridized to similarly sized EcoRI or HindIII restricted genomic fragments. However, the pattern obtained with HaeIII (BmM5 has an internal HaeIII cleavage site) indicates that BmM5 and BmMI4 are separate gene products that map closely in the genome. BmM5-GST and BmM14-GST were tested in ELISA assays with well-characterized sera from patients with lymphatic filariasis. Preliminary studies showed that better signal-to-noise ratios were obtained by measuring IgG4 antibodies to the'recombinant fusion proteins. This point was confirmed by the subclass analysis which showed that antibodies to BmM5 and BmM 14 were almost exclusively limited to the IgG4 subclass. Since there was almost no background lgG4 reactivity with GST (even with sera from patients with schistosomiasis), specific lgG4 reactivity to the recombinant antigens was much stronger than total IgG-specific reactivity as measured in the assays employed. Several studies have shown that measurement of IgG4 antibodies to filarial antigens improves diagnostic specificity [4245]. IgG4 is a minor IgG subclass that comprises only 5-7% of total IgG in US adults [46]. Increased levels of lgG4 antibodies are produced in response to chronic antigenic stimuli (e.g. infections with tissue dwelling helminth parasites), and prominent IgG4 antibody responses have been reported in human lymphatic filariasis [43,45,47]. The performance of the recombinant antigenbased IgG4 antibody assays was evaluated with

R. Chandrashekar et al,/ Molecular and Biochemical Parasitology 64 (1994) 261 271

sera from patients with lymphatic filariasis from India and Egypt and with various types of control sera. Similar results were obtained with both antigens. Positive tests were observed with about 95% of bancroftian filariasis sera from India, 86% of brugian filariasis sera from India, and 86% of bancroftian filariasis sera from Egypt. Nonendemic sera obtained from Egypt and the USA were uniformly nonreactive in the assay, as were sera from patients with schistosomiasis or intestinal helminthic infections. These results compare favorably with results of prior studies of recombinant antigens for diagnosis of lymphatic filariasis [20,21,48-50]. The crossreactivity to BmM5 and BmM14 observed with sera from patients with onchocerciasis will limit the value of these antigens for diagnosis of bancroftian filariasis with sera from areas where both infections occur (parts of sub-Saharan Africa). These recombinant antigens may be more useful for diagnosis of B. malayi infection, because to date there is no sensitive antigen test available for that infection [51]. Nonlymphatic human filarial infections (e.g. onchocerciasis) do not occur in areas that are endemic for brugian filariasis. Additional studies with brugian filariasis sera are needed. A practical feature of these antibody assays is that they are easier to perform than parasite antigen assays, and they require smaller amounts of serum. Thus, assays based on BmM5 and BmM14 may be useful for large scale screening programs, even in W. bancrofti-endemic areas, as an alternative to M F testing or antigen detection to obtain a rough index of endemicity in previously unstudied areas. In conclusion, we have cloned and characterized two recombinant B. malayi antigens with immunodiagnostic potential. Field studies are in progress to further d e f n e the practical value of these antigens for diagnosis of lymphatic filariasis.

5. Acknowledgements This work was supported in part by U S A I D N I A I D regional project entitled 'Epidemiology and Control of Vector Borne Diseases in Egypt' NO1 AI-22667 and N I H grant AI-22488.

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