The Immunodominant Brugia malayiParamyosin as a Marker of Current Infection with Wuchereria bancrofti Adult Worms

Study populations.A total of 123 patients were recruited among the adult population (>20 years old) of the island Tahaa, Society Archipelago, French Polynesia. This island has ca. 4,000 inhabitants, and a recent survey, conducted in the course of a large-scale chemotherapy assay, indicated that 22% of adult individuals had microfilariae (Mf) (13); 46% had Og4C3 circulating antigen and were thus suspected of harboring W. bancrofti adult worms (17). The patients were surveyed for a history of filariasis, given a physical examination, and screened for microfilaremia by filtration of 1 ml of venous blood. Blood was collected during the day, since W. bancrofti var.pacifica is subperiodic in French Polynesia (14). Sera were separated by centrifugation and frozen at −30°C until use. All sera were screened for the presence of adult worm circulating filarial antigen (CFA) by an Og4C3 enzyme-linked immunosorbent assay (ELISA) (12) as recommended by the manufacturer (JCU Tropical Biotechnology Pty Ltd., Brisbane, Australia). The limit for positivity was 100 U of Og4C3 antigen per ml. All endemic individuals recruited were checked for positive antifilarial serology (anti-adultBrugia malayi immunoglobulin G (IgG) (7) to determine their exposure to W. bancrofti parasitism. Nine sera from European adults who had never lived in a filariasis-endemic area and were negative for the three markers were used as the negative control (nonendemic subjects). Endemic individuals were classified into three groups: (i) Mf carriers (Mf and CFA positive), (ii) amicrofilaremic adult worm carriers (Mf negative but CFA positive), and (iii) unparasitized subjects (Mf and CFA negative). The mean age of all individuals enrolled was 48.1 years old (range, 21 to 81 years), the sex ratio was 0.48 (female/male), and the three groups were not significantly different for these two parameters.

All individuals were treated twice with a single annual dose of ivermectin at 400 μg/kg plus diethylcarbamazine at 3 mg/kg (13). Blood was collected before the first treatment (month 0), 1 year after the first treatment and just before the second treatment (month 12), and 1 year after the second treatment (month 24). Mf and CFA levels were determined at each of the three time points.

Parasite extracts and antisera.Protein extracts were prepared from Mf or L₃ larvae of W. bancroftiand from B. malayi adult worms as follows. W. bancrofti L₃ larvae were obtained from Aedes polynesiensis mosquitoes previously fed on human blood collected from a microfilaremic individual. Mf were purified from human blood on a Percoll gradient (Sigma Co., St. Louis, Mo.). B. malayiadult worms were provided by E. A. Ottesen (National Institutes of Health, Bethesda, Md.). All parasites were kept in phosphate-buffered saline (PBS) and stored at −30°C before use. Crude protein extracts were made by sonicating the parasites in PBS, and the protein concentration was determined after centrifugation by the bicinchoninic acid protein assay (Pierce, Rockford, Ill.). Mouse polyclonal serum was raised against each filarial stage in BALB/c mice by one intraperitoneal injection of 50 μg of protein extract in complete Freund’s adjuvant, followed by three intraperitoneal injections of 50 μg of protein in incomplete Freund adjuvant at 2- to 3-week intervals. The mice were bled 1 week after the last injection.

Isolation of paramyosin cDNA.A B. malayiL₃ cDNA library constructed in the lambda UniZap XR vector (Stratagene) was provided by S. A. Williams, Smith College, Northampton, Mass. (SAW94WL-BmL3 library) (2) and screened with the antiserum raised against W. bancroftiL₃. Immunoscreening was performed with horseradish peroxidase-labelled goat anti-mouse IgG heavy plus light chain (Biosys) as the secondary antibody and developed with α-chloronaphthol. The positive clones were purified by repeated cycles of immunoscreening, and the size of the B. malayi cDNA inserts was determined by PCR with T3 and T7 pBluescript primers. Recombinant pBluescript plasmids were excised from positive phages, and partial nucleotide sequencing of the 5′ and 3′ ends of the inserts was performed with the Circumvent thermal cycle DNA sequencing kit (New England Biolabs, Beverly, Mass.).

Sequence analysis.Two inserts of 3 kb (BmILM5) and 2.1 kb (BmILM6), which showed high homology to paramyosins from other filarial species, were selected, and their nucleotide sequence was determined with the Circumvent thermal cycle DNA sequencing kit and ExoVent polymerase (New England Biolabs). Sequencing of full-length cDNA in both directions was done with synthetic primers (Genosys, Cambridge, United Kingdom) designed from the paramyosin nucleotide sequences ofOnchocerca volvulus and B. malayi cDNAs. Initial assembly of the cDNA sequences and amino acid translations were performed with the sequence analysis software PC-Gene (IntelliGenetics Inc., Mountain View, Calif.). The nucleotide and protein sequences were compared with all other sequences in database searches with the BLASTN and the BLASTX search programs (1). One of the cDNAs (BmILM5) encoded the full-length paramyosin of B. malayi.

Expression and purification of recombinant paramyosins.The coding sequences of BmILM5 and BmILM6 cDNAs were amplified by PCR with the forward primer 5′-GCGGATCCGATGTCCGGTTCAC-3′ and the reverse primer 5′-CGCGAAGCTTGGGTACCGGGCCC-3′. The PCR products were subcloned between the BamHI andHindIII sites of the expression plasmid vector pGEX-FA (derived from pGEX-3T, a gift from F. Laurent, Nouzilly, France), downstream of the glutathione S-transferase (GST) coding sequence. Sequences at the sites of ligations were checked by sequencing with 3′-pGEXRev and 5′-pGEXFor primers (Pharmacia, Uppsala, Sweden). Recombinant GST fusion proteins were expressed inEscherichia coli DH5α after induction with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG). Expression of recombinant fusion proteins was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (11% polyacrylamide gels) of lysed bacterial cell pellets. Recombinant proteins were visualized by Coomassie blue staining and by using anti-GST polyclonal rabbit antibody after transfer onto nitrocellulose membranes (Hybond-ECL; Amersham, Arlington Heights, Ill.). Bacterial cultures were sonicated, and GST fusion proteins (and control GST) were purified by affinity chromatography with glutathione agarose by the protocols described by the manufacturer (Pharmacia). The homogeneity of purified proteins was checked by SDS-PAGE and Coomassie blue staining, and the protein concentration was determined by the bicinchoninic acid protein assay (Pierce).

ELISAs with recombinant paramyosin and human sera.The sera from endemic and nonendemic individuals were screened for total IgG or IgG isotype reactivity to recombinant BmILM6 truncated paramyosin as follows. Microtiter plates (Luxlon@ M29 LES; CEB) were coated in parallel with purified GST-BmILM6 fusion protein or purified GST at 0.1 μg/well for IgG assays or 0.5 μg/well for IgG subclass assays. The plates were incubated for 2 h at 37°C, stored overnight at 4°C, and blocked before use in ELISA by a 2-h incubation in 0.05% Tween 20–10% goat serum in PBS. After the plates were washed with 0.05% Tween 20 in PBS, patient sera diluted 1/100 in the same diluent (ELISA diluent) were added in duplicate, and the plates were incubated for 2 h at 37°C. The plates were washed before the addition of murine monoclonal anti-human IgG Fc (clone HP 6017; Sigma) diluted 1/1,000. After a 2-h incubation at room temperature (ca. 25°C), all the wells were washed and received a 1/2,000 horseradish peroxidase-conjugated rabbit anti-mouse reagent (Biosys); the plates were then incubated for 2 h at room temperature. After the plates were washed, o-phenylenediamine substrate (OPDA; Sigma) was added to each well for 30 min and the optical density (OD) was recorded at 492 nm. Preliminary assays to determine the appropriate dilutions of patient serum, antibodies, horseradish peroxidase and conjugates were carried out with a pool of 15 sera from microfilaremic Polynesian individuals used as positive controls and a pool of 5 sera from nonendemic European individuals as negative controls. The ELISA for IgG isotype analysis was carried out similarly, with the following isotype-specific mouse monoclonal antibodies (Sigma): IgG1, clone HP-6001; IgG2, clone HP-6002; IgG3, clone HP-6047; and IgG4, clone HP-6023 (all used at a dilution 1/2,000).

Positive and negative sera were included on all plates and used to adjust for minor plate-to-plate variations. The arithmetic mean of the OD from duplicate wells coated with GST were subtracted from the OD means from wells coated with GST-BmILM6 to give a net OD. The cutoff levels were defined as the arithmetic means ± 2 SD of the antibody activities of sera from nonendemic individuals. Anti-paramyosin IgG4 titers of positive sera were measured at a range of four or more dilutions by comparison to a standard curve obtained with the same pool of 15 microfilaremic individuals mentioned above.

Statistical analysis.Differences in net OD means between patient groups were analyzed by the Student t test or analysis of variance. Correlation between covariates was analyzed by Spearman’s correlation test. Statistical significance by any of these methods was inferred as P < 0.05.

Nucleotide sequence accession number.The sequence of the BmILM5 cDNA was submitted to GenBank under accession no. U77590 .