BmR1, a recombinant B. malayi antigen of ~30 kDa expressed by Bm17DIII DNA coding sequence (cds), has been consistently shown to be a sensitive and specific antigen for the immunodiagnosis of brugian filariasis in studies employing either ELISA or immunochromatographic rapid test (Brugia Rapid) formats [9, 11–13, 15]. When compared with the DNA sequences in GenBank, Bm17DIII cds exhibited 94% homology with the reported EST sequence derived from B. malayi microfilaria cDNA (GenBank AW244981). Southern blot hybridization assays performed on cDNA libraries of L3, L4, mf, adult male and adult female B. malayi showed that it is present in all of the above stages (Rahmah et al., unpublished data). Bands of the correct molecular weight were observed in a Western blot of B. malayi mf, adult male and adult female soluble antigens probed with monopurified antibody to BmR1 (Rahmah et al., unpublished data).
Multicenter evaluations performed with Brugia Rapid showed variable reactivity of BmR1 to sera of W. bancrofti-infected patients. Reactivity to sera from Chennai, India was 54.5% (12/22); from Indonesia was 70% (14/20) and from the Cook Islands was 90% (9/10) [12, 15]. The wide variation in the reactivity of the assay in Bancroftian filariasis in the above three geographical areas prompted us to undertake the current investigation. The present study has shown that the homolog in W. bancrofti is identical to the cDNA of BmR1 – irrespective of the source of the parasites – whether the mf were isolated from the individuals whose sera showed positive or negative reactivity with the Brugia Rapid test. Thus the observed differences in the reactivity of BmR1 antigen with W. bancrofti sera collected from different geographical regions does not appear to be due to genotypic variability between different isolates of mf. Further studies are currently underway to determine if the variability in the expression of the gene could account for the variability in the Brugia Rapid results with serum samples collected from W. bancrofti infected individuals.
PCR experiments were performed on the W. bancrofti genomic DNA samples to obtain an amplicon with a size greater than 618 bp (since an intron is expected to be present in genomic material). However, only one prominent band of 618 bp was obtained (very occasionally a faint band of >1 kb was observed which was shown later to be due to unspecific amplification). PCR on W. bancrofti genomic DNA to amplify the intron sequence (using primers based on the Bm17DIII intron) produced a sequence that shared ~75% homology to the intron of Bm17DIII. This is believed to be an amplification on another part of W. bancrofti genome, since PCR using a pair of internal primers that flank the possible intron site produced a PCR product of ~300 bp (a size that is expected if there was no intron). Conversely, amplification of B. malayi genomic material produced two kinds of amplicons: 618 bp and 1010 bp. The latter was comprised of an intron (393 bp) and two flanking exons (237 bp and 381 bp), the sequences of which were consistent with B. malayi data at TIGR website http://www.tigr.org/. Thus at Universiti Sains Malaysia, genomic DNA of Wb17DIII was found to be intronless, whereas genomic DNA of Bm17DIII was shown to have two variants (i.e one with and one without an intron). These results, though seemingly controversial, were a result of exhaustive efforts with appropriate PCR controls. Data from other laboratories will hopefully confirm these results.
Anti-BmR1 IgG4 was detected in 84.6% (44/52) of L. loa sera but generally not detected in O. volvulus serum samples [11, 16]. Ov-BmR1 and Ll-BmR1 were identical to each other and 99.7% similar to BmR1 (and to Wb-BmR1) on the nucleotide level (Figure 1). Ov-BmR1 and Ll-BmR1 were found to display identical reactivity compared to BmR1 when tested with IgG4-ELISA on a panel of serum samples (Tables 1 &2). Therefore, the difference of one amino acid between BmR1 and its homologs (Ov-BmR1 and Ll-BmR1) did not alter their antigenicity. It is interesting to note that although IgG4 has been shown to be elevated in onchocerciasis with assays using other recombinant antigens [18, 19], the IgG4 reactivity to BmR1 or Ov-BmR1 in O. volvulus was generally negative. One possible explanation for this is that adult worms mostly express Ov-BmR1 and the immune response to O. volvulus is predominantly due to mf . This may explain the very poor IgG4 response to BmR1 and Ov-BmR1. It is possible that the uptake of antigen from lymphatic filariae by antigen presenting cells is significantly different compared to O. volvulus (where adult worms and mf reside either in sub-dermal nodules or in the skin).
The BmR1, Ov-BmR1 and Ll-BmR1 recombinant antigens were also used to determine if IgG1, IgG2 or IgG3 antibodies in O. volvulus and L. loa serum samples were reactive with the recombinant proteins. In addition, the three IgG subclasses were also tested with BmR1 on assays using sera collected from patients with B. malayi and W. bancrofti infections. In all cases only anti-filarial IgG1 was reactive, while anti-filarial IgG2 and IgG3 assays were consistently negative. It is important to note that IgG1 antibodies to BmR1 and its homologs are unspecific and without any diagnostic value. The BmR1 antigen obviously contains widespread epitopes that are recognized by IgG1 antibodies.
Thus based on the current study, BmR1 and its homologs in W. bancrofti, O. volvulus and L. loa induce IgG antibody responses restricted to IgG1 and IgG 4 subclasses only. Unlike the anti-filarial IgG4 response in B. malayi infection, the IgG4 response to BmR1 in W. bancrofti and L. loa was not consistently detected in all infected individuals, indicating that this recombinant antigen will not be of much use in the diagnosis of these two filarial infections. Although IgG1 response to BmR1 was observed in all the filarial infections tested, it lacks specificity since it was also positive when tested with serum samples from normal individuals and with those infected with other parasites.