Fig 1.
Twelve potential biomarkers identified through bioinformatic analysis.
Antigens (labeled Wb1-Wb12) in red were potential pan-LF targets, while those in blue were potentially W. bancrofti specific. The heatmap depicts the expression of these antigens in each of the stages for which transcriptomic data were available, with red indicating high level of expression and blue indicating little expression during that stage (EMB – embryonic, IMF – immature microfilaria, MF – microfilaria). The predicted stage in which each antigen is most enriched is noted.
Fig 2.
Initial screening of potential targets.
A Luciferase Immunoprecipitation System (LIPS) assay was used to screen the IgG reactivity to the twelve identified antigens for reactivity with pooled sera from filaria infected and uninfected individuals. Reactivity of the antigens was tested using pooled sera from patients infected with W. bancrofti (Wb), O. volvulus (Ov), L. loa (Ll), and uninfected blood bank donors (Bb). Pooled Wb sera had high levels of anti-Wb5 IgG, while pools of Ov, Ll, and Bb sera had minimal anti-Wb5 reactivity. Cut-off values were based on 2.5x the average of blank controls.
Fig 3.
Screening of Wb5 with individual samples.
Individual samples positive for W. bancrofti (Wb), L. loa (Ll), O. volvulus (Ov), S. stercoralis (Ss), and uninfected blood bank donors (Bb) were tested for anti-Wb5 IgG levels using a LIPS assay. W. bancrofti samples were from two locations: Cook Islands (red) and India (blue). The horizontal bar within each data set represents the geometric mean. W. bancrofti from Cook Islands (n = 24), W. bancrofti from India (n = 24), O. volvulus (n = 11), L. loa (n = 24), S. stercoralis (n = 12), uninfected controls (n = 12).
Fig 4.
The monomeric structure of Wb5 was predicted by AlphaFold2 (a) and the multimeric form by AlphaFold2-multimer (ipTM: 0.524) (b, c). The model coloring is based on the pLDDT confidence measure in the B-factor field of AlphaFold predictions.
Fig 5.
Screening of different recombinant Wb5 constructs.
The reactivity of Wb5 constructs made in different expression systems (E. coli, mammalian, baculoviral) and with different purification steps (Fc-tag and His-tag cleaved and uncleaved) was compared in ELISA using a pool of. W. bancrofti samples (Wb) and a pool of uninfected blood bank controls (Bb).
Fig 6.
ROC curves constructed for mWb5 and Wb123.
Individual samples infected with W. bancrofti (Wb) and O. volvulus, L. loa, S. stercoralis, M. perstans (Other Helminths) and filaria uninfected samples were used to compare the IgG4 reactivity of Wb5 (a) and Wb123 (b) in Luminex assays. W. bancrofti samples were characterized as either positive (red) or negative (blue) using the cut-offs established by the ROC curves for Wb5 and Wb123 (c). A subset of these samples (n = 12) was negative for anti-Wb123 IgG4 antibodies, but positive for anti-Wb5 IgG4 antibodies. W. bancrofti (n = 231), O. volvulus (n = 21), L. loa (n = 22), S. stercoralis (n = 22), M. perstans (n = 22), uninfected donors (n = 148).
Fig 7.
Longevity of anti-Wb5 and anti-Wb123 IgG4 antibodies following treatment.
A LIPS assay was used to see the trend in IgG4 reactivity of Wb5 and Wb123 to samples over time from a W. bancrofti positive individual following definitive treatment (a) and in separate samples (n = 10) prior to and six months following treatment (Wb5: p = 0.0039, b; Wb123: p = 0.0020, c).
Fig 8.
Detection of antibodies against Wb5 in patients with brugian filariasis.
A LIPS assay was used to screen the IgG reactivity of Wb5 with B. malayi (n = 18) and B. timori (n = 20) samples (a). The IgG4 reactivity of Wb5 was also screened using B. timori samples (n = 19) (b).