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Characterization of a novel microfilarial antigen for diagnosis of Wuchereria bancrofti infections [1]

['Sarah E. Greene', 'Infectious Diseases Division', 'Department Of Pediatrics', 'Washington University School Of Medicine', 'St Louis', 'Missouri', 'United States Of America', 'Kerstin Fischer', 'Young-Jun Choi', 'Kurt C. Curtis']

Date: 2022-07

Abstract Background Lymphatic filariasis (LF) is a neglected tropical disease caused by the filarial nematodes Wuchereria bancrofti, Brugia malayi and Brugia timori. The Global Program to Eliminate LF uses mass drug administration (MDA) of anti-filarial drugs that clear microfilariae (Mf) from blood to interrupt transmission by mosquitos. New diagnostic tools are needed to assess the impact of MDA on bancroftian filariasis, because available serologic tests can remain positive after successful treatment. Methodology/Principal findings We identified Wb-bhp-1, which encodes a W. bancrofti homologue of BmR1, the B. malayi protein used in the Brugia Rapid antibody test for brugian filariasis. Wb-bhp-1 has a single exon that encodes a 16.3 kD protein (Wb-Bhp-1) with 45% amino acid identity to BmR1. Immunohistology shows that anti-Wb-Bhp-1 antibodies primarily bind to Mf. Plasma from 124 of 224 (55%) microfilaremic individuals had IgG4 antibodies to Wb-Bhp-1 by ELISA. Serologic reactivity to Wb-Bhp-1 varied widely with samples from different regions (sensitivity range 32–92%), with 77% sensitivity for 116 samples collected from microfilaremic individuals outside of sub-Saharan Africa. This variable sensitivity highlights the importance of validating new diagnostic tests for parasitic diseases with samples from different geographical regions. Individuals with higher Mf counts were more likely to have anti-Wb-Bhp-1 antibodies. Cross-reactivity was observed with a minority of plasma samples from people with onchocerciasis (17%) or loiasis (10%). We also identified, cloned and characterized BmR1 homologues from O. volvulus and L. loa that have 41% and 38% identity to BmR1, respectively. However, antibody assays with these antigens were not sensitive for onchocerciasis or loiasis. Conclusions Wb-Bhp-1 is a novel antigen that is useful for serologic diagnosis of bancroftian filariasis. Additional studies are needed to assess the value of this antigen for monitoring the success of filariasis elimination programs.

Author summary Lymphatic filariasis (LF) is a highly disabling and stigmatizing disease caused by parasitic worms that are transmitted by mosquitoes. There is a coordinated global effort to eliminate LF based on mass drug administration (MDA) of donated anti-filarial medications. Improved methods are needed to determine when transmission of the infection has been interrupted in previously endemic areas so that MDA can be safely stopped. This paper reports the discovery and characterization of a novel W. bancrofti antigen, Wb-Bhp-1, which is a homologue of the Brugia malayi protein used in antibody tests to monitor filariasis elimination in areas of Asia where LF is caused by Brugia species. We show that a test for IgG4 antibodies to Wb-Bhp-1 was fairly specific for W. bancrofti infection. However, the sensitivity of this test varied by the geographic origin of the samples. Sensitivity was highest for samples collected in the Indo-Pacific region and lowest for samples collected in Côte d’Ivoire. Geographic differences in the parasite or the human immune responses to infection may account for this variability. This range in sensitivity highlights the importance of validating new diagnostic tests for parasitic diseases with samples from different geographical regions.

Citation: Greene SE, Fischer K, Choi Y-J, Curtis KC, Budge PJ, Mitreva M, et al. (2022) Characterization of a novel microfilarial antigen for diagnosis of Wuchereria bancrofti infections. PLoS Negl Trop Dis 16(5): e0010407. https://doi.org/10.1371/journal.pntd.0010407 Editor: Katharina Röltgen, Stanford University, UNITED STATES Received: January 6, 2022; Accepted: April 8, 2022; Published: May 23, 2022 Copyright: © 2022 Greene et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the manuscript and its Supporting information files. Funding: This work was financially supported by the Bill and Melinda Gates Foundation (https://www.gatesfoundation.org/) with grant INV-021433 (PUF) and grant OPP1201530 (GJW). The findings and conclusions contained within are those of the authors and do not necessarily reflect positions or policies of the Bill & Melinda Gates Foundation. It was supported by the Foundation for Barnes-Jewish Hospital (https://www.foundationbarnesjewish.org/) with grant 4618 (GJW). It was supported by the National Institute of Allergy and Infectious Disease (https://www.nih.gov/) with grant R01AI144161 (MM). It was supported by the National Institute of Allergy and Infectious Diseases grant K08AI121422 (PJB). SG was supported by the National Institute of Health Training of the Pediatric Physician Scientist grant T32 HD043010. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Lymphatic filariasis (LF) is a deforming and disabling disease caused by parasitic nematodes that are transmitted by mosquitoes. Since its inception in 2000, the Global Programme to Eliminate LF has made impressive progress by reducing the estimated numbers of infected people from 120 million to 51 million [1]. This accomplishment demonstrates the effectiveness of the mass drug administration (MDA) strategy, which involves distributing anti-filarial medications to kill the larval stage of the parasite (microfilariae, Mf). The combination of ivermectin, diethylcarbamazine (DEC) and albendazole (IDA) is currently the most effective MDA regimen for clearing Mf, but results in slow clearance of filarial antigenemia [2,3]. The drug combinations used for MDA depend on whether there are other co-endemic parasitic infections in an area. Improved diagnostic tests are needed to better identify areas where MDA has been successful enough that it can be halted. Indeed, the World Health Organization (WHO) lists the development of improved diagnostics as an important priority in their 2020 Roadmap for LF elimination by 2030 [4]. Current guidelines call for transmission assessment surveys (TAS) after five or more rounds of MDA to determine whether MDA can be halted in a given area. TAS rely on detection of circulating filarial antigen (CFA) in the blood with tests such as the Filariasis Test Strip (FTS) or, in areas with brugian filariasis, on detection of anti-filarial antibodies with the Brugia Rapid test [5]. CFA tests are useful for mapping endemic areas and for assessing the impact of MDA on filariasis transmission in sentinel groups. However, TAS surveys have been shown to be insensitive tools for this purpose in some settings [6–8]. Furthermore, CFA tests in adults are a lagging indicator of MDA success, because CFA often remains detectable in human blood for years after treatment has cleared Mf [3,9]. Antibodies to some filarial antigens become detectable in blood sooner after exposure or infection than Mf or CFA [10]. Furthermore, antibody assays may be more sensitive than tests for CFA or Mf for detecting filarial infections or exposure to infection in children [9,11]. Usually, school-aged children are sampled during TAS. Therefore, antibody tests might be useful to detect whether children have been exposed to filarial parasites. Similarly, an antibody test that correlates well with the presence of microfilaremia could be useful for use in post-MDA population surveys. There are several available antibody tests for LF; the most commonly used commercially available tests detect antibodies to the filarial antigens Bm14, BmR1 or Wb123 [12–16]. The Brugia Rapid test, which is used in Brugia endemic areas, detects IgG4 antibody to the Brugia malayi protein BmR1 [16]. The function of BmR1 is unknown, but it is expressed in Mf and female worms [17,18]. The main drawback of the Bm14 or Wb123 antibody tests is that antibodies to those antigens remain detectable for years after clearance of the infection [13,15]. In contrast, antibodies to BmR1 often clear by 2 years after treatment, making anti-BmR1 antibody testing especially useful for MDA stopping decisions in areas with brugian filariasis [15,19]. The development of an antibody test for W. bancrofti that cleared within a few years after treatment could be very useful for MDA stopping decisions for bancroftian filariasis. Based on the utility of the BmR1 antibody test, we hypothesized that antibody tests based on BmR1 homologues might be useful for diagnosis of other filarial parasites that infect humans, such as W. bancrofti, Onchocerca volvulus, and Loa loa. Approximately 40% of the global LF burden is in sub-Saharan Africa, where the latter two parasites are often co-endemic with W. bancrofti [20]. Indeed, some antibody tests for LF detect antibodies in serum samples from patients with onchocerciasis and/or loiasis that could lead to inaccurate assessments of LF endemicity in co-endemic areas, similar to what has been reported for CFA tests in Central Africa [21–23]. Thus, species specificity is especially important for diagnostic tests in areas of Africa with multiple co-endemic filarial infections. Previously identified BmR1 homologues had 99–100% sequence identity to BmR1, and antibodies to these homologues did not prove to be sensitive or specific for bancroftian LF, onchocerciasis or loiasis [24]. This is a much higher degree of sequence identity than found in many other homologous genes in W. bancrofti, B. malayi and O. volvulus. Because there has been further sequencing of filarial parasite genomes since that publication, we hypothesized that additional BmR1 homologues might be present that have less sequence identity and that might be useful for sero-diagnosis. This paper reports the identification and partial characterization of these newly identified BmR1 homologues and early studies of their diagnostic potential.

Methods BmR1 homologue identification and characterization The BmR1 protein is encoded by the B. malayi gene Bm17DIII [16]. We identified a W. bancrofti BmR1 homologue, Wb-bhp-1, with a BLAST search for homologues of BmR1 on the WormbaseParasite database (Parasite.wormbase.org last accessed 3/18/21). We identified a second W. bancrofti BmR1 homologue, Wb-bhp-2, by an analysis of recently published W. bancrofti genomes [25]. BmR1 homologues from O. volvulus and L. loa were likewise identified using BLAST for homologues of BmR1 on WormbaseParasite. Amino acid alignment for these BmR1 homologues was conducted by ClustalV in MegAlign version 15 (DNAStar, Madison WI, USA). Percent identity was calculated for Wb-Bhp-1, Ov-Bhp-1 and Ll-Bhp-1 based on the amino acids conserved with BmR1 in the region of overlap. Genetic variation analysis We analyzed previously published W. bancrofti genomic data from Haiti, Mali, Kenya, and PNG to assess sequence variation in Wb-Bhp-1 and Wb-Bhp-2 [25]. Sequencing reads were retrieved from the SRA (accession SRP056210 and SRP168632), adapter-trimmed using Trimmomatic v0.39 [26], and aligned to the W. bancrofti genome assembly (GenBank accession: GCA_005281725.1) using BWA-MEM v0.7.17 [27]. Polymerase chain reaction and optical duplicates were removed using Picard tools v2.22.0 and single-nucleotide variants were called via local de novo assembly of haplotypes using GATK v4.2.2 [28,29]. Variants were quality-filtered as previously described, and coding effects were predicted using SnpEff v5.0 [30,31]. VCFtools was used to summarize the variant allele frequencies in each population [32]. Cloning and protein expression All kits were used according to the manufacturer’s instructions. PCR amplification of each BmR1 homologous gene was conducted using blunt end PCR with Phusion DNA polymerase (New England Biolabs, Ipswich MA, USA), with an annealing temperature of 52°C and using the primers listed in S1 Table. Wb-bhp-1 was amplified from W. bancrofti Mf genomic DNA obtained in Côte d’Ivoire from a person with W. bancrofti infection and no other filarial infection. It was possible to amplify this gene from genomic DNA as it had only 1 exon. Wb-bhp-2 was amplified from double-stranded DNA synthesized by Integrated DNA Technologies based on available sequence for Wb-bhp-2 in WormbaseParasite (IDT, Coralville IA, USA). Ov-Bhp-1 was amplified from O. volvulus adult worm complementary DNA (cDNA) from Uganda. Ll-Bhp-1 was amplified from double-stranded DNA synthesized by Integrated DNA Technologies based on available sequence for Ll-bhp-1 in WormbaseParasite (IDT). PCR products were visualized by agarose gel electrophoresis, and ligated into linearized pET100D plasmid, which incorporates an amino-terminal polyhistidine tag and an 8 amino acid Xpress tag (Invitrogen, Waltham MA, USA). The resultant vectors (S1 Table) were sequenced (Genewiz, South Plainfield NJ, USA), with sequence analysis on SeqManPro (DNAStar). Each plasmid was transformed into the BL21 E. coli strain for protein production. These strains were grown at 37°C in Luria broth with 50 ug/ml ampicillin to optical density at 600nm of 0.6, then induced with 1mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) for 2 hours. Cells were collected by centrifugation at 4700rpm then subjected to a freeze/thaw cycle at -80°C. Cell pellets were then lysed with CelLyticB (Sigma, St Louis MO, USA) and benzonase. Protein was purified from the clarified cell extract using cobalt or nickel His-select affinity gel purification column (Sigma). Protein was eluted in 250mM imidazole and further purified using an Electro-Eluter (Bio-Rad, Hercules CA, USA). Dialyzed eluted fractions were concentrated with an Amicon Ultra 3.5 kD MWCO cutoff filter (MilliporeSigma, Burlington MA, USA). Protein purity was assessed by SDS-PAGE electrophoresis followed by staining with SimplyBlue SafeStain (Invitrogen). Purified protein was quantified by bicinchoninic acid analysis (Bio-Rad). Antibody production One BALB/c mouse was immunized with 20 μg of recombinant Wb-Bhp-1 in complete Freund’s adjuvant and boosted 1 month later with 20 μg of Wb-Bhp-1 in incomplete Freund’s adjuvant. Immune serum was collected 9 days after the boost. Serum from an unimmunized BALB/c mouse serum was used as a negative control. Tissue fixation and immunohistochemistry Immunochemistry was performed on fixed and sectioned B. malayi parasites recovered at various stages of development from intraperitoneally infected jirds, as well as on fixed and sectioned O. volvulus nodules, which were originally obtained from an onchocerciasis patient in Ghana [33,34]. The parasites were fixed in either 4% formaldehyde or in 80% ethanol then embedded in paraffin. Immunostaining was conducted with the alkaline phosphatase anti-alkaline phosphatase (APAAP) method as previously described [33]. Primary antibody dilutions of 1:100 to 1:1000 were assessed, and the dilutions 1:200 and 1:500 were found to provide the best signal over background. Polyclonal rabbit anti-mouse IgG 1:25 (Dako, Santa Clara CA, USA) was used as the secondary antibody, then mouse APAAP of at a dilution of 1:40 (Sigma) was applied. The chromatogen Fast Red (Sigma) was used as the substrate and the slides were counter stained with hematoxylin (Merck, Darmstadt Germany). Slides were examined on an Olympus -BX40 microscope and photographed on an Olympus DP70 microscope digital camera (Olympus, Tokyo Japan). Immunoblot analysis Purified recombinant proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad). Membranes were blocked with 5% milk, then were probed with either antisera to the Xpress epitope (Invitrogen) at 1:4000 dilution or with patient sera diluted at 1:100. Blots were then incubated with secondary antibody anti-mouse IgG-alkaline phosphatase (Promega, Madison WI, USA), or anti-human IgG4-pFc’- alkaline phosphatase (Southern Biotech, Birmingham AL, USA) at 1:4000. Antibody binding was visualized by incubating blots in SigmaFast BCIP/NBT alkaline phosphatase substrate (Sigma). Indirect ELISA 96 well vinyl round bottom plates were coated with 100ul of 0.5 ug/ml of Wb-Bhp-1 in 0.06M carbonate buffer pH 9.6, covered and incubated at 37°C overnight in a humidified box. Plates were washed twice in PBS-Tween (PBST), then blocked with PBST-5% heat inactivated fetal calf sera (FCS) at 37°C for 1 hour. Human sera diluted at 1:100 in PBST-5% FCS was then added and plates were incubated at 37°C for 2 hours. Plates were washed 5 times with PBST, then anti-human IgG4-pFc’-HRP (Southern Biotech) diluted at 1:4000 in PBST-5% FCS was added and plates were incubated at 37°C for 1 hr. Plates were washed in PBST 5 times before adding the substrate o-phenylenediamine dihydrochloride (Thermo Fisher Scientific, Waltham MA, USA). The colorimetric reaction was stopped with 4M H 2 SO 4 and plates were read at 490 nm with a BioTek ELx808 plate reader (Thermo Fisher Scientific). A positivity cutoff of OD 490 > 0.2 was chosen to maximize sensitivity and specificity. Data were analyzed in Excel (Microsoft, Redmond WA, USA) and Prism version 9 (GraphPad, San Diego CA, USA). Biotinylated Wb-Bhp-1 peptide fragments (synthesized by LifeTein, Somerset NJ, USA) were used for ELISA as described above, on a plate coated with 10ug/ml streptavidin (Sigma). Human samples De-identified sera and plasma samples were from individuals infected with a single filarial parasite unless otherwise noted. We tested sera or plasma from patients infected with W. bancrofti, B. malayi, O. volvulus and L. loa (Table 1). De-identified non-endemic control sera were obtained from Barnes Jewish Hospital clinical lab in St. Louis, Missouri. Since the St. Louis population has low rates of travel or emigration from regions endemic for filarial infections, these samples were presumed to be from non-exposed individuals. PPT PowerPoint slide

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TIFF original image Download: Table 1. Sera characteristics. https://doi.org/10.1371/journal.pntd.0010407.t001 Ethics statement Sera from patients infected with W. bancrofti were collected during the studies cited in Table 1. All clinical samples were de-identified and data regarding the samples and infections were labeled by study identification number only. De-identified non-endemic control sera samples were obtained de-identified from the Barnes-Jewish-Christian Hospital Clinical laboratory in St Louis. The Washington University in St Louis Human Research Protection Office (an institutional review board) determined that work with these de-identified samples did not constitute human subjects research. Statistical analysis Statistical analysis was conducted in Prism Version 9 (GraphPad). Comparisons between antibody levels from groups of patients utilized Kruskal-Wallis one-way analysis of variance.

Discussion Antibody-based diagnostic tests could be quite useful as assessment tools for LF elimination programs. They can be formatted into inexpensive lateral flow assays that are especially useful in low resource settings. Antibodies to some filarial antigens could be more sensitive than CFA tests or Mf for demonstrating infection or exposure in surveys of school children [9,11]. However, children are sometimes not valid sentinels for this purpose in areas where filarial infections are much more common in adults than in children. Antibody tests that correlate well with the presence of microfilaremia would be quite useful for assessing the risk of ongoing transmission in areas that have received several rounds of MDA. This is especially true for areas that have received MDA with the triple drug regimen IDA, because CFA tends to persist long after Mf have been cleared by IDA. Assays like the Brugia Rapid test that reverts to negative relatively quickly after effective treatment might be especially useful for assessing the risk of ongoing transmission after MDA with IDA [16]. In this study, we identified, cloned, purified and characterized Wb-Bhp-1, a BmR1 homologue from W. bancrofti. Immunolocalization studies showed that anti-Wb-Bhp-1 antibodies bind to protein in B. malayi Mf. This result was consistent with the expression patterns reported for Bm17DIII and BmR1 [17,18,51–53]. Anti-Wb-Bhp-1 antibodies bound to a target in O. volvulus Mf, reinforcing the link between this antibody and filarial Mf. The variable sensitivity of the Wb-Bhp-1 ELISA with W. bancrofti sera from different countries was unexpected, and it is only partially explained by the relationship between ELISA results and Mf density. More work is needed to explain why the ELISA was less sensitive for W. bancrofti samples from Côte d’Ivoire, and samples from other areas in sub-Saharan Africa need to be tested to verify whether this low sensitivity is a problem in other parts of Africa. Geographic variability in Wb-bhp-1 might explain this finding. However, our analysis of Wb-bhp-1 sequences in parasites collected in diverse locations did not reveal enough sequence variation to explain the variability in antibody responses that we observed. Côte d’Ivoire is the only country with samples in this study where LF is co-endemic with onchocerciasis. However, it is unclear why coinfection with onchocerciasis would reduce antibody responses to Wb-Bhp-1. On the positive side, the Wb-Bhp-1 ELISA had a sensitivity of 77% for samples from microfilaremic individuals in Sri Lanka, India, Papua New Guinea and Egypt, which are all areas that do not have co-endemic onchocerciasis. Therefore, Wb-Bhp-1 serology may be especially useful in countries where IDA can be used to accelerate LF elimination. Additional studies will be needed to assess the value of this test for population-based sero-surveys with samples from children and adults before and after MDA. Specificity testing showed that the Wb-Bhp-1 ELISA had low-level cross-reactivity with samples from people with onchocerciasis or loiasis. This is essential for an assay to be useful for LF serology in many areas in sub-Saharan Africa. The relatively low sequence identity of homologues in O. volvulus and L. loa likely contributes to the specificity of the Wb-Bhp-1 ELISA. Interestingly, few sera from people with onchocerciasis or loiasis contained antibodies to the homologous proteins Ov-Bhp-1 or Ll-Bhp-1. It is possible that there are differences in localization, or expression of these proteins in those parasites that impact host antibody responses. In conclusion, these studies have shown that Wb-Bhp-1, a W. bancrofti homologue of BmR1, is a promising microfilarial antigen for diagnosis of bancroftian filariasis. Additional studies are needed to characterize the effect of treatment and MDA on antibody levels in individuals and on antibody prevalence in populations. Results of those studies will determine the practical value of Wb-Bhp-1 antibody testing as a surveillance tool for LF elimination programs.

Supporting information S1 Table. Strains produced and primers used for cloning. https://doi.org/10.1371/journal.pntd.0010407.s001 (DOCX) S1 Fig. Single nucleotide polymorphisms in Wb-bhp-1. (A) Graph demonstrates the 9 missense polymorphisms identified in Wb-Bhp-1. The amino acid with the mutation is plotted against the frequency with which each mutation is identified in W. bancrofti worms from the indicated country. The shaded regions represent 2 of the 3 putative antigenic surface epitopes identified in BmR1 (48). (B) Table indicates the specific polymorphisms identified relative to Wb-bhp-1 sequence, as well as the frequency with which each polymorphism is identified in each country. https://doi.org/10.1371/journal.pntd.0010407.s002 (TIF)

Acknowledgments We thank Yuefang Huang for expert technical assistance. We thank the late Dr. Büttner for the O. volvulus nodules used in this study.

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