Kurt C. Curtis

Molecular cloning of Brugia malayi antigens for diagnosis of lymphatic filariasis

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 BmM14) 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-1), and it has 62% homology (deduced amino acid sequence) with a previously described Onchocerca volvulus clone, lambda RAL-2. Glutathione S-transferase fusion proteins of BmM5 and BmM14 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.

Laboratory and Field Evaluation of a New Rapid Test for Detecting Wuchereria bancrofti Antigen in Human Blood

Global Program to Eliminate Lymphatic Filariasis (GPELF) guidelines call for using filarial antigen testing to identify endemic areas that require mass drug administration (MDA) and for post-MDA surveillance. We compared a new filarial antigen test (the Alere Filariasis Test Strip) with the reference BinaxNOW Filariasis card test that has been used by the GPELF for more than 10 years. Laboratory testing of 227 archived serum or plasma samples showed that the two tests had similar high rates of sensitivity and specificity and > 99% agreement. However, the test strip detected 26.5% more people with filarial antigenemia (124/503 versus 98/503) and had better test result stability than the card test in a field study conducted in a filariasis-endemic area in Liberia. Based on its increased sensitivity and other practical advantages, we believe that the test strip represents a major step forward that will be welcomed by the GPELF and the filariasis research community.

A multicenter evaluation of a new antibody test kit for lymphatic filariasis employing recombinant Brugia malayi antigen Bm-14

Antibody tests are useful for mapping the distribution of lymphatic filariasis (LF) in countries and regions and for monitoring progress in elimination programs based on mass drug administration (MDA). Prior antibody tests have suffered from poor sensitivity and/or specificity or from a lack of standardization. We conducted a multicenter evaluation of a new commercial ELISA that detects IgG4 antibodies to the recombinant filarial antigen Bm14. Four laboratories tested a shared panel of coded serum or plasma samples that included 55 samples from people with microfilaremic Wuchereria bancrofti or Brugia infections and 26 control samples. Qualitative results were identical in all four test sites. In addition, each laboratory tested samples from their own serum banks. The test detected antibodies in 32 of 36 samples (91%) from people with Brugian filariasis and in 96 of 98 samples (98%) from people with Bancroftian filariasis. Specificity testing showed that many serum or plasma samples from patients with other filarial infections such as onchocerciasis had positive antibody tests. Specificity was otherwise excellent, although 3 of 30 samples from patients with ascariasis and 4 of 51 with strongyloidiasis had positive antibody tests; it is likely that some or all of these people had previously lived in filariasis-endemic areas. Antibody test results obtained with eluates from blood dried on filter paper were similar to those obtained with plasma tested at the same dilution. This test may be helpful for diagnosing LF in patients with clinical signs of filariasis. It may also be a useful tool for use in LF endemic countries to monitor the progress of filariasis elimination programs and for post-MDA surveillance.

Immune responses to Brugia malayi paramyosin in rodents after DNA vaccination

Immunization with recombinant Brugia malayi paramyosin protein (BM5) induces partial immunity to this filarial nematode in jirds. The present study examined the effects of intramuscular immunization with plasmid DNA that encodes BM5. DNA-immunized mice produced strong antibody and cell-mediated responses to paramyosin. The protective activity of DNA vaccination with BM5 was tested in jirds. Vaccinated jirds produced strong antibody responses to paramyosin, but adult worm recoveries after challenge were not decreased in vaccinated animals relative to controls. These studies show that DNA vaccination can induce immune responses to filarial antigens in rodents. Further efforts will be needed to achieve the goal of inducing protective immunity to filariasis with this promising new technology.

Antibody responses to Brugia malayi antigens induced by DNA vaccination.

Background DNA vaccination is a convenient means of immunizing animals with recombinant parasite antigens. DNA delivery methods are believed to affect the qualitative nature of immune responses to DNA vaccines in ways that may affect their protective activity. However, relatively few studies have directly compared immune responses to plasmids encoding the same antigens after injection by different routes. Therefore, the purpose of this study was to explore the influence of the route of administration on antibody responses to plasmids encoding antigens from the filarial nematode parasite Brugia malayi. Methods Four B. malayi genes and partial genes encoding paramyosin (BM5), heat shock protein (BMHSP-70), intermediate filament (BMIF) and a serodiagnostic antigen (BM14) were inserted in eukaryotic expression vectors (pJW4303 and pCR™3.1). BALB/c mice were immunized with individual recombinant plasmids or with a cocktail of all four plasmids by intramuscular injection (IM) or by gene gun-intradermal inoculation (GG). Antibody responses to recombinant antigens were measured by ELISA. Mean IgG1 to IgG2a antibody ratios were used as an indicator of Th1 or Th2 bias in immune responses induced with particular antigens by IM or GG immunization. The statistical significance of group differences in antibody responses was assessed by the non-parametric Kruskal-Wallis test. Results Mice produced antibody responses to all four filarial antigens after DNA vaccination by either the IM or GG route. Antibody responses to BM5 paramyosin were strongly biased toward IgG1 with lower levels of IgG2a after GG vaccination, while IM vaccination produced dominant IgG2a antibody responses. Antibody responses were biased toward IgG1 after both IM and GG immunization with BMIF, but antibodies were biased toward IgG2a after IM and GG vaccination with BMHSP-70 and BM14. Animals injected with a mixture of four recombinant plasmid DNAs produced antibodies to all four antigens. Conclusions Our results show that monovalent and polyvalent DNA vaccination successfully induced antibody responses to a variety of filarial antigens. However, antibody responses to different antigens varied in magnitude and with respect to isotype bias. The isotype bias of antibody responses following DNA vaccination can be affected by route of administration and by intrinsic characteristics of individual antigens.

Molecular Characterization of the North American Lung Fluke Paragonimus kellicotti in Missouri and its Development in Mongolian Gerbils

Human paragonimiasis is an emerging disease in Missouri. To characterize local parasites, we examined crayfish from three rivers. Metacercaeriae consistent with Paragonimus kellicotti were detected in 69%, 67%, and 37% of crayfish from the Big Piney, Huzzah, and Black Rivers, respectively. Sequencing of the second internal transcribed spacer and other DNA markers confirmed the species identification and the presence of identical parasite sequences in clinical specimens from two human cases. Mongolian gerbils were infected by intraperitoneal injection with 3-8 metacercariae. Most gerbils died 15-49 days post-infection. Necropsies showed pulmonary hemorrhage with necrosis, and flukes as long as 8 mm were recovered from intrathoracic tissues. Western blot analysis using P. kellicotti antigen showed a strong antibody response in gerbils 39 days post-infection. These results demonstrate that P. kellicotti is common in Missouri crayfish. The gerbil model may be useful for research on the pathogenesis, immunology, and treatment of paragonimiasis.

Transcriptomic and Proteomic Analyses of a Wolbachia-Free Filarial Parasite Provide Evidence of Trans-Kingdom Horizontal Gene Transfer

Most filarial parasites in the subfamilies Onchocercinae and Dirofilariinae depend on Wolbachia endobacteria to successfully carry out their life cycle. Recently published data indicate that the few Wolbachia-free species in these subfamilies were infected in the distant past and have subsequently shed their endosymbionts. We used an integrated transcriptomic and proteomic analysis of Onchocerca flexuosa to explore the molecular mechanisms that allow worms of this species to survive without a bacterial partner. Roche/454 sequencing of the adult transcriptome produced 16,814 isogroup and 47,252 singleton sequences that are estimated to represent approximately 41% of the complete gene set. Sequences similar to 97 Wolbachia genes were identified from the transcriptome, some of which appear on the same transcripts as sequences similar to nematode genes. Computationally predicted peptides, including those with similarity to Wolbachia proteins, were classified at the domain and pathway levels in order to assess the metabolic capabilities of O. flexuosa and compare against the Wolbachia-dependent model filaria, Brugia malayi. Transcript data further facilitated a shotgun proteomic analysis of O. flexuosa adult worm lysate, resulting in the identification of 1,803 proteins. Three of the peptides detected by mass spectroscopy map to two ABC transport-related proteins from Wolbachia. Antibodies raised to one of the Wolbachia-like peptides labeled a single 38 kDa band on Western blots of O. flexuosa lysate and stained specific worm tissues by immunohistology. Future studies will be required to determine the exact functions of Wolbachia-like peptides and proteins in O. flexuosa and to assess their roles in worm biology.

Serological diagnosis of North American Paragonimiasis by Western blot using Paragonimus kellicotti adult worm antigen.

Abstract. We studied the value of an IgG Western blot (WB) with Paragonimus kellicotti (Pk) antigen for diagnosis of North American paragonimiasis. The test was evaluated with sera from patients with Pk and Paragonimus westermani infections, with control sera from patients with other helminth infections, and sera from healthy Americans. All 11 proven Pk infection sera and two samples from suspected cases that were negative by P. westermani WB at the Centers for Disease Control and Prevention (CDC) contained antibodies to antigens at 34 kDa and at 21/23 kDa. Seven of 7 P. westermani sera contained antibodies to the 34 kDa antigen, but only 2 recognized the 21/23 kDa doublet. No control samples were reactive with these antigens. Antibody reactivity declined after praziquantel treatment. Thus, the P. kellicotti WB appears to be superior to P. westermani WB for diagnosing Pk infections, and it may be useful for assessing responses to treatment.

Systems Biology Studies of Adult Paragonimus Lung Flukes Facilitate the Identification of Immunodominant Parasite Antigens

Background Paragonimiasis is a food-borne trematode infection acquired by eating raw or undercooked crustaceans. It is a major public health problem in the far East, but it also occurs in South Asia, Africa, and in the Americas. Paragonimus worms cause chronic lung disease with cough, fever and hemoptysis that can be confused with tuberculosis or other non-parasitic diseases. Treatment is straightforward, but diagnosis is often delayed due to a lack of reliable parasitological or serodiagnostic tests. Hence, the purpose of this study was to use a systems biology approach to identify key parasite proteins that may be useful for development of improved diagnostic tests. Methodology/Principal Findings The transcriptome of adult Paragonimus kellicotti was sequenced with Illumina technology. Raw reads were pre-processed and assembled into 78,674 unique transcripts derived from 54,622 genetic loci, and 77,123 unique protein translations were predicted. A total of 2,555 predicted proteins (from 1,863 genetic loci) were verified by mass spectrometric analysis of total worm homogenate, including 63 proteins lacking homology to previously characterized sequences. Parasite proteins encoded by 321 transcripts (227 genetic loci) were reactive with antibodies from infected patients, as demonstrated by immunoaffinity purification and high-resolution liquid chromatography-mass spectrometry. Serodiagnostic candidates were prioritized based on several criteria, especially low conservation with proteins in other trematodes. Cysteine proteases, MFP6 proteins and myoglobins were abundant among the immunoreactive proteins, and these warrant further study as diagnostic candidates. Conclusions The transcriptome, proteome and immunolome of adult P. kellicotti represent a major advance in the study of Paragonimus species. These data provide a powerful foundation for translational research to develop improved diagnostic tests. Similar integrated approaches may be useful for identifying novel targets for drugs and vaccines in the future.

Localization of Wolbachia-like gene transcripts and peptides in adult Onchocerca flexuosa worms indicates tissue specific expression

BackgroundMost filarial species in the genus Onchocerca depend on Wolbachia endobacteria to successfully carry out their life cycle. O. flexuosa is a Wolbachia-free species, but its genome contains Wolbachia-like sequences presumably obtained from Wolbachia via horizontal gene transfer. Proteogenomic studies have shown that many of these Wolbachia-like sequences are expressed in adult worms.MethodsSix Wolbachia-like sequences in O. flexuosa were chosen for further study based on their sequence conservation with Wolbachia genes, length of predicted open reading frames, and expression at the RNA and/or protein levels. In situ hybridization and immunohistochemical labeling were used to localize Wolbachia-like transcripts and peptides in adult worm tissues.ResultsRNA probes representing three of the six target sequences produced hybridization signals in worm tissues. These probes bound to transcripts in the intestine and lateral chords of both sexes, in the hypodermis, median chords and uteri in females, and in sperm precursor cells in males. Antibodies raised to three peptides corresponding to these transcripts bound to specific bands in a soluble extract of adult O. flexuosa by Western blot that were not labeled by control antibodies in pre-immune serum. Two of the three antibodies produced labeling patterns in adult worm sections that were similar to those of the RNA probes, while the third produced a different pattern.ConclusionsA subset of the Wolbachia-like sequences present in the genome of the Wolbachia-free filarial species O. flexuosa are transcribed in tissues where Wolbachia reside in infected filarial species. Some of the peptides and/or proteins derived from these transcripts appear to be concentrated in the same tissues while others may be exported to other regions of the worm. These results suggest that horizontally transferred Wolbachia genes and gene products may replicate important Wolbachia functions in uninfected filarial worms.