Recombinant antigens used as diagnostic tools for lymphatic filariasis.

Lymphatic filariasis (LF) is a parasitic disease caused by the worms Wuchereria bancrofti, Brugia malayi, or Brugia timori. It is a tropical and subtropical illness that affects approximately 67 million people worldwide and that still requires better diagnostic tools to prevent its spread and enhance the effectiveness of control procedures. Traditional parasitological tests and diagnostic methods based on whole protein extracts from different worms are known for problems related to sample time collection, sensitivity, and specificity. More recently, new diagnostic tools based on immunological methods using recombinant antigens have been developed. The current review describes the several recombinant antigens used as tools for lymphatic filariasis diagnosis in antigen and antibody capture assays, highlighting their advantages and limitations as well as the main commercial tests developed based on them. The literature chronology is from 1991 to 2021. First, it describes the historical background related to the identification of relevant antigens and the generation of the recombinant polypeptides used for the LF diagnosis, also detailing features specific to each antigen. The subsequent section then discusses the use of those proteins to develop antigen and antibody capture tests to detect LF. So far, studies focusing on antibody capture assays are based on 13 different antigens with at least six commercially available tests, with five proteins further used for the development of antigen capture tests. Five antigens explored in this paper belong to the SXP/RAL-2 family (BmSXP, Bm14, WbSXP-1, Wb14, WbL), and the others are BmShp-1, Bm33, BmR1, BmVAH, WbVAH, BmALT-1, BmALT-2, and Wb123. It is expected that advances in research with these antigens will allow further development of tests combining both sensitivity and specificity with low costs, assisting the Global Program to Eliminate Lymphatic Filariasis (GPELF).

Immunomodulatory potential of recombinant filarial protein, rWbL2, and its therapeutic implication in experimental ulcerative colitis in mouse.

OBJECTIVE: Immunomodulation by helminth proteins has potential therapeutic implications in inflammatory bowel disease. In the present study, we have explored the therapeutic effect of a RAL family protein of filarial parasite Wuchereria bancrofti i.e., rWbL2 protein against DSS induced colitis in a mouse model. MATERIALS AND METHODS: Anti-inflammatory activity of rWbL2 on mice peritoneal exudate cells was analyzed under in vitro condition. The colitis mice were treated intraperitoneally (i.p.) with rWbL2 in increasing doses (10 µg, 25 µg, and 50 µg) on days 4, 5, and 6. Disease severity was assessed by disease activity index (DAI), macroscopic and histopathological scores, and enzyme myeloperoxidase activity (MPO) in the colon. The response of the cultured splenocytes from treated mice to Con-A stimulation, in terms of ELISA-based assessment of the protein followed by the assessment of mRNA expression of cytokines, was measured by real-time PCR analysis. RESULT: rWbL2 protein showed anti-inflammatory activity in vitro. Treatment with rWbL2 (at 25 µg/dose) effectively attenuated disease severity by reducing weight loss, DAI, mucosal edema, colon damage, and MPO activity. This therapeutic effect was found to be associated with increased release of anti-inflammatory cytokine IL-10 and decreased release of pro-inflammatory cytokine IFN-γ and TNF-α by the splenocytes of treated mice followed by stimulation with Con-A. CONCLUSIONS: These results provide evidence of the strong immunomodulatory potential of rWbL2 protein implicating its therapeutic application against ulcerative colitis.

A multi-center field study of two point-of-care tests for circulating Wuchereria bancrofti antigenemia in Africa.

BACKGROUND: The Global Programme to Eliminate Lymphatic Filariasis uses point-of-care tests for circulating filarial antigenemia (CFA) to map endemic areas and for monitoring and evaluating the success of mass drug administration (MDA) programs. We compared the performance of the reference BinaxNOW Filariasis card test (ICT, introduced in 1997) with the Alere Filariasis Test Strip (FTS, introduced in 2013) in 5 endemic study sites in Africa. METHODOLOGY: The tests were compared prior to MDA in two study sites (Congo and Côte d'Ivoire) and in three sites that had received MDA (DRC and 2 sites in Liberia). Data were analyzed with regard to % positivity, % agreement, and heterogeneity. Models evaluated potential effects of age, gender, and blood microfilaria (Mf) counts in individuals and effects of endemicity and history of MDA at the village level as potential factors linked to higher sensitivity of the FTS. Lastly, we assessed relationships between CFA scores and Mf in pre- and post-MDA settings. PRINCIPAL FINDINGS: Paired test results were available for 3,682 individuals. Antigenemia rates were 8% and 22% higher by FTS than by ICT in pre-MDA and in post-MDA sites, respectively. FTS/ICT ratios were higher in areas with low infection rates. The probability of having microfilaremia was much higher in persons with CFA scores >1 in untreated areas. However, this was not true in post-MDA settings. CONCLUSIONS/SIGNIFICANCE: This study has provided extensive new information on the performance of the FTS compared to ICT in Africa and it has confirmed the increased sensitivity of FTS reported in prior studies. Variability in FTS/ICT was related in part to endemicity level, history of MDA, and perhaps to the medications used for MDA. These results suggest that FTS should be superior to ICT for mapping, for transmission assessment surveys, and for post-MDA surveillance.

Therapeutic potential of the immunomodulatory proteins Wuchereria bancrofti L2 and Brugia malayi abundant larval transcript 2 against streptozotocin-induced type 1 diabetes in mice.

Epidemiological and experimental evidence has supported the concept of using helminths as alternative bio-therapeutic agents in the treatment of type 1 diabetes (T1D). In the current study, two filarial proteins, recombinant Wuchereria bancrofti L2 (rWbL2) and Brugia malayi abundant larval transcript 2 (rBmALT-2) have been investigated, individually and in combination, for their therapeutic potential in streptozotocin (STZ)-induced T1D. The rWbL2 and rBmALT-2 proteins, when administered individually or in combination, have resulted in lowering of the blood glucose levels and reducing the incidence of T1D in mice. In addition, these proteins have led to reduced lymphocytic infiltration and decreased islet damage and inflammation. The curative effect was found to be associated with the suppression of release of tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), and increased production of interleukin (IL)-4, IL-5 and IL-10 cytokines by the splenocytes of the diabetic mice. Insulin-specific IgG1 and antigen-specific IgE antibodies were found to be elevated in the sera of mice treated with rWbL2 and rBmALT-2 proteins. From the findings in this study, it can be envisaged that both of these filarial immunomodulatory proteins have the potential to ameliorate T1D by altering the regulatory immune responses.

The nematode parasite Onchocerca volvulus generates the transforming growth factor-beta (TGF-beta).

Transforming growth factor-beta (TGF-beta) is a highly conserved cytokine that has a well-known regulatory role in immunity, but also in organ development of most animal species including helminths. Homologous tgf-b genes and mRNA have been detected in the filaria Brugia malayi. The in situ protein expression is unknown for filariae. Therefore, we examined several filariae for the expression and localization of latent (stable) TGF-beta in adult and larval stages. A specific goat anti-human latency associated protein (LAP, TGF-beta 1) antibody, purified by affinity chromatography, was used for light and electron microscopic immunohistochemistry. Adult Onchocerca volvulus, Onchocerca gibsoni, Onchocerca ochengi, Onchocerca armillata, Onchocerca fasciata, Onchocerca flexuosa, Wuchereria bancrofti, Dirofilaria sp., B. malayi, and infective larvae of W. bancrofti reacted with the antibody. Labeling of worm tissues varied between negative and all degrees of positive reactions. Latent TGF-beta was strongly expressed adjacent to the cell membranes of the hypodermis, epithelia, and muscles and adjacent to many nuclei in all organs. TGF-beta was well expressed in worms without Wolbachia endobacteria eliminated by doxycycline treatment. Pleomorphic neoplasms in O. volvulus were also labeled. We conclude that latent TGF-beta protein is expressed by filariae independently of Wolbachia, possibly regulating worm tissue homeostasis.

Identification and location of albumin-like antigens in third-stage larva of W. bancrofti, in adult forms of Litomosoides chagasfilhoi and in the free-living nematode Caenorhabditis elegans.

Antigens resembling those of host proteins have been identified on the surface of several filarial parasites, such as immunoglobulins and serum albumins. The origin of albumin-like antigens on filarial parasites remains unclear. Several authors suggested that they have been adsorbed, or that they were metabolic waste products from nutritional utilization of human albumin, or perhaps a contamination with human products. This study searched for human albumin-like antigens by Western blot and ultrastructural analyses on filarial parasites, third stage of W. bancrofti and adult females of Litomosoides chagasfilhoi, and on the free-living Caenorhabditis elegans nematode. Our results showed approximately 67kDa proteins recognized by anti-human albumin antibodies on extracts and excretory-secretory (ES) products of the third-stage W. bancrofti. Similar albumin-like proteins were also detected on the filarial parasite L. chagasfilhoi and on C. elegans extracts. The immunocytochemistry analysis showed human albumin-like antigens on similar tissues of these nematodes. These results provide evidence that these proteins have antigenic similarity and similar distribution in nematodes tissues. Our observations suggest that albumin-like antigens presented on filarial parasites are not acquired from the host, but rather are shared antigenic determinants found even in the third-stage larvae recovered from the invertebrate host.

Cytochemical characterization of the third-stage larva of Wuchereria bancrofti (Nematoda: Filarioidea).

In the present paper, we report the results we obtained using several cytochemical techniques to analyze the infective larva of Wuchereria bancrofti. An imidazole osmium tetroxide solution was used to visualize unsaturated fatty acids. A highly contrasted material forming a continuous structure was observed on the larval surface and over the epicuticle. A strong reaction was observed on the esophagus and also on the inner secreted material. Carbohydrates containing vic-glycol groups were not observed on the cuticle of the third-stage larva of W. bancrofti submitted to the Thiéry technique. Using a panel of eight gold-labeled lectins, we found that the cuticle exhibited slight labeling with all lectins used, indicating residues of N-acetyl-D: -glucosamine, N-acetyl-galactosamine, D: -galactose, D: -manose, and L: -fucose. Surface anionic sites were visualized by using cationized ferritin particles. Treatment with trypsin partially inhibited the reaction, whereas the treatment with chondroitinase ABC, a specific enzyme for glycosaminoglycans, completely abolished the labeling with cationic particles.

[Proteic profile and antigenic recognition of extracts from Wuchereria bancrofti L3 infective larvae]. / Perfil protéico e reconhecimento antigênico de extratos de larvas infectantes (L3) de Wuchereria bancrofti.

A study of protein characterization and recognition of the antigenic profile was accomplished in extracts of infective larvae (L3) from Wuchereria bancrofti. Two proteins of relative molecular weight of 49 and 55 kDa were recognized as antigenic in all extracts by the tested sera. The secretory/excretory antigen presented the largest number of recognized bands (105, 100, 76, 55, 49, 39 and 32 kDa) followed by the somatic antigen (100, 76, 55 and 49 kDa) when incubated with pools of sera from healthy individuals resident in endemic areas (normal endemics). Human sera and parasitized blood used to infect mosquitoes in order to obtain L3, were collected from microfilaraemic individuals living in a filariasis endemic area. From 792 persons screened with the thick smear technique, 87 (11%) were positive. No statistical significance was observed between genders. The group between 11 and 19 years of age presented higher percentage of infection (36.8%).

Perfil protéico e reconhecimento antigênico de extratos de larvas infectantes (L3) de Wuchereria bancrofti / Proteic profile and antigenic recognition of extracts from Wuchereria bancrofti L3 infective larvae

A caracterização protéica dos extratos de larvas infectantes (L3) de Wuchereria bancrofti foi realizada por eletroforese em gel de poliacrilamida, em presença de dodecil sulfato de sódio (SDS-PAGE) e o reconhecimento antigênico das proteínas por Western-blot. O maior número de bandas protéicas reconhecidas foi evidenciado nos extratos AgSE (105, 100, 76, 55, 49, 39 e 32 kDa) e AgS (100, 76, 55, e 49 kDa) na presença de soros de indivíduos endêmicos normais. As bandas de 49 e 55 kDa foram reconhecidas pelos soros dos microfilarêmicos, endêmicos normais (residentes de área endêmica livres de infecção filarial) e portadores da forma crônica da doença. As larvas infectantes foram obtidas pela dissecção de mosquitos Culex quinquefasciatus infectados com sangue microfilarêmico de voluntários portadores de microfilaremia, residentes do Município de Olinda-PE. Nos 792 indivíduos investigados pela técnica da gota espessa mensurada (60æl de sangue) 87 foram positivos (11 por cento). A diferenca da positividade entre homens e mulheres não foi significativa e a faixa etária de 11 a 19 anos foi a de maior prevalência.

Immunocytochemical localization and distribution of human albumin in Wuchereria bancrofti adult worms.

To determine whether albumin is present on adult worms of Wuchereria bancrofti, thin sections of resin-embedded parasites were incubated with a specific antiserum to human albumin. With the exception of the epicuticle, all layers of the cuticle and the hypodermis were intensely labeled. Concentration of gold particles was observed within infoldings of the hypodermal membrane. Moderate labeling of the thin basement membrane that lines the pseudocelomic cavity and the gonoduct was also observed. Within the uterus, ovular membranes labeled intensely; groups of organized particles were seen below ovular membranes and also within invaginations of microfilarial embryos. In contrast, few gold particles were seen on the surface of mature intrauterine microfilariae. No labeling was observed in control sections incubated with antiserum preadsorbed with purified human albumin. The findings suggest that human albumin may be essential for the nutrition and development of W bancrofti microfilariae.

Ultrastructural and cytochemical aspects of the cuticle of adult Wuchereria bancrofti (Nematoda: Filarioidea).

Because of the practical limitations of obtaining viable adult forms of the Wuchereria bancrofti, the major species responsible for human lymphatic filariasis, only few ultrastructural studies were carried out. Adult worms present the cuticle as the interface structure between host and parasite. Cuticle structure and the demonstration of the presence of basic proteins, lipids, small amounts of terminal carbohydrate residues, phospholipids and collagen in the cuticle was undertaken on thin sections of embedded parasites. Using immunocytochemical methods, antigenic epitopes similar to those found in the extra cellular matrix of vertebrates were localized on thin sections of the Lowicryl embedded adult filariae.

The sheath of the microfilaria of Wuchereria bancrofti has albumin and immunoglobulin on its surface.

Using direct fluorescent antibody analysis it was shown that the sheath of live microfilariae of Wuchereria bancrofti has human albumin and the immunoglobulin G subclasses IgG1 and IgG4 on its surface.

Cytochemical localization of carbohydrate residues in microfilariae of Wuchereria bancrofti and Brugia malayi.

We investigated the localization of carbohydrate residues on the surface structures of microfilariae of Wuchereria bancrofti and Brugia malayi, using a panel of 10 different gold-labeled lectins and chitinase. The sheath, a structure that encloses the microfilariae, is not a homogeneous structure, presenting two clearly distinct layers. The outer layer is more electron dense and was not labeled with the lectins. The inner layer is less dense and was intensely labeled with lectins, especially those that recognize D-galactose and N-acetyl-D-galactosamine. Small differences were observed in the lectin labeling pattern of microfilariae of W. bancrofti and B. malayi. D-galactose and fucose were observed in the cuticle of both species. Chitin, as revealed with gold-labeled chitinase, was observed in the cuticle of microfilariae of W. bancrofti but not in B. malayi.