Recombinant antigen-based antibody assays for the diagnosis and surveillance of lymphatic filariasis – a multicenter trial

The development of antifilarial antibody responses is a characteristic feature of infection with filarial parasites. It should be possible to exploit this fact to develop tools to monitor the progress of the global program to eliminate lymphatic filariasis (LF); however, assays based on parasite extracts suffer from a number of limitations, including the paucity of parasite material, the difficulty of assay standardization and problems with assay specificity. In principle, assays based on recombinant filarial antigens should address these limitations and provide useful tools for diagnosis and surveillance of LF. The present multicenter study was designed to compare the performance of antibody assays for filariasis based on recombinant antigens Bm14, WbSXP, and BmR1. Coded serum specimens were distributed to five participating laboratories where assays for each antigen were conducted in parallel. Assays based on Bm14, WbSXP, or BmR1 demonstrated good sensitivity (>90%) for field use and none of the assays demonstrated reactivity with specimens from persons with non-filarial helminth infections. Limitations of the assays are discussed. Well-designed field studies are now needed to assess sampling methodology and the application of antibody testing to the monitoring and surveillance of LF elimination programs

Cloning, expression and characterization of Brugia malayi abundant larval protein transcript-2 (BmALT-2) expressed in Pichia pastoris

Besides mass drug administration, successful elimination of human lymphatic filariasis, a mosquito-borne tropical parasitic disease, requires developing other suitable prophylactic agents such as vaccines. The Brugia malayi abundant larval transcript-2 (BmALT-2) protein has been identified as one possible candidate. So far, it (E-ALT-2) has been expressed as a 24-kDa non-glycosylated protein in Escherichia coli. Easy and low-cost downstream purification of secreted BmALT-2 in Pichia pastoris may be a vital option to inexpensive large-scale production. This study focused on expression and molecular characterization of BmALT-2 (P-ALT-2) in P. pastoris. Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis showed that some P. pastoris colonies produced 27 and 24 kDa bands and few colonies produced a 24-kDa band. Preliminary studies confirmed glycosylation of 27 kDa P-ALT-2. The ratio of glycosylated and non-glycosylated P-ALT-2 was 20:80–70:30 in various colonies. The maximum yield of glycosylated and non-glycosylated P-ALT-2 was measured as 7.99 ± 1.12 and 9.18 ± 1.35 mg L−1 compared to 6.5 ± 1.2 mg L−1 of E-ALT-2 in shake flasks. Their overall expression was about 25% and 35% higher than that in E. coli. The glycosylated P-ALT-2 exhibited about 15% higher immunoreactivity with human endemic normal sera. The enhanced secreted production by P. pastoris may lead to cost-effective large-scale production of BmALT-2

Enhancing encapsulation of filarial antigen Brugia malayi thioredoxin in nano-liposomes: The role of lecithin composition

Background and purpose: Lymphatic filariasis is a debilitating infectious disease prevalent in endemic areas, necessitating the development of an effective vaccine for eradication. Although recombinant vaccine candidates have been deemed safe, they often fail to provide sufficient protection, which can be overcome by encapsulating them in nano-liposomes. In this study, we have optimised the liposomal composition for enhanced stability and encapsulation of filarial antigen Brugia malayi thioredoxin (Bm-TRX). Experimental approach: Nano-liposomes were prepared using egg phosphatidylcholine (EPC) and cholesterol via thin-film hydration, followed by sonication and characterizing. Encapsulation efficiency was optimised using different weight ratios of EPC to cholesterol (8:2, 7:3, and 6:4) and total lipid (EPC+Cholesterol) concentration to antigen Bm-TRX (10:1, 10:2, and 10:3) followed by release kinetics study. Key results: Optimised parameters yielded spherical liposomes measuring 209 nm in diameter with narrow polydispersity. Our findings demonstrated the highest encapsulation efficiency of 70.685 % and stability of 10 hours for an EPC to cholesterol weight ratio of 7:3. The in silico study proved the antigenic nature of TRX. Conclusion: The liposomal formulations loaded with TRX, as optimized in this study, hold promise for improving antigen efficiency by enhancing stability, bioavailability, and prophylactic effects by acting as immune potentiators

Antigen detection assay with parasite specific monoclonal antibodies for diagnosis of lymphatic filariasis

Background: Lymphatic filariasis is a painful and profoundly disfiguring disease. Infection is usually acquired in childhood but its visible manifestations occur later in life, causing temporary or permanent disability. The importance of developing effective assays to diagnose, monitor and evaluate human lymphatic filariasis has been emphasized by the WHO. Methods: High-affinity monoclonal antibodies (mAbs) specific for recombinant filarial antigen WbSXP-1 were developed. An ELISA based capture assay using monoclonal and polyclonal antibodies for WbSXP-1 was used for detection of circulating filarial antigen. Results: High-affinity monoclonal antibodies (mAbs) were developed that specifically binds both W. bancrofti and B. malayi mf antigens. Two mAbs (1F6H3 and 2E12E3) of subclass IgG2a and IgM showed high affinity, avidity and reactivity to recombinant and mf native antigen. Both the mAbs were used in combination as capture antibodies and polyclonal as detection antibody to develop the assay. The assay showed very high sensitivity towards W. bancrofti mf positive samples compared to endemic normal samples (P<0.0001). Conclusion: A capture assay using high-affinity monoclonal antibodies for WbSXP-1 was developed for the detection of filarial circulating antigen in clinical samples from bancroftian infection. Besides, this would also help in epidemiological studies in endemic areas of filarial infections. (C) 2011 Elsevier B.V. All rights reserved

Impairment of Tetanus-Specific Cellular and Humoral Responses following Tetanus Vaccination in Human Lymphatic Filariasis

To investigate the consequences of the impaired parasite-specific immune response in lymphatic filariasis, the effect of concurrent Wuchereria bancrofti infection on the immune response to tetanus toxoid (TT) following tetanus vaccination was studied in 20 asymptomatic microfilaremic (MF) patients, 20 patients with chronic lymphatic obstruction/elephantiasis (chronic pathology [CP]), and 10 endemic normal (EN) control individuals at baseline and at 3 and 6 months after TT vaccination. Peripheral blood mononuclear cell (PBMC) proliferative responses to TT before vaccination were not significantly different between the EN control and CP groups, but the MF group showed significantly lower baseline proliferative responses to TT compared with either the EN or CP group. Six months following vaccination, the change in proliferative response to TT was significantly greater in the EN and CP groups than in the MF group. This difference in proliferative response was reiterated in the gamma interferon (IFN-γ) response in the EN group, in that they increased IFN-γ production by 400% at 6 months, in contrast to that seen in the filaria-infected groups. In contrast to the IFN-γ responses, PBMCs from the MF group produced significantly increased levels of TT-specific IL-10 compared with PBMCs from the EN group. Although there was significantly greater TT-specific immunoglobulin G (IgG) production at baseline between the EN and MF groups, postvaccination IgG (and IgG1 isotype) responses did not differ among the groups, whereas TT-specific IgG2, IgG3, and IgG4 were all increased in the EN group compared with the filaria-infected groups. These studies indicate that concurrent infection with W. bancrofti can diminish the immune response to an unrelated antigen by a mechanism that is likely to involve IL-10

Supplementary Materials from Elucidation of immunological response and its regulatory network by P-TUFT-ALT-2: a promising fusion protein vaccine for human lymphatic filariasis

Human lymphatic filariasis, a mosquito-borne neglected tropical parasitic disease, needs an early development of prophylactic agents such as a vaccine for its successful elimination. Our earlier study suggested the enhanced immunological response by fusion protein (P-TUFT-ALT-2) of Tuftsin and ALT-2 in a mice model. We cultured human peripheral blood mononuclear cells (PBMCs) and treated cells with <i>Escherichia coli</i>-expressed ALT-2 (E-ALT-2) and P-TUFT-ALT-2. Real-time polymerase chain reaction was performed to identify the mRNA copy number of various cytokine and transcription factor genes. The recombinant vaccine candidate was also validated for humans by immunoreactivity with human sera samples of natural infection. In this study, P-TUFT-ALT-2 stimulated 12% higher PBMC proliferation in endemic normal (EN) individuals than E-ALT-2 alone. There was enhanced production of IFN <i>γ</i> , IL-2, IL-5 and IL-12, indicating a balanced Th1/Th2 response. However, higher expression of IL-5 and lower IL-4 validate the humoral response through an IL-5-dependent manner. Also, high level of IL-17 indicates a strong Th/Treg regulation over T-cell activation. The upregulated T-bet might have enhanced IFN-γ production, whereas GATA-3 was supposed to enhance IL-5 expression. The fusion protein also exhibited 15–16% higher reactivity with EN clinical sera, exposing the upregulation of IgG1 and IgM in natural infection. The higher reactivity of P-TUFT-ALT-2 with sera of natural infection (EN) was validated indirectly by B-cell activation through various cytokines and regulatory genes produced from different T cells. Thus, these findings endorse P-TUFT-ALT-2 as a potential vaccine candidate for human lymphatic filariasis

Molecular evolution of single chain fragment variable (scFv) for diagnosis of lymphatic filariasis

Endemic countries with lymphatic filariasis are striving towards the Global Program to Eliminate Lymphatic Filariasis (GPELF) by 2020. Efficient and cost-effective diagnostic tools to assess active filarial infection are critical to eradicate lymphatic filariasis. Detection of circulating filarial antigens in sera is one of the precise methods to identify this infection. Monoclonal antibodies and single chain fragment variable (scFv) against Wuchereria bancrofti antigen SXP1 have been developed for antigen detection. Molecular cloning of scFv for recombinant expression has laid a platform for developing novel genetic constructs with enhanced reactivity. In this study, a simple procedure is developed to create diverse libraries of scFv based on a single DNA framework with all the requisites for an in vitro protein synthesis and ribosomal display. Error Prone-PCR was performed to incorporate random mutations and screened by ribosome display technique to isolate evolved scFv. Evolved scFv with six mutations showed tenfold increase in affinity compared to wild-type scFv for rWbSXP1. In silico studies showed that four mutations introduced unique molecular interactions between the evolved scFv and SXP1. Reactivity with asserted clinical samples of endemic normals (EN), microfilariaemic (MF), chronic pathology (CP) and non-endemic normals (NEN) showed significant augment (59.69%, p < 0.0001) in reactivity to MF samples with evolved scFv in comparison to wild-type scFv. Sensitivity of scFv was increased from 15.62 ng to 195 pg by evolved scFv in serum samples. This evolutionary method coupled with ribosome display has facilitated us to improve the reactivity of the ScFv without diminishing the specificity

Evaluation of immuno diagnostic assay for the exposure of stage specific filarial infection

Lymphatic filariasis is a debilitating diseases caused by filarial parasitic nematodes. The infection may be acquired in childhood but the symptoms become apparent only in later life. To evaluate the success of any intervention, sensitive diagnostics were used to identify infection among endemic normals that are likely to develop microfilaremia in due course of time. Capture assay was standardized using the recombinant protein Brugia malayi Abundant Larval Transcript-2(ALT-2) specific monoclonal and polyclonal antibodies and evaluated with serum samples of clinical groups from high and low filarial infection area individuals (HIA/LIA), Endemic Normal (EN, n = 478), microfilaeremics (MF, n = 77), chronic pathology (CP, n = 57) and non endemic normal (NEN, n = 20). In order to assess stage-specific infection, ALT-2 capture assay was compared with the early reported Venom allergen homologue (VAH) and microfilariae specific SXP-1 capture assays. Of the 632 serum samples tested, ALT-2 and VAH capture assays detected circulating filarial antigen (CFA) in 57% and 52% of HIA-EN individuals, respectively. As expected, the VAH and SXP-1 capture assays were positive for 100 % of MF individuals. The described capture assays can be useful for the detection of early and stage-specific filarial infections in endemic regions of developing countries

Sampling method standardization with r<i>Wb</i>SXP-1 assay.

<p>Samples from (<b>A</b>) MF, (<b>B</b>) EN, (<b>C</b>) CP, (<b>D</b>) NEN were analysed. Samples collected as sera, whole blood, on filter paper and by the new slide method are shown. Each data point represents individual absorbance from the four different types of samples. The horizontal bars represents the mean value of each group. The values are expressed as mean ±SD.</p