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.

Wucherria bancrofti glutathione S-Transferase: Insights into the 2.3 Šresolution X-ray structure and function, a therapeutic target for human lymphatic filariasis.

The notoriety of parasitic nematode survival is directly related to chronic pathogenicity, which is evident in human lymphatic filariasis. It is a disease of poverty which causes severe disability affecting more than 120 million people worldwide. These nematodes down-regulate host immune system through a myriad of strategies that includes secretion of antioxidant and detoxification enzymes like glutathione-S-transferases (GSTs). Earlier studies have shown Wuchereria bancrofti GST to be a potential therapeutic target. Parasite GSTs catalyse the conjugation of glutathione to xenobiotic and other endogenous electrophiles and are essential for their long-term survival in lymph tissues. Hence, the crystal structure of WbGST along with its cofactor GSH at 2.3 Šresolution was determined. Structural comparisons against host GST reveal distinct differences in the substrate binding sites. The parasite xenobiotic binding site is more substrate/solvent accessible. The structure also suggests the presence of putative non-catalytic binding sites that may permit sequestration of endogenous and exogenous ligands. The structure of WbGST also provides a case for the role of the π-cation interaction in stabilizing catalytic Tyr compared to stabilization interactions described for other GSTs. Hence, the obtained information regarding crucial differences in the active sites will support future design of parasite specific inhibitors. Further, the study also evaluates the inhibition of WbGST and its variants by antifilarial diethylcarbamazine through kinetic assays.

Cloning, large-scale production and characterization of fusion protein (P-TUFT-ALT-2) of Brugian abundant larval transcript-2 with tuftsin in Pichia pastoris.

Lymphatic filariasis is a "disease of poor people" due to a large section of affected people with economic backwardness. Therefore, successful elimination of this disease requires a cost-effective prophylactic agent such as vaccine along with conventional drugs. The Abundant Larval Transcript-2 (BmALT-2) protein of Brugia malayi has been recognized as the most potential vaccine candidate. Tuftsin, a tetra-peptide immunopotentiator has already shown the enhanced immunogenicity of various vaccine antigens in earlier studies. This study deals with the development of tuft-alt-2 fusion construct and a suitable culture condition for its large-scale production in Pichia pastoris. The recombinant P. pastoris/tuft-alt-2 with 9-11 copies of the gene construct exhibited the highest expression level. The molecular weight of P-TUFT-ALT-2 was determined as 28 kDa in SDS-PAGE including 3 kDa due to glycosylation. The dry cell biomass was 57.4 gL-1 in the bioreactor. The P-TUFT-ALT-2 expression was measured as about 35 mg L-1, which was 102% higher than flask culture. The P-TUFT-ALT-2 produced the highest 65,000 IgG peak titer in Balb/c mice. Moreover, P-TUFT-ALT-2 exhibited about 9.46% higher splenocyte proliferation than E. coli expressed E-ALT-2 alone. The enhanced secreted production of P-TUFT-ALT-2 in bioreactor would step up its commercialization as an inexpensive commercial vaccine for human lymphatic filariasis.

Molecular characterization and evaluation of Onchocerca volvulus-secreted larval acidic protein 1 (SLAP1) as a putative vaccine candidate on endemic population of lymphatic filariasis.

Filarial parasites infected nearly 160 million of the global population with onchocerciasis and lymphatic filariasis, and further, a billion of people are estimated to be at risk of infection, rendering them among the most prevalent infectious agents in the world today. Given the complexity of their life cycle and the immune evasion mechanisms of these organisms, development of a vaccine remains to be a long-term challenge. Though a number of immunodominant antigens have been characterized, the presence of homologous proteins in humans or the allelic variants are some of the major drawbacks. One of the extensively studied vaccine candidates is abundant larval transcripts (ALT) family of proteins for the following properties: highly regulated expression, abundance, excreted-secreted product of infective stage larvae, and essentially for parasite establishment and survival in the host. In the present study, stage-specific expression of secreted larval acidic protein 1 (SLAP1) was identified; an ALT orthologue from Onchocerca volvulus was cloned, expressed, and purified as a recombinant protein. Immunogenicity of OvSLAP1 was demonstrated with sera and peripheral blood mononuclear cells from endemic regions of Brugia malayi and Wuchereria bancrofti. OvSLAP1 antibodies were predominated by IgG1 and IgG2 in endemic normal (EN) and chronic pathology (CP) subjects. It has also induced marked cellular response as observed by lymphoproliferation assay. The study revealed that OvSLAP1 can segregate humoral (EN mean optical density (OD) = 0.87 ± 0.035, CP mean OD = 0.59 ± 0.029) and cellular (EN mean stimulation index (SI) = 5.87 ± 0.167, CP mean SI = 3.5 ± 0.134) immune responses between EN and CP individuals (P < 0.001), signifying its prophylactic ability and vitality for protection from filarial infections in endemic population.

The adjuvant-free immunoprotection of recombinant filarial protein Abundant Larval Transcript-2 (ALT-2) in Mastomys coucha and the immunoprophylactic importance of its putative signal sequence.

The filarial protein Abundant Larval Transcript-2 (ALT-2) of the filarial parasite Brugia malayi has been shown to produce 74% worm clearance when administered with an adjuvant. In the present study, we show that it not only induces humoral and cell-mediated immunity, but also protection up to 71% in Mastomys coucha, a permissive animal model for filariasis, even without adjuvant. This unique feature of ALT-2 protein is highly restricted to its 21 amino acid N-terminal signal sequence, the absence of which resulted in poor immune response as well as immunoprotection (49%). Moreover, ALT-2 is likely to exert immunoprotection effects in B. malayi infection by maintaining a Th1-Th2 balance, evident from higher levels of IgG1 and IgG2a as well as IL-4 and IFN-γ. An improved understanding about the role of this filarial protein in host immunity, host-parasite interaction and worm clearance will aid in the development of good immunoprophylaxis for the disease.

Molecular characterization of a truncated antigen (Wb14) of SXP-1 of Wuchereria bancrofti from four endemic regions in India.

Wb14 of Wuchereria bancrofti, an orthologue of Brugia malayi SXP-1 and W. bancrofti SXP-1, was amplified from genomic DNA of W. bancrofti microfilaria collected from four distant geographical locations in India viz., Vellore, Bhubaneshwar, Pondicherry and Sevagram. The gene was sub-cloned in a prokaryotic vector pRSET and expressed in Escherichia coli as a truncated protein (approximately 23kDa). The nucleotide sequence of the gene is 98% similar to that of WbSXP-1 and is found to be intron-less. However, the analysis and comparison of the derived amino acid sequence with WbSXP-1 showed that Wb14 is truncated at amino acid position 153. The distribution of the two genes in the studied four geographical locations indicated that WbSXP-1 is prevalent only in parasite samples from Sevagram while Wb14 is present in parasites from all the other locations. Only a limited polymorphism was observed in both the genes among the parasites from different geographical locations.

Production and single-step purification of Brugia malayi abundant larval transcript (ALT-2) using hydrophobic interaction chromatography.

Abundant larval transcript (ALT), a novel filarial protein, has been shown to have great potential as a vaccine in the prevention of human lymphatic filariasis. In this study, we report a method for the production of recombinant ALT-2 protein, expressed in the cytoplasm of bacterium Escherichia coli in soluble form and purification in a single step using hydrophobic interaction chromatography (HIC). Fermentation was done by continuous fed-batch methodology with dissolved oxygen (DO)-controlled feed addition. The culture was induced with 1 mM isopropyl-ß-D: -thiogalactopyranoside (IPTG). Up to 9 g/l dry cell weight (DCW) of biomass was obtained from 1.6 l of Luria-Bertani (LB) broth in a bench-scale reactor. Around 200 mg/l of purified ALT-2 with a yield of about 60% was obtained. This is almost a 2.5-fold increase in final protein yield compared to purification using immobilized metal affinity chromatography (IMAC).

Immunological evaluation of fusion protein of Brugia malayi abundant larval protein transcript-2 (BmALT-2) and Tuftsin in experimental mice model.

INTRODUCTION: Filariasis, a neglected tropical helminth disease needs vaccine besides mass drug administration for its successful eradication. METHODS: An attempt was made to produce a fusion protein (P-TUFT-ALT-2) of abundant larval transcript protein-2 and Tuftsin to enhance its immunogenicity. The fusion construct was expressed in Pichia pastoris, a nonexpensive commercial expression system. This study focused on the evaluation of immunological response produced by P-TUFT-ALT-2 in Balb/c mice. RESULT AND DISCUSSION: P-TUFT-ALT-2 showed an enhanced IgG peak titre compared to E. coli expressed E-ALT-2 and P. pastoris expressed P-ALT-2. IgG2b, IgG2a and IgG1 production were predominant indicating a balanced Th1/Th2 response. P-TUFT-ALT-2 also induced about 28% and 9.5% higher splenocyte proliferation over control and E-ALT-2 respectively. Splenocytes produced predominant IFN-γ followed by IL-5, IL-2 and IL-10 specifying a balanced Th1/Th2 response. P-TUFT-ALT-2 showed 55% to 80% with an average of 65% cytotoxicity in B. malayi L3 larvae in in vitro ADCC assay. CONCLUSION: This experiment validates P-TUFT-ALT-2 as a potential vaccine candidate for human lymphatic filariasis.

Comparison of immunogenicity, protective efficacy of single and cocktail DNA vaccine of Brugia malayi abundant larval transcript (ALT-2) and thioredoxin peroxidase (TPX) in mice.

Although DNA vaccines have several advantages over conventional vaccines, antibody production and protection are often not adequate, particularly in single plasmid vaccine formulation. In the present study we evaluated the efficacy of a cocktail vaccine based on plasmids encoding larval (L3) stage-specific Brugia malayi abundant larval transcript (BmALT-2) and antioxidant detoxification enzyme B. malayi thioredoxin peroxidase (BmTPX) to induce antibodies, protective efficacy and cell-mediated immune response in mice. Mice immunized with cocktail DNA vaccines containing the pVAX ALT-2+TPX developed higher titers of anti-BmALT-2+TPX (1:5000) antibodies, compared to the mice immunized with single DNA vaccine of pVAX ALT-2 or pVAX TPX (1:2000). Correlating with this, the mice administered with cocktail vaccine induced up to 78% of cytotoxicity against B. malayi mf. This cytotoxicity was high compared to 34% induced by the pVAX-ALT2 or 37% by pVAX-TPX. Moreover, cocktail vaccination of mice resulted in significantly higher level of cellular proliferative response associated with raised levels of IFN-gamma that skewed towards Th1 type of response compared to vaccination using either of the components. Taken together, these data suggest that the combination of two or more antigens maybe an effective vaccine development strategy to improve protection and immunogenicity against human lymphatic filariasis.

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 Escherichia coli-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 γ, 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.

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 poly-clonal 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.

Immunopotentiating nano-chitosan as potent vaccine carter for efficacious prophylaxis of filarial antigens.

Lymphatic filariasis (LF), a morbid vector-borne parasitic infection affects millions in tropical areas. Complete eradication can only be achieved by the development of a potent vaccine. Among the various filarial antigens that have been characterized, antigens Brugia malayi thioredoxin (TRX) and abundant larval transcript (ALT) have produced recognizable level of protection in Jirds, thereby evidenced to be good vaccine candidates. In this study an attempt was made to enhance their immunoprophylactic activity by encapsulating them in natural polysaccharide chitosan forming nanospheres (CN). High encapsulation efficiency for TRX (93%) in CN (TCN) and ALT-2 (90%) in CN (ACN) was achieved. Morphological studies confirmed the spherical and uniform distribution of nanospheres to be 220 nm. The electrostatic interaction between chitosan and the antigens were confirmed using differential scanning calorimetry and FT-IR. The study revealed the immunostimulatory property of chitosan providing enhanced level of proliferation for encapsulated antigens in peripheral blood mononuclear cells from endemic normal personals, at low concentration (TCN mean stimulation index (SI)=4.23±0.15 and ACN (SI)=4.05±0.33) compared to stimulation obtained by antigens alone. Hence, our study demonstrated that natural macromolecule derived CN can be used as efficacious immunostimulatory vaccine carter for LF thereby diminishing pathological sequel.

Dissecting the Immune Response Elicited by WbALT-2, ALT MAP in Clinical Populations and Mouse Model: A Prophylactic Measure Against Lymphatic Filariasis.

BACKGROUND: Abundant Larval Transcript (ALT) is one of the major groups of immune-dominant proteins produced by filarial worms during their larval stage. The major B-cell and T-cell epitopic domains of the ALT-2 antigen were mapped to develop a multiple antigenic peptide (MAP) prophylactic antigen against lymphatic filariasis. METHODS AND RESULTS: ALT MAP was constructed by solid phase peptide synthesis. The reactivity of whole ALT protein and ALT MAP against clinical sera described a high reactivity of endemic normal sera against ALT MAP compared to WbALT-2 protein. The antibody isotype pattern revealed elevated levels of IgG1 and IgG2 against ALT MAP, followed by IgG3 and IgG4. In this study we also analyzed the immune response pattern elicited by ALT MAP, ALT in mice models, which revealed similar pattern of humoral response, while low T cell proliferation in ALT MAP groups. The low proliferation could be attributed to T/B epitope arrangement on the construct, MHC restriction, and incomplete signal delivery by T cell receptor. CONCLUSION: The immunodominant epitopes in ALT MAP were found to play a crucial role in inducing high antigen specific proliferation. This revealed the significance of ALT MAP in stimulating innate immunity in offering protective immune response probably through the activation of complement cascade along with stimulation of cellular response. An improved understanding, including the construction of ALT MAP and parasite challenge study in jirds to determine the worm clearance would give a better insight in the characterization ALT MAP construct as a prophylactic vaccine candidate.

Biodegradable poly-l-lactide based microparticles as controlled release delivery system for filarial vaccine candidate antigens.

Modern recombinant vaccines are less immunogenic than conventional vaccines which require adjuvants to enhance the effect of a vaccine. Alum is being used as a standard adjuvant for protein based vaccines to augment immune response in several diseases. However, the problem associated with alum is it requires multiple doses at specific time intervals to achieve the adequate level of immunity. Currently the adjuvanticity of Poly-l-lactide microparticles as single dose immunization was explored to overcome multiple immunization and reported to be effective for several diseases. In this regard we adsorbed filarial recombinant chimeric multivalent vaccine candidates such as TV and FEP on to PLA by double emulsion method and analyzed the characterization of PLA encapsulated microparticles and evaluated its immune responses in mice. The efficacy of single dose of PLA encapsulated proteins was investigated in comparison with single dose of alum or protein alone. In mice, single dose of PLA encapsulated antigens such as TV and FEP elicited significantly high antibody titer of 50,000 and 64,000 respectively than single dose of alum adsorbed TV/FEP (6000/9000) and single dose of protein TV/FEP (3000/4000) alone. Further PLA encapsulated antigens induced higher levels of cellular proliferation together with significant (P<0.0001) levels of cytokine response [PLA-TV induced high levels of IL-4(Th2) and IFN-γ (Th1) cytokines whereas PLA-FEP showed high levels of IL-5(Th2) and IFN-γ (Th1)] indicating a balanced response elicited by PLA antigens. Overall strong humoral and cellular responses were observed for PLA encapsulated antigens compared with single dose of alum adsorbed or protein alone.

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.

Immunomodulation of ALT-2 and TLR may collude in antigen specific T cell hyporesponsiveness: proposed mechanism for elevated IL-10 levels in Balb/C mice.

ALT-2, a novel antigen belonging to the chromadorea ALT-2 family of the filarial nematode is proved to clear filarial parasites in Jirds. In order to increase the protection efficacy by stimulating the cell mediated immunity, MPLA a detoxified derivative of LPS known to induce the cellular response, was used in this study as an adjuvant on mice models. ALT-2+MPLA formulation elicited a high titer of total IgG antibody, with profoundly increased levels of IgG2b. Reduced splenocyte proliferation was observed in immunized group when compared to control groups which could be attributed to many in vivo factors. The levels of IFN-γ were high in unstimulated MPLA group compared to ALT-2 stimulated MPLA group, suggesting that the ALT-2 antigen suppressed the IFN-γ levels. A high level of IL-10 was induced by the ALT-2+MPLA formulation, which inhibited the production of Th2 cytokines (IL-4, IL-5) and also reduced the Th1 cytokine (IFN-γ, IL-2) levels which are not in vogue with the classical MPLA adjuvant formulation. We propose a mechanism for this immunomodulation which involves a diminished expression of TLR-4, by which the filarial parasites have evolved to evade host immune mechanism.

Functional analysis of a highly conserved abundant larval transcript-2 (alt-2) intron 2 repeat region of lymphatic filarial parasites.

The filarial-specific protein abundant larval transcript-2 (ALT-2) is expressed exclusively in the infective larval stage (L3) and is a crucial protein for establishing immunopathogenesis in human hosts. The alt-2 gene has a conserved minisatellite repeat (29 or 27bp) in intron 2 (IR2) whose significance within lymphatic filarial species is unknown. Here, we report the role of IR2 in the regulation of alt-2 gene expression using an in vitro model. Using electrophoretic mobility shift assays, we identified the presence of a putative nuclear protein binding region within IR2. Subsequent transient expression experiments in eukaryotic cell lines demonstrated that the IR2 downregulated the expression of a downstream luciferase reporter gene, which was further validated with RT-PCR. We therefore identify IR2 as a suppressor element that regulates L3 stage-specific expression of alt-2.

Immunological characterization of recombinant Wuchereria bancrofti cuticular collagen (COL-4) as putative vaccine candidate for human lymphatic filariasis.

OBJECTIVE: To elucidate immunoprophylactic potential of recombinant Wuchereria bancrofti (W. bancrofti) cuticular collagen (COL-4) in BALB/c mice and filarial clinical samples. METHODS: col-4 gene was PCR amplified from W. bancrofti L3 cDNA library and cloned in pRSET B vector. Recombinant COL-4 was over expressed in salt inducible system and was purified by nickel affinity chromatography. Humoral and cellular responses were measured by ELISA and peripheral blood mononuclear cells (PBMC) of various filarial clinical samples respectively using purified recombinant COL-4 antigen. Then the protective immune responses of COL-4 immunized BALB/c mice were characterized. RESULTS: Sequence analysis of COL-4 with human host proteins reveals lack of homology. The recombinant COL-4 was found to be at 15 kDa fusion protein. The affinity purified COL-4 showed significant reactivity with putatively immune sera and in a similar fashion it demonstrated marked proliferation in PBMC samples. Immunization studies in experimental filarial host (mice) elicited significant titers with protective antibody isotype profile (IgM and IgG). Cellular immune responses were also significant in terms of splenocytes proliferation assay on mice samples. CONCLUSIONS: Our immunological findings in experimental host suggest Th2 mediated immune response. Hence, we propose that W. bancrofti COL-4 could be an efficacious vaccine candidate against lymphatic filariasis.

Repeat region of Brugia malayi sheath protein (Shp-1) carries Dominant B epitopes recognized in filarial endemic population.

Transmission of lymphatic filariasis is mediated through microfilariae (L1 stage of the parasite) which is encased in an eggshell called sheath. The sheath protein Shp-1 stabilizes the structure due to the unique repeat region with Met-Pro-Pro-Gln-Gly sequences. Microfilarial proteins could be used as transmission blocking vaccines. Since the repeat region of Shp-1 was predicted to carry putative B epitopes, this region was used to analyze its reactivity with clinical samples towards construction of peptide vaccine. In silico analysis of Shp-1 showed the presence of B epitopes in the region 49-107. The polypeptide epitopic region Shp-149-107 was cloned and expressed in Escherichia coli. Antibody reactivity of the Shp-149-107 construct was evaluated in filarial endemic population by ELISA. Putatively immune endemic normals (EN) showed significantly high reactivity (P < 0.05) when compared to all the other categories. Antibody reactivity of Shp-1 repeat region was similar to that of whole protein proving that this region carries B epitopes responsible for its humoral response in humans. Thus this can be employed for inducing anti-microfilarial immunity in the infected population that may lead to reduction in transmission intensity and also it could be used along with other epitopes from different stages of the parasite in order to manage the disease effectively.

Evaluation of rapid blood sample collection in the detection of circulating filarial antigens for epidemiological survey by rWbSXP-1 capture assay.

BACKGROUND: Lymphatic filariasis is a neglected tropical disease leading to profound disfiguring causing socio economic burden in the tropics. Current diagnosis strategies available during field surveys and epidemics are based on traditional microscopic detections and a few antigen/antibody assays. We have compared different sampling methodologies and standardized the highly sensitive and reliable rWbSXP-1 antigen detection assay to our new sampling methodology. METHODOLOGY: Samples collected as serum, whole blood, whole blood on filter paper and whole blood on microscopic slides from patients belonging to various clinical groups of filariasis [endemic normal(EN), chronic pathology(CP), microfilaraemic(MF) and non-endemic normal(NEN)] were collected and standardized the rWbSXP-1 antigen detection assay using monoclonal antibody raised against rWbSXP-1 protein. The whole blood collected on microscopic slide based sampling method was employed in the field and the presence of circulating filarial antigen (CFA) was assessed using the rWbSXP-1 assay. PRINCIPAL FINDINGS: The sampling methods were compared and no significant difference was observed for the detection of CFA (MF, P = 0.304, EN, P = 0.675, CP, P = 0.5698, NEN, P = 0.4494). Further the optimized sampling method was utilized to collect the 1106 samples from Polur, Tiruvannamalai. The rWbSXP-1 assay gave 98 antigen positive results whereas the microscopic method gave only 17. CONCLUSIONS: Four sampling methodologies were analyzed and the new sampling methodology of whole blood collected on microscopic slide was found to be convenient for the detection of CFA using rWbSXP-1 antigen detection assay. The 1106 samples from Polur were collected using the new method. The rWbSXP-1 antigen assay perceived a 7.32% increased result which was read as false negatives on the conventional microscopic staining method. This new sampling methodology coupled with the rWbSXP-1 antigen assay can be used in epidemiological surveys for lymphatic filariasis and the same sampling methodology can be expanded to other antigen based high affinity assays.