Perumal Kaliraj

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.

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

ABSTRACT 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.

Novel Phage Display-Based Subtractive Screening To Identify Vaccine Candidates of Brugia malayi

ABSTRACT This study describes a novel phage display method based on an iterative subtraction strategy to identify candidate vaccine antigens of Brugia malayi. A cDNA library of the infective larval stage of B. malayi expressed on the surface of T7 phage was sequentially screened with sera samples from human subjects showing different manifestations of the disease. Antigens that selectively and specifically bind to immune sera were then enriched using a multi-step panning procedure. This strategy identified five antigens, four of which were previously reported (ALT-2, TPX-2, VAH and COX-2) and the other one was a novel cuticular collagen (Col-4). Sera from immune individuals specifically recognized all the five antigens. However, ALT-2 appeared to be the most predominantly recognized antigen by the immune sera. Therefore, it was decided to evaluate the vaccine potential of recombinant ALT-2 (rALT-2) in a mouse and jird model. The results presented show that immunization with rALT-2 conferred over 73% protection against a challenge infection in the jird model and over 64% protection in the mouse model. The present study suggests that phage display-based cDNA screening may be a powerful tool to identify candidate vaccine antigens of infectious agents.

Molecular characterization of a calcium binding translationally controlled tumor protein homologue from the filarial parasites Brugia malayi and Wuchereria bancrofti

We have cloned homologues of the mammalian translationally controlled tumor protein (TCTP) from the human filarial parasites Wuchereria bancrofti and Brugia malayi. TCTP genes from B. malayi and W. bancrofti were expressed in a T7 promoter vector as histidine tagged fusion proteins. Both the recombinant B. malayi TCTP (rBm-TCTP) and recombinant W. bancrofti TCTP (rWb-TCTP) have a molecular mass of approximately 28 kDa with the histidine tag. Sequence analyses showed that there is a 98% similarity between the two filarial TCTPs at amino acid levels and are immunologically cross-reactive. Analysis of soluble proteins from various lifecycle stages of B. malayi suggested that the expression of Bm-TCTP might be differentially regulated and occurs in multimeric form. Recombinant TCTP were found to form multimers in solution under non-reducing conditions. The tendency for filarial TCTPs to become multimers was predicted by the presence of the Lupas coiled coil structure in their sequence. Despite the absence of a signal sequence, Bm-TCTP is present abundantly in the excretory/secretions (ES) of microfilariae. Characterization studies showed that both Bm- and Wb-TCTPs are calcium-binding proteins and have histamine-releasing function in vitro. When injected intraperitoneally both the filarial TCTPs induced inflammatory infiltration of eosinophils into the peritoneal cavity of mice suggesting that the filarial TCTPs may have a role in the allergic inflammatory responses associated with filarial infections.

The Wuchereria bancrofti orthologue of Brugia malayi SXP1 and the diagnosis of bancroftian filariasis

The gene encoding the Wuchereria bancrofti orthologue of the Brugia malayi-derived diagnostic antigen SXP1 was identified from a W. bancrofti L3 cDNA library and characterized. The Wb-sxp-1 cDNA encoded a basic protein with a calculated molecular mass of 20.8 kDa. Wb-SXP-1 was 85% identical to the SXP1 protein described from B. malayi (Bm-SXP-1). The Wb-SXP-1 sequence also showed significant identity with proteins described from B. pahangi, Onchocerca volvulus, Acanthochilonema vitea, Ascaris suum, Loa loa, Litomosoides sigmodontis and Caenorhabditis elegans. The presence of a number of invariant and conserved residues in all of these nematode-derived molecules suggests that Wb-SXP-1 is a member of a new protein family. A recombinant form of Wb-SXP-1 was produced and it was determined that the anti-Wb-SXP-1 antibody response in patients with W. bancrofti infections was restricted to the IgG4 subclass. An anti-Wb-SXP-1 IgG4 ELISA was developed and this assay was found to be 100% sensitive for patients with patent W. bancrofti infection. Sera from individuals experiencing chronic pathology, endemic normals or patients with non-filarial nematode infections had no detectable IgG4 against Wb-SXP-1. While patients with patent Onchocerca volvulus infections were uniformly negative in the Wb-SXP-1 assay, 40% of sera from patent Loa loa infections were positive. When Bm-SXP-1 was used as the antigen under identical conditions, the assay was 88% specific for patent W. bancrofti infections and the antigen was recognized by antibodies from both O. volvulus and L. loa infections. The results strongly suggested that, for certain diagnostic filarial antigens, the use of same-species molecules can enhance the specificity of diagnostic tests.

The larval specific lymphatic filarial ALT-2: Induction of protection using protein or DNA vaccination

Genes from the infective stage of lymphatic filarial parasites expressed at the time of host invasion have been identified as potential vaccine candidates. By screening an L3 cDNA library with sera from uninfected longstanding residents of an area endemic for onchocerciasis, so‐called “endemic normals” (EN), we have cloned and characterized one such gene termed the abundant larval transcript two (ALT‐2). The stage specificity of ALT‐2 gene transcription and protein synthesis was confirmed by PCR using gene‐specific primers, and by western blot analysis of protein extracts from various stages of the parasite life cycle using specific antisera. Significant differences in antibody response to the recombinant ALT‐2 were observed in endemic populations with differing clinical manifestations of lymphatic filariasis with an antibody response present in sera from 18 of 25 (72%) EN subjects compared to 9 of 25 (36%) with subclinical microfilaracmia (MF) and 14 of 25 (52%) of those with chronic lymphatic obstruction (CP) (P=0.01 for comparison of EN to CP or to MF). This differential responsiveness suggests that the protective immunity postulated to account for their uninfected status might be associated with a response to this protein. When the utility of ALT‐2 as a vaccine candidate was tested in a murine model using either recombinant protein or a DNA vaccine construct, statistically significant protection was observed when compared to a control filarial gene product expressed across all stages of the parasite lifecycle (SXP‐1; P=0.02 for protein and P=0.01 for the DNA vaccine) or compared to adjuvant alone. This level of protection indicates that this vaccine is a promising candidate for further development.

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.

Quantitative assessment of circulating antigens in human lymphatic filariasis: a field evaluation of monoclonal antibody‐based ELISA using blood collected on filter strips

objective    To quantify circulating antigens in individuals with lymphatic filariasis by means of an ELISA using blood on filter strips.method  Circulating antigens in filarial patients and normal individuals living in an area endemic for W. bancrofti infection in Madras, India were estimated using a monoclonal antibody‐based ELISA. results  All microfilaraemics showed positivity to circulating antigens whereas people with chronic pathology and 80% of the endemic normals tested negative. The antigen levels in the blood collected in the night and during day time showed positivity and there was no difference in the antigen concentration. The results of the antigen levels collected onto filter strips correlated with their corresponding plasma antigen levels (r= 0.83). In microfilaraemics, DEC treatment did not alter the levels of circulating antigens for up to one month. conclusions  We conclude that this monoclonal antibody‐based ELISA using filter strips may be used in day time and replace the existing routine night blood surveys in our endemic area in India.

Thioredoxin from Brugia malayi: Defining a 16-Kilodalton Class of Thioredoxins from Nematodes

ABSTRACT Thioredoxins are a family of small redox proteins that undergo NADPH-dependent reduction by thioredoxin reductase. This results in a supply of reducing equivalents that cells use in a wide variety of biological reactions, which include maintaining reduced forms of the enzymes important for protection against damage from high-energy oxygen radicals, the regulation of transcription factor activity, and the inhibition of apoptosis. Here we report on a new member of the thioredoxin family of proteins from the filarial nematode Brugia malayi, Bm-TRX-1, which defines a new subclass of 16-kDa thioredoxins that occur widely in nematodes, including Caenorhabditis elegans. In addition to being larger than the thioredoxins found in mammalian and bacterial species, the putative active site sequence of Bm-TRX-1, WCPPC, does not conform to the highly conserved WCGPC reported for thioredoxins from mammals to bacteria. Interestingly, an allelic form of Bm-TRX-1 was identified with an active site sequence WCPQC, which appears to be unique to the thioredoxins from filarial species. Bm-TRX-1 was between 98% and 35% identical to thioredoxins from other nematodes and ≈20% identical to the thioredoxins from mammals and Escherichia coli. Bm-TRX-1 was constitutively transcribed throughout the B. malayi life cycle, and Bm-TRX protein was detectable in somatic extracts and excretory-secretory products from adults and microfilariae. Recombinant Bm-TRX-1 had thiodisulfide reductase activity, as measured by the reduction of insulin, and protected DNA from the nicking activity of oxygen radicals. Overexpression of Bm-TRX-1 in a human monocyte cell line negatively regulated tumor necrosis factor alpha-induced p38 mitogen-activated protein kinase activity, suggesting a possible role of the 16-kDa Bm-TRX-1 in immunomodulation.

Immune response studies with Wuchereria bancrofti vespid allergen homologue (WbVAH) in human lymphatic filariasis

A homologue of Brugia malayi venom allergen (BmVAH) was cloned from the infective stages (L3) of Wuchereria bancrofti. Sequence analysis showed 90% sequence identity between WbVAH and BmVAH. Recombinant WbVAH was then expressed and purified. VAH from other nematode parasites is being evaluated as potential vaccine candidates. Because W. bancrofti infections are more prevalent than B. malayi, it will significantly benefit using W. bancrofti antigens for vaccine development. In this study, we have evaluated the human immune responses to rWbVAH in putatively immune individuals who live in the endemic regions (endemic normal, EN) to determine the vaccine potential of WbVAH. These responses were then compared to those in infected individuals (microfilaraemic, MF and chronic pathology, CP). Results show that EN subjects carry WbVAH-specific IgG1, IgG2, and IgG3 circulating antibodies. It is interesting to note that CP patients also carried antibodies against WbVAH that was mainly of the IgG3 isotype. Peripheral blood mononuclear cells (PBMC) from EN individuals responded strongly to rWbVAH by proliferating and secreting IFN-γ. PBMC from MF patients also proliferated in response to rWbVAH but secreted mainly IL-10. Thus, there was a clear dichotomy in the cytokine production by infected patients vs individuals who are putatively immune (EN). Although vaccine potential of WbVAH has not been established yet, our findings suggest that WbVAH mediated immune responses in EN individuals is primarily Th1-biased. Further vaccination studies are underway in animal models to determine the role of WbVAH in protective immunity against W. bancrofti and B. malayi infections.