Jayaprakasam Madhumathi

Protective immune responses of recombinant VP2 subunit antigen of infectious bursal disease virus in chickens.

Infectious bursal disease virus (IBDV) is the causative agent of Gumboro disease and poses a huge threat to poultry industry. The risks associated with conventional attenuated viral vaccines make it indispensable to probe into the development of novel and rationally designed subunit vaccines which are safer as well as effective. VP2 is the major host-protective antigen found in IBDV capsid. It encompasses different independent epitopes responsible for the induction of neutralizing antibody. Here, we report the efficacy of the immunodominant fragment of VP2 which induces both humoral and cellular immunity against infectious bursal disease. A 366 bp fragment (52-417 bp) of the VP2 gene from an IBDV field isolate was amplified and expressed in Escherichia coli as a 21 kDa recombinant protein. The efficacy of rVP2(52-417) antigen was compared with two commercial IBDV whole virus vaccine strains. The rVP2(52-417) induced significantly high antibody titres in chicken compared to commercial vaccines and the anti-rVP2(52-417) sera showed reactivity with viral antigens from both commercial strains (P<0.0001) and field isolates. Also, the chicken splenocytes from rVP2(52-417) immunized group showed a significantly high proliferation (P<0.01) compared to other groups, which implies that the rVP2(52-417) fragment contains immunogenic epitopes capable of eliciting both B and T cell responses. Further, rVP2(52-417) conferred 100% protection against vIBDV challenge in the immunized chickens which was significantly higher (P<0.001) compared to 55-60% protection by commercial vaccine strains. Hence, the study confirms the efficacy of the immunodominant VP2 fragment that could be used as a potent vaccine against IBDV infection in chicken.

DNA vaccination with VP2 gene fragment confers protection against Infectious Bursal Disease Virus in chickens.

Infectious Bursal Disease Virus (IBDV) causes immunosuppression in young chickens by destruction of antibody producing B cells in the Bursa of Fabricius and poses a potential threat to the poultry industry. We have examined the protective efficacy of a subunit DNA vaccine against IBDV infection in chickens in this study. An immunodominant VP2 gene fragment (VP252-417) was cloned into CMV promoter based DNA vaccine vector pVAX1 and in vitro expression of the DNA encoded antigens was confirmed by transfection of CHO cells with vaccine constructs followed by RT-PCR and western blot analysis using IBDV-antiserum. Two weeks old chickens were immunized intramuscularly with pVAXVP252-417 and the in vivo transcription of the plasmid DNA was confirmed by RT-PCR analysis of DNA injected muscle tissue at different intervals of post immunization. Tissue distribution analysis revealed that the plasmid DNA was extensively distributed in muscle, spleen, kidney, liver, and bursa tissues. Chickens immunized with pVAXVP252-417 developed high titer (1:12,000) of anti-VP252-417 antibodies. Further, chicken splenocytes from pVAXVP252-417 immunized group showed a significantly high proliferation to the whole viral and recombinant antigen (P<0.01) compared to control groups, which implies that pVAXVP252-417 codes for immunogenic fragment which has epitopes capable of eliciting both B and T cell responses. This is evident by the fact that, pVAXVP252-417 immunized chicken conferred 75% protection against virulent IBDV (vIBDV) challenge compared to the control group. Thus, the present study confirms that the immunodominant VP2 fragment can be used as a potential DNA vaccine against IBDV infection in chickens.

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.

Crucial epitopes of Wuchereria bancrofti abundant larval transcript recognized in natural infection

Lymphatic filariasis is a tropical disease, with over 40 million people seriously incapacitated due to lymphangitis and elephantiasis. Over 99% of infections are caused by the nematode Wuchereria bancrofti. Expressed sequence tag (EST) analysis of filarial genome identified novel infective larval (L3) stage-specific antigen, abundant larval transcript (ALT-2), which was shown to be highly essential for parasite establishment and survival in the host. The unique structural features and immunological characteristics of ALT-2 indicate the presence of critical motifs that needs to be explored in natural human infection for understanding and management of the disease. In order to dissect the epitopes of ALT protein recognized in humans, eight peptides spanning the entire protein sequence were selected based on in-silico epitope prediction and synthesized. Analysis of the reactivity of W. bancrofti ALT-2 synthetic peptides with clinical sera (n = 40) from endemic areas identified epitopes recognized by putatively immune sera, of which two comprise the highly variable acidic domain (21–60). Interestingly, our study also revealed crucial epitopes recognized by microfilaremic (MF) sera with significantly high IgG4 isotype (p < 0.001), implicated in immunomodulation. The epitope peptides identified were highly specific for MF sera and, thus, have the potential to be exploited as diagnostic markers.

Proliferative responses of Brugia malayi TPX-1 and its epitopic peptide29–43 in an endemic population of human lymphatic filariasis

Although the antioxidant thioredoxin peroxidase (TPX) is a putative target exploited in vaccine studies of lymphatic filariasis, the high sequence homology with host peroxiredoxins remains a great concern. The emergence of immunomics offers a powerful tool for novel vaccine design. Further, due to the cellular hypo-response in filariasis, analysis of T epitope repertoire becomes imperative in disease control. Here, we report the cellular responses of filarial TPX-1 and the identification of T epitope (29-43) in the host non-homologous region. The strong proliferative responses induced by the peptide mimetic in mice splenocytes and human PBMCs prove the existence of T epitope recognized in endemic population.

Chimeric Epitope Vaccine from Multistage Antigens for Lymphatic Filariasis

Lymphatic filariasis, a mosquito‐borne parasitic disease, affects more than 120 million people worldwide. Vaccination for filariasis by targeting different stages of the parasite will be a boon to the existing MDA efforts of WHO which required repeated administration of the drug to reduce the infection level and sustained transmission. Onset of a filaria‐specific immune response achieved through antigen vaccines can act synergistically with these drugs to enhance the parasite killing. Multi‐epitope vaccine approach has been proved to be successful against several parasitic diseases as it overcomes the limitations associated with the whole antigen vaccines. Earlier results from our group suggested the protective efficacy of multi‐epitope vaccine comprising two immunodominant epitopes from Brugia malayi antioxidant thioredoxin (TRX), several epitopes from transglutaminase (TGA) and abundant larval transcript‐2 (ALT‐2). In this study, the prophylactic efficacy of the filarial epitope protein (FEP), a chimera of selective epitopes identified from our earlier study, was tested in a murine model (jird) of filariasis with L3 larvae. FEP conferred a significantly (P < 0.0001) high protection (69.5%) over the control in jirds. We also observed that the multi‐epitope recombinant construct (FEP) induces multiple types of protective immune responses, thus ensuring the successful elimination of the parasite; this poses FEP as a potential 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.

Immune responses of B. malayi thioredoxin (TRX) and venom allergen homologue (VAH) chimeric multiple antigen for lymphatic filariasis

Although multiple vaccine strategy for lymphatic filariasis has provided tremendous hope, the choice of antigens used in combination has determined its success in the previous studies. Multiple antigens comprising key vaccine candidates from different life cycle stages would provide a promising strategy if the antigenic combination is chosen by careful screening. In order to analyze one such combination, we have used a chimeric construct carrying the well studied B. malayi antigens thioredoxin (BmTRX) and venom allergen homologue (BmVAH) as a fusion protein (TV) and evaluated its immune responses in mice model. The efficacy of fusion protein vaccine was explored in comparison with the single antigen vaccines and their cocktail. In mice, TV induced significantly high antibody titer of 1,28,000 compared to cocktail vaccine TRX+VAH (50,000) and single antigen vaccine TRX (16,000) or VAH (50,000). Furthermore, TV elicited higher level of cellular proliferative response together with elevated levels of IFN-γ, IL-4 and IL-5 indicating a Th1/Th2 balanced response. The isotype antibody profile showed significantly high level of IgG1 and IgG2b confirming the balanced response elicited by TV. Immunization with TV antigen induced high levels of both humoral and cellular immune responses compared to either cocktail or antigen given alone. The result suggests that TV is highly immunogenic in mice and hence the combination needs to be evaluated for its prophylactic potential.

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.

Identification of a highly immunoreactive epitope of Brugia malayi TPx recognized by the endemic sera.

Abstract Filarial thiordoxin peroxidase is a major antioxidant that plays a crucial role in parasite survival. Although Brugia malayi TPx has been shown to be a potential vaccine candidate, it shares 63% homology with its mammalian counterpart, limiting its use as a vaccine or drug target. In silico analysis of TPx sequence revealed a linear B epitope in the hosts nonhomologous region. The peptide sequence (TPx peptide27–48) was synthesized, and its reactivity with clinical sera from an endemic region was analyzed. The peptide showed significantly high reactivity (P < 0.05) against the sera of putatively immune individuals compared to the nonendemic control sera. It also showed high reactivity against the sera of patients with chronic pathology and patent infection. The high reactivity of the peptide with endemic immune sera equivalent to that of whole protein shows that it forms a dominant B epitope of TPx protein and thus could be utilized for incorporation into a multiepitope vaccine construct for filariasis.