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A Comparative Evaluation of the Potency and Durability of DNA- and Protein-based Vaccines against Experimental Visceral Leishmaniasis

IR@IICB: CSIR-Indian Institute of Chemical Biology, Kolkata

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Title A Comparative Evaluation of the Potency and Durability of DNA- and Protein-based Vaccines against Experimental Visceral Leishmaniasis
Creator Mazumder, Saumyabrata
Subject Infectious Diseases and Immunology
Description Leishmaniasis is a vector-borne infectious disease with a wide spectrum of pathologies depending on the species of Leishmania. There are at least two million cases each year in 88 countries, with 367 million people at risk. The pathologies of human leishmaniasis range from selflimiting cutaneous infection to fatal infection of the visceral organs, making the disease an important health problem in the world. Conventional chemotherapies are often inadequate, toxic and expensive or are becoming less effective because of the emergence of numerous resistances, a clear risk for human health. Moreover, vaccination against VL has received limited attention compared with cutaneous leishmaniasis, although the need for an effective vaccine is pressing for the control of this disease. Since SLA in liposomes induces protective immunity and provides immunotherapy against experimental VL, we sought to investigate the feasibility of protection with this vaccine in association with non-coding pDNA in the first chapter. We demonstrate that intraperitoneal immunization with liposomal SLA in association with ISS enriched pDNA either mixed or co-entrapped within showed substantial immune responses. Furthermore, a comparison of these two formulations revealed that co-entrapment of pDNA and SLA within the liposomes was significantly effective than liposomal SLA mixed with pDNA against experimental VL in BALB/c mice resulting in almost complete protection. Since 63 kDa glycoprotein or gp63 is recognised by human VL sera, we have used this protein as a subunit vaccine in its recombinant form for the first time against VL in murine model. To obtain the recombinant gp63 from L. donovani, the full-length gp63 was cloned into bacterial expression vector pET16b, expressed, purified, and sequenced under the Gen Accession Number GQ301544. The part of the thesis work with gp63 has been divided into (i) biochemical characterization, and (ii) subunit vaccination and immunological studies. The second chapter represents the role of C-terminal domain in catalytic activity of Ldgp63. The study demonstrates for the first time that the deletion of 180-211 amino acids from the C-terminal end resulted in almost 50% reduction in activity towards substrates like azocasein, casein and gelatin. In addition, all the deletion mutant showed reduced activity towards human IgG. Using homology modeling, we identified S446 and F448 of CTD located near the active site groove and confirmed that S446 is involved in proteolysis. CD spectrometric analysis were compared between WT, 389, and S446A, and showed a structure not greatly affected by truncation or mutation. In the third chapter, the immunogenicity and protective efficacy of gp63 in its recombinant form was investigated through subcutaneous vaccination using DSPC-bearing cationic liposomes and MPL-TDM as adjuvants. The study described comparable levels of immune responses in BALB/c mice receiving either liposomal rgp63 in presence of MPL-TDM during both prime-and boost, or liposomal rgp63 along with MPL-TDM during priming, and boosting thereafter with liposomal rgp63. However, with L. donovani challenge, the group of mice receiving liposomal rgp63 in presence of MPL-TDM during both priming and boosting showed substantially enhanced immune responses and conferred higher levels of protection. The enhancement of Th1 cytokines IFN-, IL-12 and, IL-2, with a down-regulation of Th2 cytokine IL-4, along with immunosuppressive IL-10, may be involved for the protection. Furthermore, detailed immunological mechanisms demonstrate the role of CD11c+ cells in the generation of early immune responses, and macrophages in the leishmaniacidal activity. Moreover, we observed that the protection could be transferred adoptively through antigen-specific CD4+ and CD8+ T cells. To conclude, these data encourage the development of effective protein-based vaccinations that can induce strong cellular and humoral responses and might be useful for human use in clinical trials. Since liposomal SLA in association with ISS enriched pDNA conferred almost complete protection against VL (Chapter 1), we sought to investigate a comparative study to evaluate the potency and durability of DNA/DNA, DNA/Protein and Protein/Protein based vaccination using gp63 as a candidate vaccine and CpG as adjuvant. To obtain gp63 in its DNA form, a full-length gp63 from L. donovani was sub-cloned into mammalian expression vector (pcDNA3.1), transfected in CHO cell line and the level of expression at the protein level was confirmed by the western blot analysis. Although, vaccination with gp63 DNA either alone, mixed with CpG- ODN or heterologously prime-boosted with CpG- ODN showed comparable levels of protection at short-term protection study, DNA-prime/Protein-boost in presence of CpG significantly reduced hepatic and splenic parasite compared to DNA/DNA and Protein/Protein vaccination, in the long-term study. These results emphasize the potential of DNA-prime/Protein-boost vaccination over DNA/ DNA and Protein/Protein based vaccination in maintaining long-term immunity against intracellular pathogen like Leishmania.
Date 2010
Type Thesis
Format application/pdf
Identifier http://www.eprints.iicb.res.in/492/1/SAUMYABRATA_MAZUMDER_THESIS_FINAL_(DR._NAHID_ALI).pdf
Mazumder, Saumyabrata (2010) A Comparative Evaluation of the Potency and Durability of DNA- and Protein-based Vaccines against Experimental Visceral Leishmaniasis. PhD thesis, Jadavpur University.
Relation http://www.eprints.iicb.res.in/492/