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
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| Creator |
Mazumder, Saumyabrata
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| Subject |
Infectious Diseases and Immunology
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| 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.
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| Date |
2010
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| Type |
Thesis
NonPeerReviewed |
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| Format |
application/pdf
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| 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. |
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| Relation |
http://www.eprints.iicb.res.in/492/
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