Potency, Efficacy and Durability of DNA/DNA, DNA/Protein and Protein/Protein Based Vaccination Using gp63 Against Leishmania donovani in BALB/c Mice

Background: Visceral leishmaniasis (VL) caused by an intracellular protozoan parasite Leishmania, is fatal in the absence of treatment. At present there are no effective vaccines against any form of leishmaniasis. Here, we evaluate the potency, efficacy and durability of DNA/DNA, DNA-prime/Protein-boost, and Protein/Protein based vaccination against VL in a susceptible murine model. Methods and Findings: To compare the potency, efficacy, and durability of DNA, protein and heterologous prime-boost (HPB) vaccination against Leishmania donovani, major surface glycoprotein gp63 was cloned into mammalian expression vector pcDNA3.1 for DNA based vaccines. We demonstrated that gp63 DNA based vaccination induced immune responses and conferred protection against challenge infection. However, vaccination with HPB approach showed comparatively enhanced cellular and humoral responses than other regimens and elicited early mixed Th1/Th2 responses before infection. Moreover, challenge with parasites induced polarized Th1 responses with enhanced IFN-c, IL-12, nitric oxide, IgG2a/IgG1 ratio and reduced IL-4 and IL-10 responses compared to other vaccination strategies. Although, vaccination with gp63 DNA either alone or mixed with CpG- ODN or heterologously prime-boosting 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 load by 10 7 fold and 10 10 fold respectively, in long-term study. The extent of protection, obtained in thi

Disruption of Coronin 1 Signaling in T Cells Promotes Allograft Tolerance while Maintaining Anti-Pathogen Immunity

The ability of the immune system to discriminate self from non-self is essential for eradicating microbial pathogens but is also responsible for allograft rejection. Whether it is possible to selectively suppress alloresponses while maintaining anti-pathogen immunity remains unknown. We found that mice deficient in coronin 1, a regulator of naive T cell homeostasis, fully retained allografts while maintaining T cell-specific responses against microbial pathogens. Mechanistically, coronin 1-deficiency increased cyclic adenosine monophosphate (cAMP) concentrations to suppress allo-specific T cell responses. Costimulation induced on microbe-infected antigen presenting cells was able to overcome cAMP-mediated immunosuppression to maintain anti-pathogen immunity. In vivo pharmacological modulation of this pathway or a prior transfer of coronin 1-deficient T cells actively suppressed allograft rejection. These results define a coronin 1-dependent regulatory axis in T cells important for allograft rejection and suggest that modulation of this pathway may be a promising approach to achieve long-term acceptance of mismatched allografts

Potentiating Effects of MPL on DSPC Bearing Cationic Liposomes Promote Recombinant GP63 Vaccine Efficacy: High Immunogenicity and Protection

Visceral leishmaniasis (VL), a vector-transmitted disease caused by Leishmania donovani, is potentially fatal if left untreated. Vaccination against VL has received limited attention compared with cutaneous leishmaniasis, although the need for an effective vaccine is pressing for the control of the disease. Earlier, we observed protective efficacy using leishmanial antigen (Ag) in the presence of either cationic liposomes or monophosphoryl lipid A-trehalose dicorynomycolate (MPL-TDM) against experimental VL through the intraperitoneal (i.p.) route of administration in the mouse model. However, this route of immunization is not adequate for human use. For this work, we developed vaccine formulations combining cationic liposomes with MPL-TDM using recombinant GP63 (rGP63) as protein Ag through the clinically relevant subcutaneous (s.c.) route. Two s.c. injections with rGP63 in association with cationic liposomes and MPL-TDM showed enhanced immune responses that further resulted in high protective levels against VL in the mouse model. This validates the combined use of MPL-TDM as an immunopotentiator and liposomes as a suitable vaccine delivery system

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

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

The Effect of C-terminal Domain Deletion on the Catalytic Activity of Leishmania Donovani Surface Proteinase GP63: Role of Ser446 in Proteolysis

The kinetoplastid protozoan Leishmania encodes major surface glycoprotein GP63, a zinc metallopeptidase (EC.3.4.24.36) expressed both in promastigote and amastigote life stages. In the present study, we explored for the first time the role of C-terminal domain (CTD) in proteinase activity by serial truncation of Leishmania donovani GP63 (LdGP63) from carboxyl terminal end (CTend). Deletion of 180e211 amino acids from CTend (D420 and D389) resulted in almost 50% loss of catalytic activity against azocasein, casein and gelatin. Moreover, all the truncated constructs showed reduced activity towards immunoglobulin (IgG). Upon homology modeling, we identified two residues, S446, and F448 in CTD, conserved in different Leishmania species, which were positioned 6.8e11 Å apart from the active site. To ascertain the role of S446 and F448 in catalysis, we replaced S446 with Ala and Thr, and F448 with Val and Tyr by site-directed mutagenesis. The variant enzymes (S446T, F448V, and F448Y) maintained near wild-type activity, whereas S446A demonstrated 50% loss of catalytic activity towards the cleavage of various biological substrates. Kinetic analysis of S446A resulted in a 2.6-fold decrease in the affinity, 10-fold decrease in turn-over rates, and large increase in transition-state binding energy (1.4 kcal/mol) for the quenched peptide substrates. These results emphasize the relevance of CTD in the proteolytic activity of LdGP63. Fluorescence spectroscopy, and CD analysis however, indicated that the reduced activities showed by D389 and S446A were not due to global changes in the enzyme structures. Indeed, identification of S446 and its possible role in the stabilization of transition-state binding between enzyme and substrate can be exploited towards understanding of structureefunction relationship of GP63

Non-Coding pDNA Bearing Immunostimulatory Sequences co-Entrapped with Leishmanial Antigens in Cationic Liposomes Elicits almost Complete Protection Against Experimental Visceral Leishmaniasis in BALB/c Mice.

The difficulty in making successful vaccines against leishmaniasis is partly due to lack of an appropriate adjuvant. Non-coding plasmidDNA (pDNA) bearing immunostimulatory sequences (ISS) is a potent activator of innate immunity, and can thus act as an adjuvant with vaccine antigen. We therefore evaluated the efficacy of pDNA and soluble leishmanial antigens (SLA) to protect against challenge with Leishmania donovani infection. We demonstrate that immunomodulatory activity of pDNA, which potentiated a Th1 immune responses, led to enhanced protection with SLA. Importantly, adding cationic liposomes as vehicle to the antigen, with pDNA either complexed or entrapped within, significantly increased the potentiating effect of pDNA. Further, comparison of the two vaccine formulations demonstrated an impressive increase in the protective efficacy up to two folds when both antigen and pDNA were within the vehicle. Thus, these studies establish the utility of non-coding pDNA bearing ISS as strong promoters of vaccine potency of liposomal antigens especially when co-entrapped with the antigen in cationic liposomes

Ag-specific NO levels in vaccinated mice before and after 3 months challenge infection.

<p>Ten days, short-term protection (STP), and twelve weeks, long-term protection (LTP) after final boosting (post-vaccination), and 3 months after challenge infection (post-infection), splenocytes from different vaccinated mice were isolated and stimulated with rgp63 (5 µg/ml) for 96 h and the level of NO was determined in supernatants by Griess reagent. Figures (A, B) and (C, D) represent levels of NO before (post-vaccination) and after 3 months of <i>L. donovani</i> infection (post-infection) respectively. The results are shown as the mean absorbance values ± S.E. of five individual mice per group, representative of two independent experiments with similar results. OV- only vector. * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001 as assessed by one-way ANOVA and Tukey's multiple comparison test.</p

Cloning and expression of <i>L. donovani</i> gp63 in mammalian expression vector and purification from <i>E. coli</i>.

<p>(A) Clone confirmation of gp63 in pcDNA3.1 (−/−) vector. M, λDNA digested with HindIII marker; lane 1, PCR of cloned construct; lane 2, BamHI/HindIII digested pcDNA3.1-gp63 construct. (B) Expression of gp63 in transfected CHO cell line. Lane 1, western blot of pcDNA3.1-gp63 transfected construct in CHO cell line; lane 2, western blot of pcDNA3.1 transfected vector in CHO cell line. (C) Silver nitrate staining of 10% SDS-PAGE, M, molecular mass marker; lane 1, Purified recombinant gp63.</p

IFN-γ responses in BALB/c mice vaccinated with different vaccine approaches before and after 3 months challenge infection.

<p>Levels of IFN-γ ten days, short-term protection (STP), and twelve weeks, long-term protection (LTP) after final boosting (post-vaccination) (A, B), and 3 months after challenge infection (post-infection) (C, D). Splenocytes were isolated from vaccinated mice, stimulated with rgp63 (5 µg/ml) and were cultured for 96 h. The supernatants were collected, and assayed for IFN-γ through ELISA. Figures (E, F) represent <i>in vitro</i> blocking experiments either with anti-CD4⁺ or anti-CD8⁺ or both mAbs before (post-vaccination) and after <i>L. donovani</i> infection (post-infection). The results are shown as the mean absorbance values ± S.E. of five individual mice per group, representative of two independent experiments with similar results. OV- only vector. * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001 as assessed by one-way ANOVA and Tukey's multiple comparison test.</p