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

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

Vaccination with Liposomal Leishmanial Antigens Adjuvanted with Monophosphoryl Lipid−Trehalose Dicorynomycolate (MPL-TDM)Confers Long-Term Protection against Visceral Leishmaniasis through a Human Administrable Route

The development of a long-term protective subunit vaccine against visceral leishmaniasis depends on antigens and adjuvants that can induce an appropriate immune response. The immunization of leishmanial antigens alone shows limited efficacy in the absence of an appropriate adjuvant. Earlier we demonstrated sustained protection against Leishmania donovani with leishmanial antigens entrapped in cationic liposomes through an intraperitoneal route. However, this route is not applicable for human administration. Herein, we therefore evaluated the immune response and protection induced by liposomal soluble leishmanial antigen (SLA) formulated with monophosphoryl lipid−trehalose dicorynomycolate (MPL-TDM) through a subcutaneous route. Subcutaneous immunization of BALB/c mice with SLA entrapped in liposomes or with MPL-TDM elicited partial protection against experimental visceral leishmaniasis. In contrast, liposomal SLA adjuvanted with MPL-TDM induced significantly higher levels of protection in liver and spleen in BALB/c mice challenged 10 days post-vaccination. Protection conferred by this formulation was sustained up to 12 weeks of immunization, and infection was controlled for at least 4 months of the challenge, similar to liposomal SLA immunization administered intraperitoneally. An analysis of cellular immune responses of liposomal SLA + MPL-TDM immunized mice demonstrated the induction of IFN-γ and IgG2a antibody production not only 10 days or 12 weeks post-vaccination but also 4 months after the challenge infection and a down regulation of IL-4 production after infection. Moreover, long-term immunity elicited by this formulation was associated with IFN-γ production also by CD8+ T cells. Taken together, our results suggest that liposomal SLA + MPL-TDM represent a good vaccine formulation for the induction of durable protection against L. donovani through a human administrable route

Ag-specific IgG isotype responses in mice vaccinated with different vaccine regimens in long-term protected group (twelve weeks post boost) before and after 3 months <i>L. donovani</i> challenge infection.

<p>Twelve weeks after final immunization and 3 months after challenge infection, blood serum samples were collected and assayed for IgG2a, IgG1, and IgG2a:IgG1 by ELISA. 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. <i>p</i> values were calculated using one-way ANOVA and Tukey's multiple comparison test.</p>a<p>Significantly higher than gp63 DNA either free or in presence of CpG (<i>p</i><0.001).</p>b<p>Significantly higher than gp63 DNA either alone or in association with CpG, and rgp63 plus CpG (<i>p</i><0.001).</p>c<p>Significantly higher than DNA-prime/Protein-boost (<i>p</i><0.001).</p

IL-12, IL-4 and IL-10 responses in BALB/c mice vaccinated with different vaccine regimens 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 were collected from vaccinated mice, stimulated with rgp63 (5 µg/ml) and were cultured for 96 h. The supernatants were collected, and assayed for IL-12 (A–D), IL-4 (E–H), and IL-10 (I–L) through ELISA. 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

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

Ag-specific IgG isotype responses in mice vaccinated with different vaccine regimens in short-term protected group (ten days post boost) before and 3 months after <i>L. donovani</i> challenge infection.

<p>Ten days after final immunization and 3 months after challenge infection, blood serum samples were collected and assayed for IgG2a, IgG1, and IgG2a:IgG1 by ELISA. 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. <i>p</i> values were calculated using one-way ANOVA and Tukey's multiple comparison test.</p>a<p>Significantly higher than gp63 DNA either free or in presence of CpG (<i>p</i><0.001).</p>b<p>Significantly higher than DNA-prime/Protein-boost (<i>p</i><0.001).</p>c<p>Significantly higher than gp63 DNA (<i>p</i><0.05).</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