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. 2013 Apr 18;7(4):e2174.
doi: 10.1371/journal.pntd.0002174. Print 2013.

Development of novel prime-boost strategies based on a tri-gene fusion recombinant L. tarentolae vaccine against experimental murine visceral leishmaniasis

Affiliations

Development of novel prime-boost strategies based on a tri-gene fusion recombinant L. tarentolae vaccine against experimental murine visceral leishmaniasis

Noushin Saljoughian et al. PLoS Negl Trop Dis. .

Abstract

Visceral leishmaniasis (VL) is a vector-borne disease affecting humans and domestic animals that constitutes a serious public health problem in many countries. Although many antigens have been examined so far as protein- or DNA-based vaccines, none of them conferred complete long-term protection. The use of the lizard non-pathogenic to humans Leishmania (L.) tarentolae species as a live vaccine vector to deliver specific Leishmania antigens is a recent approach that needs to be explored further. In this study, we evaluated the effectiveness of live vaccination in protecting BALB/c mice against L. infantum infection using prime-boost regimens, namely Live/Live and DNA/Live. As a live vaccine, we used recombinant L. tarentolae expressing the L. donovani A2 antigen along with cysteine proteinases (CPA and CPB without its unusual C-terminal extension (CPB(-CTE))) as a tri-fusion gene. For DNA priming, the tri-fusion gene was encoded in pcDNA formulated with cationic solid lipid nanoparticles (cSLN) acting as an adjuvant. At different time points post-challenge, parasite burden and histopathological changes as well as humoral and cellular immune responses were assessed. Our results showed that immunization with both prime-boost A2-CPA-CPB(-CTE)-recombinant L. tarentolae protects BALB/c mice against L. infantum challenge. This protective immunity is associated with a Th1-type immune response due to high levels of IFN-γ production prior and after challenge and with lower levels of IL-10 production after challenge, leading to a significantly higher IFN-γ/IL-10 ratio compared to the control groups. Moreover, this immunization elicited high IgG1 and IgG2a humoral immune responses. Protection in mice was also correlated with a high nitric oxide production and low parasite burden. Altogether, these results indicate the promise of the A2-CPA-CPB(-CTE)-recombinant L. tarentolae as a safe live vaccine candidate against VL.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Expression of the A2-CPA-CPB-CTE-EGFP tri-fusion gene by L.tarentolae.
(A) Expression of EGFP by recombinant L. tarentolae-EGFP promastigotes (left) and L. tarentolae-A2-CPA-CPB-CTE-EGFP promastigotes (right) before and after glinting of fluorescence. (B) Percentage of the EGFP positive population in L. tarentolae transfected with either pLEXSY-EGFP (left) or pLEXSY-A2-CPA-CPB-CTE-EGFP (clone #5, right) as determined by flow cytometry. (C) Western blot analysis for evaluating expression of the A2-CPA-CPB-CTE-EGFP fusion protein. A 102.56 kDa band corresponding to the A2-CPA-CPB-CTE-EGFP protein was detected in the recombinant L. tar-A2-CPA-CPB-CTE-EGFP by western blotting using an anti-CPB antibody. No band was seen in lane 1 representing a negative control (L. tarentolae wild type).
Figure 2
Figure 2. Liver and splenic parasite burden in all groups following immunization and infectious challenge with L. infantum.
The parasite number in the liver (A, B) or spleen (D, E) was evaluated by Limiting Dilution Assay (LDA) at 2, 4, 8 and 12 weeks post-infection. The parasite burden in the liver was compared between groups G1 and G3, G4 (controls) (A) and between G2 and G3, G5 (controls) (B). Areas contained under the curves (AUC) obtained in A and B were compared between groups G1, G2 and their related controls groups G4 and G5, respectively (C). The parasite burden in spleen was compared between groups G1 and G3, G4 (controls) (D) and between G2 and G3, G5 (controls) (E). Areas contained under the curves (AUC) obtained in D and E were compared between groups G1, G2 and their related controls groups G4, G5, respectively (F). The number of two independent repeats is shown here as mean±S.E. of measures obtained from 4 mice of each group (see Materials and methods for more detail about the groups). G1 [vaccinated with DNA A2-CPA-CPB-CTE-cSLN (prime) and Live L. tarentolae-A2-CPA-CPB-CTE (boost)]; G2 [vaccinated with Live L. tarentolae-A2-CPA-CPB-CTE (prime) and Live L. tarentolae-A2-CPA-CPB-CTE (boost)]; G3 (control PBS); G4 [(DNA vector alone (prime) and Live L. tarentolae wild type (boost)], and G5 [Live L. tarentolae wild type (prime) and Live L. tarentolae wild type (boost)]. Only significant differences are shown in the graphs. The asterisk sign (*) indicates the significant difference between G1 and G2 with their respective controls G4 and G5, respectively and the plus sign (+) indicates the significant difference between G1 and G2 with G3 control at the indicated time points as determined by Student's test (p<0.05 denoted as *, p<0.01 denoted as **and p<0.001 denoted as ***).
Figure 3
Figure 3. Cytokine production by splenocytes in vaccinated and control mice.
IFN-γ (panel A), IL-10 (panel B), IFNγ/IL-10 ratio (panel C), IL-2 (panel D) and nitrite (NO2) (panel E) production after stimulation with rA2-rCPA-rCPB (column 1), F/T L. tarentolae A2-CPA-CPB-CTE-EGFP (column 2) and F/T L. infantum WT (column 3) as recall antigens. G1 to G5 groups are as indicated in Figure 2. Each bar represents a mean±S.E. in pg/ml (for all cytokines) or in μm for nitrite. The number of independent repeats was two and all tests were done in triplicates (number of mice per group/time point n = 3) and the results are pooled and shown as mean±S.E. of measures obtained from 6 mice in different groups. The asterisk indicates the significant difference between values at the indicated time points as determined by Student's test (p<0.05 denoted as *, p<0.01 denoted as **, p<0.001 denoted as *** and n.s. denoted as non significant).
Figure 4
Figure 4. Analysis of the specific humoral response induced in vaccinated and control mice before and after challenge.
Mice were bled after two vaccinations before challenge and 5 weeks after challenge. Sera were obtained from individual mice from each group and pooled (n = 15). G1 to G5 groups are as indicated in Figure 2. Before challenge sera were tested for anti rA2-rCPA-rCPB (A) and F/T L. tarentolae A2-CPA-CPB-CTE-EGFP (B) and after challenge sera were tested for anti F/T L. infantum (C) antibodies by an isotype-specific ELISA. The asterisk indicates the significant difference between values at the indicated time points as determined by Student's test (p<0.05 denoted as *, p<0.01 denoted as **, p<0.001 denoted as *** and n.s. denoted as non significant).
Figure 5
Figure 5. Liver and spleen histological sections stained with hematoxylin and eosin (H&E).
(A) Splenic architecture of normal (non-immunized) mice in comparison with groups G1 to G5 as indicated in Figure 2 at 4th week post-infection with 107 L. infantum stationary promastigotes. Disruption of the splenic architecture accompanied by lymphoid depletion could be seen in control mice infected with 107 L. infantum promastigotes after the first month. (B) Presence of parasites in the spleen tissue of non-vaccinated groups at 4th week after challenge with 107 L. infantum. (C) The liver granuloma reaction at 8 weeks following L. infantum challenge infection in all groups compared to the non-infected mice. (D) Mature granuloma assembly in control group 3, resulting in the attraction of lymphocytes and monocytes at 14th week after challenge The number of independent repeats was two and number of mice per group/time point n = 2.
Figure 6
Figure 6. Degree of inflammatory cell infiltration in liver parenchyma of all groups at 4 weeks after challenge.
G1 to G5 groups are as indicated in Figure 2. The number of independent repeats was two and all tests were done in duplicate (number of mice per group/time point n = 2) and the results are pooled and shown as mean±S.E. of measures obtained from 4 mice of each group. The asterisk indicates the significant difference between values at the indicated time points as determined by Student's test (p<0.05 denoted as *, p<0.01 denoted as **, p<0.001 denoted as *** and n.s. denoted as non significant).

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