Investigating the Existence of Ribosomal Protein L5 Gene in Syrian Strain of Leishmania tropica Genome: Sequencing It and Evaluating Its Immune Response as DNA Vaccine

Abstract

Cutaneous leishmaniasis in Syria is caused mainly by Leishmania tropica. It represents a serious health problem, which has aggravated further after the civil war in the country. Until now, there are no effective protective strategies, safe therapy, or efficacious vaccine to protect from this infection. DNA vaccines represent a promising approach for achieving protection against leishmaniasis. The L5 ribosomal protein plays fundamental roles in the assembly process of the ribosome subunits, so this study has chosen the ribosomal protein L5 gene to design a DNA vaccine against Leishmania tropica infection. After proving the existence of the ribosomal protein L5 gene in a Syrian strain of Leishmania tropica (LCED Syrian 01), it was sequenced and cloned into a pCI plasmid, and the designed DNA vaccine was administered to BALB/c mice. The protective response was evaluated by measuring lesion development in immunized BALB/c mice for 6 weeks after challenging mice with the parasite. RT-qPCR was used to quantify IL-12, IFN-γ, and IL-4 in draining lymph nodes (DLNs) of immunized mice. In the final week, the parasite burden was determined in footpad lesions and local draining lymph nodes (DLNs). This study demonstrated the presence and expression of the ribosomal protein L5 gene in the Syrian strain of Leishmania tropica promastigotes. The sequence of the ribosomal protein cDNA L5 gene was determined and published in Genbank. The gene size was 918 bp. Expression was also demonstrated at the level of cDNA. This study also demonstrated that vaccination with the ribosomal protein L5 gene induces TH1 response in immunized mice. This response prevents the partial development of a skin lesion of Leishmania.

Figure 1

(a) Electrophoresis of extracted L. tropica genomic DNA. Lane 1: DNA ladder 1 kb. Lanes 2 and 3: extracted L. tropica genomic DNA. (b) Electrophoresis of extracted L. tropica total RNA.

Figure 2

Amplification products of L. tropica ribosomal protein L5 on 1% agarose gel electrophoresis stained with ethidium bromide. Lanes 1 and 3: DNA ladder 1 kb. Lane 2: ribosomal protein L5-DNA amplification product approximately 918 bp. Lane 4: ribosomal protein. L5-cDNA amplification product approximately 918 bp.

Figure 3

1% agarose gel electrophoresis stained with ethidium bromide of the extracted, double-digested plasmid. Lane 1: DNA ladder 1 kb. Lane 2: undigested plasmid band. Lanes 3 and 4: digested plasmid shows a band of the ribosomal protein L5 gene and a band of the plasmid.

Figure 4

Sequencing of the ribosomal protein L5 gene in the Syrian strain of L. tropica.

Figure 5

Footpad swelling in BALB/c mice immunized intramuscularly in the left posterior thigh muscle 3 times at 2-week intervals, with 100 μg of empty plasmid as the control group pCI and 100 μg of pCI-L5 vaccine as the study group pCI-L5. After challenge in the right footpad with 10⁶Leishmania tropica promastigotes 2 weeks after the last immunization. As geometric mean ± SD. ^∗P < 0.05, significant increase in footpad swelling in the vaccinated group compared to the control group.

Figure 6

Log 10 of parasite burden in the site of inoculation and lymph nodes six week after the challenge. Parasite load was determined by limiting dilution after S.C. inoculation of 10⁶Leishmania tropica promastigotes into the right footpad. As geometric mean ± SD. ^∗P < 0.05, significant decrease in the site of inoculation and lymph nodes parasite burden in the vaccinated group compared to the control group.

Figure 7

(a) IFN-γ, (b) IL-12, and (c) IL-4 gene expression in the draining lymph nodes obtained from BALB/c mice inoculated with 100 μg pCI-L5 vaccine. The messenger RNA (mRNA) for IFN-γ, IL-12, and IL-4 gene were determined by RT-qPCR. As geometric mean ± SD (three DLNs). The relative quantification was performed by the comparative Ct method (∆∆Ct), using the lymph node from the control group as a calibrator (fold change = 1). ^∗P < 0.05, significant decrease or increase in cytokine gene expression in the vaccinated group draining lymph nodes compared to the control group.

Figure 8

IFN-γ/IL-4 ratio in draining lymph nodes (DLNs) obtained from BALB/c mice inoculated with 100 μg pCI-L5 vaccine before and after challenge with L. tropica. The IFN-γ/IL-4 ratio in response to pCI-L5 and pCI is compared (^∗P < 0.05).