ABSTRACT

Objective

Selecting an effective target for Leishmania vaccination requires identifying a protein antigen with low or no genetic diversity. The cysteine proteinase B (CPB) gene has emerged as a promising immunogenic target, though its diversity requires evaluation across different regions. Given Iran’s status as a hyperendemic region for cutaneous leishmaniasis, this study aimed to collect and analyze 30 Leishmania major isolates from various areas of Khuzestan Province in Southwestern Iran.

Methods

The CPB gene was amplified via polymerase chain reaction, and its nucleotide sequence was determined. Sequence analysis was performed using MEGA5 software, with subsequent comparison to National Center for Biotechnology Information database entries. A phylogenetic tree was constructed to compare isolated strains with reference strains from other geographic regions and species.

Results

The phylogenetic analysis demonstrated that the CPB gene of the isolated strains in different regions of Khuzestan province formed a clade with certain Leishmania major strains from various parts of the world. Overall, the genotypic analysis of the CPB gene in Khuzestan province indicated genetic similarity among 29 isolates. In contrast, one sample from Dezful (north of Khuzestan Province) exhibited a significant difference from the reference strain, resulting in notable amino acid changes.

Conclusion

Based on these findings, the CPB gene holds promise as a potential candidate for vaccination development against cutaneous leishmaniasis in this region.

INTRODUCTION

Leishmania major, the causative agent of cutaneous leishmaniasis, places nearly 10% of the global population at risk of infection. Annually, approximately two million new leishmaniasis cases are reported worldwide (1), with Iran representing a key endemic region where the disease constitutes a significant public health burden. The World Health Organization has prioritized leishmaniasis as an emerging and uncontrolled disease, prompting extensive efforts to develop effective vaccines, including the exploration of numerous candidate proteins (2).

Among these, cysteine proteinase B (CPB) stands out due to its critical role in parasite immune evasion (3). This 36-kDa protein, encoded by a 12,939-nucleotide gene on chromosome 8, is expressed in both promastigote (flagellated) and amastigote (non-flagellated) life cycle stages of Leishmania. CPB facilitates disease pathogenesis and serves as a key antigenic target. Phylogenetically, three cysteine proteinase types A, B, and C have been identified in Leishmania, with CPA and CPB homologous to mammalian Cathepsin-B, and CPC resembling Cathepsin-L (4).

Notably, CPB exhibits marked interspecies variability. For instance, Leishmania mexicana harbors 19 tandemly repeated CPB gene copies, while Leishmania donovani and Leishmania infantum possess five and one copies, respectively (5). Functional studies reveal that CPB deficiency in L. mexicana disrupts glycoprotein 63 (GP63) expression and impairs phagolysosomal activity (6). Furthermore, Rafati et al. (6) demonstrated that natural CPB isoforms in Leishmania major (L. major) elicit immune responses in human lymphocytes, infected murine models, and cutaneous leishmaniasis patients. Genetic polymorphisms in CPB contribute to phenotypic and genotypic diversity across Leishmania strains, underscoring its evolutionary plasticity (7).

Despite these advances, no provincial-level analysis of CPB genetic diversity has been conducted in Khuzestan, a hyperendemic tropical region in Iran. This study addresses this gap by performing molecular and genotypic analyses of CPB in L. major isolates from diverse geographic locations within the province. Identifying structural homogeneity in CPB across isolates would support its candidacy for region-specific vaccine development, offering a targeted strategy to mitigate cutaneous leishmaniasis in this high-risk population.

METHODS

Ethical Approval and Sample Collection

This study received ethical clearance from the Medical Ethics Committee (IR.TUMS.MEDICINE.REC.1400.014). Thirty L. major isolates were obtained from cutaneous leishmaniasis patients across five geographic subdivisions of Khuzestan Province (north, south, east, west, and central; six samples per region). Promastigotes were cultured in biphasic NNN medium supplemented with RPMI 1640 until reaching stationary phase, followed by cryopreservation at -20 °C. Genomic DNA extraction was performed on 3×10⁶ promastigotes using the Qiagen DNA Mini Kit (catalog no. 51304, South Korea), with PBS-washed parasites processed according to manufacturer protocols. DNA concentration was quantified spectrophotometrically at 260 nm.

Primary Polymerase Chain Reaction (PCR)

Primary PCR amplification targeting kinetoplast DNA minicircles (kDNA) was conducted to differentiate L. major from L. tropica, utilizing a Sinaclone PCR Kit (Shiraz, Iran). The species-specific 620-bp amplicon was generated using primers:

Forward: 5’-GGGGTTGGTGTAAAATAGGG-3’

Reverse: 5’-TTTGAACGGGATTTCTG-3’

Reactions (25 µL total volume) contained 12.5 µL master mix, 2.5 µL primer pair (10 pmol/µL), 5 µL template DNA, and 5 µL DNase-free water. Thermocycling parameters included: Initial denaturation: 95 °C for 3 min

35 cycles of:

Denaturation: 93 °C for 40 s

Annealing: 63 °C for 40 s

Extension: 72 °C for 1 min

Final extension: 72 °C for 5 min

Amplification products were resolved on 2% agarose gels stained with ethidium bromide (0.5 µg/mL) and visualized under ultraviole illumination.

CPB Proliferation

CPB gene was used to design primers amplification 1100 bp nucleotide band. Primers included:

Forward: 5’-ACGGTCTTAGCGTGCGAGTTGTG-3’

Reverse: 5’-TCGTGCAGCACATGTCGCTTG-3’

PCR reactions (25 µL) comprised 12.5 µL master mix, 1 µL each primer (10 pmol/µL), 5 µL DNA template, and 5.5 µL DNase-free water. Thermal cycling conditions were:

5 minutes at 95 °C for initial denaturation and 35 cycles including 94 °C for 1 min, 57 °C 30 seconds for annealing, 72 °C 1 minute for extension. The PCR process was ended at 72 °C 10 min for final extension. The electrophoresis of PCR products was performed using gel agarose 2% and ethidium bromide, and the bands appeared by gel documentation apparatus.

Phylogeny Analysis

Sequence alignments and phylogenetic trees were constructed using MEGA5 software. Evolutionary relationships were inferred via the Neighbor-joining algorithm with Kimura 2-parameter distance modeling. Topological robustness was evaluated through 1,000 bootstrap replicates, with clade stability thresholds set at ≥70%. Nucleotide substitution patterns were analyzed under a gamma-distributed rate variation model (shape parameter α=0.5).

Statistical Analysis

Statistical analysis was performed using SPSS software, version 22 (SPCC Inc., Chicago, IL, USA). Descriptive analysis was used to calculate the mean and standard deviation and parametric tests (ANOVA) and non-parametric tests (Mann-Whitney and Wilcoxon) were used. Significant differences were considered at a p-value of less than 0.05.

Demographic data

The study cohort comprised 30 cutaneous leishmaniasis patients, with a male-to-female ratio of 12:18 (40% vs. 60%). Age distribution revealed a predominance of cases in the 21-30-year demographic, though infections spanned ages 2 to 80 years. Cutaneous lesions localized predominantly to the hands (21/30 cases, 70%), followed by facial involvement (9/30, 30%). Four patients exhibited lesions at multiple anatomical sites, and 50% of cases presented with more than one lesion.

Primary PCR

Primary PCR amplification targeting kDNA minicircles confirmed L. major as the sole causative agent across all isolates, evidenced by a species-specific 620-bp amplicon (Figure 1). Electrophoretic differentiation from L. tropica, which produces an 800-bp fragment under identical conditions, excluded coinfection or misclassification. Subsequent amplification of the CPB gene yielded a conserved ~1,170 bp product in all isolates (Figure 2), confirming target gene integrity across geographically distinct L. major strains from Khuzestan Province.

Sequencing Analysis

Sequencing of 10 representative isolates (GenBank accessions OM037016-OM037025) revealed minimal nucleotide divergence from the L. major Friedlin reference strain, with one notable exception: Isolate OM037023 exhibited 14 nonsynonymous substitutions, resulting in substantial amino acid alterations within the CPB catalytic domain (Figures 3, 4). Clinically, infections involving OM037023 correlated with atypical presentations, including larger lesion sizes (>5 cm diameter), multidermatomal spread, and reduced responsiveness to antimonial therapy. The clinical changes in this study may be due to the presence of Leishmanis RNA virus 2 (LRV2). Çulha et al. (8) in Hatay-Türkiye failed to show LRV2 in 20 isolates of Leishmania included 17 L. tropica, 2 L. major and 1 L. infantum. Mirabedini et al. (9) in Iran, out of four isolates of L. major, three samples were positive for LRV2. The changes mentioned are described in Table 1.

Using the neighbour-joining method, the phylogenetic analysis of the gene encoding CPB DNA sequence was performed in L. major, along with the rest strains available in Genebank. The sequence patterns of the CPB gene in L. major isolated from patients in this study were compared to those of species in GenBank, demonstrating that all isolates, except for isolate number 8, exhibit a high degree of similarity with all isolates and standard Friedlin strain (Figure 5).

DISCUSSION

CPB constitutes a critical virulence factor in Leishmania spp., mediating parasite survival through differentiation roles in host cell invasion, immune evasion, and nutrient acquisition (10-12). Experimental validation of CPB’s pathogenicity was demonstrated by Denise et al. (13), who observed attenuated cutaneous lesion progression in BALB/c mice infected with CPB-deficient L. mexicana mutants. This phenotype correlated with enhanced Th1-polarized immunity (e.g., elevated IFN-γ), whereas CPB gene complementation restored lesion expansion and parasite proliferation via Th2 axis activation, evidenced by upregulated interleukin (IL)-4, immunoglobulin (Ig)G1, and IgE titers (13, 14). Such immunological modulation underscores CPB’s dual role as both a virulence effector and immunomodulator, motivating ongoing efforts to develop CPB-targeted chemotherapeutics and subunit vaccines (15, 16).

Based on this, much attention has been paid to this enzyme for therapeutic purposes as well as vaccine production. Denise et al. (13) demonstrated that in Balb/C mice infected with Mexican L. mexicana strains with CPB gene disruption, disease severity was reduced, and induction of protective TH1 response led to weak expansion of cutaneous lesions. Reintroducing the CPB gene into the mutant strains increased lesion growth and parasite proliferation and led to TH2 axis activity, as evidenced by increased levels of IL4, IgG1, and IgE (13).

Prophylactic strategies leveraging CPB have shown promise in preclinical models. Doroud et al. (17) reported sustained protective immunity in BALB/c mice immunized with a CPB DNA vaccine adjuvanted with nanoparticles, manifesting as reduced lesion sizes and robust Th1 responses (IFN-γ/IL-5 ratio >3.2) compared to controls.

The existence of homogeneity with low diversity in the CPB gene in Leishmania species gives hope that the production of an immunogenic vaccine against the CPB gene can be used against all Leishmania species. Still, due to the molecular diversity of the CPB enzyme in different Leishmania species, it is hindered for this purpose. Gomes et al. (18) showed that the supply of CPB gene is different in various developmental stages of the parasite and identified stage-specific CPB expression dynamics, with promastigotes producing 1.5-fold higher protein levels than amastigotes, modulated by environmental pH and temperature fluctuation.

Saadati et al. (19) were able to isolate L. donovani from 3477 mosquitoes. Examining the CPB gene in this parasite species showed the difference between the genotypes of this gene in L. donovani and other species of Leishmania; therefore, they concluded that one type of vaccine could not be used to protect this parasite from other species. Mohammadpour et al. (20) in Fars province examined the CPB gene in 100 clinical samples of Leishmania cutaneous and showed many differences in the CPB gene sequence in clinical samples. Their results are different from the present study, which showed that only one of the 10 sequenced samples had genotypic and clinical differences with other isolates, proving the importance of making vaccines based on local species. The Chaouch et al. (5) study showed that the sequences of L. tropica and L. major have the least diversity, and intraspecies nucleotide polymorphisms largely lead to silent mutations, especially in the case of L. major. However, in L. donovani, there are many differences as a result of the deletion of a 39 nucleotide segment in the cysteine proteinase genotype (5).

In the present study, out of the ten samples examined in the nucleotide sequence, nine isolates were similar to the Friedline standard strain, except in a few areas that led to changes in the nucleotide sequence, and no differences were observed, and these changes did not lead to amino acid changes. Therefore, there was no change in the parasite’s behaviour. Only in sample no. OM037023, significant nucleotide sequence alterations were identified when compared to the Friedline reference strain and other samples isolates. These genetic modifications induced substantial amino acid substitutions, resulting in altered parasite pathogenicity manifested through increased lesion severity, prolonged disease duration, and reduced therapeutic efficacy. This divergence parallels reports of LRV2-associated hyperpathogenicity, though LRV2 detection protocols were not explicitly performed here. The observed genetic homologous in the CPB gene of L. major highlights its potential utility as a molecular target for region-specific vaccine development.

However, the outlier OM037023 underscores the necessity for pan-regional genomic surveillance to capture emergent virulent strains. Future studies should integrate proteomic profiling to quantify CPB expression variability across parasite life stages and assess cross-reactivity of CPB-directed antibodies against heterologous Leishmania species.

CONCLUSION

Vaccination in Leishmaniasis is the important and safty method for controlling the disease in endemic areas. To select of appropriated target for candidate vaccine need to identification and characterization of immunogenic and homologous gene or proteines. This study presented that CPB gene and protein are suitable target for designing an effective vaccine in this region. Further study is needed for evaluation LRV2 detection gene in isolated samples and increasing sample size for reaching to confidental results.