Leishmaniasis is a vector-borne infection caused by Leishmania—an obligate intracellular protozoan parasite. The two morphologically distinct forms of this are the promastigote, which is passed on by female Phlebotomine sandflies, and the amastigote, which occurs in mammalian hosts. The several clinical forms of the disease can be grouped into three main clinical forms: visceral (VL), mucocutaneous (MCL), and cutaneous leishmaniasis (CL) (1). CL is a painless and chronic ulcer at the site of sandfly bites and is the most common clinical syndrome in many affected regions, especially in the Middle East, where it has been reported in two main forms: zoonotic CL (ZCL) caused by Leishmania major and anthroponotic CL (ACL) caused by Leishmania tropica and mixed infection with them, which is high there (2, 3). In 2022, WHO reported that 85% of the global CL incidence occurred in eight countries, Afghanistan, Iran, Iraq, Syria, Algeria, Brazil, Colombia, and Peru (4). New outbreaks in the Middle East in recent years have been linked to wars in Syria, Yemen, Turkey, and Iraq. Refugee migration from endemic to non-endemic areas and vice versa, poor hygiene, malnutrition, weak immune systems, poor housing, lack of resources, environmental conditions, climate change, poor urbanization management, use of agricultural lands for residential purposes, and changes in vector populations link to a substantial rise in CL prevalence, which are present circumstances in most of the Middle East (3, 5, 6) (Figure 1). Although the first line of treatment of leishmaniasis with pentavalent antimonials is affordable and generally available in many endemic countries in the Middle East, economic sanctions, war, and counterfeit drug markets make access to the standard treatment difficult. In addition, the efficacy of this type of treatment is variable due to drug resistance and induction of organ toxicity (2, 3).

Fortunately, the development of immunity to the parasite in infected individuals following rehabilitation has highlighted the role of vaccination in disease management (5). In addition, the partial understanding of the immunopathogenesis of leishmaniasis has motivated immunologists and researchers in the leishmaniasis field to investigate and develop the different types of vaccines required. In the early 20th century, controlled inoculation of live virulent L. major promastigotes was used to immunize people in hyperendemic regions, preventing parasite infection in up to 80% of people. However, leishmanization as effectively powerful to control CL had several disadvantages that led to its abandonment (except in Uzbekistan, where this method is still used), including permanent skin lesions, safety concerns about HIV transmission, limitations in immunosuppressed people, and issues with Good Manufacturing Practice (GMP) standards (7, 8). Given these challenges, vaccine development shifted to inactivated vaccines. Due to the simplicity and cost-effectiveness of the production process, inactivated vaccines have been developed in various formulations. They are considered safe human vaccines and have been used as an alternative medication for drug-resistant type CL (9). Inactivation of the parasite while preserving the antigenic structures has been achieved by physical methods such as heat, chemicals, sonication, or UV radiation. This category has been studied in many clinical trials, but none of them have been approved by the World Health Organization (WHO) due to the lack of remarkable efficacy and the need for multiple vaccine doses (7, 8).

In addition to extended vaccines based on whole organism components, purified immunogenic fragments of the parasite have been developed as vaccine candidates, reducing the possibility of adverse reactions. Leishmune^®–a commercial canine vaccine consisting of fucose–mannose ligand (FML) from Leishmania donovani adjuvanted with QuilA saponin shows moderate clinical signs and lesions in vaccinated/infected dogs (8, 10). Remarkable advances in molecular biology have led to a new generation emergence of subunit or synthetic leishmaniasis vaccines based on membrane or soluble parasite proteins, replacing the previous native form vaccines. Cost-effectiveness and a straightforward manufacturing process allow their large-scale production. There is no live pathogen and no risk of infection in immunosuppressed individuals. With all these advantages, there are also some disadvantages, including an attempt to escape immune system deactivation and increased immunogenicity. Variations in the final conformation and structure of peptides occur due to heterologous expression systems, which could almost be related to post-translational modifications. Also, the epitopes could be selected to induce the desired immune response, and a particular antigenic arrangement could be chosen to induce a milder immune response (8, 11).

The Leish-111f vaccine is a tandem combination of three highly conserved Leishmania antigens, thiol-specific antioxidant (TSA), L. major stress-inducible protein 1 (LmSTI1), and Leishmania elongation initiation factor (LeIF), resulting in a 111 kDa polyprotein. In addition, studies indicated that Leish-111f formulated in IL-12 induces antibody response and IFN-γ production as well as soluble Leishmania antigen (SLA), but MPL-SE is considered a suitable alternative due to problems related to the manufacturing process and uncertainty of safety (12). Leish-111f is the first leishmaniasis vaccine to demonstrate immunogenicity in human clinical trials (12). In human clinical trials, Leish-111f is the first leishmaniasis vaccine that has demonstrated immunogenicity. A total of 77 healthy Indian subjects, with or without previous exposure to Leishmania, were administered three doses of Leish-F1+MPL-SE and followed for 168 days. Results showed safe and mild reactions associated with an increase in Th1-type cytokines (13). Purified peptides from different hosts administered with CpG adjuvant in BALB/c mice and eukaryotically expressed vaccine resulted in greater immune protection than the prokaryotic vaccine due to critical modifications that occur during protein construction in L. tarentolae, such as glycosylation, which involves the attachment of carbohydrate molecules to the N- or C-terminus of proteins, responsible for efficient peptide folding and interaction. Moreover, many studies have shown that glycosylation improves the immunogenicity and duration of conjugated vaccines compared to non-glycosylated vaccines (14, 15). Recently, significant advances in gene editing tools and Leishmania genome manipulation and generation of mutant weakened parasites have been explored as a desirable means of disease management. In this paper, we have reviewed the development of genetically live attenuated Leishmania vaccines.

Following the entry of Leishmania promastigotes into the host's dermal layer via the sandfly bite, the parasites reside in phagocytic cells such as tissue macrophages and dendritic cells or neutrophils. Leishmania GP63 directly uses complement C3 cleavage to prevent complement-mediated lysis, allowing C3bi to interact with the phagocytic cell receptor CR3 for facilitating attachment and uptake. Activated dendritic cells migrate from sites of antigen acquisition to draining lymph nodes and present Leishmania antigens to naive T cells, accompanied by the production of cytokines leading to CD4+ and CD8+ activation. The future fate of the parasite depends on the polarization and the final phenotype of the macrophages. The differentiation of macrophages into pro-inflammatory (M1) or anti-inflammatory (M2) phenotypes, known as macrophage polarization, plays a critical role in the immune response to leishmaniasis. Resistance to Leishmania infection is associated with the M1 phenotype, whereas the M2 phenotype dominates in susceptible environments. The balance between M1 and M2 macrophage polarization can be regulated by cytokines produced by CD4+ Th1 and Th2 lymphocyte subpopulations. The M1 macrophage polarization is mainly due to LPS, IFN, TNF, and GM-CSF, which also activates the complement system and recruits the immune cells. The polarization of macrophages into the M2 subset by the Leishmania parasites under secretion of Th2 cytokines and reduction of dendritic cells results in a decrease in antigen presentation and an immunosuppressive environment that supports their survival (16–18). Toll-like receptors (TLRs) play a key role in enhancing the immune response in the context of cutaneous vaccination by identifying pathogens. Some TLR ligands, such as prokaryotic CpG oligodeoxynucleotide (ODN) motifs, are considered effective adjuvants identified by TLR9. CpG ODNs induce the production of pro-inflammatory cytokines, including IL-12 and IFN-γ, which promote the development of a Th1 immune response (19). In a case-control study, gene expression measurement of IL-12 P40, IFN-γ, IL-1β, IL-4, and IL-10 from peripheral blood mononuclear cells (PBMC) of patients with anthroponotic cutaneous leishmaniasis (ACL) who responded and those who did not respond to meglumine antimoniate treatment showed a significant increase in Th1 cytokines (IL-12 P40, IFN-γ, and IL-1β) in the responsive group and Th2 cytokines (IL-4 and IL-10) in the non-responsive group (20). It has also been reported that the CD4+ T-cell response weakens in people with symptomatic visceral leishmaniasis but could return along with central memory T-cells that induce immunity after medication (21).

Attenuated vaccines could be produced by limiting the pathogenicity of the parasite through some techniques (Table 1). Weakened pathogens as whole-organism vaccines could present a set of antigens to the immune system, limiting the effect of antigenic polymorphism and genetic variation (22). It could also simulate actual infection and potentially activate the Th1 immune response. But sometimes, depending on the attenuation method, important immunogenic epitopes cannot be generated. This is a major drawback that limits the use of attenuated vaccines in immunosuppressive conditions such as HIV infection, organ transplantation, chemotherapy, or pregnancy. Strategies used to attenuate parasites based on defined and undefined genetic alterations include chemical, physical, and genetic attenuation (Table 1).

Physical methods include techniques such as prolonged subculture, use of radiation (gamma rays or UV), and temperature sensitivity. Treatment with mutagenic agents or promastigote culture under antibiotic pressure is considered chemical attenuation. The gentamicin-attenuated L. major vaccine is now in clinical trials and has shown promising results in mice and humans.

On the other hand, it also defined modifications that lead to the knocking out of genes responsible for pathogenicity. Today, this approach could be a suitable alternative that reduces the potential for reversibility (23–25). In addition, unlike the old method of leishmanization, mutant parasites altered using precise gene manipulation tools led to the appearance of an improved leishmanization in terms of non-pathogenicity and protection against all divergent Leishmania species (26).

The development of genetically modified live attenuated L. major Centrin-deleted parasites as a second method of leishmanization could induce protection via the action of IFN-γ-secreting Ly6+CD4+ T effector cells and multifunctional T cells that secrete cytokines such as IFN-γ, which is necessary for their production and survival. The LmCen–/– vaccine could also generate CD4+ skin tissue-resident memory (TRM) T cells that proliferate at the site of infection and secrete more IFN-γ and granzyme B in immunized animal models (46, 48). Central memory T cells (TCM) and skin TRM have been characterized as Leishmania-independent memory T cells (Figure 3). TRM cells are particularly suitable for protection, probably due to their localization and recruitment following vaccination or Leishmania infection. Following the parasite challenge, TRM cells immediately begin to reduce parasite loads, and it has been suggested that development strategies involving these cells will be helpful in pursuit of a leishmaniasis vaccine (84).

In addition, Greta Volpedo et al. reported that immunization with Centrin-deficient L. mexicana also results in higher levels of IL-12 and generation of central memory T cells (CD4+CD44+CD62L+) and significantly higher Th1 immune responses in the skin and lymph nodes of BALB/c mice compared to non-immunized mice. Overall, the ratio of IFN-γ/IL-10 to IFN-γ/IL-4 represents the physiological balance between Th1 and Th2 responses that determines disease outcome and can make the difference between resistance and susceptibility. However, when compared to the New World Leishmania strains that cause cutaneous disease, L. major exhibits different immunological characteristics and pathologies. Analysis of metabolic responses in immune cells following immunization with LmexCen–/– revealed increased aspartate metabolism and pentose phosphate pathway (PPP), which induce M1 polarization in macrophages, and PPP also promotes nitric oxide production. In addition, increased taurine/hypotaurine metabolism at the site of infection and linoleic acid in lymph nodes could motivate macrophage and T-cell activation against the parasite. In addition, arachidonic acid (AA)—an endocannabinoid metabolite with significant anti-inflammatory properties—showed an escalation in the course of infection in vivo. In general, the discovery of metabolic and immunological interactions following Leishmania vaccination could improve the development of innovative strategies in vaccine formulation (67). Given the endemicity of CL, a vaccine that prevents severe disease could have a significant impact on public opinion. However, a live attenuated vaccine that could also block parasite infection and thus prevent both cutaneous manifestations would have a much greater impact by reducing community transmission and potentially establishing herd immunity. Advances in molecular parasitology, creating deleterious gene mutations, altering replication fidelity, optimizing codons, and exerting control through genetic engineering tools, particularly the CRISPR/Cas9 system, which offers new ways to control L. major infection and replication, are renewing interest in a new generation of live attenuated vaccines, although potentially safer and more broadly applicable live vaccines require further testing before further advancing to human trials.

The spectrum of leishmaniasis varies due to host genetics and situation, parasite strain, and climate change. However, enough studies have shown that different forms of leishmaniasis can be prevented by vaccination. Unfortunately, there is currently no vaccine approved for human immunization on the global market. The development of an effective vaccine depends on its profitability for key stakeholders, vaccine developers, and manufacturers. Vaccine production requires a high level of trust in the public interest. Of course, government support attention to public health problems and international reflection are considered effective. Great advances have been made in the field of biological technologies to expand the range of vaccines. Recombinant multi-peptide adjuvanted vaccines such as Leish-F1 + MPL-SE and adenovirus-based DNA vaccines such as ChAd63-KH are now available. The priority of live attenuated Leishmania vaccines is considered to be a strong technique for the control of leishmaniasis, which has gained great attention due to the improvement of genetic engineering technologies such as the CRISPR/Cas system. The evaluation of gene candidates in terms of efficacy and immune response against the wild parasite has shown that Centrin1 is the most encouraging and is recognized as a good option for genetically live attenuated Leishmania vaccines. As we know, all the in vivo studies have been performed in animal models, which represent the early stages of the development of genetically attenuated vaccines and have not yet reached human clinical trials. In general, confirmation of logical guidelines related to live attenuated Leishmania development could administer a fine direction to major studies before handling human clinical trials and seriously reorganize the timeline of vaccine candidates.

ZR: Investigation, Visualization, Writing – original draft. HD-M: Conceptualization, Investigation, Project administration, Supervision, Validation, Visualization, Writing – review & editing. ME: Conceptualization, Investigation, Writing – review & editing. HK: Funding acquisition, Project administration, Resources, Supervision, Writing – review & editing.