Leishmaniasis Vaccine Development: A Review of Current Candidates and Cross-Species Protection Potential

Abstract

Leishmaniases are infections caused by Leishmania parasites and transmitted through the bite of infected female Phlebotomus (Old World) and Lutzomyia (New World) sandflies. The disease disproportionately affects marginalized communities with limited healthcare access. With no approved human vaccines available, leishmaniasis treatment and prevention depend heavily on chemotherapeutics that face growing drug resistance challenges alongside toxicity concerns. The development of safe, effective and affordable vaccines against human leishmaniasis remains a global health priority for disease control and elimination, mostly in resource-limited settings. This review synthesizes progress in leishmaniasis vaccine platforms including live-attenuated parasites, whole-killed parasites, DNA, protein subunit, peptide-based and chimeric/multiepitope vaccines and their homogenous and heterogenous efficacy. Live-attenuated and whole-parasite vaccines have been accounted to elicit robust cellular immunity but pose safety risks, particularly in immunocompromised hosts. While both second- and third-generation vaccines exemplified by LEISH-F1/F3 polyproteins, elicit strong Th1-biased T cell responses in preclinical models, their efficacy in humans remains limited. However, the highlighted collective efforts are pivotal in steering the rational development of future research using various formulations for multiple management of leishmaniasis through cross-protection. Furthermore, emerging strategies including mRNA platforms, nanoparticle delivery, reverse vaccinology, and immunoinformatics offer promising avenues for accelerating vaccine discovery and advancing the development of novel and effective human vaccines.

1. Introduction

1.1. Background

Leishmaniasis is a vector-borne disease caused by flagellate protozoa of the genus Leishmania parasite (family Trypanosomatida). Leishmaniasis is a prioritized neglected tropical disease (NTD) that occurs in more than 98 countries worldwide, affecting more than 1 billion people in endemic areas. It is reported to affect populations in southern Europe, Asia, Africa, the Middle East, and Central and South America, with over 1 million new cases and 20,000–40,000 deaths reported each year [1,2]. With its diverse clinical manifestation and widespread distribution, linked with malnutrition, conflicts, population displacement, poor housing, weakened immunity and limited financial resources, leishmaniasis remains a challenging disease [3,4].

Leishmaniasis is transmitted by the bite of infected female sandflies of the genus Phlebotomus in the Old World, and of the genus Lutzomyia in the New World, with at least 93 sandfly species being proven or probable vectors worldwide [5,6]. Presently, there are 20 different Leishmania species that are described as pathogenic for humans (

Table 1. Leishmania species causing infections in humans: transmission cycles, clinical diseases and epidemiological characteristics of the main Leishmania species worldwide.

Subgenus	Species	Old/New World	Clinical Disease	Geographical Distribution	Main Reservoir	Vector
Leishmania	L. aethiopica	OW	LCL, DCL	East Africa (Ethiopia, Kenya)	Hyraxes (dassie)	Phlebotomus spp.
	L. amazonensis (syn L garnhami)	NW	LCL, DCL, MCL	South America (Brazil, Venezuela, Bolivia)	Rodents, possums	Lutzomyia spp.
	L. donovani (syn L archibaldi)	OW	VL, PKDL	Central Africa, South Asia, Middle East, India, China	Human	Phlebotomus spp.
	L. infantum	OW	CL	Mediterranean countries (North Africa and Europe), Southeast Europe, Middle East, Central Asia, North, Central and South America (Mexico, Venezuela, Brazil, Bolivia), China	Dog, hare	Lutzomyia spp.
	L. infantum chagasi	NW	VL	Central and South America	Dog, fox	Lutzomyia spp.
	L. major	OW	CL	North and Central Africa, Middle East, Central Asia	Rodents	Phlebotomus spp.
	L. mexicana (syn. L. pifanoi)	NW	LCL, DCL	USA, Ecuador, Venezuela, Peru	Rodents, marsupials	Lutzomyia spp.
	L. tropica	OW	LCL, VL	North and Central Africa, Middle East, Central Asia, India	Human	Phlebotomus spp.
	L. venezuelensis	NW	LCL	Northern South America, Venezuela	Human, cats	Lutzomyia spp.
	L. waltoni	NW	DCL	Dominican Republic	Rodents	Lutzomyia spp.
Viannia	L. braziliensis	NW	LCL, MCL	Western Amazon Basin, South America (Guatemala, Venezuela, Brazil, Bolivia, Peru)	Rodents, dogs	Lutzomyia spp.
	L. guyanensis	NW	LCL, MCL	Northern South America (French Guinea, Suriname, Brazil, Bolivia)	Possums, sloths, and anteaters	Lutzomyia spp.
	L. lainsoni	NW	LCL	Brazil, Bolivia, Peru	Rodents, sloths	Lutzomyia spp.
	L. lindenbergi	NW	LCL	Brazil	Rodents, sloths	Lutzomyia spp.
	L. naiffi	NW	LCL	Brazil, French Guinea	Armadillos	Lutzomyia spp.
	L. panamensis	NW	LCL, MCL	Central and South America (Panama, Columbia, Venezuela, Brazil)	Sloths	Lutzomyia spp.
	L. peruviana	NW	LCL, MCL	Peru, Bolivia	Humans, dogs. rodents	Lutzomyia spp.
	L. shawi	NW	LCL	Brazil	Sloths, nonhuman primates	Lutzomyia spp.
Mundinia	L. martiniquensis	NW, OW	LCL, VL	Martinique, Thailand	Human, rodents	Vectors unknown, possibly Sergentomyia spp. and Culicoides spp. (OW), Lutzomyia spp. (NW)
Sauroleishmania	L. tarentolae		Unknown	North Africa, southern Europe, Middle East	Lizards	Sergentomyia spp.

Abbreviations: NW—New World, OW—Old World, CL—cutaneous leishmaniasis, VL—visceral leishmaniasis, LCL—localized cutaneous leishmaniasis, MCL—mucocutaneous leishmaniasis, PKDL—post-kala-azar, spp.—species.

) [6,7]. Leishmaniasis is classified into several forms, depending on the disease manifestation due to the species of infecting parasite. However, the main three forms are visceral leishmaniasis (VL), cutaneous leishmaniasis (CL) and mucocutaneous leishmaniasis (MCL). The tegumentary forms of leishmaniasis contribute to around two-thirds of the global disease burden, whereas VL is responsible for most reported deaths globally. Traditionally, leishmaniasis has been divided into two groups, which are the Old World and New World leishmaniasis. The classification refers to the geographic region where the infection is acquired, whereby Old World leishmaniasis are found in Middle East, the Mediterranean basin and Africa, while New World leishmaniasis have been documented in Mexico and Central and South America.

In addition to traditional endemic patterns, contemporary drivers including human migration, armed conflict and climate change significantly reshape leishmaniasis epidemiology [8,9]. Mass displacement from conflict zones, such as Syria’s civil war, has fueled CL outbreaks among refugees in Turkey (2956 Syrian cases, 2013–2023, predominantly L. tropica) and neighboring Lebanon/Jordan, exacerbated by crowded camps and disrupted vector control [10,11]. Furthermore, climate change expands vector habitats; for example, in Morocco, warming scenarios predict L. major range extension into High Atlas and Rif regions (1.5–1.6% gain by 2050), while Phlebotomus papatasi proliferates in southern areas. In Europe, L. infantum transmission risks rise northward, with Greece’s Epirus showing 9.6% increased suitability by 2080 and Italy/France reporting canine expansions linked to pet travel [12]. The Mediterranean basin exemplifies these shifts, with L. infantum and L. tropica cases surging in Turkey (emerging VL foci) and Spain amid urbanization and sandfly altitudinal migration. These dynamics underscore the urgency of vaccines adaptable to evolving transmission landscapes.

While vaccines for canine leishmaniasis exist, no prophylactic vaccines are available for human leishmaniasis, despite extensive research efforts and testing of clinically prepared vaccines in various human trials (Table 2) [13]. Current control strategies for leishmaniasis rely primarily on chemotherapeutic interventions. However, these regimes have limited efficacy stemming from adverse side effects, toxicity, emerging drug resistance, unaffordable prices and suboptimal clinical outcomes. Moreover, development of a safe and effective vaccine remains elusive due to the parasite’s mechanisms to evade the host immune system. The current human vaccine candidates are still in experimental stages, primarily focused on enhancing immune responses to prevent infection or disease progression. The focus of control strategies continues to rely heavily on vector control, early diagnosis and treatment of active infections. This scoping review serves to underscore the importance of elucidating vaccine platforms, adjuvants, trials and cross-protection strategies for the prospect of effective leishmaniasis management and new treatments.

1.2. Lifecycle: Parasite and Vector

Leishmania is a wide genius featuring a complex life cycle with two main stages: the promastigote and the amastigote. The stages alternate depending on the host, promastigotes develop in the invertebrate vector (sandfly), while amastigotes reside in vertebrate hosts (human and other animals) (Figure 1). Although sandflies were long suspected as vectors for Leishmania transmission, it was not until 1921 that this was proven by suspension of ground sandflies causing Oriental sore [14]. In the Old World, Leishmania species are transmitted by sandflies of the Phlebotomus genus, while those in the New World are transmitted by the Lutzomyia genus vector (Figure 2). Transmission can follow anthroponotic (human to human) or zoonotic (animal to human) cycles depending on the region [15].

The cycle begins when a female sandfly takes a blood meal from an infected vertebrate host, ingesting host cells that contain amastigotes, which are the intracellular, non-motile form of the parasite and are mostly found in macrophages. Within the sandfly’s midgut, the amastigotes transform into procyclic promastigotes (Figure 1), the flagellated, motile forms of the parasite with a slender body measuring 15–20 μm in length and 1.5–3.5 μm in width. In the sandfly’s midgut, procyclic promastigotes divide and develop into non-dividing nectomonad promastigotes, which migrate to the anterior midgut and transform into leptomonad promastigotes. These then differentiate into infective metacyclic promastigotes, which migrate to the sandfly’s proboscis for transmission to a mammalian host during a sandfly bite, a process known as metacyclogenesis over 7–10 days [16,17,18].

The transferred metacyclic promastigotes in the host along with the vector’s saliva trigger a phagocytic process by adhering to the plasma membrane. The promastigotes are rapidly engulfed by the host’s immune cells, particularly macrophage, and infect the parasitophorous vacuole, where they differentiate into ovoid amastigotes with a size of about 2–4 μm in diameter [16,18]. Inside macrophages, amastigotes replicate via binary fission in the phagolysosome, eventually rupturing the host cell to release parasites that infect neighboring macrophages. This intracellular cycle of amastigote replication continues, leading to the development of symptoms based on the Leishmania species and host response: VL, CL or MCL [19]. The infected macrophages circulating in the host’s blood or skin will then serve as a source of infection for the vector [20]. When another sandfly bites the infected mammal, it ingests the amastigotes present in the host’s blood or tissues, which will lead to the starting of the cycle again. The amastigotes transform into promastigotes within the sandfly, and the life cycle of Leishmania is perpetuated.

Table 2. Commercial and clinical trial Leishmania vaccine along with the mechanism, classification and expected outcomes.

Vaccine Name	Mechanism	Classification	Clinical Trial Phase	Vaccine Antigen	Adjuvant	Target	Findings	Reference
Leishvaccine (L. amazonensis)	CD4+, CD8+, and B cell activation	First generation	III	Whole-killed promastigotes of L. amazonensis	BCG	Dogs	Innate immunity (especially neutrophils and eosinophils) and activated CD4+T, CD8+T, and B cells. Safe but no immune response in humans.	[21]
L. mexicana + BCG	T cell activation	First generation	III	Killed Leishmania	BCG	Humans	Low levels of Leishmania skin test (LST) conversion. Decrease in parasite incidence.	[22]
L. mexicana + L. amazonensis + BCG	T cell activation	First generation	–	Killed Leishmania	BCG	Humans	Cost- effective CL infection treatment. No side effects.	[23]
ALM + BCG	T cell activation	First generation	II	Killed Leishmania	BCG	Humans	Treatment of CL and VL, with decrease in VL incidence in LST-converted individuals.	[24]
Leishmune (L. donovani)	T cell activation	First generation	III	Fractioned vaccine (FML)	Saponin	Dogs	Long lasting and strong protection against VL in dogs.	[25,26,27]
CaniLeish (L. infantum)	Th1 induction	First generation	III	LiESP	Saponin	Dogs	Only approved vaccine in France with high protection against canine leishmaniasis. Block Leishmania transmission from dogs to human.	[28,29]
Gentamicin- attenuated L. infantum	T and B cell activation	First generation	–	Live-attenuated Leishmania	None	Dogs	Strong protective effect against CVL in dogs. Reduced the occurrence of VL in human population.	[30]
Gentamicin- attenuated L. major	Th1 induction	First generation	III	Live-attenuated Leishmania	None	Humans	Safe and protective against CL in human population	[31]
LEISH-F1	T cell activation	Second generation	I	TSA, LmSTI1, and LeIF	MPL-SE	Humans	Protection against VL in humans. The vaccine was safe and tolerable, inducing T cell production of IFN-γ and other cytokines in response to stimulation with the antigen.	[32]
LEISH-F2	T cell activation	Second generation	II	TSA, LmSTI1, and LeIF	MPL-SE	Humans	Protection against CL in humans when combined with the adjuvant.	[33]
LEISH-F3	T cell activation	Second generation	I	NH36 and SMT	MPL-SE and GLA-SE	Humans	Protection against VL in humans. Subjects vaccinated with Leish-F3 and GLA-SE had significant levels of antigen-specific IgG antibodies in their serum, along with IFN-γ, TNF, and IL-2 cytokines.	[34]
Leish-Tec	T cell activation	Second generation	III	L. donovani A2 protein	Saponin	Dogs	Protection against canine leishmaniasis. Vaccination of infected healthy animals significantly reduced clinical progression and decreased mortality.	[35]
LetiFend	Second generation	Second generation	III	L. infantum proteins (H2A, LiP2a, LiP2b, and LiP0)	None	Dogs	Overall efficacy in the prevention of confirmed cases of canine leishmaniasis in endemic areas with high disease pressure was shown to be 72%.	[36]
Chad63-KH	Cd8+ T cell activation	Third generation	II	KMP-11 and HASPB	None	Humans	Safe and protective against VL and PKDL in humans through innate and acquired immunity. It elicited a variety of CD8+ T cells specific to Leishmania antigens, IFN-γ and the activation of dendritic cells.	[37,38]

Abbreviations: BCG, Bacillus Calmette–Guérin; FML, fucose–mannose ligand; LiESP, L. infantum excreted–secreted protein; TSA, thiol-specific antioxidant; LmSTI1, L. major stress-inducible protein 1; LeIF, L. braziliensis elongation and initiation factor; MPL-SE, monophosphoryl lipid A; NH, nucleoside hydrolase; SMT, sterol 24-c-methyltransferase; GLA-SE, glucopyranosyl lipid A stable oil-in-water nano-emulsion; KMP-11, kinetoplastid membrane protein 11; HASPB, hydrophilic acylated surface protein B.

Table 2. Commercial and clinical trial Leishmania vaccine along with the mechanism, classification and expected outcomes.

Vaccine Name	Mechanism	Classification	Clinical Trial Phase	Vaccine Antigen	Adjuvant	Target	Findings	Reference
Leishvaccine (L. amazonensis)	CD4+, CD8+, and B cell activation	First generation	III	Whole-killed promastigotes of L. amazonensis	BCG	Dogs	Innate immunity (especially neutrophils and eosinophils) and activated CD4+T, CD8+T, and B cells. Safe but no immune response in humans.	[21]
L. mexicana + BCG	T cell activation	First generation	III	Killed Leishmania	BCG	Humans	Low levels of Leishmania skin test (LST) conversion. Decrease in parasite incidence.	[22]
L. mexicana + L. amazonensis + BCG	T cell activation	First generation	–	Killed Leishmania	BCG	Humans	Cost- effective CL infection treatment. No side effects.	[23]
ALM + BCG	T cell activation	First generation	II	Killed Leishmania	BCG	Humans	Treatment of CL and VL, with decrease in VL incidence in LST-converted individuals.	[24]
Leishmune (L. donovani)	T cell activation	First generation	III	Fractioned vaccine (FML)	Saponin	Dogs	Long lasting and strong protection against VL in dogs.	[25,26,27]
CaniLeish (L. infantum)	Th1 induction	First generation	III	LiESP	Saponin	Dogs	Only approved vaccine in France with high protection against canine leishmaniasis. Block Leishmania transmission from dogs to human.	[28,29]
Gentamicin- attenuated L. infantum	T and B cell activation	First generation	–	Live-attenuated Leishmania	None	Dogs	Strong protective effect against CVL in dogs. Reduced the occurrence of VL in human population.	[30]
Gentamicin- attenuated L. major	Th1 induction	First generation	III	Live-attenuated Leishmania	None	Humans	Safe and protective against CL in human population	[31]
LEISH-F1	T cell activation	Second generation	I	TSA, LmSTI1, and LeIF	MPL-SE	Humans	Protection against VL in humans. The vaccine was safe and tolerable, inducing T cell production of IFN-γ and other cytokines in response to stimulation with the antigen.	[32]
LEISH-F2	T cell activation	Second generation	II	TSA, LmSTI1, and LeIF	MPL-SE	Humans	Protection against CL in humans when combined with the adjuvant.	[33]
LEISH-F3	T cell activation	Second generation	I	NH36 and SMT	MPL-SE and GLA-SE	Humans	Protection against VL in humans. Subjects vaccinated with Leish-F3 and GLA-SE had significant levels of antigen-specific IgG antibodies in their serum, along with IFN-γ, TNF, and IL-2 cytokines.	[34]
Leish-Tec	T cell activation	Second generation	III	L. donovani A2 protein	Saponin	Dogs	Protection against canine leishmaniasis. Vaccination of infected healthy animals significantly reduced clinical progression and decreased mortality.	[35]
LetiFend	Second generation	Second generation	III	L. infantum proteins (H2A, LiP2a, LiP2b, and LiP0)	None	Dogs	Overall efficacy in the prevention of confirmed cases of canine leishmaniasis in endemic areas with high disease pressure was shown to be 72%.	[36]
Chad63-KH	Cd8+ T cell activation	Third generation	II	KMP-11 and HASPB	None	Humans	Safe and protective against VL and PKDL in humans through innate and acquired immunity. It elicited a variety of CD8+ T cells specific to Leishmania antigens, IFN-γ and the activation of dendritic cells.	[37,38]

Abbreviations: BCG, Bacillus Calmette–Guérin; FML, fucose–mannose ligand; LiESP, L. infantum excreted–secreted protein; TSA, thiol-specific antioxidant; LmSTI1, L. major stress-inducible protein 1; LeIF, L. braziliensis elongation and initiation factor; MPL-SE, monophosphoryl lipid A; NH, nucleoside hydrolase; SMT, sterol 24-c-methyltransferase; GLA-SE, glucopyranosyl lipid A stable oil-in-water nano-emulsion; KMP-11, kinetoplastid membrane protein 11; HASPB, hydrophilic acylated surface protein B.

1.3. Pathogenesis

Leishmaniasis pathogenesis has classically been described as a consequence of an imbalance between Th1 and Th2 CD4⁺ T helper cell responses, profoundly influencing disease progression and clinical manifestation [39]. A Th1-dominant immune response marked by secretion of interferon-γ (IFN-γ), tumor necrosis factor (TNF) and interleukin-12 (IL-12) promotes macrophage activation and nitric oxide (NO)-dependent elimination of intracellular parasites, thereby achieving robust parasite suppression and minimal parasitemia. However, excessive or dysregulated Th1 responses can drive tissue damage through heightened cellular inflammation, predisposing individuals to mucocutaneous leishmaniasis, where parasite numbers are low but immune-mediated destruction is extensive [39].

In contrast, Th2-skewed responses driven by IL-4, IL-5, IL-10 and IL-13 compromise macrophage effector activity and foster parasite persistence and dissemination. Antibody responses alone are insufficient for protection, reinforcing the requirement for robust cell-mediated immunity. Importantly, excessive or dysregulated Th1 responses can drive immune-mediated tissue damage, particularly in mucocutaneous disease, highlighting the need for vaccines that elicit balanced but durable Th1 immunity rather than uncontrolled inflammation. Increasing evidence further indicates that disease severity is not solely determined by Th1/Th2 polarization but also involves regulatory pathways, including IL-10-producing regulatory T cells, dysfunctional CD8⁺ T cell responses, and chronic immune activation that contribute to parasite persistence and tissue pathology.

2. Materials and Methods

2.1. Data Source and Search Strategy

This review was guided by the following questions: (1) what are the existing formulations of available vaccines for management of leishmaniasis? (2) what are the key advancements, challenges, and comparative efficacy of current vaccine strategies (for example leishmanization, DNA, protein subunit) for cross-protection against multiple Leishmania species? (3) what immunological barriers (e.g., antigenic diversity, weak CD8⁺ responses) hinder cross-protective vaccine development, and how can they be overcome? and (4) which future strategies (e.g., prime-boost approaches, advanced delivery systems, adjuvants) hold the most potential for achieving durable cross-protective immunity in leishmaniasis? A literature search was conducted using online databases including PubMed, Web of Science, SciELO and HINARI to identify relevant studies (

Table 3. The search strings as used in databases studied and the total number of articles included for screening.

Database	Search String	Total Results
Pubmed	(“cross protection”[MeSH Terms] OR (“cross”[All Fields] AND “protection”[All Fields]) OR “cross protection”[All Fields]) AND (“leishmaniasis”[MeSH Terms] OR “leishmaniasis”[All Fields] OR “leishmaniases”[All Fields] OR “leishmaniasis vaccines”[MeSH Terms] OR (“leishmaniasis”[All Fields] AND “vaccines”[All Fields]) OR “leishmaniasis vaccines”[All Fields])	107
Web of Science	ALL = (cross-protection and leishmaniasis)	121
SciELO	Leishmania AND (vaccine OR vacuna OR vacina) AND (“cross protection” OR “protección cruzada” OR “proteção cruzada”)	981
HINARI	((cross-protection) OR (cross immunity) OR (cross resistance)) AND ((leishmaniasis or Leishmania) OR (cross-protection and Leishmania) OR (cross immunity and leishmaniasis))	405
Google scholar	((cross-protection) OR (cross immunity) OR (cross resistance)) AND ((leishmaniasis or Leishmania) OR (cross-protection and Leishmania) OR (cross immunity and leishmaniasis))	40
Total		1654

). The choice of databases considered the research questions and the percentage of the relevant documents from the primary search results. Respective index terms and search queries were used to gather relevant studies. An additional search was conducted using Google Scholar, Google search engine and manual search from the reference list of the screened articles. Other sources were the World Health Organization (WHO) and the United States Centre for Disease Control and Prevention (CDC).

2.2. Studies Screening

Articles were downloaded from the search databases in RIS format, which were then imported to Rayyan for duplicate check (https://www.rayyan.ai/). The review involved two-stage screening, title/abstract screening and full-paper screening, which was performed by two independent reviewers (Clara Yona (CY) and Amit Kumar (AKD)). Further exclusion of the articles was performed during data collection process after full-text review (MM, EM and AL). Data related to cross-protection of Leishmania infections using various vaccines regimes were summarized using the thematic analysis method.

2.3. Inclusion and Exclusion

In this review, studies published from January 2000 to June 2024 were included based on the following specific criteria to ensure relevance and quality; articles only written in English and available as full-text publications were considered, while abstracts, conference proceedings and unpublished data were excluded unless they provided substantial and verifiable findings. The primary focus was on studies investigating the function ability of vaccine candidates from both in vitro and in vivo experiment cross-cutting both preclinical and clinical trials and potentiality of cross-protection between different Leishmania species, including those assessing immune responses, vaccine-induced protection, or previous infection-mediated immunity.

2.4. Data Extraction

Relevant data from the selected studies were extracted to ensure a comprehensive analysis of Leishmania cross-protection using a standardized data extraction form created in Microsoft Excel 2013 (Microsoft Corporation, Washington, DC, USA). Data extraction was independently performed by two reviewers (CY and AK) to ensure accuracy and minimize bias. Any discrepancies in data extraction were resolved through discussion or consultation with a third reviewer. Essential study details such as author names, publication year, study location, and study design were extracted. For experimental and/or immunization studies, information on the Leishmania species involved, duration of the study, vaccine type, adjuvants, administration route, duration of immunity and type of animal model used were collected. In studies assessing immune responses, details on cytokine profiles, T cell responses, antibody production, and other immunological markers indicative of cross-protection were extracted.

3. Leishmania Vaccines

3.1. First-Generation Vaccines

3.1.1. Live-Attenuated Parasites

Live-attenuated vaccines, also termed as first-generation vaccines involve the use of weakened parasites. This approach has been an appealing as they recapitulate natural infection, which potentially triggers a similar immune response but without the risks associated with exposure to fully virulent parasites [40,41]. Considering the development procedure, live-attenuated vaccines are categorized by development method into genetically defined or undefined parasites, while considering reproducibility, efficacy and safety as key criteria. Early “leishmanization” was documented in Israel, Russia and Uzbekistan, which is the sole country to use a licensed live and dead L. major vaccine for high-risk groups [41,42].

Leishmanization with wild types has been addressed with protection against both homogenous and heterogenous leishmaniasis in animal models. Leishmanization with L. major provided robust protection against visceral infection with L. infantum in C57BL/6 mice. The protection was associated with a rapid CD4⁺ T cell response producing IFN-γ at both the dermal challenge site and viscera, with majority of IFN-γ cells trapped in the vasculature in the viscera [43].

Nevertheless, a conventional high-dose intravenous challenge approach would neglect the crucial initial step of skin deposition of parasites, which mimics the natural infection process in humans. This similar concern was raised from an earlier cross-protective high dosage of L. braziliensis against L. major infection in mice models. Attenuated L. major reduced parasite load in mice challenged with wild-type L. infantum, demonstrating potential for leishmanization [44]. The route of administration influenced cross-protection, aligning with other studies showing that intravenous irradiated L. donovani could protect the BALB/c mice, but not by subcutaneous administration [45]. In another study on experimental infection in monkeys (Macaca mulatta), L. major induced cross-protection against L. amazonensis and L. guyanensis, yet failed to protect against L. braziliensis [46].

The potential of genetically modified parasites as candidate vaccines has expanded in recent years, owing to the practicality of scalable and cost-effective production. With the recent advancement of various approaches, including homologous recombination and CRISPR/Cas technology, modified parasites have been studied with the development of marker-free parasites through specific mutagenesis [47,48,49,50]. In most cases, deletion and/disruption of one or more essential genes related to parasite survival and/or virulence is performed while maintaining the antigenic repertoire of wild-type counterparts and thus the ability to induce a broad-spectrum immune response [51,52].

The Centrin 1 (cen) gene codes for a cytoskeletal (calcium-binding) protein, which is important for eukaryotic cellular division, is necessary for amastigote replication in Leishmania [53]. Early investigation of centrin-deficient L. donovani (LdCen^−/−) infection in animal models induced protective T cell response during VL by L. donovani and MCL by L. braziliensis [53]. Over the past years, similar studies of cen1 knockout parasite strains including L. major (LmCen^−/−), L. mexicana (LmexCen^−/−) and L. donovani (LdCen^−/−) have shown to elicit protective immunity both in murine and hamster models [40,47,54]. Volpedo et al. [55] demonstrated that live-attenuated L. mexicana cen1 knockout (LmexCen^−/−) parasites reduced parasitic burdens and increased protective cytokine levels both in vitro in bone marrow-derived macrophages (BMDMs) and dendritic cells (BMDCs), and in vivo in BALB/c models of CL. Similarly, LmexCen^−/− immunization suppressed the disease promoting IL-10 and IL-4 cytokines while boosting IFN-γ production, resulting in higher IFN-γ/IL-10 and IFN-γ/IL-4 ratios relative to non-immunized animals, and conferred protection against VL from needle-delivered L. donovani [55].

Due to safety concerns with leishmanization, attenuated Leishmania spp. cell lines serve as a promising alternative, delivering the parasites’ near-complete antigen repertoire alongside robust immune responses. The LdCen^−/− cell lines conferred a long lasting cross-protection against L. mexicana with increased IFN-γ, IL-2 and TNF-α production in the immunized groups [51]. Similar findings by Avendaño-Rangel et al. [56] showed that LdCen^−/− effectively induced cross-protection against L. braziliensis infection. On the contrary, the LbCen^−/− cell line with impaired survival failed to confer protection against homologous infection with a reported Th2-biased immune response 17 [56]. In another experiment, LmCen^−/− vaccine elevated Th1-related cytokines IFN-γ and TNF-α while suppressing anti-inflammatory IL-10 and IL-21, conferring protection against sandfly-transmitted VL in a preclinical hamster model [57].

Deletion of MIF gene, L. donovani biopterin transporter (LdBT1^−/−), A2–rel gene in L. donovani from both live and attenuated parasites [58,59], silent information regulatory two single allele deletion (LiSIR2⁺) in L. infantum [60], cysteine proteinases in L. mexicana [61] and LiHsp70^−/− [62] resulted in protective immunity against various Leishmania infections. Additionally, the study showed that LiHsp70^−/− would be a safe live vaccine following lack of pathological signs in infected immunodeficient SCID mice and hamsters (Mesocricetus auratus) [62]. The Ldp27^−/− was reported to induce cellular immunity protection against challenge with homogenous L. donovani [63] and heterologous L. major, L. braziliensis infections in mice [63], as well as L. mexicana in immunized animals [51]. Furthermore, Lmp27^−/− was able to induce significant levels of IFN-γ that protected mice challenged with both L. major and L. infantum [64]. Genetically modified live-attenuated parasites have been shown to elicit long-term protective immunity in animal models. A recent evaluation showed that transgenic L. tarentolae expressing gamma-glutamyl cysteine synthetase (γGCS) conferred protection against both CL and VL infection, whereas wild-type L. tarentolae exerted no significant impact on parasite burden relative to infected controls in BALB/c mice and hamsters [65].

3.1.2. Whole-Killed Parasites

First-generation vaccines, developed from whole-killed or live-attenuated parasites eliciting broad immunity gradually replaced leishmanization [66]. Since Leishmania can be easily cultured in cell-free media, whole-killed parasites were initially used in Leishmania skin tests (LST) for diagnosing human infections. Early vaccine formulations for leishmaniasis, which primarily focused on whole-killed parasites for New World infections, were explored as early as the late 1930s. Trials of different preparations of polyvalent, whole-killed parasites by Sales-Gomes were the first-generation vaccine developed against CL in Brazil in 1939. Later studies supported the immunogenic ability of a killed vaccine of five Brazilian isolates by a positive LST in vaccinated individuals [67]. The absence of circulating antibodies suggests preferential induction of protective cell-mediated immunity over a protective humoral response. In randomized, double-blind clinical trials, whole-cell autoclaved L. amazonensis strain’s (BIOBRAS^®, Brazil) immunogenicity and safety was confirmed in Colombia; however, no protection was conferred [23,68].

In an attempt to increase the immune response, clinical studies of Leishmania killed vaccine have been investigated with Bacillus Calmette–Guérin (BCG) as an adjuvant [69,70]. Clinical studies of a trivalent vaccine comprising killed L. amazonensis, L. guayanensis and L. braziliensis effectively controlled CL in Ecuador by modulating the innate immune response through neutrophils and eosinophils, alongside activating CD4⁺ T cells, CD8⁺ T cells and B lymphocytes. Both groups receiving monovalent L. amazonensis vaccine + BCG and placebo ID groups presented high skin test conversion in Colombia [23]. Leishvacine—a trivalent L. amazonensis vaccine—further supported the role of BCG in significantly reducing the incidence of CL in patients [69]. Clinical trials with a cocktail vaccine of L. mexicana and L. amazonensis demonstrated protection with minimal side effects and favorable production economics in Venezuela [71]. Likewise, pasteurized-killed L. braziliensis promastigotes combined with BCG successfully treated severe, chemotherapy-refractory CL in Venezuela [72]. In the Old World, autoclaved L. major (ALM) + BCG showed promising results in managing CL in Iran. Meanwhile, alum, as an adjuvant, enhances the immunogenicity of alum-precipitated ALM + BCG vaccine against CL and VL in primate models [73]. A single dose of alum-precipitated ALM + BCG increased antigen-specific proliferation, elevated IFN-γ levels, and robust positive delayed-type hypersensitivity (DTH) in Indian Langur monkeys. In dogs, it reduced CVL incidence and lowered human transmission in Iran [74]; however, other studies indicate that booster doses are required to sustain immunity [69,75].

The ALM + BCG vaccine trial in Sudan could not offer significant protective immunity against VL compared to BCG alone in human [76]. While the vaccine efficacy was not significant, individuals who had a LST conversion experienced a notably lower incidence of VL compared to non-responders. Similar findings of reduced incidence in LST-converted participants were consistent with findings in Brazil and Esfahan studies [75]. The persistence in LST conversion in the absence of parasites suggested the necessity of a booster dose to maintain the test positivity. A phase II of alum–ALM + BCG vaccine mixture confirmed the safety of the vaccine through a cross-protection in human volunteers against VL [77], and another trial against chronic PKDL had promising results [78]. A killed but metabolically active (KBMA) L. infantum chagasi vaccine offered protection against virulent L. infantum chagasi comparable to that of live parasites. Both formulations elicited mixed Th1 and Th2 cytokine profiles [79]. Consequently, KBMA technology emerged as a potential safe and efficacious vaccine approach against Leishmania.

3.2. Second-Generation Vaccines

The second-generation vaccine consists of defined antigens and native proteins expressed in heterologous microbial vectors. Native proteins extracted from Leishmania can be used in either purified or crude forms to elicit a protective immune response. The second-generation vaccines benefit from a high level of purification, which helps with standardization and large-scale production. Recombinant approaches also employ engineered cells/proteins with complementary epitopes into unified polyprotein constructs. Although a wide range of Leishmania vaccines are under investigation, the majority target parasite-derived antigenic proteins or recombinant products from defined antigens [13,80].

While few recombinant vaccine candidates have advanced to clinical trials, many subunit and recombinant formulations have undergone extensive preclinical evaluation as potential Leishmania vaccines [81]. Over 30 distinct protein antigens including lipophosphoglycan (LPG), thiol-specific antioxidant antigen, glycoprotein 63 (gp63), Leishmania homolog of receptors for activated C-kinase (LACK), L. major stress-inducible protein 1 (LmSTI1), and diverse combinations have been explored for vaccine development against Leishmania, serving as either second- or third-generation candidates.

3.2.1. gp63 (63 kDa Surface Glycoprotein)

Leishmanolysin, also known as 63 kDa surface glycoprotein (gp63), stands as the most extensively investigated second-generation Leishmania vaccine candidate. Purified gp63 has shown inconsistent protective efficacy across diverse experimental models, varying with Leishmania strains and adjuvant combinations. Recombinant gp63 expressed in E. coli either failed to protect mice against L. major infection or conferred only partial protection in primate challenge models. On the contrary, native gp63 purified from L. major promastigotes, formulated with BCG or liposomes, protected mice against subsequent L. major or L. mexicana challenges [82]. Lymphocytes from gp63-immunized animals showed robust activation upon stimulation with antigen-pulsed macrophages, surpassing responses to infected cells. Moreover, gp63 delivered via Salmonella typhimurium transformants or BCG expression conferred resistance to L. major and L. donovani infection in mice, marked by robust T cell proliferation alongside elevated IFN-γ and IL-2 production.

Unlike previous formulations, liposomal recombinant gp63 elicited robust DTH responses and sustained long-term immunity. Moreover, dendritic cells (DCs), key initiators of immune priming, were harnessed with gp63 synthetic peptides or native gp63; notably, DCs pulsed with certain gp63-derived peptides conferred protection, whereas others exacerbated disease progression [83]. Also, immunization with transfected cells co-expressing gp63 and CD40 ligand (CD40L) preferentially induced Th1 responses and significant protection against virulent L. major and L. amazonensis challenges [84].

3.2.2. Hypothetical Proteins

In Leishmania, hypothetical proteins (LiHyD) identified by immunoproteomics have served as effective diagnostic markers and/or vaccine candidates against tegumentary and VL [85,86,87,88,89,90]. A L. braziliensis hypothetical protein (LbHyp) and the eukaryotic initiation factor 5a (EiF5a) from L. braziliensis elicited cross-protection in mice against L. amazonensis infection, driving a robust Th1 response that reduced parasite load in infected tissues and organs, alongside minimal inflammation and swelling, compared to control groups [91].

A hypothetical protein LiHyD- (LinJ.33.3150) is conserved across Leishmania species including those causing tegumentary leishmaniasis (TL), with identity of 80% with L. major homolog (LmjF.33.2990) and 56% with L. braziliensis (LbrM.33.3270). Initially evaluated against L. infantum, it conferred protection in homologous challenge models. Lage et al. [88] reported a significant reduction in footpad swelling and parasite burden in the infection sites, liver, spleen and draining lymph nodes of rLiHyD/saponin-immunized mice. High levels of IFN-, IL-12 and GM-CSF were observed in immunized mice after in vitro stimulation with rLiHyD, as well as by using L. major or L. braziliensis protein extracts. Partial protection in immunized BALB/c mice was associated with parasite-specific IL-12-dependent IFN-γ secretion, which was mainly produced by CD4⁺ T cells against tegumentary leishmaniasis caused by L. major and L. braziliensis [87]. In context and as described in other studies [86,87,89], the use of Th1 adjuvant improved the protective efficacy of the protein. Immunization by LbHyp and the EiF5a from Leishmania braziliensis along with saponin as adjuvant produced significantly higher levels of protein and parasite-specific IFN-γ, IL-12 and GM-CSF, conferring cross-protection against L. amazonensis infection [91]. Also, a Th1 response was induced in L. infantum infected mice using both a DNA LiHyR and rLiHyR/saponin vaccines [92].

A Leishmania-specific protein, LiHyT, induced cross-protection against tegumentary leishmaniasis by L. major and L. braziliensis, with marked reductions in footpad swelling and parasite burden compared to the control groups [86]. Elevated reports of parasite-specific GM-CSF, a molecule linked to the macrophages activation and resistance against infection, have been reported across multiple Leishmania species, including L. major [93], L. donovani [94], L. infantum [85,95] and L. amazonensis [96]. In the case of L. amazonensis, protection of mice correlated with an IL-12-dependent IFN-γ production, driven predominantly by CD8⁺ T cells [96].

3.2.3. LRP (Leishmania ribosomal Proteins)

Leishmania ribosomal proteins combined with CpG-ODN conferred protection against L. major in both susceptible BALB/c and resistant C57BL/6 mice through IL-12 dependent of IFN- γ production in BALB/c models [97]. Similarly, L. major ribosomal proteins L3 (LmL3) and L5 (LmL5) with CpG ODN protected L. major reinfected BALB/c mice eliciting high parasite-specific IFN-γ and elevated IgG2a/IgG1 ratios for anti-Leishmania antibodies [98]. However, no LmSLA-specific cytokines were produced, which was related to lower ribosomal protein content in total extracts versus purified LmLRP. Across formulations, Th1-biased responses correlated with antigen-specific IFN-γ production alongside suppressed parasite-induced IL-4 and IL-10 responses. A combined LmL3/LmL5 vaccine with CpG-ODN, aTh1 adjuvant, induced LRP-specific IFN-γ, protecting both C57BL/6 and BALB/c mice against L. major [99]. Given the high conservation of LmL3/LmL5 across Leishmania species, their formulation with CpG ODN drove parasite antigen-specific IFN-γ, yielding cross-protection -against L. amazonensis and L. chagas infection [99].

3.2.4. NH36 (Nucleoside hydrolases)

Nucleoside hydrolases (NHs), absent in mammals yet essential for nucleotide salvage in Leishmania, are highly conserved across Leishmania species (84–99% homology) serving as phylogenetic markers. The L. donovani NH36 protein and its C-terminal F3 domain elicited cross-protection against L. chagasi, L. amazonensis and L. braziliensis. The vaccine induced Th1-polarized immunity marked by high IgG2a/IgG2b, IFN-γ/TNF-α-producing CD4⁺/CD8⁺ T cells, and multifunctional T cells correlated with sustained parasite clearance and persistent DTH [100]. Additionally, L. donovani NH36 cross-protected mice against both L. braziliensis [101] and L. amazonensis infections [102], characterized by high expression of IFN-γ and TNF-α. Moreover, an F1F3 chimeric vaccine amplified cross-protection through conserved epitopes promoting polyfunctional T cell responses, positioning NH36 as a pan-Leishmania vaccine candidate [100,102].

3.2.5. TLR (Toll Like Receptor)

A multicomponent vaccine Leish-11f, is the first vaccine to be assessed in clinical trials. Leish-111f, a fusion of three Leishmania antigens conserved across various Leishmania spp.: L. major homolog of eukaryotic thiol-specific antioxidant (TSA), L. braziliensis elongation initiation factor (LeIF) and LmSTI1 [81]. The combinination of Glucantime^® with Leish-110f^® + MPL-SE^® effectively protected dogs against VL caused by L. chagasi. This resulted in lower mortality, improved survival and enhanced cellular responses to leishmanial antigens, in comparison to Glucantime monotherapy [103]. Polyprotein fusion of multiple recombinant antigens streamlines immunochemotherapy by simplifying production, cutting costs, facilitating distribution in resource-limited settings, serving as an alternative when conventional chemotherapy is ineffective [103].

The Leish-111f polyprotein has undergone modifications for manufacturing and regulatory purposes, like amino acid modifications without affecting its immunogenicity, as demonstrated by both cellular and humoral responses [104]. When formulated with adjuvants like MPL-SE, it enhances Th1 responses, promoting macrophage activation and parasite clearance [105]. Leish-111f combined with rIL-12 or MPL-SE showed protective efficacy in experimental models, inducing strong humoral and T cell responses leading to parasite load reduction against VL in animal models [106,107]. Intranasal immunization with Leish-111f plus CT provided Th1-mediated protection against L. major, whereas Leish-111f alone showed no efficacy [108]. Leish-111f efficacy has been presented in phase I and II trials tested on healthy volunteers and CL patients in Brazil [109] and Peru [110], as well as recovered VL patients in India [32].

Furthermore, LEISH-F1, a modified recombinant Leish-111f, has shown promise for leishmaniasis control, as the first candidate to advance to phase I/II trials, exhibiting strong efficacy in preclinical models [104,106,109]. Elsewhere, Leish-110f conferred protection against VL by L. infantum, which was linked to IFN-γ, TNF and IL-2 production [104]. Both natural (Leish-110f + MPL-SE) and synthetic (Leish-110f + EM005) TLR4 agonists improved their immunogenicity and protective efficacy [104]. Additionally, LEISH-F2, an improved LEISH-F1 variant, demonstrated safety and efficacy in adults and adolescent patients with CL, progressing to phase II trials [111].

3.2.6. SMT (Sterol 24-C-Methyltranferase)

Sterol 24-c-methyltransferase (SMT) and ergosterol, which are absent in mammals, represent potential anti-leishmanial vaccine and drug targets. It is essential for sterol biosynthesis in trypanosomatid parasites, fungi, and higher plants, and is highly conserved across Leishmania species, making it a strong candidate for cross-protection [112]. Recombinant L. infantum SMT (rSMT) with monophosphoryl lipid A, which is a potent TLR 4 agonist formulated in a stable emulsion (MPL-SE) induced a Th1 immune response, reducing ear lesions in mice challenged with VL [112]. This formulation also conferred cross-protection against L. major through multifunctional CD4⁺ T cells producing IFN-γ, IL-2, and TNF-α, underscoring SMT’s broad-spectrum potential [113]. Th1-skewing adjuvants, like MPL-SE, enhanced immunity, as seen in Leish-111f and KSAC-based vaccines [106,112,114]. Similarly, the multicomponent LEISH-F3, a fusion of NH and SMT from L. donovani and L. infantum with GLA-SE, induced Th1 immunity in mice and healthy humans, with phase I clinical trials confirming its safety and efficacy against Leishmania infection [34,115].

3.2.7. CPs (Cysteine Proteinases)

Cathepsin L-like cysteine proteinases (CPs) predominantly expressed and active in extended lysosomes of amastigotes, emerge as key vaccine candidates. The CPs play a significant role in suppressing host Th1 responses, potentially by impaired antigen presentation and T cell proliferation, fostering chronic non-healing lesions. Leishmania CPs comprise three classes: Type I (CPB), with C- terminal extension (CTE) encoded by multicopy genes, and Type II (CPA) and Type III (CPC), encoded by a single copy gene. Leishmania mexicana deletion mutant (Δcpb) infection yielded lower parasite burdens and Th1 response, fully healing lesions in C3H mice and attenuating growth in BALB/c models. Similarly, mice vaccination with CPs elicited protective T cell proliferation against L. major infection [116]. Also, the hybrid fusion protein of CPA/B induced partial protection in experimental cutaneous infection, while rCPB with poloxomer 407, but not rCPA, enabled mice to achieve partial protective immunity through high IFN-γ producing CD8⁺ T lymphocytes against L. major [117,118].

3.2.8. HASPB (Hydrophilic Acylated Surface Protein B)

Hydrophilic acylated surface protein B (HASPB), a member of a family of surface proteins, which are expressed in metacyclic and amastigote stages, has shown protection in experimental models against leishmaniasis [119]. The protection that was conferred by rHASPB1 immunization correlated with rHASPB1-specific IFN-γ producing CD8⁺ T cells. Antibodies rise against hydrophilic antigens; rK9 and rK26 present potential tools for the serodiagnosis of both CL and VL, particularly in endemic regions [120,121]. A lentiviral KMP11-HASPB vaccine expressing the KMP11-HASPB fusion protein of L. infantum could induce the immune system to produce IFN-γ against L. infantum, which results in low parasite burden [122].

3.2.9. KMP11 (Kinetoplastid Membrane Protein-11)

Kinetoplastid membrane protein-11 (KMP11), a highly conserved surface membrane protein across Kinetoplastidae, expressed in both Leishmania promastigotes and amastigotes stages, exhibits 97% nucleotide homology between L. major and L. donovani [123]. With minimal homology with human proteins, KMP11 has been reported to elicit both innate and acquired immunity against Leishmania, positioning it as a prime vaccine candidate. Extensive studies confirm its robust protective potential in mice [123,124]. Notably, a study by Moroof et al. [125] reported that adenoviral KMP11 and HASPB vaccine conferred a protection against L. donovani in mice. Both antibody responses and IFNγ + CD8⁺ T cell responses were boosted, supporting the use of adenoviral vectors in delivery of leishmanial antigens. In another study, immunization with a KMP11-HASPB fusion protein showed a significant increase in IFN-γ, IgG2a indicating the activation of Th1 cells, macrophages and cellular immunity against L. major [121].

3.2.10. PHB (Prohibitin)

PHB, a key protein involved in parasite cell proliferation, aging, B cell maturation and mitochondrial integrity, with the presence of anti-PHB antibodies in VL patients, showed potential as a diagnostic marker of both canine and human VL [126]. Recombinant PHB (rPHB) demonstrated protective efficacy in immunized animals, with Th1 immune responses with high IFN-γ, IL-12 and GM-CSF levels. Additionally, rPHB elicited IFN-γ production in the immunized mice against VL, as well as a proliferative response specific to the protein and higher IFN-γ levels induced in PBMCs from recovered individuals [127]. Collectively, these findings position PHB as a promising diagnostic marker and vaccine candidate for canine and human VL.

3.2.11. LACK (Leishmania Analog of the Receptor Kinase C)

LACK, a highly conserved Leishmania antigen, has also been widely studied as a vaccine candidate. Plasmid-based LACK vaccination, with or without IL-12, conferred long-term protection against L. major [128] and L. infantum [129]. Also, intranasal immunization with a LACK-encoding plasmid protected mice [130] and hamsters [131] against L. chagasi infections. Oral delivery via Lactococcus lactis expressing LACK/IL-12 induced a Th1 response and partial protection against L. major [132]; however, rLACK/rIL-12 failed to protect mice against L. amazonensis [133]. Heterologous prime-boost regimes with L. infantum p36/LACK vectors protected against L. major [134]. Furthermore, in dogs, a recombinant canine distemper virus (rCDV) expressing LACK, TSA and LmSTI1 yielded strong protection, with rCDV-LACK dogs developing smaller and non-ulcerated nodules [135].

3.2.12. Histone Proteins

Histone proteins-H2A, H2B, H3 and H4, which are conserved across Leishmania species, play an important role in DNA packaging, transcription and gene regulation. Histone proteins of L. major and L. infantum have been reported as relevant immunogens for the host immune system during both Leishmania infection and disease [136]. Recombinant H2B protein, along its amino and carboxyl-terminal regions has been evaluated against both CL and VL [136,137]. A combination of recombinant L. donovani histone proteins rLdH2-4 consisting of rLdH2B+rLdH3+rLdH2A+rLdH4, conferred protection for hamsters against L. donovani challenge with increased inducible NO (iNO) synthase mRNA transcripts and Th1-type cytokines—IFN-γ, IL-12 and TNF-α and down-regulation of IL-4, IL-10 and TGF-β [136]. An investigation on recombinant L. major H2B protein, along its amino and carboxyl-terminal regions revealed an efficacy of only divergent amino-terminal region in protecting BALB/c mice against a virulent L. major challenge [137]. In another study, H2B protein induced a mixed Th1/Th2 response in CL-healed individuals [138], while H1 elicited in vivo humoral responses against L. braziliensis [139]. In another study, overexpression of Leishmania histone H1 mRNAs in the L. major promastigotes correlated with reduced parasite infectivity in vitro [140].

3.2.13. A2 Proteins

The A2 protein, exclusive to the amastigote stage and vital for Leishmania persistence in mammalian hosts, was originally identified in L. donovani and displays a marked conservation across Leishmania species, particularly in the L. mexicana and L. major complexes. Preclinical studies in mice demonstrated that A2-based vaccination elicits protective immune responses against L. donovani, L. amazonensis, and L. mexicana infections [133,141,142,143,144]. Leish-Tec^®, a second-generation L. infantum A2-based vaccine licensed in Brazil, effectively prevented canine leishmaniasis in field trials when combined with saponin as adjuvant [145,146]. Immunization with rA2 plus IL-12 elicited a mixed Th1/Th2 profile that conferred protection against VL [141]. Intraperitoneal delivery of recombinant L. tarentolae expressing L. donovani A2 protein elicited a Th1 response against L. infantum, in contrast to intravenous administration, which induced a weaker Th2-skewed response in BALB/c mice [143,147]. A tri-fusion vaccine combining L. tarentolae expressing A2, CPA and CPB(-CTE) provided protection against L. infantum, driven by a high IFN-γ/IL-10 ratio and strong humoral immunity [148]. Additionally, a L. infantum polyepitope vaccine (Poly-T Leish) and its adjuvanted form (Poly-T Leish/SM) activated multifunctional T cells (IFN-γ, TNF-α, IL-2) while reducing IL-4 and IL-10 levels [149]. Similarly, rA2 + alum/CpG and live T. cruzi CL-14 vaccines elicited IFN-γ-driven protection in mice, highlighting the potential of rA2-based strategies for human trials and dual protection against leishmaniasis and Chagas disease [150].

3.2.14. PSA-2 (Promastigote Surface Antigen)/gp46/M-2

Another promising vaccine target, the GPI-anchored membrane glycoprotein gp46/M-2, was originally identified as a promastigote-specific antigen unique to L. amazonensis [129]. Immunization of mice with the membrane glycoprotein gp46/M-2 induced significant protection against L. amazonensis, evidenced by reduced parasite burden and improved disease outcomes [128,132,133]. Following immunization with the native parasite surface antigen PSA-2 formulated with Corynebacterium parvum, mice were able to develop a Th1-mediated response that conferred protection [151]. In contrast, vaccination with recombinant E. coli expressed PSA-2 combined with C. parvum failed to provide protective immunity in mice, despite also inducing Th1 responses [152]. Vaccination using recombinant PSA-2 expressed in L. mexicana promastigotes presented a cross-protection against L. major challenge, while in vitro exposure of PBMCs from previously exposed CL patients with PSA-2 stimulated high IFN-γ and TNF-β secretion; subsequent work also showed efficacy against L. infantum in dogs, natural hosts [153,154].

3.2.15. LPG (Lipophosphoglycan)

Immuno-ability of LPG3, which is an endoplasmic reticulum chaperone in the HSP90 family has also been studied as a potential vaccine [155,156,157,158]. Recombinant L. major LPG3 (rLPG3) induced a Th1 response in human cells, characterized by IFN-γ secretion with no effect on Th2 or Th17 cells, even at high concentrations [159]. Similarly, LPG3 fragments enhanced TNF-α and IFN-γ by NK cells [160]. Immunization of BALB/c mice with rLPG3 plus CpG-ODN elicited a Th1 response offering partial protection against L. chagasi [161]. A LPG3 plus IFA conferred a mixed Th1/Th2 response, which partially protected BALB/c mice, while LPG3 + saponin (LPG-SAP) achieved a reduction in splenic and hepatic parasitism, by a Th1/Th17 response [162].

3.2.16. LdγGCS (L. donovani γ-Glutamylcysteine Synthetase)

Immunization with a plasmid containing L. donovani γGCS (rLdγGCS) gene protected BALB/c mice against L. donovani infection, boosting cell-mediated immunity, which was presented with increased nitrite levels. However, the protection level was lower than likely to be clinically useful, suggesting the use of other formulations [163]. Immunization of mice with rLdγGCS (rLdγGCS) alone or incorporated into a non-ionic surfactant vesicle (NIV) as adjuvant conferred homologous protection against L. donovani infection with IFN-γ production compared to the control mice [164].

3.3. Third-Generation Leishmania Vaccines

3.3.1. DNA Vaccines

In recent years, a third-generation vaccination approach has emerged to enhance the accuracy and effectiveness of management of leishmaniasis. These innovative vaccines utilize nucleic acid-based formulations, specifically those derived from DNA or RNA, typically with prime-boost vaccination protocols [165,166]. Plasmid DNA vaccines that encode key protective Leishmania antigens represent a promising approach for immunization against leishmaniasis, leveraging their inherent adjuvant effects. Additionally, these vaccines stimulate both humoral and cellular immunity while enabling economical production, making them suitable particularly for resource-limited settings with constrained healthcare infrastructure [167]. Over the years, DNA vaccines have offer greater stability with considerable flexibility for combining multiple genes in a single construct [168,169]. The DNA-LACK/MVA-LACK stands out as one of the most thoroughly evaluated vaccines in murine and canine models of CL and VL. In mice, LACK DNA immunization, with or without IL-12, elicited durable protection against L. major infection, though depleting CD4⁺ and CD8⁺ T eliminated this effect [170]. Similarly, intranasal delivery of LACK DNA protected mice against L. amazonensis and L. chagasi infections yet failed when delivered intramuscularly despite IFN-γ induction; the heterologous prime-boost strategy elicited robust protection through long-term CD4⁺ T cells and polyfunctional CD8⁺ effector memory cells in BALB/c mice [171]. These findings align with prior research emphasizing the role of CD8⁺ memory cells in long-term protection alongside CD4⁺ T cells’ pivotal immune function.

Jorjani et al. [172] evaluated DNA vaccine encoding three L. major genes, revealing that a pCAGGS-IL-12 cocktail boosted IFN-γ in BALB/c mice. However, no significant difference was observed between groups with or without the adjuvant. Additionally, a LACK-DNA formulated in chitosan microparticles (CMCs) delivered prolonged protection against L. infantum, evidenced by vaccine mRNA in peripheral tissues. The enhanced immunity was also linked to elevated IFN-γ, suppressed IL-10, improved systemic health markers and markedly lower parasite loads persisting six months post-vaccination [173].

Several other candidates have been tested as DNA vaccines against various forms of leishmaniasis, including TSA. Immunization and co-immunization of mice with pcTSA and pcTSA + AlPO4 enhanced spleen cell proliferation and induced Th1-specific immune responses in mice. Antibody and IFN-γ titers were higher in the pcTSA + aluminum phosphate (AlPO4) group than in the pcTSA alone group. However, there was no significant difference between the immunized groups, which was a similar case with IL-4 titer [174]. DNA vaccination using NH36 hydrolase, the main component of FML, as an immunizing agent elicited a strong Th1 response that conferred protection in mice against L. chagasi, L. mexicana [175] and L. amazonensis [176], with protective efficacy further enhanced by co-administration of aluminum phosphate [176]. However, NH36 failed to confer protection against L. amazonensis, exhibiting higher IL-4 and IL-10 levels, increased edema, and higher parasite loads when co-administered with A2 [142]. The DNA-encoding N-terminal domain of the proteophosphoglycans (PPG) vaccine conferred protection against L. donovani challenge in golden hamsters (Mesocricetus auratus), with the rise in NO synthase, IFN-γ, TNF-α presenting them as potential vaccine against VL [177]. Immunization of BALB/c mice using MAPK10, one of the 15 mitogen-activated protein kinases (MAPKs) protected BALB/c mice against L. major infection [178]. Proof of cross-protection using LmjMAPK10 vaccine was later reported in BALB/c mice against L. donovani infection [179,180].

Vaccination with DNA encoding P4 nuclease and IL-12 (P4/IL-12 regimen) developed a Th1 response and protected mice against L. amazonensis infection but failed to against L. major infection. Campbell et al. [181] underscored the species-specificity cross-protection, pointing that variations in P4 protein sequence between Leishmania species contribute to the differential protection. In golden hamsters, the KMP-11 DNA vaccine conferred long-term sterile protection against both antimony-sensitive and antimony-resistant L. donovani strains. This protection was associated with a mixed Th1/Th2 response, a significant increase in IFN-γ, TNF-α, and IL-12 levels, and marked downregulation of IL-10 [182]. Notably, exogenous IL-12 enhanced the efficacy of the KMP-11 DNA vaccine against L. major by inducing a polarized Th1 response. However, the same IL-12 adjuvant reduced protection against L. donovani, suggesting that while a mixed Th1/Th2 response benefits L. donovani infection, a strong Th1 response is crucial for L. major protection [182,183]. These findings underscore the importance of tailoring immune strategies based on Leishmania species and immune response polarization. A lentiviral vaccine expressing L. infantum KMP11-HASPB induced cytokine production and cross-protected BALB/c mice against L. major [121]. However, multigenic vaccine containing KMP-11, Tryparedoxin peroxidase (TRYP), LACK and gp63 did not protect dogs against L. infantum challenge infection [184].

Evidence indicates that the simian adenovirus (ChAd63) is highly effective in eliciting a broad spectrum of CD8⁺ T cells, particularly in response to Leishmania antigens. The ChAd63-KH vaccine replication-defective simian adenoviral vector expressing L. donovani KMP-11 and HASPB, when administered intramuscularly, induced CD8+ T cell responses with IFN-γ, TNF, and IL-2 production, along with DC activation [37]. A CD4⁺ and CD8⁺ T cell expansion was also pronounced in pHisAK70-vaccinated mice upon L. infantum challenge, whereas only CD8⁺ T cells increased in L. major [185,186,187] and L. amazonensis infections [188]. The L. infantum LPG3 DNA vaccine produced a mixed Th1/Th2 response in BALB/c mice, increasing IFN-γ and IL-10 levels but showing weak humoral immunity [189]. Co-administration of L. infantum HSP70 with the LPG3 DNA did not protect susceptible BALB/c and resistant C57Bl/6 with a presentation of a mixed in vivo Th1/Th2 response [190]. Unlike L. donovani LPG, which conferred protection against VL, L. donovani LPG + BCG failed to protect BALB/c mice against L. major, likely due to structural differences in LPG across species [191].

3.3.2. mRNA Vaccines

The success of mRNA vaccines against COVID-19 has prompted many researchers to explore this platform for developing vaccines against parasitic diseases, including leishmaniasis. Effective vaccines should enhance both innate and adaptive immune responses while generating long-term memory immunity against infection, capabilities that recent studies demonstrate mRNA vaccines substantially improve [192]. The mRNA platform also allows simultaneous expression of multiple proteins, eliciting immunity against different epitopes from different targets [193]. For the purpose of building on a long-term antigen selection initiative that included patients with cutaneous and visceral leishmaniasis from both the New and Old World and their corresponding Leishmania species, the LEISH-F2 and LEISH-F3+ fusion protein sequences were identified for evaluation as repRNA vaccine candidates, demonstrating protection in two-thirds of animals against VL. Direct comparison of subunit and replicon RNA (repRNA) vaccines bearing identical antigen-inserts, revealed divergent T cell responses: repRNA uniquely elicited a CD8⁺ T cell response absent in subunit immunized mice [194]. Further, priming with the repRNA followed by boosting with subunit vaccine generated extremely potent CD4⁺ T cell responses and protected mice against L. donovani challenge. In silico fusion of LmSTI1 from L. major and SP15 from Phlebotomus papatasi (PpSP15) protein on alphavirus-derived self-amplifying mRNA in the form of viral replicon particles presented effectiveness of the antigenic combination [195]. Furthermore, Savar et al. [196] demonstrated high expression of PpSP15-LmSTI1 through a self-amplifying mRNA (SAM) vaccine platform in Baby Hamster Kidney fibroblast (BHK-21) cells. These findings presented SAM platform as an alternative to conventional plasmid DNA-based vaccines against leishmaniasis.

4. Discussion

The development of safe, effective and affordable vaccines against leishmaniasis has been a long-standing challenge, despite decades of research and promising preclinical findings. Leishmania species exhibit substantial genetic and antigenic diversity across Old World and New World strains, creating challenges for species-specific vaccines. Co-circulation of multiple species like L. infantum and L. tropica in Mediterranean foci heightens reinfection risks in overlapping endemic areas. Thus, a single cross-protective vaccine could streamline control efforts and reduce development costs for poly-endemic regions. The candidates reviewed in the current analysis, including TSA, LmSTI1, KMP-11, GP63 and others, represent important strides toward immunoprophylaxis against Leishmania [32,37,38]. However, research evidences the complexity of inducing durable and cross-protective immunity across diverse Leishmania species [197]. Additionally, analysis of cross-protection in the reviewed studies was limited by heterogeneity study designs, animal model variability, and routes of administration for both the vaccines and parasites, as well as diverse immunological endpoints—such as cytokine profiles, parasite burdens and lesion sizes—and poor preclinical-to-human translation [198,199,200,201,202,203].

Few studies in murine models have shown that antigens such as TSA and LmSTI1 can induce Th1-skewed immune responses characterized by IFN-γ production, DTH responses and reduced lesion size conferring both homogenous and heterogeneous protection.

However, efficacy remains inconsistent, particularly in models mimicking natural infection via sandfly challenge, where parasite load reduction does not always correlate with complete protection. Additionally, the variability likely reflects differences in antigenic conservation across species, strain heterogeneity and host immune backgrounds. While TSA and KMP-11 are relatively among conserved proteins across Leishmania species, proteins such as gp63 display more polymorphism, potentially limiting their universal applicability [204,205]. Moreover, immunodominant epitopes may vary between species, raising the possibility that vaccines targeting a narrow antigen repertoire may offer incomplete coverage [198].

Most data supporting leishmaniasis vaccine candidates are derived from murine studies, particularly BALB/c and C57BL/6 animal models. These models provide valuable immunological insights into Th1/Th2 polarization and cellular responses. However, they fail to fully recapitulate human disease dynamics [197]. Canine models, particularly for zoonotic VL by L. infantum, have been more predictive and have already supported the licensure of veterinary vaccines, including Leishmune^®, Leishtec^®, CaniLeish^®, and Letifend^®. Only two licensed vaccines, Leishmune^® (FML-saponin) in Brazil and CaniLeish^® (LiESP/QA-21) in Europe, have demonstrated significant protection against severe disease progression and mortality under natural field conditions [206,207]. However, neither has translated to human use or trials due to species-specific immune divergences and safety concerns over antibody-mediated exacerbation in human VL pathogenesis. Despite progression of LEISH-F1 (Leish-111f) and LEISH-F3/F3+ to phase I/II human trials and phase I, respectively, they failed to advance to successful phase III prophylactic trials. Factors such as long-term protection against natural infection, lack of validated correlations and high transmission pressures in field settings challenge the efficacy of trials.

The absence of validated immune correlations of protection hampers candidate prioritization and trial endpoint selection. Although Th1-biased CD4⁺ T cell responses—for example, IFN-γ and IL-2 production—are associated with resistance in murine models, human VL patients often display mixed Th1/Th2 profiles, with a potential exacerbation of the disease by antibody responses. Furthermore, the Leishmania diversity and clinical heterogeneity across Old and New World regions demand tailored antigens, but multi-epitope vaccines like LEISH-F3+ still falter under strain-specific immune evasion or antigenic variation.

Recent advances in Leishmania genome and proteome databases have enabled bioinformatics-driven epitope selection [208,209,210]. The identification of immunodominant T and B cell epitopes through predicted MHC binding and antigenicity, marks a major advance, expediting and simplifying vaccine designing [211,212]. These reverse vaccinology approaches hasten candidate selection by prioritizing conserved, high-affinity epitopes essential for cross-species Th1 immunity. However, in vitro and in vivo validation remains essential to verify that predicted epitopes elicit protective responses effectively. Additional limitations encompass inaccurate prediction of polysaccharide or glycolipid antigens, inconsistent simulation of antibody responses, and unreliable immunogenic peptide ranking; moreover, this approach has been reported as unsuitable for highly variable, structurally complex, or unstable vaccine antigens.

5. Conclusions

Translating preclinical data from animal models to human applications poses a major challenge, as animal immune responses to Leishmania infection often diverge substantially from humans profiles. Sandfly saliva immunomodulation, host immune complexity, and variable clinical manifestations further hinder cross-protection, as highlighted in the reviewed studies. Failure of human vaccine trials to confer a long-term immunity against Leishmania without geographical limitations is attributed to the parasite’s antigen diversity due to the existence of multiple species globally. Moreover, limited consideration on endemic realities like co-infections (HIV, helminths), vector variability, drug resistance and cold-chain logistics in resource-poor settings hampers field deployment of thermostable, effective Leishmania vaccines. Prioritizing standardized correlates of protection, such as durable Th1 responses, alongside region-specific formulations, will bridge these gaps and accelerate viable cross-protective vaccines. This review underscores the need for human-centric trials, standardized correlates of protection and multi-species platforms to advance cross-protective Leishmania vaccines.