From infection to vaccination: reviewing the global burden, history of vaccine development, and recurring challenges in global leishmaniasis protection

ABSTRACT

Introduction

Leishmaniasis is a major public health problem and the second most lethal parasitic disease in the world due to the lack of effective treatments and vaccines. Even when not lethal, leishmaniasis significantly affects individuals and communities through life-long disabilities, psycho-sociological trauma, poverty, and gender disparity in treatment.

Areas covered

This review discusses the most relevant and recent research available on Pubmed and GoogleScholar highlighting leishmaniasis’ global impact, pathogenesis, treatment options, and lack of effective control strategies. An effective vaccine is necessary to prevent morbidity and mortality, lower health care costs, and reduce the economic burden of leishmaniasis for endemic low- and middle-income countries. Since there are several forms of leishmaniasis, a pan-Leishmania vaccine without geographical restrictions is needed. This review also focuses on recent advances and common challenges in developing prophylactic strategies against leishmaniasis.

Expert opinion

Despite advances in pre-clinical vaccine research, approval of a human leishmaniasis vaccine still faces major challenges – including manufacturing of candidate vaccines under Good Manufacturing Practices, developing well-designed clinical trials suitable in endemic countries, and defined correlates of protection. In addition, there is a need to explore Challenge Human Infection Model to avoid large trials because of fluctuating incidence and prevalence of leishmanasis.

KEYWORDS:

• Human Leishmaniasis
• Leishmania
• vaccines
• prevention
• neglected tropical diseases
• live-attenuated vaccines
• DNA vaccines
• genetically modified parasites

1. Introduction

Due to the increased recognition of neglected tropical diseases (NTDs) as major public health problems, great strides have been made toward reducing the burden of these infectious diseases in developing countries around the world. However, leishmaniasis remains the second most lethal parasitic disease due to the substantial obstacles to universal treatment in many endemic areas and the lack of an approved vaccine [1].

2. Methods

Pubmed and GoogleScholar databases were searched to compile the most relevant and up-to-date information on global leishmaniasis etiology, burden, prevention and treatment alternatives to vaccination, historical vaccine candidates, and previous vaccine candidate performance. Due to the scope of the article, not all significant studies in Leishmania literature could be cited.

3. Leishmaniasis, a global public health problem

3.1. Pathogenesis of Leishmaniasis

Leishmaniasis encompasses multiple disease presentations in humans caused by unicellular, motile protozoan parasites of the genus Leishmania. Leishmania species have two major life stages – motile promastigotes and immotile amastigotes. Promastigotes reside in sandflies and are spread by at least 98 sandfly species (from the subfamily Phlebotominae) which serve as intermediate host and vector [2], whereas amastigotes are responsible for disease symptomology in infected people via intracellular replication, manipulations of the host immune system, and secondary infections [3].

Although the fundamentals of the dimorphic lifecycle of Leishmania in host and sandfly is the same for all forms of leishmaniases, disease pathology differs by Leishmania species and geographical location of infection. Clinical presentation of leishmaniasis is classified into three main types: cutaneous leishmaniasis (CL), visceral leishmaniasis (VL; also known as Kala-azar), and mucocutaneous leishmaniasis (MCL) [4]. VL is the most serious public health concern among the leishmaniases as it is highly lethal if left untreated due to its systemic effects. Initial symptoms include fever, hepatosplenomegaly, and low levels of erythrocytes and leukocytes as well as platelets. However, the most deadly effects include later sepsis development from secondary infection, excessive bleeding from loss of platelets, severe anemia, and rapid weight loss [5]. Depending on geographic distribution of species, VL can be caused by L. donovani or L. infantum – the latter of which can also infect dogs in Europe, Brazil, and North Africa [5]. Along with dogs, other animals can develop infections from multiple human Leishmania spp. including cats, horses, sheep, hyrax, snakes, lizards, sloths, foxes, and numerous rodents such as the fat sand rat [6–15]. Furthermore, with some regional differences, roughly 10% of all dogs in Southwestern Europe and 2–50% of dogs in various regions of Brazil are estimated to be carriers of canine leishmaniasis, therefore public health strategies to control human leishmaniasis must and do consider the possibility of zoonotic reservoir hosts for Leishmania species [16–18].

Unlike VL, MCL and CL are not lethal but still cause severe disease which can result in social stigma for patients due to serious disfigurement and permanent scars. Skin malformities in CL patients are diverse – often simulating herpes zoster or leprosy lesions, among other potential misdiagnoses [19]. Although this diversity in clinical manifestations can make diagnosis exceedingly difficult, symptoms typically begin with a small papule at the site of infection which then grows into a larger skin nodule and subsequently to an open ulcer [19]. Depending on the causative species of CL, lesions can be localized, diffuse, or disseminated, and can be self-healing in 3–24 months or become chronic in <10% of patients (as in leishmaniasis recidivans) [20]. Each distinct CL lesion has a unique immune response profile [21]. Another distinct form of leishmaniasis is MCL. Similarly, to CL, MCL causes destructive changes to skin around mucous membranes of the host, especially the nose and mouth [22].

3.2. Diagnosis and treatment of Leishmaniasis

Although preventive measures through government and individual action can be effective, they require consistent investment to reduce disease risk for local populations, therefore rapid diagnosis is equally important to high-endemic regions. The gold-standard for VL and CL diagnosis remains microscopy of biopsy or aspirate samples in the case of VL or histology/direct microscopy in the case of CL [23,24]. Other tests are becoming more common such as rapid diagnostic antibody/antigen tests, latex agglutination, and PCR which are further discussed here [25,26]. These alternatives are especially appealing to remote medical centers seeking decentralized point-of-care options, however antibody tests in particular are limited in their utility for relapsed infections [27]. Furthermore, PCR is increasingly important to identify specific species of infection for tailored treatment of patients [28].

After diagnosis, treatment of leishmaniasis must also contend with multiple challenges. Unfortunately, although affordable, the WHO-standard pentavalent antimonial combination regimen can cause severe side effects such as liver, heart, and kidney toxicity [1,22,27,29,30]. In addition, drug efficacy varies by region due to parasite resistance to first line antimonials which has been observed as high as 40–60% in Algeria and Bihar, India [31,32]. Intravenous application of liposomal amphotericin B represents the most significant advance from this paradigm in recent years as it can clear all systemic leishmaniases with low levels of toxicity – however, at much greater expense [27,29,33]. Additional treatment alternatives have been under investigation including surgery [34], cryosurgery [35,36], heat therapy [37–39], as well as a variety of localized drug-delivery strategies for localized lesions [22]. Lastly, plastic surgery can attempt to rectify disfiguring scars that often remain following clearance of CL and MCL [40].

3.3. Immunology of Leishmaniasis

Given the array of parasite species which cause leishmaniases around the globe and diverse forms of presentation, the immunology of these diseases is complex. As such, here we review only the `main concepts of immunology for the major forms of leishmaniases while providing more in-depth resources on each species here [21,41–47].

Leishmania parasites can be acquired by a broad range of phagocytic cell populations including neutrophils, dendritic cell, and monocytes; however, Leishmania’s preferred host cell is the macrophage. Regardless of locality, these cells phagocytize the promastigotes deposited by the sandfly and facilitate parasite differentiation into amastigotes as well as parasite replication within the host cell phagolysosome. Leishmania parasites prevent the infected cell from undergoing apoptotic clearance to allow the host macrophage to assist the amastigote’s spread into new cells via membranous extrusions, which attract and are internalized by nearby uninfected macrophages [48]. This colonization of macrophages occurs most heavily in bone marrow, spleen, liver, and lymph nodes for VL, primarily in the skin for CL, or mucosa for MCL [19,41]. In all leishmaniases, heavily parasitized macrophages can finally become successful in killing amastigotes primarily following activation by CD4+ Th1 cells [41,49]. In response to MHC Class II-facilitated antigen presentation from the infected macrophage phagolysosomes, CD4+ Th1 cells release IFN-γ. This activates the infected M1 macrophages to produce reactive oxygen species, nitric oxide and increase phagolysosome fusions to overcome the parasite defense mechanisms in both mouse and human infections [50,51]. Th2 immune response mechanisms characterized by the cytokines IL-4, IL-13, and improved antibody production also play roles in leishmaniasis; Th2 activation is generally counterproductive to CL clearance [52,53], but IL-4 production may be required for clearance in L. donovani infection during VL [54,55]. Paradoxically, both Th1 and Th2 responses can have mixed results for hosts depending on the overall balance [56]. For example, although TNF-α and IFN-γ are beneficial Th1 cytokines in CL, excessive Th1 cytokine production can result in a hyperinflammatory state which enables the CL lesions to spread metastatically to nearby tissues [57].

Regardless of the exact benefit to host for clearance, antibodies are inevitably made during Leishmania infection; this allows for seropositivity to be used for studying past infections. PCR and ELISA evidence from hyperendemic Bihar, India, revealed surprisingly high rates (~20%) of healthy individuals with no history of VL tested positive for both tests [58]. This result can only be explained by previously unreported asymptomatic VL infections.

3.4. Biological risk factors for Leishmaniasis

Although many people are resistant, patients with confounding factors are especially at risk for leishmaniasis. Risks for susceptibility include acute and chronic malnourishment, HIV co-infection, host genetics, or individuals with prescribed medication for other health issues; these all involve immunosuppression and can enable dormant Leishmania to reactivate as well as present new opportunities for acute cases [59–62]. In the case of HIV coinfection, the mode of action is predictable – HIV-mediated loss of CD4+ Helper T cells make clearance of disease more difficult through the loss of a core pathway for macrophage activation [63], however the relationship between immunosuppression and leishmaniasis disease progression is still not fully understood [62,64].

As with host susceptibility, Leishmania virulence is also increasingly recognized for its variability even within a single species. Naturally occurring Leishmania strains with enhanced virulence have been found to distinguish themselves not only via typical single nucleotide polymorphisms (SNP) or indel type mutations in virulence proteins, but also via significant copy number variation of important genes [65–68]. Copy number variation is the more unique challenge to leishmaniasis treatment as Leishmania parasites are fully capable of possessing variable chromosome ploidy and they regularly exchange genetic information – even across species such as in the creation of hybrid promastigotes or amastigotes [69–72].

3.5. Epidemiology of Leishmaniasis

Given the diversity of host and pathogen biological concerns, research into demographic and geographic risk factors for leishmaniasis becomes even more compelling to fully understand the scope of at-risk populations. The burden of leishmaniasis is especially concentrated among 6 countries (Bangladesh, Brazil, Ethiopia, India, South Sudan, and Sudan) which contain >90% of VL cases and 10 countries (Afghanistan, Algeria, Brazil, Colombia, Costa Rica, Ethiopia, the Islamic Republic of Iran, Peru, Sudan, and the Syrian Arab Republic) with >70% of CL cases [73]. Detailed maps classified by clinical form, Leishmania species vectors, reservoirs and regions, as well as interactive maps can be consulted in the following references [74–76]. Per World Health Organization (WHO) 2016 estimates, 616 million people and 431 million people live in these high-burden endemic countries and are therefore at risk of VL and CL respectively [73]. Furthermore, despite much work on improved access to diagnosis and treatments, 1.3 million total leishmaniasis cases were reported in 2015–1 million CL, 300,000 VL, and 20–50,000 lethal VL cases [1,77].

Among this large, susceptible population, demographic and environmental risk factors for VL and CL are diverse and complicated around the world [78]. For example, VL risk factors were characterized in Ethiopia as biased toward the elderly, people who sleep outdoors for their occupation such as herders, displaced populations, as well as involving a variety of environmental factors [79]. Other such studies in Kenya and Argentina also found that risk factors for vector contact strongly correlate to CL disease risk such as people who live near or work in forests, reside in homes with cracked walls or permanent openings from windows and roofing, reside near waterways or ponds, do not perform any type of insect control at their home, or are younger in age (<15) [80,81].

In aggregate, Hotez et al. estimated leishmaniases to be responsible for over 2 million Disability-Adjusted Life Years (DALYs) lost from death and disfigurements – 9^th among all infectious diseases in 2004 [82]. Although the bulk of this burden is concentrated in impoverished communities of tropical countries, significant developed countries must also be concerned with leishmaniases. Southern Europe such as Italy and Spain have always been endemic, however, following expansion of native sandfly ranges, the US has now also reported autochthonous cases in Texas, Oklahoma, Florida, and Arizona [83–87]. Alongside this increased attention and awareness, significant progress has been made in reducing the number of deadly VL cases around the world – especially in India, Nepal, and Bangladesh – yet VL and CL remain serious public health threats globally due to the lack of a protective vaccine [88,89].

4. Critical need for a Leishmania vaccine

4.1. Strategies for the management of Leishmaniasis

NTDs such as leishmaniasis exert a heavy socio-economic burden on endemic countries with an already precarious health system and financial status [90,91]. Even when it is not lethal, this disease can have long lasting effects on affected individuals and communities including life-long disability, psycho-sociological trauma, poverty, and enhancing gender disparities [92]. On an individual level, these issues impact the health, quality of life, and the ability of a person to work [92,93]. However, they can also present a significant financial burden for the government, as the implementation of effective disease prevention and control strategies can be prohibitively expensive, especially for low- and middle-income countries (LMIC) [92,94]. Despite the significant impact that these diseases have on public health and the economy of endemic countries, their global impact is limited, and they are often ignored by the international community [94].

Over the years, the World Health Organization (WHO) has worked with the international community, as well as local governments, to manage human leishmaniasis by developing roadmaps with clear yearly goals for control and elimination. Some of the strategies implemented by this organization include developing preventive chemotherapy, intensifying disease management, strengthening vector control, and improving water availability and sanitation [95]. Recently, WHO put forward a new approach highlighting the Sustainable Development Goals for each NTD for the years 2021–2030 [96,97]. The proposed target goal for VL is to eliminate this disease as a public health problem (<1% case fatality rate due to primary disease) in 64 countries. For CL, 85% of the cases have to be detected and reported in 87 countries, and 95% of these cases need to receive treatment by 2030 [97].

Leishmania-specific strategic plans tailored to different geographical areas have been developed with the goals of improving surveillance, outbreak preparedness and response, diagnosis, treatment, rehabilitation, nutritional support, training and resources for medical professionals, control of sand fly vector and reservoir hosts, as well as reducing transmission, morbidity, and mortality in humans and dogs [98,99]. Unfortunately, many of these target goals have not yet been achieved, despite the efforts of the international community.

4.2. Challenges in the management of Leishmaniasis

Even with strong public health policies set in place in endemic areas, there are additional external factors that can hinder the achievement of the target goals by promoting leishmaniasis emergence and dissemination. For instance, military unrest and unstable political conditions can contribute to the limited implementation of preventive and curative measures [100,101]. Berry, et al. have described leishmaniasis as a ‘disease of guerrilla warfare,’ and showed an increased risk for CL and VL in areas with civil turmoil, ethnic conflicts, foreign military interventions, and political terror [101]. Furthermore, the upsurge of international travel in the recent decades has increased the risk of exposure to leishmaniasis and other NTDs. In North, Central, and South America, 27% of CL cases have been recorded in areas bordering different countries [102]. Tourists and military personnel traveling to endemic areas are especially at risk [103]. Surveillance strategies such as GeoSentinel (Global Surveillance Network of the International Society of Travel Medicine in partnership with the US CDC), have been developed to monitor travel-related infections such as leishmaniasis [104].

Numerous studies have also identified climate change as a predictor of leishmaniasis emergence and geographical distribution [105–109]. Purse, et al. created a model to predict and quantify the role of climate change and other factors in determining the distribution of CL and VL in the Americas. Their results showed that climatic factors can explain up to 80% of the past disease variance [106]. Another study predicted the ecological risk of contracting leishmaniasis to expand from Central America toward the United States and Southern Canada due to climate change [109]. A similar trend was found in Europe using a forecasting niche modeling approach to investigate changes in disease dissemination. This model predicted that the range of sandfly distribution will expand north from the Mediterranean region as climatic conditions change [105].

Animal reservoirs are another factor affecting disease emergence and transmission. Dogs remain the main reservoir for many forms of leishmaniasis, although other animals such as carnivores, rodents, and cattle can also be affected [110,111]. The seroprevalence of Leishmania in dogs ranges from 3 to 50% depending on the endemic areas, although PCR-based diagnostic methods suggest this percentage might be even higher [112]. Culling seropositive dogs is recommended in many endemic countries, as treatment could lead to the development of drug resistance in Leishmania. However, culling has not been shown to be particularly effective to reduce incidence of human leishmaniasis, and it presents economic and ethical concerns [113,114]. Furthermore, almost half of all infected dogs do not present clinical symptoms, while the other half presents a wide range of nonspecific symptoms, making surveillance and diagnosis very difficult [115]. Developing strategies to prevent and control canine leishmaniasis is not only a veterinary issue, but also a public health goal, as dogs can facilitate transmission to humans in endemic as well as Leishmania-free areas [116]. This has been a particular problem in the Mediterranean region and South America [112]. Although no vaccine has yet been approved for human use several vaccines are available for canine leishmaniasis, such as CaniLeish® and LetiFend® in Europe, and Leishmune® (withdrawn in 2014) and Leish-Tec® in Brazil. Safety and efficacy of these vaccines are reviewed in this reference [117]. These observations suggest that improving surveillance and treatment of the disease might not be enough to significantly slow down the spread of leishmaniasis, and that there is a critical need for a strong campaign of prevention in the future decades.

4.3. Developing prophylactic strategies for the prevention of Leishmaniasis

The preventive strategies for human leishmaniasis to date are restricted to limiting exposure to the vector and preventing bites by using bed nets and insect repellent, wearing clothes that cover usually exposed areas of the body, and avoiding the outdoors at dusk and dawn when sand flies are most active [91]. The development of effective prophylactic vaccines is imperative to control leishmaniasis. It is known that patients who recover from leishmaniasis are protected against subsequent infection [118]. Moreover, prophylactic vaccination with a low dose of L. major in non-exposed areas of the body (leishmanization) has been previously employed in endemic areas as it elicits protective immunity against reinfection [119,120]. Although this practice remains unfeasible due to safety and standardization issues, leishmanization suggests that a wide variety of Leishmania antigens, as well as a low level of parasitic persistence, might be needed to provide long lasting immunity and indicates the feasibility of a vaccine [120].

A recent paper used a mathematical transmission model to predict the impact of different vaccine candidates on human VL transmission and incidence in the Indian subcontinent. Overall, development of protective immunity leads to the fastest reduction in VL incidence. Assuming that 100% of the population is vaccinated and only VL and PKDL patients act as reservoirs, an annual incidence of less than 1 VL case per 10,000 individuals (elimination target) can be achieved in a moderately endemic area with a vaccine efficacy of about 60% [121].

An important factor to consider when developing an effective human vaccine are asymptomatic individuals. From a surveillance and diagnostic standpoint, asymptomatic infections are difficult to discover and identify. Several studies have identified asymptomatic individuals as potential carriers for different species of Leishmania [122–124]; however, a recent study conducted in the Indian state of Bihar shows that asymptomatic individuals do not play a major role in maintenance and transmission of L. donovani [125]. Nevertheless, asymptomatic individuals infected with other species of Leishmania might display a greater degree of transmissibility to the sand fly. More research must be conducted to determine the impact these individuals have on disease transmission. Assuming that asymptomatic individuals are the main reservoir for human VL, a vaccine with only 37% efficacy could reduce infectiousness, although an efficacy of 70% would be needed to reduce the development of symptoms. To reach the elimination target, a vaccine must reduce infectivity of asymptomatic individuals by 50% for 11 years, or reduce infectivity of all affected individuals (symptomatic and asymptomatic) by 50% for 4 years. Furthermore, a vaccine that can reduce the chance of developing symptoms by 50% can eliminate VL in 10 years if only symptomatic individuals are responsible for disease transmission, and in 19 years if asymptomatic individuals are the main reservoir [121].

Taken all together these studies highlight the difficulty of controlling NTDs such as leishmaniasis. Even when diagnosed in time, leishmaniasis is difficult to treat, as the therapeutic options currently available are limited and often present toxic side effects and increased parasitic resistance [100]. Interestingly, individuals who recover from leishmaniasis acquire lifelong immunity against subsequent infections, indicating that a vaccine is feasible [118]. Research also shows that development of an effective vaccine would be more economical than maintaining long-term vector control, diagnostic, and treatment strategies. Bacon, et al. used a computer model to assess the economic value of a CL vaccine in endemic Latin American countries. They have determined that even a vaccine with a limited efficacy of 50% and a modest duration of protection of 5 years would be more cost-effective than the current available chemotherapies [126]. Similar results were found in relation to a VL vaccine in the Indian state of Bihar [127]. A vaccination campaign would additionally prevent individuals from developing long-lasting disabilities, reducing the economic burden by lowering health care costs and eliminating lost productivity – elements that are especially important for financially disadvantaged governments and workers. This highlights the need for a pan-Leishmania vaccine effective against all (or most) species of Leishmania without geographical restrictions. Vector-based vaccines could help achieve this goal [128]. Over the years the scientific community has developed different vaccine candidates, which can be classified into: killed or live-attenuated parasites, recombinant proteins, and DNA vaccines and are discussed below [129].

5. Leishmania experimental vaccines to date

5.1. Killed Leishmania vaccines

First generation vaccines utilize antigens derived from the whole parasite, either in a killed or live-attenuated form. Leishmania can be cultured easily in cell-free media, which allowed for the initial use of whole killed Leishmania parasites in Leishmanin skin tests (LST) as a method to diagnose human patients [130]. This previous use of Leishmania, as well as its simple and low cost of production in developing countries, spurred Sales-Gomes to explore different formulations of polyvalent, whole-killed Leishmania as a first-generation vaccine against CL in Brazil as early as 1939. Pessoa and Curban followed in the 1940s, observing 80% efficacy in clinical trials for a polyvalent vaccine consisting of 18 Leishmania strains [131]. Later, in the 1970s, investigation was continued by Mayrink and colleagues in Brazil with a killed vaccine composed of five different non-specified Brazilian Leishmania isolates. These early studies showed the killed vaccine to be safe and immunogenic after two intramuscular injections, observed by a positive LST in 78.4% of vaccinated volunteers within 3 months. However, serological tests displayed a lack of circulating antibodies, indicating the ability of the vaccine to induce a cell-mediated response over a protective humoral response [131–134]. First-generation Leishmania vaccines similar to this are the only vaccine candidates that have progressed to Phase 3 clinical trial evaluation [135].

In Venezuela, Convit and colleagues introduced the use of Bacillus Calmette Guerin (BCG) as an adjuvant to autoclaved L. amazonensis to treat CL [136]. Later studies utilized BCG as an adjuvant with killed Leishmania vaccines in an attempt to increase the cell-mediated response. Clinical studies have been conducted in Ecuador on a trivalent vaccine of L. braziliensis, L. guyanensis, and L. amazonensis [137,138] and a trivalent L. amazonensis vaccine known as Leishvacin [139]. In Colombia, studies of a single strain L. amazonensis vaccine produced by BIOBRAS in Brazil have been investigated [135,140]. In Iran and Sudan, a L. major killed vaccine produced by the Razi Institute has also been tested [141–147]. These candidates exemplify the ability of a killed Leishmania vaccine adjuvanted with or without BCG to be a safe option – safety being an advantage of killed vaccines. Many of these studies, as well as others that have been summarized [131,148,149], have exhibited an induction of cell-mediated responses in vitro (high IFN-γ level) and in vivo (positive LST). However, in all cases except for the study by Armijos, et al. [137], protection against future infection was not associated with vaccination; therefore, the lack of protection has been a disadvantage of killed vaccine designs. Thus, first generation of killed Leishmania parasites remain an inadequate vaccine candidate against leishmaniasis.

5.2. Subunit and recombinant vaccines

Second generation vaccines focus on pathogen antigens including purified native protein fractions, synthetic or recombinant subunits, and recombinant viruses or bacteria possessing antigen DNA [148]. In the case of Leishmania, conservation of antigens amongst the various isolates and species, low antigenic variation, and the availability of the complete Leishmania genome sequence has allowed for the use of Leishmania proteins as vaccine candidates [150]. Suitable peptide antigens can be identified through peptide mapping in which the whole parasite proteome, known peptide libraries, and known immunogenic proteins are screened in silico or in vitro [151,152]. The use of animal models such as mice, hamsters, dogs, and non-human primates have further aided in the discovery of many parasite antigens that induce protective immunity in such models [153].

The numerous subunit and recombinant Leishmania vaccine candidates have been extensively reviewed [148,149,153,154]. These candidates utilize notable Leishmania antigens including gp63, p36/LACK, A-2, FML, PSA-2/gp46/M-2, LCR1, ORFF, KMP11, LeIF, LmSTI1, TSA, HASPB1, protein Q, and cysteine protease B(CPB) and A (CPA) [148]. Many of these adjuvanted second-generation vaccine candidates have induced protection against reinfection in cutaneous animal models of L. major, L. mexicana, L. amazonensis, and L. pifanoi, as well as in visceral animal models of L. donovani, L. infantum, L. chagasi, and are therefore more efficacious than their killed Leishmania predecessors. The Infectious Disease Research Institute (IDRI) has generated a recombinant vaccine candidate, LEISH-F1, which was the first to progress to phase I and phase II clinical trials where it displayed safety and immunogenicity against L. major and L. infantum. Similar candidates produced by IDRI, LIESH-F2 (against CL) and LEISH-F3 (against L. donovani and L. infantum), have also entered clinical trials with promising results [155–157]. Since pure proteins generally induce a weak cell-mediated response, adjuvants and multiple doses are often needed for these candidates to initiate and tailor long lasting vaccine-induced immunity – a disadvantage to subunit vaccines. In particular, VL patients who show an immunosuppressed state can be benefited using adjuvants. Aluminum salts seem to increase immunogenicity in murine models and in patients treated with sodium stibogluconate and have been proposed since the 1920s [158]. Also, some liposomal mediators can be used to deliver antigens to antigen-presenting cells (APCs) [159]. More recently IL-12, IFN-α, IFN-γ, and TLR antagonists have been shown to mediate the immune responses, specifically during chemotherapy against leishmaniasis [158,160]. The success of these adjuvants in traditional drug treatment could be translated into vaccine development to promote the induction of a strong Th1 cell-mediated response, crucial for protection and resistance to Leishmania infection [161]. Another limitation to subunit vaccines is that isolation of enough purified protein is time consuming and difficult. Target molecules may also become misfolded, or errors may occur in the post-translational processing, transport, and localization process of the protein. However, subunit vaccines can also be advantageous since they do not include live component. Therefore, they pose no risk of infection and are suitable for immunocompromised individuals.

Interestingly, investigations involving Leishmania sand fly vector saliva and promastigote secretory gel (PSG) have revealed its impact on modulating the bite site immune environment and development of disease [162–164]. Leishmania parasites have shown increased virulence when transmitted by sand fly vector, this virulence being associated with proteins in the saliva of the New World vector species Lutzomyia longipalpis [165,166]. Vector-derived factors of transmission are rarely considered in vaccine development, however, due to these observations, sand fly salivary proteins have been investigated as potential vaccine candidates either alone or combined with Leishmania proteins. It has been reported that individual saliva antigens are able to elicit paradoxical effects on disease outcome than the whole saliva. The three most promising candidates to date are LJM19 and LJL143 from the L. infantum vector Lutzomyia longipalpis, and PdSP15 from the L. major vector P. duboscqui. It is hypothesized that these proteins normally function to recruit phagocytic macrophages and neutrophils to the bite site by increasing vasodilation, in turn promoting the infection. However, studies of these candidates on mice, hamsters, dogs, and non-human primates revealed their immunogenicity and effectiveness in protecting against infection [167–172]. Furthermore, salivary proteins have been shown to induce the cell-mediated immune response without the addition of an adjuvant, unlike recombinant protein candidates [170]. Thus, investigation of sand fly salivary proteins in Leishmania vaccine development continues to be a growing area of the field.

5.3. DNA vaccines

More recently, genetic immunization strategies have arisen as third generation vaccines that utilize direct injection of nucleic acids, either as mRNA, naked plasmid DNA, or encapsulated in a viral vector. An RNA vaccine expressing LEISH-F2 followed by a subunit vaccine was shown to protect against L. donovani in murine models [173]. Furthermore, interest in DNA vaccines began after observation that intramuscular injection of plasmid DNA stimulated the production of its encoded protein in vivo [174]. In the case of Leishmania, a large repertoire of antigens have been investigated, with the most prevalent DNA vaccines encoding proteins previously tested as recombinant vaccine candidates [148]. Such candidates include single vaccines consisting of one or more protein genes fused on a single plasmid [175–181], or multigenic vaccines possessing multiple protein genes in a cocktail of separate plasmids [178,182–185]. These vaccines remain in pre-clinical trials and have been tested against the CL strains of L. major, L. mexicana, and L. amazonensis in mice, as well as the VL strains of L. donovani, L. infantum, and L. chagasi in mice, hamster, and dog models [148].

The first DNA vaccine developed for leishmaniasis was composed of the gene encoding gp63, which has been shown to induce a Th1 response and significantly protect against L. major infection [177,186,187] and partially protect against L. mexicana infection in BALB/c mice [188,189]. Another DNA vaccine known as LACK has been the most extensively studied Leishmania DNA vaccine, delivering various degrees of protection in cutaneous (L. major) and visceral (L. donovani and L. infantum) infections in mouse and dog models [175,190–193]. Additional DNA vaccine candidates that have induced protective immunity in animal models include genes encoding for proteins such TSA, KMP11, PSA-2, A-2, NH36, LmSTI1, cysteine proteases, and histones and have been previously reviewed [148,149,194]. Heterologous prime boosting has also been utilized to increase the immunogenicity of these vaccines [195–197]. In this method, the DNA vaccine is first used to prime, followed by a booster of the corresponding protein expressed on a recombinant viral vector such as vaccinia virus (VV) or modified vaccinia virus Ankara (MVA). Vaccine efficacy can thus be improved by enhancing the humoral and cell-mediated responses, as well as increasing the production of protective cytokines [198].

DNA vaccines are appealing due to their ease of production, stability, and safety. They are also known to elicit a stronger Th1 immune responses than their protein vaccine counterparts [199]. Expression of the encoded antigen occurs over the course of several days and allows for induction of a humoral and cell-mediated response leading to long-term immunity. The fusion of multiple antigen genes onto a single plasmid is an additional advantage of DNA vaccines. Furthermore, naked DNA vaccines lacking a viral coat are resistant against neutralizing antibody reactions toward viral coat proteins, a problem observed with recombinant vaccines. On the other hand, the lack of this protein coat remains as a disadvantage to naked DNA vaccines since it usually serves to protect against DNA degradation and aiding delivery to host cells [200]. Overall, DNA vaccines have been successful in animal models, although, translation to human has been more difficult owing to their poor immunogenicity and inability to generate significant clinical benefits. Thus, there is currently no DNA vaccine approved for human use not only for leishmaniasis but also against other infections [201]. However, optimization strategies continue to be investigated, such as optimization of transfection and the use of a prime-boost strategy that has improved the efficacy in larger animals and humans [194,201].

5.4. Live-attenuated vaccines

Live-attenuated vaccines involve the genetic deletion of genes responsible for the virulence and/or survival of an organism. They are able to induce an immune response like that of a natural infection without the risk of disease. The live parasite is properly delivered and processed through its natural infection pathway, and all parasite antigens remain expressed for presentation to the host immune system. This allows for the development of specific effector cells and long-term protective immunity against reinfection [202]. Attenuation can be accomplished by two different methods: undefined attenuation from long-term in vitro culture by using irradiation, chemical mutagenesis, or selective pressure, or alternatively, defined attenuation by specifically removing a target virulence gene [203]. One of the historical ways of disrupting Leishmania virulence genes is by homologous recombination of the two alleles with selectable marker genes [204]. Suicide cassettes have also been utilized to introduce drug sensitive genes into the Leishmania genome to control infection and induce immunity [205–207].

In the case of Leishmania, the availability of the complete Leishmania genome sequence has facilitated the effort to identify and analyze genes involved in virulence of the parasite to produce defined vaccine candidates [150]. Multiple defined live-attenuated vaccines generated from L. major, L. mexicana, L. amazonensis, and L. donovani have been developed that display significant protection against CL and VL in susceptible mice [208–212]. In particular, Biopterin transporter 1 (BT1)-deleted L. donovani parasites have been shown to be less infective than their WT counterpart, and to promote protective immunity characterized by IFN-γ. However, BT1 null parasites were still detected in the spleen and liver after 4 weeks of injection, and could pose safety risk [213]. Another L. donovani genetically modified strain was generated by deleting the gene coding for Ldp27, a protein of the cytochrome c complex abundantly expressed in amastigotes. Immunization with Ldp27-deficient parasites resulted in reduced parasitic loads and enhanced Th1 long term protection in CL and VL challenge models [214,215]. Another group created a heat shock protein (HSP)-70-II null L. infantum vaccine candidate. This strain also showed lower parasitic burdens and enhanced protective immunity against a model of CL, but not VL [216–219]. Furthermore, Santi, et al. showed that L. infantum KHARON1 (KH1) null mutants present growth defects and are unable to infect macrophages in vitro, however long-lasting protection has still not been demonstrated [220]. In general, many gene targets have been the focus of defined attenuated Leishmania vaccines, such as dhfr, lpg2, cpa, cpb, Ufm1, p27, SIR2, BT1, HSP70, centrin, and the paraflagellar rod-2 locus which are reviewed in these references [148,149,202,203]. However, evaluation of these genetically modified candidates in animal models has exhibited mixed results of protection.

Safety is of paramount importance to vaccine development. Although live-attenuated vaccines best mimic natural infection and induce protective immunity, safety constraints limit their advantage. Potential reversion to a wildtype virulent form, presence of the antibiotic resistant genes or the risk associated with vaccinating immunosuppressed individuals are relevant concerns [149]. However, advances in genetic manipulation such as the CRISPR/Cas9 system can overcome some of these barriers. Utilizing CRISPR/Cas9 technology to generate attenuated Leishmania vaccine candidates is advantageous since it does not require the use of antibiotic-resistance selection genes, a requirement for advancement to human trials. Vaccines generated in this manner, namely the centrin-deleted (L. donovani Centrin-/- was not generated with the CRISPR/Cas9 technique) L. major vaccine candidate, has proven to be safe, immunogenic, and protective in mice and hamsters [118,222]. Furthermore, it has also been shown that centrin-deleted L. major parasites do not survive inside the sand fly gut. This removes the risk of attenuated parasites being picked up by the sand fly and regaining virulence through recombination with wild-type parasites in the sand fly gut [222]. Thus, live-attenuated Leishmania vaccines have a promising future in the field.

A subset of the above-mentioned vaccines is shown in Table 1; exemplifying the advantages and disadvantages of different vaccination methods.

Table 1. Diverse vaccination approaches tested against Leishmania

Type of vaccine	Mechanism	Manifestation	Challenge	Advantages	Disadvantages	References
Killed Leishmania	Polyvalent whole killed Leishmania	CL	Made with 5 to 18 species of Leishmania	Safe and immunogenic. Positive LST in 78.4% within 3 months	Lack of circulating antibodies	[131–134]
	BCG as adjuvant to killed Leishmania	CL	L. braziliensis, L. guyanensis, L. amazonensis, L. major	Safe. IFN-γ induction in vitro. Positive LST in vivo	Future protection not associated with vaccination	[135–147]
Subunit and Recombinant Vaccines	Recombinant LEISH-F1, LIESH-F2 and LEISH-F3	VL and CL	L. donovani, L. infantum, L. major	Safe and immunogenic	Weak cell-mediated response. Need to be administered together with an adjuvant	[155–157].
	Sand fly saliva antigens: LJM19 and LJL143 from L. longipalpis and PdSP15 from P. duboscqui	VL and CL	L. infantum, L. major	Immunogenic and effective. Induction of cell-mediated immune response without the addition of an adjuvant	Not reported	[167–172]
DNA Vaccines	RNA vaccine expressing LEISH-F2	VL	L. donovani	Protection against infection. Induction of potent local innate and Th1 immune responses	Need for a careful formulation to protect RNA from degradative enzymes and allow its delivery across the cell membrane. Possibly liposome formulation	[173]
	gp63 DNA vaccine	VL and CL	L. donovani, L. major, L. mexicana	Safe. Induction of Th1 response. Protection against L. major and partially against L. mexicana in murine models	Weak immunogenicity in canine VL	[177,184,186,187]
	LACK DNA vaccine	VL and CL	L. donovani, L. infantum and L. major	Varied protection in canine and murine models. Induction of Th1 response	Does not protect against L. donovani	[175,190–193]
Live-attenuated Vaccines	BT1-/- L. donovani	VL	L. donovani	Protective immunity, IFN-γ production. Less infective than the WT	Still detected in liver and spleen after 4 weeks	[213]
	Ldp27-/- L. donovani		L. donovani, L. brazilensis and L. major	Reduction of parasite loads. Long- and short-term protection. Increased Th1 response, protective immunity against CL and VL. Parasites fail to survive inside the macrophages	Few surviving parasites in liver after 13 weeks	[214,215]
	HSP70-II null L. infantum	VL and CL	L. infantum, L. amazonensis and L. major	Reduction of parasite loads. Induction protective inflammatory responses. Protection against CL	Inability to control the infection in spleen and BM in VL model	[216–219]
	KHARON1 null mutants L. infantum	VL	L. infantum	Cell cycle arrest from the parasite. Unable to sustain experimental macrophage infection. Production of IgG and IL-17. Protection against the WT	Long lasting protection is still not demonstrated	[220]
	Cen-/- L. major and L. donovani	VL and CL	L. donovani and L. major	Safe, immunogenic, and protective in mice and hamsters. Parasites fail to survive inside the macrophages. Does not survive in vector’s gut	There might be safety issues in immunocompromised individuals, however this has not yet been demonstrated	[118,221,222]

Select examples of Leishmania vaccines demonstrate the general mechanism, the clinical manifestation and species used for challenge, as well as the advantages and disadvantages of each approach.

CL: Cutaneous leishmaniasis; VL: visceral leishmaniasis; LST: Leishmanin skin test; BCG: of Bacillus Calmette Guerin; PSG: promastigote secretory gel; BT1: Biopterin transporter gene; HSP: Heat shock protein Cen: centrin gene.

6. Common challenges in the development of a Leishmania vaccine

In the absence of a vaccine, natural Leishmania infection is currently the main immunization process, as cohort population studies show that recovered patients are usually protected for life [223,224]. These patients also display a positive response to a leishmanin skin test (LST). LST can be used as a diagnostic method to identify previous Leishmania infections, as it consists of the development of a delayed-type hypersensitivity (DTH) skin reaction in response to intradermally injected Leishmania antigen. LST elicits swelling, inflammation and induration, accompanied by a strong Th1 and macrophage response in recovered patients of CL and VL, even after many years after the infection, which exemplifies how natural infections act as a method of efficient immunizations [225–228]. A more in-depth discussion about the relevance of LST can be found at [229].

These data have encouraged some countries, such as Israel, Iran, and Uzbekistan to use the inoculation of live L. major, a skin restricted strain, as a vaccination method until recent years. The intentional intradermal immunization in covered parts of the body with live parasites is called Leishmanization, and it has been successful in reducing the incidence of CL in target countries [230]. Nevertheless, as we learnt from the experience of Iran, besides its effectiveness, leishmanization might result in the development of chronic or temporary non-healing lesions, and there is also the risk of culture contamination with fungi or bacteria [231,232]. Even when there is a reduction of CL incidence, leishmanization does not eliminate the problem because infected individuals are also reservoirs for the parasite and contribute to L. major transmission [233]. So far, there is not an equivalent immunization process against VL or MCL, since both manifestations of leishmaniasis lead to aggressive symptoms, and in the case of VL, even death. For this reason, it is not feasible to use leishmanization for MCL or VL.

The success of Leishmanization and natural Leishmania infection in the development of protection against CL proves that vaccination is possible. As shown in the previous section, many vaccine candidates have been proposed so far, nevertheless, there are still some challenges related to the biology of the infection and the manufacture which need to be overcome before achieving a safe and efficacious vaccine.

First, there is a concern about the balance between safety and efficacy. Live attenuated vaccines can achieve attenuation by either physicochemical methods (i.e. long-term invitro culture, mutagenesis, or irradiation) or by genetic modifications. In the first case, there is the concern that the parasites could regain virulence; this is also possible for parasites with mutations in just one allele of the genes [203]. Laboratory-generated Leishmania mutants are also antibiotic resistant, which is a useful and practical method for parasite selection during scientific research. Some parasites may retain those genes, which can also lead to antibiotic resistance within Leishmania populations, making this type of vaccine not feasible for clinical use [234]. Thus, homozygous mutants without antibiotic resistance genes would be ideal candidates for safe use in humans. Additionally, live attenuated vaccines should be designed to be safe for immunocompromised patients, since vaccination is also necessary in regions with high rates of HIV-positive individuals [203]. On that regard, killed parasites may appear as a safer alternative, providing a broad range of antigens without the risk of virulence. However, they fail to provide significant protection, suggesting that a carefully designed live attenuated vaccine could be a better option [131].

Another factor to consider when testing the efficacy of a novel vaccine is the difference between needle and sand fly challenge [235]. The sand fly saliva contains molecules which facilitate the bloodmeal by dampening coagulation or modulate the host responses, either favoring immune evasion or inducing an immune response [42,236]. Differences in the protection, immune profile and time of response have been observed when immunized mice were either challenged by sand fly bite or needle injection; and the immune response also changed depending on the initial immunization (live versus killed L. major) [237,238]. Even in experimental models of sand fly infection, the dose of parasites delivered and the frequency varies within populations, making the vaccine testing process more complex [239]. Therefore, there is a critical need for testing candidate vaccines in animal models of sand fly challenge, as it more closely resembles clinical infection.

Finally, the fact that there are many different clinical manifestations and immune responses against the variety of Leishmania species, makes the development of a single vaccine protective against all the different species even more complicated [129,203].

It is noteworthy to mention that there is also an historical delay in the development of vaccines against NTD, including leishmaniasis. This could be due to the fact that vaccines are less profitable than drugs, hence, they are an unattractive market for industry [129,240].

Once the complications of developing a safe and efficacious vaccine can be overcome, the clinical trials need to be properly designed. This might not be a straightforward decision as identifying the right region and population has its own issues. For example, even in Bihar, the Indian state with the highest incidence VL rate, there are areas in the state which reported less than 1 case per 10,000. This incidence is too low to perform a clinical phase 3 including a group receiving the in-test vaccine and another receiving an alternative immunization [229]. Performing clinical trials in endemic regions is necessary to test the protection against Leishmania after challenge, nevertheless, this means that participants will be exposed to the sand flies for uncontrolled times, resulting in differences in the parasite inoculation [230].

7. Concluding remarks

Leishmaniasis affects more than 88 countries around the globe, many of which are low to middle income countries (LMIC). Despite the high morbidity and mortality of this disease there are no vaccines for human use currently available. So far, many candidate vaccines, including killed or live-attenuated parasites, recombinant proteins, and DNA vaccines have been developed [129]. However, challenges such as safety, efficacy, the selection of adjuvants, and the need for sand fly challenge testing remain. Leishmanization has been shown to be the most effective vaccination against CL. CRISPR-based methods enabled the development of Leishmania genetic mutants with precise deletions that embody the immunogenicity and protection effects observed in virulent L. major parasites, but without causing pathology [118,221]. Preclinical studies have shown that genetically modified live attenuated Leishmania parasites can also confer protection against VL, raising the prospect of a safe and efficacious pan-Leishmania vaccine for the first time [222,241].

Toward realizing this goal, several considerations must be satisfied. First, we need to collaborate and incentivize industry partners to manufacture vaccines under Good Manufacturing Practices (cGMP) conditions since such vaccines are not profitable. Secondly, a typical Phase III trial is likely to be prohibitively expensive due to the necessity to include large number of subjects, only a small fraction of which are likely to develop the disease following natural exposure. To mitigate this problem, alternative vaccine trial models, such as a Challenge Human Infection Model (CHIM), can be employed as previously shown in other disease models [242]. However careful and stringent incorporation of exploratory biomarkers, as well as the reintroduction of a standardized LST as a surrogate biomarker of immunogenicity, in Phase I studies should also be investigated to demonstrate vaccine safety and immunogenicity both in endemic and non-endemic countries. This will help to proceed to Phase III efficacy trials. Lastly, we need a vaccine which is affordable for LMICs. Despite its significant impact on public health and the economy of endemic countries, the global impact of leishmaniasis is often ignored by the international community because it is the disease of the poor [80].

8. Expert opinion

Leishmaniasis is one of the top ten Neglected Tropical Diseases and impacts large populations in LMICs. With the increased migrations from and tourism to LMICs, this disease has the potential to spread in non-endemic countries. Little is known about the global impact of this disease, pathogenesis, and treatment options. In addition, there is a lack of effective strategies to control this disease, a need for new diagnostic assay development approaches such as involving omic approaches, and a need for discovery of new drugs that would not result in parasite resistance. On the other hand, vaccination can be one of the effective measures to control and/or eliminate leishmaniasis. Previously, several efforts have been made to develop vaccines against various species of Leishmania. However, very few of the candidate vaccines have been rigorously evaluated beyond pre-clinical studies to assess safety, efficacy, select dose and adjuvants, and to satisfy the need for natural sand fly challenge essential for progress toward clinical studies and regulatory approval. It has been demonstrated that people who have recovered from leishmaniasis are protected for lifetime from future infections, a practice come to be known as Leishmanization. Leishmanization has been widely considered be the most effective vaccination against CL. However, because of the uncertain safety of this practice, it is no longer practiced widely. Recently, genetically modified Leishmania parasites as prophylactic vaccines have been tested in the pre-clinical models that showed protection similar to leishmanization. Therefore, to fully realize the potential of Leishmania parasites as safe and efficacious vaccines, several considerations must be satisfied. First and foremost, a full characterization of the vaccine strain is necessary to meet the expected standards of safety by the regulatory authorities. This includes manufacture of the vaccine strain under cGMP conditions with complete genome sequences, lot to lot consistency, well-defined growth conditions, and absence of adventitious agents toward meeting the safety, identity, purity, and potency standards. For example, some species of Leishmania parasites (both new and old world) contain endogenous Leishmania RNA viruses, which are known to promote disease exacerbation [243]. In addition, care must be taken to develop a media formulation that is free from serum containing Bovine Spongiform Encephalopathy agent. In some situations, it may be necessary to avoid use of serum altogether and use alternatives to serum because of the religious beliefs and cultural norms prevalent in some of the endemic countries. Secondly, there needs to be a well-designed plan to assess efficacy of such vaccines in endemic countries. For example, in many endemic countries there are other co-infections along with Leishmania, therefore the vaccine of choice should be efficacious under these conditions. The choice of clinical trial sites must be such that there is significant incidence and prevalence of the disease since there are variations in the incidence in the subregions of the endemic areas. This may require a large number of subjects, which may not be possible in some situations and may take years to complete the efficacy studies due to the considerable lag between exposure and development of acute disease. Recently, an alternative model of vaccine efficacy known as Challenge Human Infection Model (CHIM) is being tested for various pathogens. Vaccine developers need to explore CHIM to test Leishmania vaccine efficacy. In addition, there is a need for well defined correlates of protection. Toward that goal there is a need to explore new biomarkers of immunogenicity in addition to the markers widely used in preclinical studies. For example, one should explore the well studied Leishmania Skin Test (LST) as a surrogate biomarker of immunogenicity. However, LST is not currently being produced under cGMP conditions that would be permissible in clinical use. Similarly, use of new upcoming technologies, such as single cell RNA seq and/or NanoString RNA and DNA analysis, will help identify new biomarkers of immunogenicity in the exploratory investigations early in the clinical studies, which could be correlated with potential protection. Since Leishmania parasites cause a wide spectrum of diseases, and manufacturers generally lack market incentives to make vaccines against leishmaniasis, it may be desirable to develop a pan-Leishmania vaccine that may be efficacious against multiple species of the parasite. Live attenuated L. major parasites (LmCen^−/-) have shown early promise in affording protection against heterologous challenge with L. donovani indicating the feasibility of a pan-Leishmania vaccine [222]. Finally, we need to develop partnerships with the vaccine manufacturers and create an ongoing dialogue with the authorities in the LMICs to highlight the importance of a Leishmania vaccine and its potential in control and elimination of leishmaniasis and to achieve global public health goals.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. The statements made in this review represent the FDA authors' own best judgments. These comments do not bind or obligate the Food and Drug Administration.

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Funding

This paper was funded by National Institutes of Health, Global Health Innovation Technology Fund.