Responsible for up to 30,000 deaths annually, leishmaniasis is a complex spectrum of diseases endemic in 97 countries around the globe. Disease control relies heavily on the early diagnosis and treatment of the active cases (relevant for anthroponotic disease), although it is widely accepted that a prophylactic vaccine for human leishmaniasis is the way to achieve the successful elimination of human disease (taking in consideration the vast list of non-human reservoirs that enable the perpetuation of parasites all around the globe). The notion that infection leads to strong and long-lasting immunity against leishmaniasis supports vaccination as an achievable goal. However, and in spite of the different candidates tested along the years, till date, we still do not have an approved vaccine for humans. In this chapter, we will explore the last advances made in the field of vaccines against Leishmania without forgetting the historical perspective, essential to the understanding of the road already undergone. We will then discuss the correlates of disease and protection, still neither consensual nor definitive, as well as the issue of pre-clinical to clinical translation. The complete understanding of these issues will be essential for the approval of a successful vaccine for human leishmaniasis.
- human vaccines
- correlates of protection
- cellular immunity
Vaccination is undoubtedly one of the greatest achievements of modern medicine, responsible, together with the use of antimicrobials and access to clean water and sanitation, for the global human demographics transformation in the past two centuries [1, 2, 3]. The apparently insignificant proportion of the world population, whose lives are spared annually, thanks to vaccines (0.04 or 0.1%, if we include deaths avoided by smallpox eradication), is equivalent to up to 3 (or 8) million lives spared per year and a cumulative of more than half a billion deaths avoided just in the twentieth century [4, 5]. Nevertheless, and notwithstanding the significant and successful global efforts toward the goal of universal health protection/promotion, the picture could be much better. On the one hand, just by improving global vaccination coverage, an additional 1.5 million deaths could be avoided yearly . On the other hand, there are still many deadly infectious diseases, whose prevention through vaccination is theoretically possible but for which there are no vaccines approved [6, 7, 8]. There are different compatible explanations/hypothesis that together justify it. The first one has to do with legal and ethical reasons: to test/approve/administer a pharmaceutical product nowadays is harder than it was 100 years ago . Also in a chronologic point of view, it is not surprising that there are still no vaccines available for emerging diseases (e.g., Zika or MERS-CoV) [10, 11]. Other reasons have to do directly with the convergence of the nature of the pathogens with the evolution of vaccine technologies : (i) almost all vaccines available till date are humoral based, which is not the best option against intracellular pathogens (e.g.,
Fortunately, with the arrival of the new millennium, WHO/UN initiatives such as the Millennium Development Goals (Goal 6, Target 3) and more recently the Sustainable Development Goals (Goal 3, Target 3.3) contributed to an increase in the awareness on the NTDs and consequently the investment on strategies to control them [18, 19]. The best example of concrete measures undertaken to “end the neglect” is given by the London Declaration on NTDs, signed in 2012 by 20 parties (including governmental organizations, non-profits and pharmaceutical companies) and endorsed thereafter by many others, that proposes to meet the goals set by the WHO Roadmap to overcome the global burden of NTDs (2012–2020), that include the elimination of five diseases and the control of five others. One of the potential short-term controllable NTDs is the fatal form of leishmaniasis .
Endemic in 97 countries around the globe, leishmaniasis is a complex spectrum of diseases [21, 22]. The first layer of complexity is given by its vector-borne nature, which introduces an extra variable (the phlebotomine vector) to the binomium host pathogen. The second one is given by the 20
With this chapter, we propose to explore the broad anti-
2. Vaccines for human leishmaniasis: where do we stand?
2.1. Leishmanization as the proof of principle of vaccines against leishmaniasis
The close relation of the human host and
Leishmanization was no more than the controlled induction of the cutaneous disease to prevent the consequences of natural infection, such as the scarification of exposed body parts (particularly the face) and the consequent life-long psychosocial impact and simultaneously to decrease the disease incidence in hyperendemic areas [32, 33]. In the 1970s and 1980s, several trials were performed using live virulent
|Type||Approach/vaccine candidate(s)||Disease form||Vaccine development pipeline||Efficacy/outcome||Reference|
|Inoculation of live, virulent ||CL||Effective clinical use in the former Soviet Union, Israel and Middle East* (discontinued)||About 80% efficacy||[31, 34, 36]|
|Heterologous protection mediated by inoculation of live, virulent ||VL||Pre-clinical studies in mice||No effect in BALB/c mice; protection in C57Bl/6 mice||[46, 47, 48]|
|Live vaccines||Inoculation of a dermotropic ||VL||Pre-clinical studies in mice||Protection against challenge with viscerotropic |||
|Physically attenuated parasites||CL/MCL/VL||Pre-clinical studies in mice and hamsters||Homologous protection for ||[49, 50, 51]|
|Non-defined composition; live, attenuated and/or drug-sensitive parasites (through culture, chemical, radiation or genetic manipulation)||Chemically attenuated parasites (N-nitrosamines/antibiotic pressure)||CL/MCL/VL||Pre-clinical studies in mice and dogs||Homologous protection for |||
|Genetically attenuated parasites (||CL/MCL/VL||Pre-clinical studies in mice/hamsters/dogs/macaques||Homologous protection for ||[51, 52, 53, 54, 55]|
|Genetically modified parasites (gain of function)—suicide mutants: ||CL/VL||Pre-clinical studies in mice and hamsters||Homologous long-term protection (lesion free) in mice for ||[50, 56, 57]|
|Immunization with non-pathogenic ||VL||Pre-clinical studies in mice and dogs||Promising results in mice and dogs||[50, 60, 61]|
|First generation vaccines||ALM adjuvanted with BCG||CL/VL||Pre-clinical and human clinical studies||Protection in macaques against ||[54, 65, 67, 68]|
|Alum-ALM adjuvanted with BCG||CL/VL||Pre-clinical and human clinical studies||Immunogenic and safe in humans; protective (single dose) in macaques challenged with ||[54, 56, 69]|
|Autoclaved ||VL||Pre-clinical studies in mice||Significant levels of homologous protection|||
|Phenol or Heat inactivated ||CL/MCL||Human clinical studies||52% Efficacy in endemic area (phenol inactivation); no protection against ||[65, 67]|
|Merthiolate-killed ||CL||Pre-clinical and Human clinical studies||Protection in mice not reproduced in humans||[65, 73, 74]|
|Non-defined composition; whole killed parasites or parasite fractions||Sonicated ||VL||Pre-clinical studies in mice hamsters and monkeys||Good homologous protection in all species; liposomal formulation elicits the best protection in mice||[36, 54, 71, 72]|
|Liposomal ||CL||Pre-clinical studies in mice||Significant levels of homologous protection|||
|Fucose-Manose ligand adjuvanted with saponin||VL||Pre-clinical studies in mice and hamsters; “clinical” studies in dogs||Protection in mice and hamsters challenged with ||[66, 75]|
|VL||Pre-clinical/“Clinical” studies in dogs||Significant, long-lasting protection against canine VL in a field trial in an endemic area (||[54, 66, 76, 77]|
|Second generation vaccines||Membrane proteins: native ||CL/VL||Pre-clinical studies in mice, dogs and macaques||Promising results regarding homologous protection in mice; dubious protection in monkeys against ||[50, 54, 71, 83, 84, 85]|
|“Soluble proteins”: recombinant ||CL/MCL/VL||Pre-clinical studies in mice, hamsters and dogs; ||Promising results in mice and hamsters; a major limitation is that most of the antigens were not tested in superior models; positive response in ||[36, 50, 54, 67, 84, 85, 87, 88, 89, 90]|
|Defined antigens: (native) or produced through DNA recombinant technology (more frequent)||Peptides: CPA, GP63, LmSTI1, LiKMP-11, PEPCK; often associated DC-based vaccination or nano-sized vaccine-delivery systems; adjuvanted (MPLA, CpG-ODN)||CL/VL||Pre-clinical studies in mice||Partial protection for ||[71, 89, 93, 94]|
|Fusion protein/polyprotein: Q protein, Leish-F1 (Leish 110-f), Leish-F2 (Leish 110-f), Leish-F3, Leish-F3+, KSAC, 8E + p21 + SMT, KMP-11 + LJL-143 + Leish-F3 + (in virosomes), rLiHyp1 + rLiHyp6 + rLiHyV+rHRF multiepitope; adjuvanted (BCG, Saponin, CpG-ODN, GLA-SE, MPLA, ALD and MPL-SE)||CL/VL||Pre-clinical studies in mice, hamsters, dogs and macaques; human clinical studies||Promising results in mice (CL and VL) and hamsters; protection conferred to dogs against challenge with ||[54, 84, 85, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105]|
|Third generation vaccines||DNA plasmidic vaccines (usually self adjuvanted): LdPDI, tuzin, HbR, A2, Histones+p36, LACK, TSA + LmSTI1, gp63, KMP-11, CPB, ORFF, NH36, TRYP, PSA-2, γGCS, PEPCK, LeIF, GP63 + HSP70, LeIF+/orTSA; MIDGE-Th1 vectors encoding conserved T-cell epitopes from KMP11, TSA, CPA, CPB, and P74||CL/VL||Pre-clinical studies in mice, hamsters, dogs and macaques; ||Generally good protective responses in mice and hamsters correlated with the induction of Th1 immunity; partial (Histones+p36) and good (LACK, cysteine proteinase) protection in dogs; protection in macaques (TSA + LmSTI1); effective in mice in immuno-chemotherapeutic approaches (MIDGE Th1); strong possibility of human immunogenicity (MIDGE Th1)||[36, 50, 54, 84, 85, 89, 110, 111, 112, 113, 114, 115, 116, 117]|
|DNA vaccination and/or modified expression systems||Recombinant viral vectors: recombinant/modified vaccinia virus expressing TRYP, LACK, KMP-11; recombinant Influenza virus expressing LACK; (non-replicative) recombinant adenovirus expressing A2, Leish-F3 or KMP-11-HASPB; recombinant lentivirus expressing KMP11-HASPB||CL/VL/PKDL||Pre-clinical studies in mice, dogs and macaques; human clinical studies||Promising results obtained in all animal models; vaccine safe and immunogenic in humans (replication defective adenovirus coding for KMP-11-HASPB)||[50, 83, 119, 120, 121, 122, 123]|
|Live recombinant bacterial vectors: ||CL/VL||Pre-clinical studies in mice||Different results obtained, varying from disease exacerbation (A2 ||[51, 124]|
|Vector-derived vaccines||Th1 immunity inducing sand fly salivary proteins: recombinant or DNA encoding LJM-19 (SALO), PdSP-15, PpSP15 (also ||CL/MCL/VL||Pre-clinical studies in mice, hamsters, dogs and macaques||Evidences or described effect in protection from (natural) infection in all animal models, except with PpSP-44 which leads to exacerbation of cutaneous disease||[128, 129, 130, 131, 132, 133, 134, 135]|
|Recombinant or DNA coding for sand fly derived proteins (including heterologous expression systems)||Insect-based transmission blocking vaccine: anti ||CL||86% reduction of sand fly-midgut ||[136, 137]|
2.2. An overview of the vaccine candidates against human leishmaniasis explored since leishmanization until the present day
The knowledge produced by leishmanization trials and campaigns conducted at the end of the last century is the most important evidence that the development of a vaccine against leishmaniasis is quite far from being impossible. The quest for such an essential pharmaceutical, indispensable for the achievement of global disease control, has been continuous (in a scale proportional to the funding for NTD research) and fruitful if we consider the number of candidates and different approaches tested. Here we will separate them into five major groups: live vaccines (“leishmanization like”), first-, second-, and third-generation vaccines, and vector-derived vaccines. Table 1 compiles the information to be discussed in the next sub-headings, presenting not only the different candidates/approaches tested along the years but also the disease form they were destined to prevent, their placement in the vaccine development pipeline, and the main findings reported.
2.2.1. Live vaccine candidates
The success of leishmanization is still used to support the investigation of vaccine approaches based on live parasites (called by some as leishmanization revisitation ), that according to the authors have the advantage of at least partially reproducing the normal infectious process (and consequently induce a “close-to-natural” anti-
2.2.2. First-generation vaccine candidates
Together with live attenuated vaccines, killed whole pathogens or fractions of them (inactivated and fraction vaccines) comprise a large proportion of the approved vaccines for humans today . In line with what happened chronologically in modern vaccinology, killed/fractionated vaccines against leishmaniasis were developed both contemporarily and posteriorly to the “leishmanization era,” to answer to the safety concerns associated with live virulent/attenuated vaccines. The main advantage of first-generation vaccines in relation to the live vaccine counterparts is consequently their innocuity: the pool of antigens in its native form will still be “delivered” and elicit a specific memory response (diversity in antigenic
Although it is a topic we do not explore in this chapter, it is important to stress that killed vaccines, different from what was observed in the prophylactic context, have shown great promise in a therapeutic context (revised in ).
2.2.3. Second-generation vaccine candidates
The birth and evolution of the molecular biology field contributed immensely to the rhythm of science in general. Today, the production of a single antigen is usually easily achievable, as it is the possibility of scaling-up the process to an industrial level. Second-generation vaccines are a consequence of this scientific evolution (although some are native proteins, most of them are recombinant antigens) and consist of defined antigens, generally together with an immune response enhancer. They are usually accepted by the scientific community, as well as by the regulatory entities that so far have approved three vaccines for human use (including the hepatitis B recombinant vaccine that replaced the traditional plasma-derived one ). The main advantage of these vaccines in relation to the ones earlier discussed is the defined composition that allows an easier standardization. Another advantage we can think of is the elimination of immuno-dominance events that invariably occur if a complex antigen mixture is used as a vaccine and may hinder the potential of good vaccine candidates . As disadvantages, the following should be considered: (i) the limited duration of antigen availability might impact the memory pool and limit the “protection window”  (more complex immunization schemes have to be used) and (ii) recombinant proteins, usually expressed in heterologous systems, may be slightly different from native proteins (particularly concerning post-translational modifications ) which might impact their immunogenic potential (more relevant for humoral responses, considering conformational epitopes).
Second-generation vaccines against leishmaniasis are the group with higher representativeness. Here, for the sake of clarity, we separate them into four different groups: membrane and soluble proteins (full single recombinants), peptides, and polyproteins (multivalent), whose main candidates are enumerated in Table 1. The studies from fractionated parasites postulated that parasite membrane proteins had a good vaccine potential. Because of that, and also due to their relative abundance, relevant in terms of antigen presentation, many membrane proteins were explored as vaccine candidates in the pre-clinical context for both CL and VL with promising results [51, 55, 72, 84, 85, 86]. Among these is the well-known, and extensively studied in the context of anti-
We cannot end this sub-section without stressing that generally these second-generation vaccine candidates require the co-administration of adjuvants to warrant their efficacies as vaccines for leishmaniasis. Table 2 resumes the relevant information on the topic, extensively covered by two recently published reviews [107, 108].
|Adjuvant||Class||Mechanism of action||Type of immune response||Licensed for use in human vaccines|
|Aluminum mineral salts||Particulate formulation; antigen depot||NALP3, ITAM, antigen delivery, IL-1 secretion, necrosis, inflammasome||Antibody, Th2||✓ (adjuvant of different commercially available vaccines)|
|Simple or emulsified Lipid A analogues (e.g., GLA, MPL)||Immuno-modulatory molecule||TLR-4 agonists||Antibody, Th1||✓ (in combination with Alum in HBV and HPV vaccines)|
|Imidazoquinolines (e.g., Imiquimod, R848)||TLR-7, TLR-8 agonists||Antibody, Th1||X (clinically tested in cancer immuno-therapy)|
|CpG-ODN||TLR-9 agonists||Antibody, Th1, Th2, CD8+ T cells||X (clinically tested in HBV, malaria, influenza and anthrax vaccines and in cancer immuno-therapy)|
|Saponins (e.g., QuilA, QS21)||Unknown||/||X (clinically tested in combination with cholesterol in HCV, influenza and HPV vaccines and in cancer immuno-therapy)|
|Nanoparticles (e.g., Virosomes*, Liposomes)||Particulate formulation||Antigen delivery; cross-presentation enhancer*||Antibody, Th1, Th2||✓ (HAV and |
2.2.4. Third-generation vaccine candidates
The notion that intradermal or intramuscular injection of a plasmid into an animal model would be enough to generate antigen-specific immune responses was responsible for the creation of a new arm in the vaccine research field. Although Initially DNA vaccines were not as well accepted as first- and second-generation vaccines, not only due to potential ethical implications (injection of foreign genetic material into humans that could, for instance, integrate within the human genome) but also due to safety concerns such as the possible generation of autoimmune pathologies initiated by the generation of anti-DNA immune responses . However, these potential issues of DNA vaccines were, with time, shown to be irrelevant, both through extensive pre-clinical research and through several clinical trials performed that confirmed DNA vaccines as safe and immunogenic in humans (although for some candidates, the immunogenicity data was not as promising as expected) [109, 110]. Yet, contrary to the other vaccine approaches discussed earlier, we still have no data from phase IV studies of DNA vaccination, since till date there is no third-generation vaccine approved for human use (although there are already four approaches approved for veterinary use) [109, 110]. However, considering that third-generation vaccines are the most recent approaches (studies started in the 1990s), it is likely a matter of time until the approval of the first DNA vaccines considering some advantages attributed to them: (i) they are easy to design, produce, and scale up (potentially more cost-effective); (ii) they are quite stable, which minimize distribution and logistics-related complications; and (iii) they can induce both humoral and cellular immune responses (including CD8+-mediated cytotoxicity) . Here, we categorize third-generation vaccines in three clusters: DNA vaccines, viral heterologous expression systems, and live bacterial expression systems. DNA vaccines are the more expressive in respect of the number of candidates explored, containing the simplest vaccine candidates: consist of usually non-adjuvanted plasmids (the “real DNA vaccines”). Similar to what was described for second-generation vaccines, both membrane (e.g., KMP-11 and gp63) and non-membrane antigens (e.g., NH, CPB, HSP70, and A2) were explored in the context of plasmid vaccine candidates (Table 1), pre-clinically, using animal models for both CL and VL [37, 51, 55, 85, 86, 90, 111, 112, 113, 114, 115, 116, 117, 118]. Interestingly, many of the candidates tested as second-generation vaccines (and particularly those that have shown some degree of promise) were retested as DNA vaccines, either individually or in “multi-antigen” approaches (e.g., KMP-11, A2, LACK, and TSA+LmSTI1), showing the adoption of a rationale-based vaccine development . The general reproduction of the results obtained with second-generation vaccines, after immunization with their DNA counterparts (CL and VL models, including mice, hamsters, dogs, and macaques), validated these approaches as potentially effective agents in the context of anti-
2.2.5. Vector-derived vaccine candidates
It has become clear that to consider the sand fly vector only from the perspective of vector-control strategies would be not only reductive but also contribute to a major delay in the achievement of the disease elimination objective. The anti-
2.3. Questions that deserve to be answered
As a connecting point between the current and subsequent sections, we raise some questions for which we still do not have a clear answer today. The first one is if the development of a pan-
3. Vaccines for human leishmaniasis: what is still missing?
So far, and consciously, we described the different vaccine candidates explored till the present days as vaccines against leishmaniasis, highlighting only their effectiveness in a qualitative way (effective/non-effective, promising or not) and not discussing the immune mechanisms linked to those results: first, because Table 1 contemplates vaccine candidates developed for the different leishmaniasis forms, whose pathogenic mechanisms are distinct (and not completely understood)  and additionally, because the correlates of protection (that may also be distinct, depending on the disease form) are still far from being well established (they are neither consensual nor definitive). Such facts may have different justifications, as (i) we are still missing key insights concerning vector-parasite-host interactions (both in disease and in health states); (ii) the translation value of the animal models used is limited; or (iii) the models used are not adequate.
3.1. From “mice to man”: the issue of animal models, correlates of protection, and translation
What is known today regarding cellular immune responses to
In respect of these three subjects (correlates of protection, animal models, and translatability of pre-clinical studies to humans) that have major overlaps and cannot be separated, there are still too many shades of gray to account for. As a way to eliminate the fogginess, it will be important to identify the divergent and common points of many anti-
3.2. From “man to mice”: the insufficiency of prospective studies
Leishmaniasis animal models have been undoubtedly an extremely useful tool to understand better the host–parasite interactions that influence either resistance to infection or disease development [157, 182]. This is true for both cutaneous and visceral diseases, although much more relevant in the latest. It would be both unethical and dangerous to biopsy diseased individuals spleen, liver, or bone marrow (target organs of the viscerotropic
Today we still do not have a vaccine approved for human leishmaniasis (regardless of the disease form). Many candidates were tested in the last century, and up to nowadays only vector-derived vaccines were not tested in the clinical context; for all the other parasite-derived candidates, regardless of the vaccine generation they are part of, we have proof of principle of at least immunogenicity and safety (in human healthy individuals) and therefore a precedent is open. Yet, the efficacy clinical trials performed so far (the last more than 50 years ago), excluding leishmanization, were overall disappointing. Such information is however as valuable as any positive result and should be used from a perspective of “learning from our mistakes.” There are still many questions to be answered in the anti-
This work was funded by the project NORTE-01-0145-FEDER-000012, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). Additionally, this work was funded in part by the Intramural Research Program of the NIH, National Institute of Allergy and Infectious Diseases. PC was supported by Foundation for Science and Technology (FCT), Portugal, through the individual grant SFRH/BD/121252/2016.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.