INTRODUCTION
Leishmaniasis, a neglected infectious disease, accounts for approximately 700,000 to 1 million cases reported annually in India, Iran, Afghanistan, Iraq, Brazil, Colombia, and East African countries including Ethiopia, Sudan, and Somalia [1]. Leishmaniasis can manifest in various forms, including cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), visceral leishmaniasis (VL), and post kala-azar dermal leishmaniasis (PKDL). These conditions are caused by at least 20 different species of the obligate intracellular parasite Leishmania, which is transmitted to humans by phlebotomine sandflies in the Old World and Lutzomyia in the New World [2]. Parasites known as dermotropic Leishmania infect skin resident macrophages in CL, and a viscerotropic Leishmania spreads through the bloodstream and invades organs including the liver, spleen, bone marrow, lymph nodes, and intestines in VL [3]. During Leishmania infection, immune cells such as neutrophils, macrophages, natural killer cells, dendritic cells, and CD4+ and CD8+ T cells have major roles in host-parasite interactions. Various pro-inflammatory cytokines such as Interferon-γ (IFN-γ), Interleukin-12 (IL-12), and Tumor necrosis factor-α (TNF-α), secreted by cells of the immune system, support host protection, whereas anti-inflammatory cytokines such as Transforming growth factor-β (TGF-β) and Interleukin-10 (IL-10) promote parasite persistence [2].
Existing drugs such as pentavalent antimonials (Sb5+) are currently considered the first line therapeutics, whereas liposomal amphotericin B, miltefosine, and paromomycin are used as second line therapeutic drugs. However, their long-term effectiveness is compromised, by toxicity and drug resistance [4]. Consequently, interest in developing affordable treatments with low toxicity and strong immune-boosting properties has renewed. Efforts have focused on developing prophylactic vaccines, on the basis of the observation that individuals who were previously infected with Leishmania spp. and recovered are resistant to re-infection [2]. The practice of leishmanization with intradermal administration of low-dose live and virulent parasites, mainly Leishmania major, for protection against Leishmania started around the year 1940 [4,5]. Because leishmanization has various complications and adverse effects, other methods are necessary. First-generation vaccines for leishmaniasis include whole parasites that may be killed, live attenuated, or fractionated with or without adjuvant [5]. However, killed Leishmania offers relatively less protection because of a weak T helper1 (Th1) response, thus necessitating the use of adjuvants for optimal effectiveness, whereas live attenuated vaccines trigger both humoral and cell-mediated immunity for extended periods [4,6]. First-generation vaccine candidates continue to evolve, and second-generation vaccines (various recombinant components and purified fractions of native proteins as parasite antigens, and synthetic peptides as antigen epitopes) and third-generation vaccines (nucleic acid-based vaccines) are being introduced. Subunit and recombinant Leishmania vaccines confer protection through robust cell-mediated and antibody-mediated immunity, and are suitable for individuals with compromised immune systems. However, difficulty in developing purified fractions in sufficient amounts remains a constraint. Nucleic acid vaccines are advantageous in several aspects: they generate prolonged antigen exposure, thereby activating CD4+ and CD8+ T cells, dendritic cells, and antibody-producing cells, and inducing IFN-γ production. However, because DNA is inserted into the host genome, other gene interruptions and consequently, autoimmune disease may occur. Therefore, safety is a major concern, and a feasible alternative approach is urgently needed [4,6,7]. Interestingly, targeted deletion of a particular gene can affect parasite growth, virulence, and survivability without altering its immunogenic properties, thus eliciting a strong host-protective immune response without pathogenicity (Fig 1). Moreover, antigenic similarities with the respective wild-type strains evoke B cell and T cell mediated immunity similar to that in wild-type infection, and generate long-lasting immunological memory [7]. Here, we focus on vaccine strategies based on gene modification in parasites and their influence on the host’s protective immunity.
GENETICALLY MODIFIED VISCEROTROPIC LEISHMANIA
Vaccines targeting the genes encoding metabolic enzymes such as arabino-1, 4-lactone oxidase (ALO), and fructose 1–6 bisphosphate (FBP) have shown significant efficacy in experimental VL models. ALO is involved in the biosynthesis of ascorbate, which protects Leishmania by metabolizing reactive oxygen species (ROS). Vaccination with ALO-deficient Leishmania donovani (Ld) protects hosts against wild-type (WT) Ld infection, through the secretion of pro-inflammatory cytokines including IFN-γ and TNF-α [8]. Deletion of another enzyme, FBP, involved in gluconeogenesis and glycogenesis pathways, is effective against L. donovani challenge, by inducing NO (nitric oxide), IFN-γ, and IL-12 [9]. Deletion of genes for transporter proteins, such as biopterin transporters or BT1, disrupts the transport of pterin, which in turn is involved in folate biosynthesis, thus enabling generation of attenuated Leishmania for use as a vaccine candidate against leishmaniasis. BT1 null mutants confer protection by upregulating IFN-γ against virulent Ld [8,10].
To date, the most promising candidate for live attenuated vaccine design against leishmaniasis is centrin which is responsible for centrosome duplication and segregation in eukaryotes. In rodents, immunization with centrin knockout Ld or LdCen(−/−) increases the ratio of T helper 1/T helper 2 cells (Th1/Th2) by upregulating IFN-γ; downregulating the secretion of IL-10 and Interleukin-4 (IL-4); and increasing CD4+ T helper17 (Th17) and CD8+ cytotoxic T cells, M1 macrophages, and Nα neutrophils. These combined effects confer protection against not only Ld but also L. mexicana and L. braziliensis challenge [7]. p27 is an amastigote-specific protein, and Ldp27(−/−) has been observed to have diminished virulence. Immunization with these null mutants protects hosts against WT Ld infection, through the upregulation of pro-inflammatory cytokines, such as IFN-γ, TNF-α, and IL-12, as well as anti-inflammatory cytokines, such as IL-4, IL-10, and Interleukin-13 (IL-13), thereby controlling spleen and liver tissue damage even after 20 weeks post-immunization [11]. Both LdCen(−/−) and Ldp27(−/−) have been found to protect experimental BALB/c mice against dermotropic L. mexicana infection even 30 weeks after immunization, through induction of pro-inflammatory cytokines, including IFN-γ and TNF-α secreted by CD4+ and CD8+ T cells, and inhibition of Th2 cytokines including IL-4, IL-13, and IL-10 [12]. Importantly, LdCen(−/−) and Ldp27(−/−) immunization has been found to significantly increase secretion of the protective cytokines IL-12, IFN-γ, TNF-α, Interleukin-6 (IL-6), and Interleukin-17 (IL-17) from human peripheral blood mononuclear cells (PBMCs) in healed patients with VL (HVL) and patients with PKDL, through the expansion of IFN-γ secreting CD4+, CD8+ T cells, and IL-17 secreting CD4+ T cells [13]. LdBPK061160, a PBN1 orthologue in L. donovani, encodes a non-catalytic subunit of the glycosylphosphatidylinositol (GPI) anchored mannosyl transferase I (GPI-MTI) complex, which is important for parasite persistence and infection. LdPBN1(−/−) immunization effectively reduces the parasite load between 28 and 56 days of needle challenge, as compared with that in naive animals [14]. DDX3 DEAD-box RNA helicase, also known as Hel 67, plays a crucial role in RNA metabolism, promastigote to amastigote transformation, and virulence in Leishmania. Hel 67(−/−) parasites lower the parasite burden of the WT Ld strain in the spleen, liver, and bone marrow in infected hamsters, by inducing NO production [8]. Gene-deleted L. infantum has also shown promise in promoting an anti-leishmanial host environment. The essential cytoskeleton protein KHARON1 (KH1) and histone deacetylating protein Silent information regulator 2 (SIR2) play crucial roles in parasitic sustenance. KH1 double knockout Likh1(−/−) and SIR2 single knockout L. infantum Li SIR(+/−) have been found to protect against WT infection in BALB/c mice, in vaccinated versus naive groups; protection is mediated by a high IL-17 and high IFNγ/IL-10 ratio respectively [8,15]. Another protein, heat shock protein 70-II (HSP70-II), is crucial for adaptation in the host system and hence in infection establishment. HSP70-II mutant HSP70-II(−/−) L. infantum has been found to protect L. major infected experimental animals. HSP70-II(−/−), with either intravenous or subcutaneous inoculation sites, results in a decline in infection, and induces central memory T cells and effector memory T cells in the spleen. Vaccination also elicits IFN-γ production by CD4+ and CD8+ T cells [16].
GENETICALLY MODIFIED DERMOTROPIC LEISHMANIA
Dihydrofolate reductase and thymidylate synthase (DHFR-TS) enzymes (involved in pyrimidine biosynthesis) and the Golgi GDP-mannose transporter LPG2 are also crucial for Leishmania infectivity. Vaccination with dihydrofolate reductase thymidylate synthase double knockout or dhfr-ts (−/−) L. major confers protection against the WT by controlling lesion development and decreasing parasite infection. Moreover, immunization with LPG2 knockout L. major has been found to be effective against WT challenge by inhibiting pro-parasitic IL-10 and IL-4 [8,17]. Centrin knockout L. major or LmCen(−/−) protects hosts against WT L. major infection by upregulating IFN-γ+ effector T cells. Most importantly, targeted gene deletion with the CRISPR-Cas9 technique, without any antibiotic-resistance gene insertion, makes LmCen(−/−) parasites the most potent candidate for phase I clinical trials. Immunization with LmCen(−/−) increases effector T cells and tissue-resident memory T cells in mice, thus protecting against both needle challenge and sand fly mediated virulent L. major challenge. LmCen(−/−) also confers protection against Ld infection by enhancing secretion of IFN-γ and TNF-α in the spleen [7]. Cross-protection by centrin-deleted L. mexicana LmexCen(−/−) against Old World VL has been found to efficiently control Ld infection as many as 15 months after needle challenge and 12 months after sandfly mediated infection, through upregulation of IFN-γ, TNF-α, and IL-12p40, and downregulation of IL-4 and IL-10 [18]. Modified genes of viscerotropic and dermotropic Leishmania showing vaccination efficacy are summarized in Table 1.
Immunological effects of genetically modified live attenuated Leishmania vaccines, along with their functions, animal models, modes of administration, duration of protection, benefits, and drawbacks.
| Vaccine candidate | Modification | Function | Parasite | Model | Mode of administration | Protection duration | Immune response | Benefits | Drawbacks | References |
|---|---|---|---|---|---|---|---|---|---|---|
| Arabino-1,4 lactone oxidase (ALO) | Double gene knockout | Ascorbate biosynthesis | L. donovani | BALB/c mice | Needle challenge | 20 weeks | High IFN-γ and NO production | Induces a robust cell mediated immune response specific to the antigen, regardless of whether parasite is present, and thus is a relatively safer vaccine. | Vaccines have not yet been tested in hamsters and non-human primates. Immunological characteristics of memory cells have not been investigated. | [8] |
| Fructose-1,6-bisphosphate (FBP) | Double gene knockout | Conversion of fructose-1,6-bisphosphate to fructose 6 phosphate, thus aiding in the final step of gluconeogenesis |
L. donovani
L. major | BALB/c mice | Needle challenge | 12–16 weeks | High IFN-γ, IL-12, and NO production; elevated IFN-γ/IL-10 ratio; T cell conversion from anergic state to active state | Provides long-lasting protection. Because FBP knockout Leishmania are metabolically challenged, this option might be safe. | The vaccination strategy was not evaluated in a sandfly challenge. Only BALB/c mice were used as an experimental model. | [9] |
| Folate/biopterin transporter (BT1) | Double gene knockout | Folate biosynthesis | L. donovani | BALB/c mice | Needle challenge | 8–12 weeks | High IFN-γ production and low parasite burden | Ensures long-term protection against wild type infection. | Testing against only visceral leishmaniasis was conducted. | [8,10] |
| Kharon 1 (KH 1) | Double gene knockout | Flagellar protein involved in transporting glucose transporter (LmxGT1) to the flagellar membrane |
L. mexicana
L. infantum | BALB/c and IFN-γ(−/−)
C57BL/6 mice | Needle challenge | 20 weeks | Upregulation of IL-17; high IFN-γ/IL-10 ratio; low Th2 response | Various modes of vaccine administration are being assessed, with a primary focus on their ability to stimulate the immune response. | Vaccine induced cross-protection has not been documented to date. | [8,19] |
| Centrin* | Double gene knockout | Growth regulation by centrosomal replication and separation |
L. donovani
L. mexicana L. major L. braziliensis | Golden hamsters, dogs, and BALB/c mice | Needle challenge and sandfly challenge | 12–15 months | Marked increase in IFN-γ, TNF-α, IL-1β, IL-6, and IL-23; involvement of central and tissue-resident memory cells |
Lm centrin knockout parasites provide cross-protection against needle challenge and sandfly challenge. Generation of central and tissue-resident memory is also evaluated. | Vaccination with Lm centrin knockout parasite has not been tested against new world Leishmania. Therefore, whether this knockout parasite can be used as a universal Leishmania vaccine in the future is unclear. | [7,8,18] |
| p27 | Double gene knockout | Involved in oxidative phosphorylation |
L. donovani
L. major | BALB/c mice, dogs, and human-derived monocytes | Needle challenge | Up to 30 weeks | Upregulation of IL-12, IFN-γ, TNF-α, IL-2, and IL-6 | Generation of IFN-γ+ TNF-α+ cytokine producing cells and immunological memory provides cross-protection. | Efficacy of vaccination was assessed only through needle challenge experiments. | [11–13] |
| DDX3 Dead box RNA helicase of 67 kDa (Hel67) | Double gene knockout | Regulates RNA metabolism, translation and RNA editing | L. donovani | Golden hamsters | Needle challenge | Up to 12 weeks | High NO production; delayed type hypersensitivity (DTH) response | Offers prolonged protection. | Detailed assessment of various immune cells and protective cytokines has not yet been conducted. | [8,20] |
| Heat shock protein 70 (HSP 70) | Double gene knockout | Involved in protein folding and activation, expressed in cell-stressed conditions and also responsible for parasitic virulence. | L. infantum | BALB/c and C57BL/6 mice | Needle challenge | Up to 12 weeks | Activation of CD4+ and CD8+ T cells with high IFN-γ secretion | Vaccination provides cross-protection along with homologous protection under challenge with wild-type virulent strains. | Vaccine efficacy was tested against only needle challenge. | [8,16] |
| Non-catalytic component of GPI mannosyl transferase complex (GPI-MTI) | Double gene knockout | Helps in adhesion; is required for maintaining parasite virulence | L. donovani | BALB/c mice | Needle challenge | Up to 8 weeks | Both cell-mediated and humoral immune response generated at 14 days and peaking 77 days post-infection, with the parasite clearance in 28–56 days | CRISPR/Cas9 was used to knock out the gene encoding a flippase, an element of the GPI-MTI complex. | Immunization has not been investigated in a hamster model to date. Potency against CL has not been explored. | [14] |
| Dihydrofolate reductase and thymidylate synthase (DHFR-TS) | Double gene knockout | Involved in parasitic metabolism | L. major | BALB/c mice | Needle challenge | Up to 9 months | Control lesion development | Confers protection against virulent L. major with subcutaneous, intramuscular, and intravenous routes of administration. | Immunization with a large dose (106) is needed to protect the host. Even after this high dose immunization, mild infection was seen. Testing was conducted against only CL forms and did not provide any cross-protection. | [8,21] |
| Golgi mannose transporter (LPG-2) | Double gene knockout | Helps transport GDP mannose to the Golgi apparatus | L. major | BALB/c, C57BL/6, and SCID mice | Needle challenge | Up to 12 weeks | High IFN-γ/IL-4 ratio; decreased parasitemia | Provides protection in immuno-suppressed SCID mice. IL-4 and IL-10 secretion are very low. | Relatively low amounts of IFN-γ are produced. Only needle challenge was used | [17] |
| Inhibitor of cysteine peptidase (ICP) | Overexpression | Regulates protein processing by inhibiting cathepsin L | L. mexicana | CH3 and C57BL/6 mice | Needle challenge | Up to 13 weeks | Elevated IFN-γ; low IL-4 secretion | Confers long term protection with high Th1 and low IgG1 response. | Vaccination with ICP through a subcutaneous route did not protect the host against virulent L. mexicana challenge. | [22] |
| Silent information regulator 2 (SIR2) | Single gene knockout | Play important role in chromatin condensation; important role in chromatin condensation | L. infantum | BALB/c mice | Needle challenge | 10 weeks | Elevated IFN-γ/IL-10 ratio conferring protection | Vaccination decreases splenic and hepatic parasite burden, and also confers protection. | Only needle challenge was used. | [15] |
*Current status: Only LmCen(−/−) vaccine is ready to test in humans in phase 1 clinical trials.
CHALLENGES AND PROSPECTS
Currently, several gene-modified vaccines are being designed and evaluated for their anti-leishmanial efficacy. Although some have shown promise, their translation into clinical practice has yet to be fully achieved, because of several limitations. The chance of reversal of the gene-modified parasites into the virulent WT forms because of the high plasticity of the Leishmania genome, as well as the risk of compensatory gene expression and functional activation, cannot be ignored [23]. The emergence of cross-resistance to certain conventional anti-leishmanial drugs because of retained antibiotic-resistant genes is another major concern that must be avoided [21]. The large-scale production, cultivation, and high manufacturing costs of vaccine candidates, along with proper storage, validation of the mode of administration (sandfly bite or injection), and long-term follow-up studies after challenge additionally must be addressed. In natural infection, sandfly challenge is particularly important, because it mimics bite incidence, thus recruiting neutrophils at bite sites [7]. Consequently, the assessment of a vaccine’s efficacy in protecting hosts should be verified twice through sandfly challenge and needle challenge both. If the issues are properly addressed, genetically modified live attenuated Leishmania may hold promise in the field of prophylactic vaccine development, because it induces immune responses consistently, thus supporting disease outcomes favoring the host.
The limitations of other gene-deleted live attenuated candidates, specifically involving crucial survival genes for Leishmania, must be evaluated in experimental models. Some promising potential candidates with this approach are being studied for their anti-leishmanial efficacy. L donovani with attenuation of critical enzymes in biosynthetic pathways, such as glutamine synthetase (GS), hypoxanthine-guanine phosphoribosyltransferase (HGPRT), xanthine phosphoribosyltransferase (XPRT) for purine biosynthetic pathways, spermidine synthase, and ornithine decarboxylase for polyamine biosynthetic pathway, have failed to persist in mouse models; however, these candidates are very likely to initiate immune responses targeting the induction of the memory response at early stages, thus also ensuring timely parasite clearance under challenge with a virulent wild type. Several cases of cross-protection have already been reported, as summarized above. In this context, a vaccine designed based on L. major that can protect against diverse manifestations of this disease will be valuable. New prospecting candidates continue to be reported, including genetically modified L. major with attenuated 2,4-dienoyl-coenzyme A (CoA) reductase (DECR), nucleoside transporter (nt4), Alkyl dihydroacetone phosphate synthase (ADS1), and ATP binding cassette (ABC) transporter (ABCG1/ABCG2), and L. amazonensis with attenuated mitochondrial carrier protein (MIT1) are potential candidates to be tested for restricted survival in experimental systems [8,24].
Candidates’ specificity and potential also depend on the methods chosen for gene deletion in parasites and the route of administration during immunization, which directly affect their survival and priming of the host immune system. In addition, parasites’ persistence plays a major role in their host priming ability as well as their duration of protection. In this context, the aforementioned LmCen(−/−) parasites are notable for their uniqueness and superiority to LdCen(−/−), because of their marker-free construction with the CRISPR/Cas9 method and the dermotropic nature of the parasite [7,25]. Dermotropic vaccines are advantageous because of their self-resolving nature, and their ability to generate localized immune responses and visible manifestations. In the case of LdCen(−/−), despite its potency as an effective vaccine, concerns have been raised regarding the parasite’s viscerotropicity: it can be baneful if left untreated, thus preventing further human clinical trials [20].
To progress toward clinical trials, promising candidates must be effective against sandfly challenge rather than needle challenge, without affecting the host. Dermotropic candidates are favored over the viscerotropic candidates, as discussed previously. Moreover, immunological studies should proceed beyond ex vivo experiments to human trials, and must be experimentally tested in both endemic and non-endemic areas in later trial phases, to determine their effectiveness and gain approval. In conclusion, on the basis of the analysis herein and the varying potential of the promising and presumptive candidates described above, the search for live attenuated Leishmania vaccines is not expected to end with one or a few successful candidates, new members must be reported and evaluated in the future.