Fernanda Ludolf
Fernanda F. Ramos1
Eduardo A. F. Coelho- 1Programa de Pós-Graduação em Ciências da Saúde: Infectologia e Medicina Tropical, Faculdade de Medicina, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
- 2Departamento de Patologia Clínica, Colégio Técnico (COLTEC), Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
Leishmaniasis is defined as a complex of diseases caused by protozoan parasites of the genus Leishmania, which comprises 20 parasite species pathogenic to mammalians, such as humans and dogs. From a clinical point of view, and considering the diversity and biological complexity of the parasites, vectors, and vertebrate hosts, leishmaniasis is classified according to the distinct clinical manifestations, such as tegumentary (involving the cutaneous, mucosal, and cutaneous-diffuse forms) and visceral leishmaniasis. Many issues and challenges remain unaddressed, which could be attributed to the complexity and diversity of the disease. The current demand for the identification of new Leishmania antigenic targets for the development of multicomponent-based vaccines, as well as for the production of specific diagnostic tests, is evident. In recent years, biotechnological tools have allowed the identification of several Leishmania biomarkers that might potentially be used for diagnosis and have an application in vaccine development. In this Mini Review, we discuss the different aspects of this complex disease that have been addressed by technologies such as immunoproteomics and phage display. It is extremely important to be aware of the potential applications of antigens selected in different screening context, so that they can be used appropriately, so understanding their performance, characteristics, and self-limitations.
Leishmania parasite complexity
The global burden of leishmaniasis has remained high for the past 10 years, with more than 220,000 new cases reported per year by the WHO (1). Immunization is the most effective way to prevent countless cases of the disease, while early diagnosis is essential for establishing an effective treatment strategy, limiting the pathology and improving the quality of life of patients. In this context, the development of new diagnostic tests and vaccines is essential for containing the disease, particularly considering that the currently available tools for disease control remain limited (2–4). In this scenario, many issues and challenges remain unaddressed due to the complexity and diversity of factors associated with this disease.
Leishmaniasis is a complex of diseases caused by more than 20 pathogenic species of protozoan parasites of the genus Leishmania, which are transmitted to humans through the bite of infected sandflies. The range of clinical manifestations mainly depends on the parasite species and host factors, in which the immune response plays a central role. According to the clinical manifestations, leishmaniasis can be divided into tegumentary leishmaniasis, which comprises cutaneous (CL), mucosal (ML), and cutaneous-diffuse forms (CDL), and visceral leishmaniasis (VL). CL is usually limited to an ulcer that is self-curing; however, it can evolve into severe manifestations, such as ML and CDL, which cause morbidity in patients. VL can be fatal if acute and untreated, while Post-kala-azar leishmaniasis is a skin manifestation that can occur in the populations of some countries after VL treatment (5–7).
The transmission of the parasites can occur by zoonotic or anthroponotic cycles, depending on the infecting Leishmania species and host background. Leishmaniasis is primarily zoonotic, with the exception of Leishmania donovani and Leishmania tropica. VL caused by Leishmania infantum is a zoonotic disease, in which domestic dogs are important parasite reservoirs (6, 8). Canine infection by L. infantum often shows a variety of cutaneous and visceral manifestations (9).
The Leishmania life cycle involves two main parasite forms: promastigote and amastigote. Promastigotes, the infective stage for mammalians, are inoculated into the host skin by the proboscis of the sandfly vector during the blood meal. Then, promastigotes are captured by macrophages and differentiate into amastigotes, which are the intracellular stage responsible for the development of disease (10). Leishmaniasis is a disease caused by an immunological imbalance between the host immune system and the parasite virulence factors (11).
Leishmaniasis vaccine
Given the complexity of the disease, with many Leishmania species involved in different disease clinical forms, it remains unclear whether a universal vaccine or distinct vaccines are required for effective protection. The point is that, to date, a vaccine to protect against human TL and VL is not available, and the few canine vaccines available present problems and/or controversial results. The self-curing and lifelong protection that occurs in CL and in the leishmanization practice suggest that the development of a protective vaccine is feasible. However, Leishmania vaccinology still suffers from bottlenecks, which limit its progress (2).
Leishmanization, the century-old practice of vaccinating people with Leishmania major live promastigotes, was suspended by the WHO due to biosafety concerns (12, 13). Thus, developed vaccines based on crude parasite antigenic preparations achieved low efficacy in preclinical trials (2). Second-generation vaccines based on single or chimeric recombinant proteins have been tested in experimental models (14–18). However, only three Leishmania polyproteins composed of antigens, LEISHF1, LEISHF2, and LEISHF3, have recently entered human clinical trials (11, 19). Third-generation vaccines, based on DNA vaccines, have also been developed and evaluated in experimental models, but little progress has been made (20, 21).
So far, there are three commercially available canine leishmaniasis (CanL) vaccines: Leish-Tec1®, CaniLeish1®, and Letifend®. Leish-Tec1® consists of the recombinant amastigote A2 protein and has shown a protective efficacy of approximately 71% in a low-infective area and 35.7% protection in a high-transmission area. The use of Leish-Tec1® is problematic due to the use of the same antigen by some serological diagnostic tests, hampering the differentiation between infected and vaccinated animals (11, 22, 23). CaniLeish1®, which is composed of L. infantum-secreted/-excreted promastigote antigens, has shown variable protection results as it only decreased the infectiousness burden and the risk of dogs developing symptomatic disease (24–26). Letifend® is a recombinant chimeric protein (Protein Q) that has shown 72% efficacy in preventing disease (27). In this context, owing to the variable effectivity of the few available vaccines that protect against CanL, new antigens still need to be identified and tested in experimental models and then in canines.
Leishmaniasis immune diagnosis
Serological methods are routinely applied for the diagnosis of many infectious diseases, such as leishmaniasis. However, peculiarities of the antibody response against specific Leishmania antigens are crucial for the performance of serological tests. High levels of anti-Leishmania antibodies are usually found in patients with active VL, but they are scarce in asymptomatic cases or in those with TL, particularly CL cases. Therefore, a suitable serological test for diagnosing the distinct clinical forms of leishmaniasis is not available (28–30).
Serological diagnosis has proven to be useful in assisting the diagnosis of VL, although it is limited in terms of distinguishing between active and past or cured infections (30–33). In addition, in immunosuppressed individuals, antibody-detection tests have shown limited sensitivity (34, 35). The high molecular diversity of the rK39 homologous sequences among African L. donovani strains accounts for the different performances of commonly used rK39-based RDTs in different regions of the world (30). Furthermore, cross-reactivity has also been found among Leishmania and other diseases overlapping the same regions (36). In this context, there is still a need to identify new diagnostic antigens that can diagnose TL and improve the diagnosis of VL (28).
Advances in biotechnological tools: Immunoproteomics and phage display
The search for new antigenic biomarkers has been ongoing for decades and has been based on empirical science, with studies evaluating soluble, crude, and/or total antigenic extracts. Nowadays, the search for more assertive and refined antigenic biomarkers can be carried out due to advances in the field of immunology, molecular biology, and high-throughput methodologies, which confer the ability to interrogate the entire antigenic repertoire and aim to improve the sensitivity and specificity of diagnostic tests, as well as the efficacy of vaccine candidates (37–41). In this context, biotechnological tools, such as immunoproteomics and phage display, have improved the scientific process and brought about the possibility of working with a tangible number of antigens, enhancing the chance of success.
Advances in proteomics have provided valuable information about proteome-wide expression profiling and host-parasite interactions, particularly with regard to virulence factors, pathogenicity, and antigenicity (39). Immunoproteomic studies, which combine proteome and serological analysis, have focused on the evaluation of immune responses in the hosts by analyzing their specific antibody/antigenic repertoire in the disease context, refining the selection of parasite antigens to be evaluated as vaccines and/or diagnostic targets (39, 42). Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) has been explored as a proteomic approach to separate the complex mixture of protein extracts into single protein spots, which is followed by mass-spectrometry and genomic/transcriptomic data analysis to identify the proteins. Up to 1,000 protein spots can be resolved on one 2D-PAGE at a time; however, owing to some limitations of the method, the profile may be restricted to highly abundant and soluble proteins. As a protein with different post-transduction modifications (PTM) migrates as distinct spots on the gel, it is not uncommon to find spots from a single gel corresponding to identical protein IDs (39, 43). The combination of 2D-PAGE and western blot (2D-WB), known as serological proteome analysis (SERPA), improves the identification of proteins recognized by antibodies present in patients´ immune sera, and such a strategy is defined as immunoproteomic (43–46).
Phage display has also been used as a biotechnological approach with the purpose of identifying new biomarkers. This is a high-throughput technology based on the selection of phage-fused surface-exposed foreign peptide variants from a clone library, which recognize/bind specific antibodies/molecules. The approach comprises an in vitro selection process in which the binding affinity assays (biopanning) using the mimotopes exposed on the phage surface are followed by DNA sequence analysis of the corresponding phage DNA. The phage display approach demonstrates the desired functionality in terms of diagnosis and vaccine development (47, 48).
Biotechnological tools and their application for leishmaniasis
Comparative analyses of sequenced and annotated Leishmania genomes have uncovered little variation in unique gene content across parasite species. Nonetheless, large-scale genetic differences in terms of gene and chromosome copy number have been found to contribute to alterations in gene expression in response to environmental conditions in mammalian hosts (49). To figure out the functional differences of proteins among species, proteomic studies have been conducted to compare the promastigote and amastigote stages using total and/or secreted protein extracts, with results showing some modest differences in protein expression levels (50).
Leishmania immunoreactive proteins have been identified through the association of high-throughput and serology methodologies, such as immunoproteomics and phage display, when sera samples from infected canine and human patients were used in the protocols. Several Leishmania immunoproteomic and phage display studies have been conducted, each of them trying to answer specific questions regarding the complexity of the disease (Tables 1, 2). Several proteins have been identified as antigenic, in addition to antigens already described in the literature, along with a large number of hypothetical proteins that, together, represent an important contribution (66, 67). With these studies, antigens with immunogenic and/or serological diagnostic potential with human and canine disease were identified in the context of the complexity of the disease. In this review, these topics are addressed.
Table 1 Summary of Leishmania immunoproteomic studies.
Table 2 Summary of Leishmania phage display studies.
Immunoproteomics for tegumentary leishmaniasis
To solve problems associated with the serodiagnosis of TL (low levels of antileishmanial antibodies are usually found in most patients), an immunoproteomic approach involving 2D-PAGE of Leishmania braziliensis total protein extracts and sera from ML and CL patients was conducted. This study identified 20 potential candidates, which were specific to antibodies in the sera (51). Six of these identified proteins were found only at the promastigote stage, four at the amastigote-like stage, and 10 at both parasite stages. An ELISA evaluation of the tryparedoxin peroxidase recombinant protein, which was identified in this study, showed 100% sensitivity and specificity for both CL and ML (51). Proteins found in this immunoproteomic study, such as enolase protein, eukaryotic elongation factor-1 beta protein, eukaryotic Initiation factor 5a, tryparedoxin peroxidase, and hypothetical protein LbHyM, were later evaluated and showed high potential for the serodiagnosis of the disease or immunogenic efficacy against TL and/or even VL in a murine model (18, 68–72).
In another study, Lima et al. (52) conducted a 2D-WB using total protein extracts from L. amazonensis, L. braziliensis, and L. infantum against sera pools of TL or VL patients. To identify biomarkers specific to TL, spots from L. amazonensis and L. braziliensis reactive to VL sera and spots from L. infantum reactive to TL sera were all excluded. MS analysis identified nine proteins for L. amazonesis and two proteins for L. braziliensis, which were considered abundant and immunoreactive against TL sera (52). These antigens may help the development of a specific diagnostic test for TL, distinguishing it from the VL form.
Immunoproteomics for visceral leishmaniasis
An immunoproteomic approach performed with L. infantum protein extract, promastigotes, and amastigotes in reactions against sera from Brazilian HVL patients identified 29 promastigote proteins and 21 amastigote proteins. The authors suggested that such antigens could be evaluated as diagnostic or vaccine candidates against the disease (53). Two of these identified antigens, endonuclease III and GTP-binding protein, were expressed and tested in an ELISA against a serological panel, and results showed around 100% sensitivity and specificity for HVL diagnosis (53). The proteins rGTP and pyridoxal kinase and the hypothetical proteins LiHyQ and LiHyJ, identified in this immunoproteomic study, were evaluated afterwards and showed potential to be developed as a new serodiagnostic test and/or a vaccine (21, 73–76). LiHyQ was evaluated for its ability to diagnose TL, HVL and CanVL and showed a superior diagnostic performance compared with a parasite antigenic preparation and two commercial kits (73). Additionally, LiHyJ showed high sensitivity and specificity values in diagnosing HVL and canVL (74). Furthermore, rGTP protein was recently shown to be protective against experimental VL in a murine model (76). LyHyJ and pyridoxal kinase, administered either as a DNA plasmid or recombinant protein, showed potential as immunogens against HVL (21, 75).
Abánades et al. (58) conducted a Leishmania chagasi 2D-WB to investigate issues regarding the low antibody titers in asymptomatic responders and the poorly characterized serologic markers of recovery or resistance to infection. This study identified two antigens exclusively for VL pretreatment, five for VL post-treatment, five for VL treatment in resistant/asymptomatic patients, and two that were common to all.
An immunoproteomic study using L. infantum promastigote Iranian strain and pooled sera of Iran VL endemic area subjects was conducted, and 11 immunoreactive proteins were identified. A recombinant multi-epitope antigen was designed from the list of identified proteins and evaluated for its potential performance in the serodiagnosis of human VL (54).
As coinfection between VL and human immunodeficiency virus (HIV) has increased in several countries and current serological tests are not sensitive enough to detect most VL/HIV cases, new studies are needed to identify more sensitive antigens. In this context, Machado et al. (55) developed an immunoproteomic approach to search for new biomarkers that could have the capacity to diagnose VL and VL/HIV co-infection cases. Results showed that 43 protein spots were recognized by antibodies in both VL and VL/HIV sera, and 26 proteins were identified by mass spectrometry. One of them, β-tubulin, was expressed, purified, and evaluated using an ELISA as a proof-of-concept for validating their findings, and results showed significant sensitivity and specificity values in diagnosing both VL and VL/HIV patients.
Silver-stained 2D-PAGE of L. donovani crude extract revealed 1,067 protein spots, while corresponding 2D-WB identified 330 antigens. From those, 68 antigens could be assigned to the stained spots, showing that the antigenicity of these proteins did not correlate with protein expression levels. Six proteins were identified by MS. Western blot of L. donovani incubated individually with sera from 15 VL patients showed bands with a broad range of immune specificities and extensive heterogeneity of the serological response (56).
A urine-based ELISA has been suggested as a non-invasive, simple, and safe alternative for diagnosing infectious diseases, such as VL (77, 78). Urine antibodies against 51, 55, and 63 kDa proteins were found with 100% recognition by samples from VL patients. Spots with the same molecular weight size were excised from 2D-PAGE and the proteins were identified as elongation factor 1α of L. infantum, α-tubulin of L. donovani, and glycoprotein or leishmanolysin of L. donovani, respectively (57).
Immunoproteomics for canine leishmaniasis
Dogs are the main domestic reservoir of Leishmania and, as such, are important in the maintenance of the transmission cycle. As the occurrence of asymptomatic infection is considerably higher than that of apparent clinical illness in the infected animals, it is very important to have a more efficient diagnostic test. Additionally, the detection of antigens in asymptomatic dogs could lead to the identification of more assertive vaccine candidates. Machado et al. (79) reviewed the advances in CanL antigen identification using immunoproteomics and phage display and explored the results obtained with these technologies and the validation of some of the identified hypothetical proteins. Briefly, to date, immunoproteomic approaches have been performed with protein extracts from the amastigotes and promastigotes of L. infantum and L. chagasi in reactions against sera of asymptomatic and symptomatic VL dogs from different regions of the world (14, 42, 80, 81).
Phage display in Leishmania
Phage display has been used to identify Leishmania mimotopes that may have a potential application as diagnostic markers and vaccine candidates against leishmaniasis (Table 2). In a phage display study, Costa et al. (59) identified eighteen mimotopes, eight of which showed approximately 100% sensitivity and specificity in an ELISA performed against canine VL sera. Two mimotopes, as well as their synthetic peptides, were later tested for the diagnosis of human VL, and results showed also significant sensitivity and specificity values (82). Additionally, phage display technology was used to select clones of phage-exposed peptide-specific antibodies from asymptomatic and symptomatic VL patients. Eight phage clones were selected after the biopanning cycles, and their reactivity was evaluated using a phage-ELISA, with results showing they were highly efficacious at identifying VL patients, thus demonstrating the feasibility of using mimotopes for the development of a more specific and sensitive serodiagnosis of VL (61). Sera from L. infantum-infected dogs were used in another phage display biopanning, and the corresponding peptides were evaluated as vaccine candidates in a murine model, with aluminum hydroxide and a liposome formulation eliciting significant protection (60). Selected phage clones reactive to HVL sera and with a Th1 direction after stimulus of human peripheral blood mononuclear cells (PBMCs) were also used to immunize BALB/c mice, and a partial protection against the parasite challenge was observed (62). Carvalho et al. (65) used the same experimental strategy, in which sera of TL patients were used to select specific mimotopes, followed by immune stimulation of human PBMCs. Two selected clones were then tested in immunization protocols in BALB/c mice, and protection against L. amazonensis infection was achieved, with significant reductions in the parasite load and footpad swelling. In another study, Ramos et al. (63) selected nine mimotopes using sera from VL and VL/HIV patients in the biopanning cycles. The corresponding epitopes were produced as synthetic peptides and evaluated using an ELISA against a human serological panel. Three of these antigens were able to diagnose both VL and VL/HIV coinfection with approximately 100% efficacy. Three peptides were synthetized after their mimotopes where identified by phage display using antibodies from L. braziliensis patients. Antibodies produced in hamsters immunized with these peptides reacted with bands of different sizes in SDS-PAGE with L. braziliensis protein extract. Additionally, peptides were recognized by sera from CL patients in an ELISA (64).
Conclusion
With the applicability of biotechnological tools such as immunoproteomics and phage display, several antigens have been identified in different contexts of the complexity of leishmaniasis. Immunoproteomics and phage display studies are characterized by the intention of opening doors to new studies more than generating final conclusions, and in this way, the specific outcomes or identified biomarkers fall into a list of proteins/mimotopes that could be assessed in future original studies. Such strategies have showed applicability in this selection process but future studies validating their findings in target mammalian models are certainly necessary to define the protective or diagnostic status of these antigens for canine and human leishmaniasis. Some of them have been successfully evaluated in experimental trials as vaccine or diagnostic candidates. The current demand for new Leishmania antigens for the development of multicomponent vaccines and for the production of specific diagnostic tests is evident, and these processes have been aided by immunoproteomic and phage-display approaches. It is very important to be aware of the objective of screening antigens so that they are used properly and their performance, characteristics, and limitations are well understood.
Author contributions
FL designed the manuscript. FL, FFR, and EAFC wrote the manuscript. FL and EAFC revised the manuscript. All authors contributed to the article and approved the submitted version.
Funding
This work was supported by grant APQ-02167-21 from the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), grant APQ-408675/2018-7 from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) both from Brazil, and by grant MR/R005850/1 from the Medical Research Council (VAccine deveLopment for complex Intracellular neglecteD pAThogEns - VALIDATE), UK. The authors also thank the Brazilian agencies Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and CNPq for the scholarship and fellowship.
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Keywords: Leishmania, biomarkers, immunoproteomics, phage display, vaccine, diagnostic
Citation: Ludolf F, Ramos FF and Coelho EAF (2023) Immunoproteomics and phage display in the context of leishmaniasis complexity. Front. Immunol. 14:1112894. doi: 10.3389/fimmu.2023.1112894
Received: 30 November 2022; Accepted: 23 January 2023;
Published: 08 February 2023.
Edited by:
Joseli Lannes-Vieira, Oswaldo Cruz Foundation (FIOCRUZ), BrazilReviewed by:
Fariborz Bahrami, Pasteur Institute of Iran, IranCopyright © 2023 Ludolf, Ramos and Coelho. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Fernanda Ludolf, feludolf@gmail.com