Apoptotic blebs from Leishmania major-infected macrophages as a new approach for cutaneous leishmaniasis vaccination
Introduction
The Neglected Tropical Disease (NTD) leishmaniasis is endemic in 98 countries affecting approximately 12 million people per year with 350 million people living at risk of disease (World Health Organization). Causative Leishmania species are sand fly borne kinetoplastid parasitic protozoa [1] and infection can lead to a wide spectrum of clinical pathologies, from self-healing but scarring cutaneous leishmaniasis (CL) to fatal visceral disease (VL). Amongst other factors, this diversity of disease is dependent on the parasite species, host immunity, and genetic background [2]. Largely due to elimination efforts in south Asia, the global burden of VL has decreased substantially in the past decade. However, due to forced migration, the cases of CL have substantially increased in the same period [3]. Approximately, 75% of reported CL cases occur in 10 countries with an incidence rate of 0.7–1.3 million cases annually. However, the actual prevalence of this disease is estimated to be 6 to 10 times higher [4,5].
Controlling reservoirs and vectors, providing diagnostics and treatment, and the emergence of drug-resistant species are serious challenges for the control of CL. These issues illustrate the need for new drugs for CL or effective vaccines [[6], [7], [8]]. There are three generations of vaccine used against leishmaniasis, 11 of which entered in clinical trials. These include Leishvaccine, ALM, Leishmune, CaniLeish, and GALM for first generation, LEISH-F1, LEISH-F2, LEISH-F3, Leish-Tec, and SMTγ+NHμ for second generation, and ChAd63-KH for third generation. Among these, Leishmune®, CaniLeish®, and Leish-Tec were approved to be used as vaccine in animal [9]. Therefore, there is still no appropriate vaccine available for human use.
In Iran in the 1970s and 1980s vaccination against CL widely used intradermal inoculation of live promastigotes of L. major (2–3 × 105), leishmanization. However, this practice was discontinued due to drawbacks such as ulcer development in a few susceptible individuals [10]. Immunity on leishmanization has been proposed to be due to vaccinated people harboring live parasites in their skin which release excretory/secretory antigens (ESAs), stimulating the host immune system and induce protection [11]. Accordingly, researchers have focused on Leishmania spp. ESAs as vaccine targets [12]. However, it is now known that the protective immunity against L. major is related to immune system memory, not the parasite presence in the skin tissue [13,14]. In light of this, studies have focused on several types of vaccine, including live or live-attenuated parasites, the whole killed parasites, Leishmania spp. antigens and naked DNA-encoding parasite antigens. However, none so far have had the efficacy to be developed for use as a vaccine in humans [15,16]. Against this backdrop, we focused on a new approach to vaccination against CL: apoptotic blebs from Leishmania major-infected macrophages.
Apoptotic blebs/bodies contain phosphatidylserine and phosphorylcholine on their surface, facilitating the clearance of these bodies by antigen presenting cells such as macrophages [17]. After phagocytosis of an apoptotic bleb, the antigen presenting cells process and present antigenic epitopes to the adaptive immune cells on class I and class II major histocompatibility complex (MHC-I and -II) molecule. However, there is preferential antigen presentation on MHC-I and therefore the cell-mediated immune response in stimulated, this is important for an effective CL vaccine [[18], [19], [20], [21]]. Therefore, in this study we evaluated apoptotic blebs from L. major-infected macrophages as a vaccine candidate for CL in a murine model.
Section snippets
Leishmania maintenance
Leishmania major (MRHO/IR/75/ER) was used in the present study. Promastigotes were cultured and maintained in RPMI-1640 medium (Gibco, Grand Island, NY, USA) supplemented by fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and penicillin-streptomycin solution (pen-strep; 100 IU/mL penicillin and 100 μg/mL streptomycin; Sigma-Aldrich, Missouri, USA). In order to reduce the proliferation of the parasite and to maintain it without passage for about 2 months, the amount of FBS was adjusted to
Quantity and quality of apoptotic blebs from L. major-infected macrophages
The blebs were quantified using flow cytometry. 86.6% of the bodies identified were apoptotic, of which 30.1% were early apoptotic bodies and 56.5% were late apoptotic bodies. The total number of these bodies was 20,606,700 per mL (Fig. 1).
The quality of the apoptotic bodies was established using SEM. The average size was 3.88 μm (95% CI 2.69–5.07), and the bodies were uniform and slightly oval (>98%) in shape. All had an intact cell membrane (Fig. 2).
Cytokine and antibody assay
IFN-γ, IL-4, and total IgG levels were
Discussion
There are many challenges for the development of a CL vaccine, including the genetic diversity in human populations and in the species and strains of Leishmania parasite, the type of vaccine, dose of vaccine, and route of vaccine administration [27]. The purpose of the vaccination is the development of immunological memory, and both CD4+ and CD8+ T cells are important for the immunity against L. major. Therefore, it is clear that an effective vaccine should not only be safe and easily available
Conclusions
In the presented study, apoptosis was induced in Leishmania-infected macrophages and the apoptotic-blebs collected. These bodies will contain fragments of the parasite and together can function as analogous to a whole Leishmania. Whilst the mechanism of inducing protective immune response has not been evaluated for the apoptotic-blebs from L. major-infected macrophages in the current study, it is likely that these ‘fragments’ constitute a complex antigenic picture necessary for immunity. The
Author statement
Roghiyeh Faridnia: Leishmania Maintenance, Apoptosis Induction, Preparation of Leishmania Lysate Antigens, Immunization of Mice. Hamed Kalani: Flow Cytometry, Scanning Electron Microscope, Lymphocyte Proliferation Assay, Design of the Study, Drafting the Manuscript. Hajar Ziaei Hezarjaribi: Immunization of Mice. Paul W. Denny: Writing-Reviewing and Editing. Alireza Rafie: Cytokine and Antibody Assay. Mahdi Fakhar: Infection Challenge, Data analysis, Writing and Editing. Stela Virgilio:
Declaration of competing interest
The authors declare that there is no conflict of interest.
Acknowledgments
The vice chancellor of research at the Mazandaran University of Medical Sciences, Sari, Iran supported this work with a grant (Grant number: 1481).
References (44)
- et al.
Cutaneous leishmaniasis
Lancet Infect. Dis.
(2007) - et al.
Leishmaniasis
Lancet
(2018) - et al.
Potent in vitro antileishmanial activity of a nanoformulation of cisplatin with carbon nanotubes against Leishmania major
J Glob Antimicrob Resist
(2019) - et al.
Mining for natural product antileishmanials in a fungal extract library
Int J Parasitol Drugs Drug Resist
(2019) - et al.
Leishmanization revisited: immunization with a naturally attenuated cutaneous Leishmania donovani isolate from Sri Lanka protects against visceral leishmaniasis
Vaccine
(2013) - et al.
IL-10 signaling in dendritic cells attenuates anti-Leishmania major immunity without affecting protective memory responses
J. Invest. Dermatol.
(2015 Nov) - et al.
Innate and adaptive immune response to apoptotic cells
J. Autoimmun.
(2007) - et al.
MHC molecules and microbial antigen processing in phagosomes
Curr. Opin. Immunol.
(2009) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Anal. Biochem.
(1976)- et al.
CD4+ T cell-mediated immunity against the phagosomal pathogen leishmania: implications for vaccination
Trends Parasitol.
(2019)