The potential of live attenuated vaccines against Cutaneous Leishmaniasis

Journal Pre-proof The potential of live attenuated vaccines against Cutaneous Leishmaniasis A. Zabala-Peñafiel, D. Todd, H. Daneshvar, R. Burchmore PII:

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YEXPR 107849

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Experimental Parasitology

Received Date: 7 October 2019 Revised Date:

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Accepted Date: 1 February 2020

Please cite this article as: Zabala-Peñafiel, A., Todd, D., Daneshvar, H., Burchmore, R., The potential of live attenuated vaccines against Cutaneous Leishmaniasis, Experimental Parasitology (2020), doi: https://doi.org/10.1016/j.exppara.2020.107849. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc.

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The poten potential of live live attenuated vaccines against Cutaneous Leishmaniasis

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Zabala-Peñafiel, A. , Todd, D. , Daneshvar, H. , Burchmore, R.

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Janeiro, Brazil; Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences,

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University of Glasgow, Glasgow, United Kingdom; Leishmaniasis Research Center, Kerman University of Medical

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Sciences, Kerman, Iran.

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Corresponding author: [email protected]

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Abstract

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Cutaneous Leishmaniasis is a serious public health problem, typically affecting poor populations with limited access

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to health care. Control is largely dependent on chemotherapies that are inefficient, costly and challenging to deliver.

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Vaccination is an attractive and feasible alternative because long-term protection is typical in patients who recover

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from the disease. No human vaccine is yet approved for use, but several candidates are at various stages of testing.

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Live attenuated parasites, which stimulate long-term immune protection, have potential as effective vaccines, and

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their challenges relating to safety, formulation and delivery can be overcome. Here we review current data on the

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potential of live attenuated Leishmania vaccines and discuss possible routes to regulatory approval.

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Keywords

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Leishmania, vaccines, live attenuated parasites, leishmanization, cutaneous Leishmaniasis.

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Leishmania and Leishmaniasis

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Leishmaniasis is a neglected tropical and emerging disease and a public health problem among some of the poorest

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populations across the world (Alvar et al., 2012). Around 1.5 million new cases occur each year, resulting in

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significant mortality and morbidity (WHO, 2017). Leishmaniasis is controlled by chemotherapies that have significant

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side effects and which may be costly and/or of limited efficacy. A zoonosis, Leishmaniasis is difficult to control

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through transmission-blocking strategies. The ongoing Syrian humanitarian crisis has resulted in displacement of

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more than 7 million people to neighbouring endemic areas, leading to a dramatic raise of Leishmaniasis cases in the

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Middle East at a time when public health systems face myriad challenges (Ozkeklikci et al., 2017).

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Leishmaniasis is caused by infection of mononuclear phagocytes with protozoa of the genus Leishmania. Infections

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can persist for months or years, causing a range of pathologies that are determined by the species of parasite but

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which are also influenced by host response (Akhoundi et al., 2016). Leishmania parasites are transmitted to a

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mammalian host through the bite of blood-feeding sandflies, which deliver the flagellated promastigote stage into

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the skin, where they may be phagocytosed by potential host cells. Promastigotes that survive the challenges

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Laboratório de Biologia Molecular e Doenças Endêmicas, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de 2

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presented by the host cell transform to an amastigote form, which can multiply and disseminate as an obligate

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intracellular form (Nylén and Gautam, 2010).

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More than 20 human-infective Leishmania species are recognised. Cutaneous Leishmaniasis (CL), associated with a

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range of pathologies that may be severe and sustained, but which are typically not fatal, is predominantly caused by

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Leishmania major and Leishmania tropica in the Old World and by Leishmania mexicana complex and subgenus

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Viannia in the New World. Visceral Leishmaniasis (VL), which is commonly fatal if untreated, is caused by Leishmania

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donovani and closely related species (Murray et al., 2005).

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CL is the most common form of Leishmaniasis, characterized by skin lesions that can be ulcerative but typically

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self-healing over a period of months or years (Murray et al., 2005). After resolution of a primary ulcer, a low number

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of parasites remain, and they can cause different outcomes depending on the parasite species and host status.

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Experiments with mice indicate that the host immune cells responsible for processing these parasites can stimulate

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naïve CD4 T cells to differentiate into either effector T cells, which tackle the current infection, or memory T cells,

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which can activate and protect the host upon a subsequent infection (Glennie and Scott, 2016). Additionally,

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immunocompromised individuals who do not exhibit a T cell-mediated response, can develop a severe diffuse CL

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(Carvalho et al., 1994; Scott and Novais, 2016). In 5-20% of CL cases, an exaggerated immune response against the

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remaining parasites can cause a disseminated muco-cutaneous Leishmaniasis (MCL), which can destroy the mucous

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membranes in the naso-oropharyngeal cavities (Handler et al., 2015; Scott and Novais, 2016). American

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Tegumentary Leishmaniasis (ATL), endemic in Latin America, is caused exclusively by Leishmania of the subgenus

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Viannia, L. amazonensis and L. mexicana complex and can lead to MCL and, sometimes, to VL (de Luca and Macedo,

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2016).

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Chemotherapy is a key tool in the control of Leishmaniasis (WHO, 2017), but the side-effects associated with the

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available drugs are such that treatment must be delivered in a clinical setting at a significant cost. Available

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chemotherapies do not result in sterile cure, probably because minimum inhibitory concentrations of drug are not

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achieved in all of the intracellular niches the parasite can access (Laskay et al., 2008; Oullette et al., 2004,

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Ponte-Sucre et al., 2017). This may promote the selection of drug resistance and allow for recrudescent infection,

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but may also sustain immunity against further infections. Thus, in this critical context for Leishmaniasis control,

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exposure to attenuated parasites through vaccination could greatly reduce the dependence on chemotherapy,

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relieving morbidity and stress on healthcare budgets, and slowing the emergence of drug resistance (De Luca and

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Macedo, 2016; Handman et al., 2001).

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The immune immune response to Leishmania

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The immune response to Leishmania depends on very complex cellular interactions between host and parasite. The

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well described TH1/ TH2 dichotomy suggests that an effective immune response against Leishmania involves a

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balance between two different populations of CD4 cells, which can be host-protective T Helper 1 (TH1) and

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disease-promoting T Helper 2 cells (TH2) (Kedzierski, 2011; Liew, 1989). This was shown in studies of mice infected

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with L. major but is less evident in cases caused by L. amazonensis and L. braziliensis (De Luca and Macedo, 2016).

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However, it is generally accepted that the functional heterogeneity of T cells is associated with macrophage

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phenotypes (Liew, 1989) which can be either classically activated (M1) to kill/inhibit parasites or alternatively

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activated (M2) to permit parasite survival and multiplication (Mills and Ley, 2014). M1 activation is mediated by

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IFN-γ that stimulates nitric oxide (NO) and reactive oxygen species (ROS) production, and enhances macrophage

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killing ability (Olivier et al., 2005). IL-12 is the main cytokine that induces IFN-γ production and TH1 cell

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differentiation, leading to further secretion of pro-inflammatory cytokines, parasite clearance and control of disease

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(Liu and Uzonna, 2012).

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Although macrophages are the main host cells up taking Leishmania promastigotes, neutrophils and dendritic cells

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(DCs) can also recognise and phagocytose these parasites. Depending on the infecting species, neutrophils can

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either protect or kill Leishmania. For example, L. major parasites are able to survive within polymorphonuclear

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granulocytes neutrophils (PMNs) using them as “Trojan horses” to subsequently and silently infect macrophages,

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inhibiting M1 and promoting their survival (Laskay et al., 2003). Conversely, L. amazonensis promastigotes are killed

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by neutrophils through the release of neutrophil extracellular traps (NETs) (Guimaraes-Costa et al., 2009).

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Neutrophils can control L. braziliensis and L. amazonensis infection by becoming apoptotic and interacting with

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macrophages to activate M1 and induce ROS production and parasite killing (Novais et al., 2009).

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DCs are part of the family of professional antigen presenting cells, along with macrophages, and capture and

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process antigens in all peripheral non-lymphoid tissues, functioning as immune system sentinels (Brandonisio et al.,

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2004). After contact with parasites, these cells mature and migrate to the T lymphocytes of the lymphoid organs,

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where they present antigens to naive T lymphocytes and modulate their response. DCs are flexible and can promote

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TH1, TH2 or a regulatory T response profile (TREG) (reviewed by Liu and Uzonna, 2012).

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ROS production is a primary defence orchestrated by the innate immune response through macrophages, but when

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Leishmania parasites evade this and survive, the adaptive immune response begins to secrete IFN-γ that stimulates

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higher ROS and NO production. However, Leishmania can elaborate several evasion mechanisms which allow them

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to inhibit ROS and NO production, alter the cytokine cascade, dysregulate the TH1/ TH2 balance and change M1/M2

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phenotypes in order to modulate the host immune response to their advantage (Figure 1).

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Figure 1. Immunomodulation induced by Leishmania infection. Metacyclic promastigotes enter the host through the

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dermis and are engulfed by phagocytes, as the sandfly vector ingests and regurgitates blood during feeding.

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Metacyclic promastigotes can encounter three host phagocytes as follows: 1) In the macrophage (MØ), intracellular

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amastigotes survive and multiply within the parasitophorous vacuole, limiting secretion of pro-inflammatory

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cytokines IL-12 and IFN-γ (black dashed arrows). 2) Metacyclic promastigotes use the polymorphonuclear neutrophil

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granulocyte (PMN) as “Trojan horse” to survive within the phagosome, limiting the secretion of pro-inflammatory

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cytokine TNF-α and increasing production of anti-inflammatory cytokine TGF-β (black solid arrows). Also, the

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infected neutrophil can become apoptotic and signal the recruitment of more MØ which will engulf it without

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recognising the parasite, silencing the MØ killing ability (M1) and inhibiting secretion of ROS and NO (red dashed

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line). 3) In the dendritic cell (DC), intracellular amastigotes survive within the phagosome and limit the secretion of

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pro-inflammatory IL-12 (black dashed arrow) while increasing secretion of anti-inflammatory IL-10 and IL-4. The low

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levels of pro-inflammatory cytokines inhibits CD4 T cell differentiation into TH1 cells – a protective response against

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infection - whereas the high secretion of anti-inflammatory cytokines leads to a polarization of CD4 T cell

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differentiation into TH2 cells (yellow solid arrows), activating the alternative MØ phenotype (M2) and allowing

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parasite survival and establishment of infection.

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Immunity and Control of Pathology

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The complex interaction between Leishmania and the host immune system means that a robust immune response

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to Leishmania infection may not lead to sterile immunity. An immune response that is sufficient to keep pathogen

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numbers supressed can effectively control pathology while permitting a stable and asymptomatic infection which

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supports concomitant immunity (Brown and Grenfell, 2001). A host that supports a sub-patent Leishmania infection

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is protected from symptoms arising from subsequent infection with a different Leishmania population, though not

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necessarily protected from a resulting mixed infection (Mandell and Beverley, 2016). Concomitant immunity has

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benefits for the parasite, since a sustained infection allows for transmission over time while providing an

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environment for parasite genetic exchange. It also has potential benefits to the host, which is protected from

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symptomatic Leishmaniasis while it supports a sub-patent infection (Mandell and Beverley, 2016). This phenomenon

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has been exploited since antiquity in a practice known as leishmanization, which involved transfer of material from a

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leishmaniasis lesion to a naïve individual (Nadim et al., 1983).

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More recently, leishmanization involved immunization with live virulent parasites from culture, usually in an

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inconspicuous area of the body, to protect the recipient from subsequent disfiguring natural infection, which

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typically occurs on the face or limbs exposed to insect bites (Nadim et al., 1983). In the 1980’s, large-scale

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leishmanization was performed in the Soviet Union and Israel with variable success, depending on the viability and

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infectivity of the parasite used (Mitchell et al., 1987). During the Iran-Iraq war, the Iranian Government launched a

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“mass leishmanization” program for soldiers and refugees, to control increased CL incidence (Nadim and Javadian,

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1988). Leishmanization is no longer employed as a public health strategy, because of potential negative outcomes

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from deliberate infection with poorly characterised and virulent parasites. Large, poorly resolving ulcers and allergic

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reactions were reported (Nadim and Javadian, 1988) but there were also concerns about the emergence of new

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cases in non-endemic areas (Mitchell et al., 1987) and the risk associated with vaccination of immunosuppressed

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individuals (Silvestre et al., 2008). Nevertheless, leishmanization demonstrates the potential to exploit concomitant

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immunity for vaccination against leishmaniasis, and has encouraged efforts to develop antileishmanial vaccines.

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Concomitant immunity decreases in hosts where sterile cure is achieved, showing that a sustained immune

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stimulation by viable parasites is required. This suggests that killed parasites or molecular vaccines may be less

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effective than live parasites (Tabbara et al., 2005). Live parasites, establishing natural infection, mean the parasite

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will present the native antigens needed to induce a TH1 protective response (Silvestre et al., 2008). CD4 T cells are

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the predominant mediators of protection and a viable parasite population may be required to sustain a pool of

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these cells and mount a protective response when challenged by a secondary infection (Glennie and Scott, 2016).

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However, a vaccine that elicits a durable memory T cell population could confer protection without requiring

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continual immune stimulation from a persistent parasite population. To this end, a live attenuated vaccine that can

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establish an infection but which ultimately succumbs may be ideal. Leishmania parasites display different levels of

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virulence and can readily become attenuated although, depending on the genetic basis, attenuation may be

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reversible (Sinha et al, 2018).

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Strategies for Attenuation of Leishmania Parasites

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Extensive research supports the use of canine and mice models along with different methods to attenuate live

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parasites, to test their efficacy and safety as vaccine candidates (Costa et al., 2011). The majority of studies have

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been done with L. mexicana and L. major for CL, and L. infantum and L. donovani for VL (Costa et al., 2011; Silvestre

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et al., 2008). This review is focussed on the development of vaccines to control CL, but we have included some

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relevant data on VL. Live attenuated parasites present some obvious hurdles as vaccine candidates, in particular the

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possibility of reversion to a virulent state (Vergnes et al., 2005); standardization and large-scale production in

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endemic areas (Cruz et al., 1991); and immune differences between clinical models and the final human host

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(Kedzierski et al., 2006).

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Live attenuated parasites can be separated into 2 groups, according to the attenuation strategy used: undefined and

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defined genetic modification (Silvestre et al., 2008). Undefined attenuation can occur through long term in vitro

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culture, with or without overt selection, through irradiation or by chemical mutagenesis. Defined attenuation

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involves specific mutagenesis to derive mutant parasites that no longer encode on or more genes important for

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virulence. The development of reverse genetic strategies for Leishmania has facilitated defined attenuation through

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targeted gene “knockout”, which has the significant advantage that parasites are unlikely to recover the deleted

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gene(s) (Saljoughian et al., 2014). Undefined attenuation may result in parasites that can readily revert to virulence

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or that display reduced immunogenicity. The remarkable plasticity of the Leishmania genome (Sterkers et al, 2012)

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presents both opportunities for the selection of attenuated lines, but also raises significant issues regarding the

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stability of such lines. The attenuation strategies discussed in this review are summarised in Table 1 and discussed

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below.

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In several earlier studies, chemical mutagenesis was used to generate Leishmania mutants that were assessed for

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attenuation. BALB/c mice, immunized with L. braziliensis promastigotes that had been cloned after mutagenesis,

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showed a 70% reduction in lesion size upon challenge with the virulent parental strain, compared with lesions in

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naïve control mice.

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Chemical mutagenesis of L. major was found to generate mutant parasites with diminished lipophosphoglycan (LPG)

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expression (Kimsey et al, 1993) The Leishmania cell surface is covered by phosphoglycans; LPGs attached by

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glycophosphoglycans anchors; proteophosphoglycans attached through proteins; and small glycoinositol

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phospholipids. Altogether, these molecules are essential for infection, virulence and pathogenesis in the mammalian

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host (Späth et al., 2003). Indeed, LPGs have a critical role in the inhibition and subversion of host killing machinery at

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early stages of infection (Olivier et al., 2005). LPG deficient L. major promastigotes were engulfed and killed by

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BALB/c macrophages and those that survived were not able to replicate. The absence of CL lesions in susceptible

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BALB/c mice showed that LPG is a virulence factor needed for establishment of L. major infection (Kimsey et al.,

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1993).

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In vitro selection with the aminoglycoside antibiotic gentamicin was used to attenuate L. major, L. mexicana and L.

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infantum (Daneshvar et al., 2003a). Gentamicin binds to the 30S ribosomal subunit and targets the RNase P complex

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in eukaryotes (Walter et al., 1999). Gentamicin-attenuated lines infected, but were unable to survive as intracellular

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amastigotes, in BABL/c macrophages. They elicited higher secretion of IFN-γ and lower IL-4 and IL-10, potentiating a

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TH1 protective immune response (Daneshvar et al., 2003a). BALB/c mice immunized with gentamicin-attenuated L.

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major were protected upon challenge with virulent L. major (Daneshvar et al., 2003b; Daneshvar et al., 2009a). In L.

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major infection in mice, IgG1 production is mediated by TH2 cytokines while IgG2 is promoted by IFN-γ, a TH1

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cytokine (Mohammadi et al., 2006).

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IgG1 levels than mice infected with virulent L. major, suggesting potentiation of a TH1 response (Daneshvar et al.,

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2009b) .A gentamicin-attenuated L. infantum vaccine proved effective in a field trial in dogs (Daneshvar et al, 2014).

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L. major is currently approved for usage in live attenuated vaccine clinical trials in humans (Alvar et al., 2013) and

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the gentamicin-attenuated L. major vaccine is currently in human trials (Daneshvar, unpublished).

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Encouraging results with gentamicin-attenuated Leishmania prompted efforts to identify the molecular lesions

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responsible for loss of virulence. Comparative proteomic analysis of virulent and attenuated L. infantum revealed a

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significant up-regulation of LiAlba13, an Alba-domain protein, in attenuated parasites, together with a dysregulation

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of tryparedoxin peroxidase (Daneshvar et al., 2012). The Alba family are RNA binding proteins found in eukaryotes

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(Goyal et al., 2016). The L. infantum genome encodes two Alba-domain proteins, Alba13 and Alba20, which

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physically interact with each other and other ribosomal subunits (Dupe et al., 2014; Dupe et al., 2015), including

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subunits of the RNase P complex known to be a target of aminoglycosides such as gentamicin (Aravind et al., 2003).

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Upregulation of LiAlba13 may thus reflect adaptation to gentamicin selection (Daneshvar et al., 2012). Modulation

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of tryparedoxin peroxidase expression may suggest a response to oxidative stress and, indeed,

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gentamicin-attenuated L. infantum were more susceptible to oxidative challenge (Daneshvar et al., 2012). Recently,

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altered protein redox state has been reported in gentamicin- attenuated L. mexicana (Prakash et al, 2018).

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Molecular characterisation of undefined attenuated lines may enable the generation of defined mutants that

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recapitulate this proven attenuated vaccine (Burgess & Burchmore, 2012).

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The ability to manipulate the Leishmania genome in a systematic manner has allowed the construction of defined

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live attenuated vaccines. Specific genes can be disrupted in the diploid Leishmania genome by knockout of one or

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both alleles (Dey et al, 2013). Mutants retaining one allele, while often displaying phenotypic differences with

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respect to wild type parasites, may be unsuitable for vaccine applications due to concern about facile reversion.

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Homozygous knockout mutants are attractive for stable attenuation of parasite virulence, but must be conditionally

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viable to permit in vitro generation and to allow at least transient survival in the recipient host. Sustained survival

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without eliciting pathology is the ideal for protective immunity, but a challenging goal to achieve, given the

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variability in host response.

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Encouraging results were obtained with one of the earliest gene knockouts reported in Leishmania (Titus et al.,

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1995), for the dihydrofolate reductase-thymidylate synthase gene (Δdhfr-ts) essential for Leishmania amastigote

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viability (Cruz et al., 1991). Mice immunized with this mutant displayed dramatically reduced lesions when

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challenged with wildtype L. major and the attenuated line appeared to be avirulent in both susceptible and

BALB/c mice immunized with gentamicin-attenuated L. major displayed lower

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immunodeficient mice (Titus et al., 1995).

However, tested in a Rhesus monkey model, Δdhfr-ts gave no significant

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protection to subsequent challenge, although it was notable that the attenuated parasites persisted in the monkey

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host for months (Amaral et al., 2002). Subsequently, a variety of other Leishmania knockout mutants have been

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tested for their potential as attenuated vaccines

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L. mexicana encodes multiple cysteine protease genes (cpa, cpb and cpc) which are thought to be responsible for

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enhancing IL-4 production and, as a result, potentiate a TH2 response promoting parasite survival and supressing M1

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macrophage activation (Alexander et al., 1998). In vivo, a cpa gene knockout (Δcpa) could simulate a natural

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infection at the phenotypic level (Souza et al., 1994), while Δcpb mutants infected macrophages with markedly

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reduced efficiency. In BALB/c mice, Δcpb mutants produced lesions, albeit at a slower rate (Mottram et al., 1996). L.

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mexicana lacking both cpa and cpb

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1998). Peripheral blood monocyte cells (PBMCs) from hamsters infected with Δcpa/cpb expressed lower levels of

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IL-10 and TGF-β levels, compared with PBMCs infected with wild type L. mexicana, suggesting induction of TH1

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protective response (Saravia et al., 2006).

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Following encouraging results from the application of chemical mutagenesis to disrupt LPG expression in L. major

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(Kimsey et al., 1993), an lpg1 gene knockout (Δlpg1) was generated and showed delayed survival and lesion

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development in BALB/c mice (Späth et al., 2000). However, some Δlpg1 amastigotes persisted and led to pathology

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(Späth et al., 2000). A LPG2 gene knockout (Δlpg2) was less virulent in vitro and, in in vivo tests, mutant parasites

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persisted at the infection site over two years without causing pathology (Späth et al., 2003). Further studies

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revealed that some Δlpg2 amastigotes that were able to regain replication ability and induce cutaneous pathology in

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mice (Späth et al., 2004). This work highlights the concern that attenuated parasites may revert to a virulent

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phenotype though compensatory mutations.

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Genes with important roles in amastigotes may be readily targeted for knockout in the promastigote stage, allowing

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the production of live attenuated Leishmania vaccines that are compromised as the intracellular form. p27 is a

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subunit of the cytochrome c oxidase (COX) complex in the mitochondrial membrane and important to maintain COX

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activity in amastigotes (Dey et al., 2010). BALB/c mice infected with L. major ΔLmp27 mutants showed no significant

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lesion development and significantly lower parasite load, compared to wild type L. major (Elikaee et al., 2018).

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major ΔLmp27 immunized mice were challenged with either L. major or L. infantum and were protected against

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both (Elikaee et al., 2019). Analysis of cytokine production and antibody response showed increased expression of

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IFN-γ and IgG2a in both immunised groups, while the IFN-γ/IL-4 and IgG2a/IgG1 ratios showed a polarization

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towards a TH1 response profile (Elikaee et al., 2019). L. major ΔLmp27 mutants showed a reduced of rate

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proliferation inside macrophages, presumably resulting in reduced virulence but sufficient persistence to mount a

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cell-mediated protective response.

(Δcpa/cpb) showed a similar phenotype (Mottram et al., 1996; Alexander et al.,

L.

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A drawback with Leishmania attenuated through targeted gene knockout, and a reason for their restriction in

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clinical studies, is the presence of antibiotic resistance genes used to select knockout mutants (Silvestre et al., 2008).

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For instance, Δcpa/cpb mutants acquired resistance to the antibiotics used to delete cpa and cpb genes (Saravia et

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al., 2006). The use of such mutants as vaccines might contribute to the emergence or spread of drug resistance,

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amongst Leishmania parsites (Denise et al., 2004), or more generally. Various strategies allow the selection of

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Leishmania knockout mutants that do not retain antibiotic resistance genes (Denise et al., 2004), that are negatively

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selected with metabolic markers (Gueiros-Filho & Beverley, 1996) or that are selected with Leishmania-derived

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resistance markers (Zhang and Matlashewski, 2015). The rapid development of CRISPR Cas9 gene editing technology

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will accelerate the production of gene knockout mutants in Leishmania (Beneke et al, 2017; Zhang and

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Matlashewski, 2015), allowing more rapid testing for attenuated phenotypes.

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Live attenuated vaccines to control other Leishmaniases with gentamicin-attenuated L. infantum in dogs show LeishmaniasesStudies ses

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no clinical signs of disease but induction of anti-Leishmania IgG (Daneshvar et al., 2009b). Subsequently, a field trial

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was established in Iran, by introducing approximately 100 leishmanaisis-naïve dogs from a non-endemic region to a

258

rural area endemic for canine Leishmaniais. Approximately half of the dogs were immunized with attenuated L.

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infantum and all were monitored over a 24 month period, during which they were potentially exposed to

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Leishmaniasis infection. After 24 months, 30% of the unvaccinated dogs had acquired Leishmaniasis while only one

261

of those vaccinated showed any clinical signs of disease (Daneshvar et al., 2014). Canine leishmaniasis is a significant

262

problem, from the perspectives of both zoonosis and animal health, but the ethical hurdles that must be overcome

263

to establish clinical trials are less daunting that for human studies.

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L. donovani lacking the centrin1 gene (Selvapandiyan et al., 2004) gave protection in BALB/c mice and hamsters

265

(Selvapandiyan et al., 2009), and subsequently in dogs (Fiuza et al., 2013; Fiuza et al., 2015). Similarly, mice

266

immunized with L. donovani lacking the p27 gene were protected (Dey et al., 2013). Importantly, centrin1 and p27

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knockout lines also conferred heterologous protection against L. major and L. braziliensis challenge (Carrion et al.,

268

2011; Selvapandiyan et al., 2009; Dey et al., 2013) and the L. donovani centrin1 mutant protected dogs against

269

challenge with L. infantum (Fiuza et al., 2015). Currently, only L. major is approved for live attenuated vaccines

270

clinical trials in humans (Alvar et al., 2013), meaning that progress will likely be more rapid with testing L. major

271

vaccines, which may prove to confer heterologous protection against other co-endemic species.

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Table 1. Strategies to attenuate CL Leishmania species. species. Attenuation Strategy Chemical mutagenesis (Kimsey et al., 1993)

Undefined genetic modifications

Leishmania species species - model

Result

L. major - BALB/c

Decreased lesion size.

L. major - BALB/c

>TH1 vs.
Gentamicin pressure (Daneshvar et al., 2003b; Daneshvar et al., 2009a; Daneshvar et al., 2009b)

Gentamicin pressure (Daneshvar et al., 2003a)

Mutagenesis and temperature pressure (Gorczynski, R., 1985) dhfr-ts gene knockout (Titus et al., 1995; Amaral et al., 2002)

Cysteine protease a or b gene knockout (Mottram et al., 1996;

L. mexicana - BALB/c

Response polarised towards TH1.

L. braziliensis - BALB/c

Decreased lesion size, antibodies related to TH1.

L. major - BALB/c

Response polarised towards TH1.

L. major - Rhesus monkey

Failed to elicit protection.

L. mexicana – BALB/c

>TH1, slower parasite growth and smaller lesions.

Alexander et al., 1998)

Defined genetic modifications

No lesions.

Cysteine protease a and b gene knockout (Alexander et al.,

L. mexicana - BALB/c

1998; Saravia et al., 2006)

L. mexicana - Hamster

Delayed lesions but disease reversion.

LPG gene knockouts (Späth et al., 2000; Späth et al., 2003, Späth et al., 2004, Liu et al., 2009)

L. major - BALB/c

Decreased lesions but disease reversion.

Paraflagellar rod-2 gene knockout (Sollelis et al., 2015)

L. major - BALB/c

Non-motile parasites, decreased lesions.

LmxM.22.0010 gene knockout (Ishemgulova et al., 2018)

p27 gene knockout (Elikaee et al., 2018; Elikaee et al., 2019)

L. mexicana - BALB/c

L. major - BALB/c

Successful colonization of Lutzonmyia longigalpis, same lesion size as Wt infection. >TH1, no lesion development, homologous protection and heterologous protection against L. infantum.

274 275

Routes to development of a live attenuated vaccine to control Cutaneous Leishmaniasis

276

Given the drawbacks with chemotherapy and vector control, a prophylactic vaccine for Leishmaniasis is an attractive

277

goal. It is feasible, given the protective immunity that can be demonstrated after infection with Leishmaniasis.

278

Efficient immunity against parasitic diseases requires sustained antigen stimulation since immunity decreases when

279

this stimulation ceases. Live vaccines can sustain antigen presentation, in a native context, but attenuation of

280

virulence is important to avoid bestowing the disease against which the vaccine is intended to protect. We have

281

outlined some of the strategies that have been employed to generate live attenuated vaccines which can control

282

Leishmaniasis in animal models. Some of the most promising results have been obtained with

283

gentamicin-attenuated and gene knockout parasites. These strategies might be combined in a “reverse vaccinology”

284

approach where protective antigens are identified in attenuated lines and then targeted by gene knockout to

285

generate attenuated lines with defined changes, possibly with an emphasis on conserved antigens that confer

286

heterologous protection.

287

Safety is the paramount consideration before such vaccines can be tested in humans. Cutaneous Leishmaniasis

288

caused by L. major is typically associated with limited pathology, and can be controlled by appropriate

289

chemotherapy, allowing the possibility that vaccine recipients can be treated if necessary. For some of the

290

attenuated Leishmania described, reversion to a virulent form seems unlikely, but concerns about the interaction

291

between an attenuated parasite and an immunocompromised host remain to be addressed. The concern that

292

attenuated parasites might recombine with virulent parasites in the host is difficult to assess (King et al., 2015) but

293

the concomitant immunity that is conferred by a persisting parasite, whether inoculated as a vaccine or through

294

natural infection, does not inhibit the establishment of further infections. Thus, in an endemic situation, it is possible

295

that attenuated parasites will share a host with virulent parasites, and could be transmitted together to a sandfly

296

vector, where recombination is known to occur (Akopyants et al, 2009). It is difficult to conceive how recombination

297

between attenuated and virulent parasites could give rise to progeny of enhanced virulence, although this is an

298

argument to avoid the use of mutants that carry drug resistance markers. Studies in dogs with

299

gentamicin-attenuated L. infantum suggest that these parasites do not multiply, are not detected beyond the site of

300

inoculation and are not detected at all months after vaccination (Daneshvar et al, 2014), so the likelihood of an

301

attenuated vaccine line encountering a virulent parasite may be minimal. An attenuated vaccine that can stimulate

302

enduring memory T cells need not persist in the host to maintain immunity.

303

Practical issues around the production and delivery of live Leishmania preparations are significant, but there are

304

existing biotechnology products based on live Leishmania (https://www.jenabioscience.com/lexsy-expression) and

305

health care, education and existing cold chain infrastructures might readily be adapted for the delivery of other

306

vaccines in most Leishmaniasis-endemic regions (Lloyd & Cheyne, 2017). Economic issues are also relevant, as the

307

cost of bringing a new vaccine to approval is significant (Srivastava et al., 2016), while the market for an

308

antileishmanial vaccine is unlikely to be profitable. Most of the research described herein has been performed in

309

academic laboratories with charity funding and progressing from proof-of-principle studies to field trails and product

310

development is challenging for such groups. The majority of attenuated vaccines have been tested only in rodent

311

models and there is a need for an improved pipeline to move to human trials.

312

development increased from 2007 to 2013, suggesting a greater interest in financing development of vaccines to

313

control important, global diseases such as Leishmaniasis (Gillespie et al., 2016).

314

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Leishmaniasis is a vaccinable disease Live attenuated Leishmania parasites can stimulate long term protection from disease We discuss challenges and possible routes to approval of live attenuated Leishmania vaccines