GHENT UNIVERSITY

FACULTY OF VETERINARY MEDICINE

Academic year 2015 - 2016

VACCINATION AGAINST LEISHMANIA: IMMUNOLOGICAL CHALLENGES AND REVIEW ON

AVAILABLE OPTIONS IN LABORATORY ANIMALS AND DOGS.

by

Nikita DE LAET

Promoters: Prof. dr. Louis Maes

Prof. dr. Edwin Claerebout

Literature Review

as part of the Master’s dissertation

© 2016 Nikita De Laet

DISCLAIMER

Universiteit Gent, its employees and/or students, give no warranty that the information

provided in this thesis is accurate or exhaustive, nor that the content of this thesis will not

constitute or result in any infringement of third-party rights.

Universiteit Gent, its employees and/or students do not accept any liability or responsibility for

any use which may be made of the content or information given in the thesis, nor for any

reliance which may be placed on any advice or information provided in this thesis.

GHENT UNIVERSITY

FACULTY OF VETERINARY MEDICINE

Academic year 2015 - 2016

VACCINATION AGAINST LEISHMANIA: IMMUNOLOGICAL CHALLENGES AND REVIEW ON

AVAILABLE OPTIONS IN LABORATORY ANIMALS AND DOGS.

by

Nikita DE LAET

Promoters: Prof. dr. Louis Maes

Prof. dr. Edwin Claerebout

Literature Review

as part of the Master’s dissertation

© 2016 Nikita De Laet

PREFACE

First of all, I would like to thank my promotor Prof. dr. Louis Maes for the incentive he gave me to write

this literature study about the immunological aspects in leishmaniasis and particular thanks for the

time he invested to bring my thesis to its present form. I would also like to thank my co-promotor Prof.

dr. Edwin Claerebout for his valuable suggestions.

Special thanks also for Prof. dr. Eric Cox for his help regarding the more difficult immunological

aspects of my literature study and Prof. dr. Guy Caljon for his additional suggestions and advice.

My friends and family were also a big help for me, especially my boyfriend Mathias Peene who has

read my thesis to check for spelling mistakes, helped me with the overall lay-out and the drawing of an

own Figure 2 summarizing the scope of my thesis.

TABLE OF CONTENTS

DISCLAIMER

FRONT PAGE

PREFACE

TABLE OF CONTENTS

ABSTRACT ......................................................................................................................................1

SAMENVATTING .............................................................................................................................2

INTRODUCTION ..............................................................................................................................4

LITERATURE STUDY ......................................................................................................................6

1. ANIMAL MODELS ....................................................................................................................6

1.1 Rodents ....................................................................................................................................... 6

1.1.1 The mouse ........................................................................................................................... 6

1.1.1.1 BALB/c mouse .................................................................................................................. 8

1.1.1.2 C57BL/6 mouse ................................................................................................................ 9

1.1.2 The hamster ....................................................................................................................... 10

1.2 Carnivores.................................................................................................................................. 11

1.3 Primates .................................................................................................................................... 13

1.4 Other animals ............................................................................................................................ 14

2. ANTILEISHMANIA VACCINES FOR DOGS ..........................................................................15

2.1 First-generation vaccines ................................................................................................................. 15

2.2 Second-generation vaccines ............................................................................................................ 15

2.3 Third-generation vaccines ............................................................................................................... 16

2.4 Commercial CVL vaccines ................................................................................................................ 16

DISCUSSION .................................................................................................................................18

REFERENCES ...............................................................................................................................19

1

ABSTRACT

Leishmaniasis is one of the more neglected tropical diseases that is gaining importance because the

parasite and its vectors are spreading. Although chemotherapeutics are available, a lot of emphasis is

currently being placed on preventive measurements such as vaccination, since the current drugs are not

devoid of adverse effects and are subject to drug resistance causing to post-treatment relapses. This

thesis reviews the general immunological aspects in Leishmania with particular focus on the animal

models, covering the BALB/c mouse (susceptible), the C57BL/6 mouse (resistant), the hamster, dogs

(important for the transmission to humans) and primates. The main immunological responses to

Leishmania infection (mostly L. major) can be summarized as follows: interleukin-12 (IL-12) modulates a T

helper 1 (Th1) response which leads to parasite killing and healing while IL-4, IL-10 and IL-13 steer a Th2

response which leads towards a non-healing response, causing progressive disease and even death.

First, second and third generation vaccines are briefly discussed. At present, second generation vaccines

are the most frequently used, for example the commercially available vaccines for dogs (Leishmune®,

Leish-Tec® and CaniLeish

®). These vaccines are not 100% effective because of the genetic differences

between Leishmania species/strains, the lack of a valid animal model and infection route that resembles

the natural way of infection and the polymorphism of the clinical outcome. There is yet no vaccine

available for humans, but one subunit vaccine is currently being developed.

Key words: animal models – immunology – Leishmania – leishmaniasis - vaccines

2

SAMENVATTING

Leishmaniasis is een vector overgedragen tropische ziekte veroorzaakt door de protozoa Leishmania.

Deze ziekte is momenteel in een opmars wegens de spreiding van de parasiet en zijn vectoren, onder

andere door de klimaatsverandering en de globalisatie. Er zijn verschillende klinische vormen van

leishmaniasis, waarbij de cutane en viscerale vorm het vaakst voorkomen. Viscerale leishmaniasis (VL)

kan fataal zijn indien niet behandeld. Momenteel is er echter geen geneesmiddel behandeling die zowel

veilig als volledig effectief is. Daarom is het des te belangrijker om profylactische benaderingen te

exploreren en vaccinatie is hier de logische eerste optie. In deze literatuurstudie wordt er dieper ingegaan

op de gebruikte diermodellen voor leishmaniasis, de immunologische reacties op de parasiet, de

verschillende categorieën vaccins en de momenteel beschikbare vaccins voor de hond.

De meest gebruikte diermodellen voor Leishmania zijn de muis, de hamster, de hond en primaten. Zowel

gevoelige als resistente muizenstammen worden gebruikt om de immunologische reacties na infectie met

Leishmania te achterhalen. BALB/c muizen zijn gevoelig voor infectie en afhankelijk van het Leishmania

species vertonen ze ernstige ongecontroleerde lesies die leiden tot een progressieve ziekte en soms zelfs

sterfte. BALB/c zijn zo vatbaar omdat ze geen IL-12 produceren, wat een ‘helende’ T helper 1 (Th1)

respons moduleert; zij produceren voornamelijk IL-4, IL-13 of IL-10, wat zorgt voor een Th2 respons wat

eerder aanleiding geeft tot een non-heling respons. Verder wordt er ook een verschil in reactie opgemerkt

naargelang welke infecterende parasietenstam. Zo is bijvoorbeeld het LACK antigen van belang voor de

reactie tegen L. major, maar niet voor de reactie tegen L. mexicana.

De C57BL/6 muis is in het algemeen vrij resistent tegen een Leishmania infectie doordat ze een vrijstelling

van IL-12 bewerkstelligen wat leidt tot een beschermende Th1 respons met de vrijstelling van interferon

gamma (IFNγ) wat de INOS expressie en NO (nitric oxide) productie in de macrofaag stimuleert waardoor

de parasiet afgedood kan worden. Ook in deze muizenstam worden parasiet-afhankelijke verschillen in

immuunrespons waargenomen. De immunologie bij deze 2 muizenstammen komt vrij goed overeen met

de immunologie bij de andere diersoorten en vormt de basis voor het al dan niet resistent zijn tegen

infectie.

De hamster is een erg goed diermodel voor onderzoek aangezien deze diersoort symptomen ontwikkelt

gelijkaardig aan de mens. Enkele nadelen aan het gebruik van hamsters is dat ze moeilijk genetisch te

manipuleren zijn, dat er weinig beschikbare reagentia (o.a. antistoffen, cytokines,…) zijn en dat ze

intraveneus, intracardiaal of intraperitoneaal geïnfecteerd moeten worden, wat verschilt van de natuurlijke

infectieroute.

Geïnfecteerde honden vormen het belangrijkste reservoir voor L. infantum en spelen een belangrijke rol in

de overdracht naar de mens. Honden geïnfecteerd met L. infantum vertonen een gelijkaardig ziektebeeld

als de mens, waardoor ze geschikt zijn voor secundaire vaccin evaluatie. Sommige honden blijven, net

3

zoals de mens, asymptomatisch. Ook hier is de immunologie T-cel gemedieerd, maar iets complexer dan

in muizen.

Primaten worden gebruikt voor tertiaire of preklinische vaccin evaluatie wegens hun nauwe fylogenetische

verwantschap met de mens. Er zijn echter geen T-cel merkers of cytokine assays beschikbaar, wat het

vaccin onderzoek erg bemoeilijkt.

Het is belangrijk om over een anti-Leishmania vaccin voor honden te beschikken aangezien ze het

reservoir zijn voor de zoönotische infectie van L. infantum naar de mens. Er zijn eerste, tweede en derde

generatie vaccins, waarvan de tweede generatie vaccins de beste keuze lijken. Momenteel zijn

Leishmune®, Leish-Tec

® en CaniLeish

® op de markt. Deze drie vaccins induceren een potente

immuunrespons, maar geen enkel biedt 100% bescherming. Voor de mens is er nog geen vaccin

beschikbaar; een sub-unit vaccin bevindt zich momenteel in klinische fase II.

De genetische verschillen tussen de verschillende Leishmania stammen, de gebruikte diermodellen en

hun infectieroute die verschilt van de natuurlijke infectie en het polymorfisme van het klinische beeld

maakt het ontwikkelen van een goed vaccin lastig. In de toekomst zal nog heel wat onderzoek moeten

gevoerd worden voordat er een volwaardig effectief vaccin voor zowel hond als mens beschikbaar zal

komen.

4

INTRODUCTION

Leishmaniasis is a vector-borne disease transmitted by female sand flies and caused by protozoan

parasites belonging to the genus Leishmania (Reithinger et al., 2007). Leishmaniasis is currently among

the six endemic diseases considered as high priorities worldwide and is still one of the most neglected

tropical diseases (Hotez et al., 2006; Rezvan and Moafi, 2015). The disease is becoming more important

because of globalization, climate change and other circumstances which allow the parasite and its vectors

to spread (Antoniou et al., 2013, Loría-Cervera and Andrade-Narváez, 2014).

Leishmaniasis consists of different clinical forms of which visceral (VL) and cutaneous leishmaniasis (CL)

are the most common (Gupta and Nishi, 2011). The visceralizing species are L. donovani and L. infantum

in the Old-World and L. chagasi in the New-World (Wilson et al., 2005). However, studies have shown that

L. chagasi and L. infantum are actually the same species (Mauricio et al., 2000). CL causing species

include L. major, L. tropica, L. pifanoi, L. garnham, L. venezuelensis, L. mexicana, L. amazonensis, L.

aethiopica, L. panamensis, L. guayanensis, L. peruviana and L. braziliensis. L. braziliensis can also cause

mucocutaneous leishmaniasis (MCL) (Ashford, 2000, Wilson et al., 2005).

Dogs are the primary domestic reservoir host for zoonotic VL caused by L. infantum, but unlike most

reservoir hosts, infected dogs can suffer from severe disease. Also cats and horses can become infected

with Leishmania species. In Europe, there are quite a number of other indigenous animals that can be

infected, such as different rodent species, weasels, etc. (Gramiccia and Gradoni, 2007; Quinnell and

Courtenay, 2009). Zoonotic CL caused by L. major is not posing a problem in Europe because the natural

reservoir hosts, namely different genera of gerbils (Rhombomys, Psammomys and Meriones), do not

occur in Europe at the moment (Ready, 2010).

The life cycle of Leishmania is presented in Fig. 1. Infection occurs when sand flies take a blood meal on

the host and transmit infective metacyclic promastigotes. These promastigotes attach to mononuclear

phagocytes or neutrophils and are taken up into a phagosome by phagocytosis. Next, the phagosome

fuses with lysosomes to form a phagolysosome. Once the promastigotes are inside the macrophage, they

transform into amastigotes which immediately start to divide by binary fission. These amastigotes are

released from bursting macrophages and will invade other macrophages, dendritic cells or fibroblasts

(Gupta and Nishi, 2011). When a sand fly takes a blood meal on an infected host, free amastigotes and

intracellular amastigotes are ingested. In the midgut, these amastigotes transform into procyclic

promastigotes which multiply and finally transform into metacyclic promastigotes which will migrate to the

proboscis to infect another vertebrate at a next blood meal (Kaye and Scott, 2011; Nieto et al., 2011).

VL can be fatal if left untreated. Pentavalent antimonials (SbV), amphotericin B (AmB), paromomycin

(PMM) and miltefosine (MIL) are the most commonly used drugs in humans. However, none of these is

devoid of toxicity and can cause adverse reactions like fever, nephrotoxicity, myocarditis, nausea and

5

abdominal pain. Some of these side effects may even cause death. In addition, most drugs have a

species-specific activity (de Menezes et al., 2015). Because of the overall lack of a safe and effective

treatment, preventive measures remain quite important (Jain and Jain, 2015) and vaccination would

indeed be the best method for prevention of all forms of leishmaniasis. Vaccination of dogs would also

reduce zoonotic VL. Quite some research already went into the development of an effective and safe

antileishmania vaccine (Dye, 1996; Rezvan and Moafi, 2015) but there are still major challenges because

of the heterogeneity of the human population or the unusual host evasive mechanisms of Leishmania

species (Joshi et al., 2014).

Since vaccine research is largely dependent on the availability of validated (laboratory) animal models,

this master thesis aims to review the current animal models together with the underlying immunological

mechanisms. Based on this information, possible difficulties and challenges will be given on the making

and success of future vaccines.

Fig. 1 L. infantum life cycle (Nieto et al., 2011).

6

LITERATURE STUDY

1. ANIMAL MODELS

Animal models are expected to imitate most pathological features and immunological responses perceived

in humans when exposed to different Leishmania species having different pathogenic characteristics.

Several animal models for leishmaniasis and antileishmanial vaccine testing are already available, but

none of them exactly mimics what happens in man. A really suitable laboratory host for Leishmania should

cover various aspects such as host-parasite interactions, pathogenesis and biochemical changes to be

used in prophylaxis, maintenance of parasites and especially evaluation of antileishmanial drugs and

vaccines (Gupta and Nishi, 2011).

Compared to in vitro systems, there are both advantages and disadvantages when using in vivo systems.

In vitro settings have the following advantages: parasites obtained from just a few animals are sufficient to

test many compounds, the requirement of test compound is small, the turnover of screening results is fast

and the results are consistent. A limitation is that in vitro test results always need to be verified in animal

models (Gupta and Nishi, 2011). Nevertheless, in vivo and in vitro experiments may have a close

correlation because the amastigote (the disease-producing stage) can be maintained in vitro as axenic

amastigotes and in macrophage culture representing a semi- in vivo condition (Gupta and Nishi, 2011).

1.1 RODENTS

Rodents are mainly used for primary evaluation. BALB/c mice (Mus) and Syrian golden hamsters

(Mesocricetus auratus) are currently most commonly used for vaccine testing against VL. The Chinese

and European hamster (Cricetulus and Cricetus), NMRI, DBA/1 and C56BL/6 mice (Mus), rats (Rattus),

multimammate rats (Mastomys), squirrels (Sciurus) and gerbils (Gerbillus) are also being used (Hommel

et al., 1995; Gupta and Nishi, 2011).

1.1.1 The mouse

One of the major advantages of mice is that they are easy to keep and breed, with a short generation time

of 10 to 12 weeks (Guénet and Bonhomme, 2003). A disadvantage is that they must be intravenously

injected with amastigotes to obtain a reproducible pattern of colonization in the liver and spleen (Bradley

and Kirkley, 1977). Mice have also been vaccinated and infected subcutaneously in the footpad with

L. infantum promastigotes (Martins et al. in 2015). It is clear that both routes of infection largely differ from

the natural route of infection by the sand fly (Handman, 2001).

7

Outbred mouse strains are susceptible to most dermatotropic Leishmania species of mammals and tend

to develop cutaneous lesions at the inoculation spot which may occasionally visceralize or metastasize.

These outbred strains are resistant to infection with L. donovani. On the other hand, the susceptibility of

inbred strains is genetically controlled and depends on host factors (Hommel et al., 1995). Mice with a wild

type Scl1 1a1 locus (CBA) show an earlier parasite growth than those with a mutated locus (BALB/c and

C57BL/6) (Gupta and Nishi, 2011). Inbred strains also have the advantage to allow many comparisons

with subsequent rigorous genetic analysis. This way, the genetic component of each phenotypic

characteristic can be demonstrated (Hommel et al., 1995; Guénet and Bonhomme, 2003).

Five regions of the mouse genome actually determine ‘susceptibility’ or ‘resistance’. These include the

macrophage resistance locus Lsh/Ity/Beg (now named Nramp) on chromosome 1, the NOS2 region on

chromosome 11 encoding for inducible NO synthase, the Scya genes, also located on chromosome 11

and encoding for the β-chemokines subfamily, the cluster of cytokine genes for IL-4, IL-5 and IL-13 and

the genes of the major histocompatibility complex locus. Also members of the JAK kinase family of

tyrosine kinases have been charted on a region of chromosome 4 (Hommel et al., 1995; Milon et al.,

1995).

Fig. 2: Leishmania immunology (based on Alexander and Bryson, 2005).

8

1.1.1.1 BALB/c mouse The commonly used BALB/c mouse is an inbred strain susceptible to Leishmania infection. Upon

infection, these mice develop severe and uncontrolled lesions that lead to progressive disease and

sometimes even death (Loría-Cervera and Andrade-Narvae, 2014). When infected with VL, BALB/c mice

develop organ-specific immune responses. The liver serves as a site for initial parasite expansion and the

spleen serves as a safe spot for long-term persistence. Resolution of VL in the liver correlates with the

formation of granulomas, making the liver refractory to reinfection. In the spleen, parasite replication starts

later and remains at a low level, but can persist for a lifetime (Wilson et al., 2005). BALB/c mice can be

used as a model for studying self-healing or subclinical infections (Loría-Cervera and Andrade-Narváez,

2014) since infection with VL is not fatal.

BALB/c mice do not produce IL-12 which modulates towards a T helper 1 (Th1) response, but they

produce IL-4 or IL-13 which steers towards a T helper 2 (Th2) response (Fig. 2). This results in

unregulated parasite replication in infected cells. This is further facilitated by IL-10 production of the host

cell. IL-10 production by CD4+ CD25+ T regulatory cells and regulatory B cells can facilitate non-healing

disease but can also maintain latent infection and concomitant immunity (Alexander and Bryson, 2005;

Ronet et al, 2010).

1.1.1.1.1 Leishmania major The inability for BALB/c mice to develop a Th1 response irrespective of a Th2 response upon infection

with L. major has been assigned to the inability to produce or respond to IL-12. T cells of BALB/c mice are

unresponsive to IL-12 because the expression of IL-12Rβ2 is suppressed by IL-4 from vβ4 Vα8 CD4+T

cells, which undergo an expansion related to the LACK (Leishmania analogue of the receptors of activated

C kinase) antigen (Himmelrich et al., 1998; Alexander and Bryson, 2005). IL-4 can be involved in this

process, but IL-4 independent processes have also been described with similar outcome (Guler et al.,

1996; Alexander and Bryson 2005).

Various BALB/c strains have CD4+ T cells with an intrinsic IL-4Rα independent Th2 effect. This is

regulated by dice (determinant of IL-4 commitment) and dice-2 located on chromosome 16 and 7, leading

to an increase of CD4+ cells that commit to IL-4 release (Bix et al., 1998). Via STAT6 (a transcription

factor) activation, IL-4 prevents the co-polarization of Interferon-γ receptor (IFNGR) with TCR (T cell

receptor), which leads to a Th2 response (Maldonado et al., 2004). IL-13 is closely related to IL-4 and has

a similar effect on Leishmania infection (Gumy et al., 2004). Besides IL-4 and IL-13, IL-10 also contributes

to susceptibility of the BALB/C for L. major (Noben-Trauth et al., 2003).

Intrinsic defects in the function of antigen presenting cells (APC), like IL-1 deficient dendritic cells and T

cell development may also be underlying reasons for the susceptibility of BALB/c mice to L. major (Filippi

et al., 2003; Alexander and Bryson, 2005). It has been shown that wild type (WT) mice secrete more IFN-γ

and less IL-4 than IL-1 type 1 receptor deficient (IL-1RI-/-) mice (Satoskar et al., 1998). IL-1 secretion also

stimulates dendritic cells to increase the production of IL-12 (Eriksson et al., 2003). Regulatory T cells

9

(Tregs) and regulatory B cells (Fig. 2) in BALB/c mice produce TGF-β which inhibits Th1 differentiation

and suppresses the production of NO by macrophages, which is important for parasite killing (Gumy et al.,

2004; Alexander and Bryson, 2005).

1.1.1.1.2 Leishmania amazonensis / L. mexicana

Leishmania species have evolved over time, so different virulence factors have been identified. This

means that not all the immunological data obtained from L. major can be directly extrapolated to the other

Leishmania species. The fact that most mouse strains are resistant to L. major but susceptible to

L. mexicana or L. amazonensis underlines this statement (McMahon-Pratt D and Alexander, 2004). When

BALB/c mice are infected with L. mexicana, IL-4 plays a very important role in the immunological

response, but the LACK antigen is not involved (Torrentera et al., 2001). The cathepsin L-like CP (CPB) is

an important virulence factor of L. mexicana which probably induces IL-4 (Pollock et al., 2003). IL-10 and

IL-13 seem to play a less prominent role during L. mexicana as compared to L. major infection (Alexander

and Bryson, 2005).

1.1.1.1.3 Non-healing visceral leishmaniasis

When BALB/c mice are infected with L. donovani, IL-12 becomes involved in the Th1 and Th2 responses

and the liver and spleen require IL-12 to control the infection. This differs in resistant mouse strains

(Engwerda et al., 1998). TGF-β inhibits the Th1 response in BALB/c mice infected with L. chagasi, which

results in difficult healing (Wilson et al., 1998). Also IL-10 is a major immunosuppressive component that

inhibits resistance to L. donovani (Murphy et al., 2001).

1.1.1.2 C57BL/6 mouse The C57BL/6 mouse is an inbred strain that is in general resistant to infection with Leishmania. This

phenotype is associated with a parasite-specific Th1 response. IL-12 is released when promastigotes

transform into amastigotes inside the macrophage and activates natural killer cells and Th1 cells. The

differentiated Th1 cells then release IFNγ which stimulates iNOS expression and NO production in the

macrophage (Fig. 2) leading to parasite killing and healing. (Sacks and Noben-Trauth, 2002; Alexander

and Bryson, 2005).

1.1.1.2.4 Leishmania major

The Th1 response induced in C57BL/6 mice is responsible for the resolution of L. major infection. IL-12

released from macrophages and dendritic cells, possibly augmented by some other cytokines, drives the

differentiation and proliferation of T helper 1 cells. IFN-γ released from these Th1 cells and probably but

less important CD8+ T cells and IL-12 activated natural killer (NK) cells mediate macrophage activation so

they can produce and release nitric oxide (NO) which leads to Leishmania killing (Cunningham, 2002).

Besides IL-12, CD40-C40 ligand interactions are also important for macrophage activation and

development of a Th1 response. TNF-α, migration inhibition factor (MIF) and type-1 interferons also

10

contribute to the activation of the leishmanicidal activity of macrophages (Kamanaka et al., 1996;

Alexander and Bryson, 2005). IL-27 (WSX-1) stimulates the IFN-γ production through STAT1 (a

transcription factor)-mediated induction of T-bet, which is a T cell related transcription factor that helps the

development of a Th1 response (Artis et al., 2004). Just like BALB/c mice, resistant C57BL/6 mice can

produce IL-4 in early infection, proving that the release of IL-4 does not always implicate the absence of a

Th1 response (Scott et al., 1996). Even when IL-12 is absent, C57BL/6 mice remain resistant to L. major.

The exact mechanisms involved in this resistance are not yet known, but it may have to do with

redundancy in the induction of type-1 responses (Alexander and Bryson, 2005). Recently, a clinical L.

major isolate (LmSd) was shown to induce chronic lesions in C57Bl/6 mice despite their intrinsic

resistance. The non-healing properties of the LmSd infection despite the induction of a sustained Th1

response, was related to increased IL1 production and the localized recruitment of neutrophils indicating

that other immunological parameters than the Th1/Th2 paradigm are involved (Charmoy et al., 2015).

Long term persistence of a patent infection with concomitant immunity (= immunity because of the

persistence of pathogens) is dependent on antigen-specific natural CD4+ CD25+ T-regulatory cells which

use IL-10 dependent and independent mechanisms to suppress CD4+CD25- effector T cells precluding

elimination of all parasites. If no persistent infection is present, protective immunity reduces significantly

(Belkaid et al., 2002).

1.1.1.2.5 Leishmania amazonensis/ L. mexicana

When C57BL/6 mice are infected with L. mexicana, IL-12 and the Th1 response play an important role in

the immunological response, but in contrast to infection with L. major, IL-12 does not resolve lesions. This

proves that there are differences between the different Leishmania strains (Torrentera et al., 2002).

Studies on L. amazonensis with STAT6-/- (C57BL/6/129/Sv) mice demonstrated that STAT6-mediated IL-

4 signaling leads to progression of infection and that a Th1 response develops in the absence of STAT6

(Stamm et al., 1998).

1.1.1.2.6 Non-healing visceral leishmaniasis

Research in C57BL/6 mice has shown that Th2 cells and their cytokines IL-4 and IL-5 do not contribute to

a long term VL infection (Kaye et al., 1991). IL-4 and IL-4 receptor deficient C57BL/6 mice are more

susceptible to L. donovani compared to wild-type mice (Satoskar et al., 1995). As in BALB/c mice, IL-10

plays an important immunosuppressive role (Murphy et al., 2001).

1.1.2 The hamster

When infected with L. donovani, L. chagasi or L. infantum, hamsters develop symptoms similar to humans

and are therefore the preferred model for the characterization of molecules and mechanisms involved in

the pathogenesis of VL (Hommel et al., 1995). The fatal outcome of VL in the hamster has been related to

11

the loss of macrophage effector function, inducible NO synthase mRNA or enzyme activity in liver or

spleen tissue, despite a strong Th1-like cytokine response (Melby et al., 2001). The lack of NO production

is due to a defect in the transcriptional activation of NOS2 and is probably the reason why hamsters

cannot control a Leishmania infection (Perez et al., 2006). The terminal phase of VL is characterized by

cachexia and because of this, the hamster has been used as a model to study the effect of cytokines and

the interactions between malnutrition and disease outcome (Pearson et al., 1992). Persistence that is

responsible for relapses in humans can also be reproduced in hamsters.

Hamsters are also very susceptible to L. braziliensis, L. panamensis and L. guyanensis which are

responsible for most of the CL- and MCL-related human pathology in South-America. Some hamsters

develop dermal metastasis or viceralization when infected with some strains of L. panamensis and L.

guyanensis, as is normally seen in L. braziliensis infection (Martinez et al., 1991; Almeida et al., 1996).

A significant disadvantage of hamsters as an animal model is that there are very few inbred strains

available from commercial sources, which makes it hard to manipulate these animals genetically (Hommel

et al., 1995). In addition, there are very few available reagents, such as antibodies to cell markers and

cytokines (de Oliveira et al., 2004). Another disadvantage of the VL model is that only high doses of SbV

can suppress established lesions (Garg and Dube 2006).

Hamsters are mostly infected with large numbers of parasites via the intravenous, intracardial or

intraperitoneal route (Stauber et al., 1958). This differs from the natural route of infection by the sand fly

bite where metacyclic promastigotes combined with saliva end up in the skin of the host. It has been

proven that a salivary protein of Lutzomyia longipalpis protects the hamster against the fatal outcome of

infection with L. infantum. Immunization with 16 DNA plasmids coding for salivary proteins led to the

identification of LJM19, a novel 11-kDa protein. This discovery reinforces the concept of using

components of sand fly saliva in experimental infection and vaccine strategies against leishmaniasis

(Gomes et al., 2008).

Hamsters are very frequently used for the collection of spleen-derived (ex vivo) amastigotes to infect mice

(Handman, 2001) since infection with metacyclic promastigotes tends to be less reproducible. Hamsters

are most used for vaccine studies and histopathological research, despite the lack of fine

immunochemicals that limits the mechanistic exploration of the immune responses. The main reason is

that VL in hamsters is most closely related to the disease in man, while mice do not develop typical VL

symptoms and are even resistant to different Leishmania species (Melby et al., 2001).

1.2 CARNIVORES

Infected canines constitute the main domestic reservoir of L. infantum and play an important role in the

transmission to humans (Alvar et al., 2004). Dogs infected with L. infantum have a similar disease pattern

as humans, making them a good animal model for secondary vaccine testing (Abranches et al., 1991a).

12

Some dogs do not show any symptoms, but this also occurs in humans (Pinelli et al., 1995). One of the

few differences is that dogs often show skin lesions, while this is rare in humans (Hommel et al., 1995).

In 1989, the first vaccination trial in dogs was performed using a partially purified L. infantum-derived

antigen preparation. The study results differed from a similar experiment in murine models, indicating the

differences between natural hosts and experimental animal substitutes (Moreno and Alvar, 2002). The

German Shepherd is a good dog breed for vaccine testing against VL because the experimental infection

has many clinicopathological similarities to the disease in man and also gave better experimental results

than Beagles (Keenan et al., 1984). Mixed breed dogs from endemic regions for canine VL have also

been used with success (Abranches et al., 1991a). Based on the prevalence of natural infections, German

Shepherds, Boxers and Dobermans carry VL more frequently than other breeds and young adults and

older dogs also show higher prevalence of infection (Abranches et al., 1991b).

Intravenous inoculation is the best way to induce disease in dogs whereby it is better to use amastigotes

to produce clinical symptoms more effectively (Moreno and Alvar, 2002). Although less successful,

infections can also be induced by intradermal inoculation of metacyclic promastigotes, infected

macrophages or infected sandflies (Abranches et al., 1991a; Killick-Kendrick et al., 1994; Pinelli et al.,

1994; Hommel et al., 1995). Foxes (Vulpes), zorros (Cerdocyon), jackals (Canis), ferrets (Mustela),

kinkajous (Potos), olingos (Bassaricyon) and cats (Felis) have also been reported as suitable animal

models for leishmaniasis in the past (Thatcher et al., 1965; Hommel et al., 1995).

Dogs infected with L. infantum may respond in two ways depending on immunological mechanisms: they

either remain asymptomatic (resistant) or they develop clinical symptoms (susceptible) (Pinelli et al.,

1994). Just as in mice, the immune responses in dogs are T-cell mediated but more complex. There is a

Th1-response in the asymptomatic phase, while in the symptomatic phase both a Th1 and Th2 response

can be observed since IL-4, IL-10, IL-12, IFN-γ and TNF-α can be found (Lage et al., 2007). It has been

shown that susceptible dogs do not respond to LSA (Leishmania soluble antigen) and that they have high

non-protective serum antibodies against Leishmania antigens. There are contradictory results regarding

the role of IL-10 in CVL. Quinnell et al. showed in 2001 that there are no high concentrations of IL-10 in

infected dogs, while Lage et al. proved the exact opposite in 2007. In resistant asymptomatic dogs, there

are higher titers of IL-2 and TNF present compared to the susceptible dogs (Pinelli et al., 1994) and

asymptomatic dogs have a Th1 response mediated by LSA stimulated cells that produce IL-2, IFN-γ and

IL-18 (Chamizo et al., 2005). CD8+ lymphocytes have been detected in asymptomatic infected dogs, but

not in susceptible animals. These T cells destruct infected macrophages and hence contribute to

resistance (Pinelli et al. 1995).

13

1.3 PRIMATES

Non-human primates are used for tertiary or pre-clinical testing because of their close phylogenetic

relation with humans (Garg and Dube, 2006). However, the use of primate models is controversial

because of expenses, difficult handling and restriction to artificial laboratory experiments (because they

cannot be used in natural challenge experiments). Primates are also immunological black boxes, which

means that there are no T-cell markers and cytokine assays available and that there are variations from

one animal to another. This makes primates a much less attractive model for antileishmania vaccine

studies (Hommel et al., 1995).

In the past, chimpanzees (pan), gibbons (hylobates), mandrils (papio), rhesus macaques (macaca), toque

macaques (macaca), kras (macaca), capuchins (cebus), bush babies (galago), sykes monkeys

(cercopithecus), mangabeys (cercocebus), patas (erythrocebus), vervets (cercopithecus), marmosets

(callithrix), baboons (papio), squirrel monkeys (saimiri), owl monkeys (aotus), indian langurs (Presbytis)

and slow loris (Nycticebus) have been used for experimental testing on leishmaniasis (Medina 1966;

Christensen and Vasquez, 1981; Githure et al., 1987; Hommel et al., 1995).

Owl monkeys and squirrel monkeys develop a short but fulminating infection and have been used for

antileishmanial screening (Chapman et al, 1981; Chapman et al., 1983). Vervet monkeys can be infected

with L. donovani and show competent humoral and cellular immune responses to homologous parasites

(Gicheru et al., 1995). Rhesus monkeys have been used to test a new vaccine based on a recombinant

A2 antigen (Grimaldi et al., 2014) and to test a sand fly salivary protein vaccine. Indian Rhesus monkeys

have been used because rhesus monkeys of Chinese origin show an enhanced resistance to CL (Oliveira

et al., 2015).

As already mentioned, both CL and VL occur in man and because of different immunological responses,

they will be discussed separately.

There are very small differences between the immunological reactions in cutaneous leishmaniasis (CL),

mucosal leishmaniasis (MCL), disseminated leishmaniasis (DL) and diffuse cutaneous leishmaniasis

(DCL) (da Silva Santos and Brodskyn, 2014), which will not be discussed in detail in this thesis. In humans

infected with CL, T cells produce IL-2, IL-4 and IL-10 but no IFN-γ during active disease (Bomfim et al.,

1996). IFN-γ and TNF-α can induce a Th1 response which is important for healing. CD8+ T cells produce

Th1 cytokines that are important for the modulation of the CD4+ T cell response (Pompeu et al., 2001). IL-

10 acts immunosuppressive and can limit collateral tissue damage (Antonelli et al., 2004). IL-10 also

decreases the production of IL-17. This cytokine normally recruits neutrophils and contributes to the

production of IL-1, IL-6 and TNF-α (Oliveira et al., 2014). Previous studies have shown that IL-27 can

enhance the IL-10 production which helps in infection control, but other studies have obtained

contradictory results (Murugaiyan et al., 2009; Oliveira et al., 2014).

14

IFN-γ is also of great importance in VL. It has been shown that people infected with L. donovani can

control the infection when Leishmania antigen stimulated mononuclear cells produce sufficient IFN-γ,

while the disease will progress in people with low IFN-γ amounts (Ribeiro-de-Jesus et al., 1998). IL-4 also

contributes to the immune response against VL and could lead to a decreased release of IFN-γ trough a

negative feedback mechanism (Zwingenberger et al., 1990). IL-12 that leads towards a Th1 response

cannot be found in patients with active VL. Once people are treated, peripheral blood mononuclear cells

(PBMC) start to produce IL-12 and IFN-γ which supports healing (Ghalib et al., 1995). As in mice and

dogs, IL-10 is found in humans infected with L. donovani. This Th2 cytokine has an immunosuppressive

role which decreases the macrophage and T cell responses against the parasite (Ghalib et al., 1993).

1.4 OTHER ANIMALS

Guinea pigs (Cavia) have been used in multiple experiments using L. major, L. infantum and L. tropica to

evaluate skin tests with intradermal antigen (Briand et al., 1999; Khabiri et al., 2006; Khabiri et al., 2007).

Goats (Capra) have been used for antileishmanial testing. For example, Anjili et al. (1994) have done an

experiment where goats were infected with L. major through bites of infected sandflies compared to

needle inoculation of culture-derived promastigotes.

Rabbits (Oryctolagus), cotton rats (Sigmodon hispidus), flying foxes (Pteropus), chinchillas (Chinchilla),

pacas (Cuniculus), agoutis (Dasyproctidae), Armadillos (Dasypus), opossums and ferrets have

occasionally been used (Medina, 1966; White et al., 1989; Hommel et al., 1995). It was shown that the

opossum is a better model for experimental VL than the ferret or armadillo, based on weight loss,

hepatomegaly, splenomegaly, amastigote densities and microscopic lesions (White et al., 1989).

15

2. ANTILEISHMANIA VACCINES FOR DOGS

Canine visceral leishmaniasis (CVL) is an important disease that does not only occur in the Mediterranean

area, but has also spread to the Middle East, Asian countries (Foroughi-Parvar and Hatam, 2014) and

Latin-America. Since dogs play a key role in the transmission of this zoonotic infection in man, it is

important to develop strategies to control CVL (Dantas-Torres et al., 2012). Insecticides, drug treatment

and elimination of infected dogs by culling have all been used (Tesh, 1995), but elimination of infected

dogs remains ethically unacceptable, chemotherapy is not fully effective and asymptomatic dogs continue

to play an important role in the transmission. It must be said that these control strategies have been

largely unsuccessful, stressing the need for an effective vaccine. The ideal vaccine should contain several

antigens that are highly expressed in the amastigote stage, but few antigens can protect against more

than one species in animal models (Foroughi-Parvar and Hatam, 2014).

The most important challenges in vaccine development is the complexity associated with antigenicity,

variable host responses, variability among the different Leishmania species and costs associated with

vaccine development (Singh and Sundar, 2012). A first vaccine for CVL was reported in 2001 and was a

fuccose mannose ligand (purified surface antigen) vaccine of L. donovani which evoked a protective

response (da Silva et al., 2000). Recently, a live attenuated L. donovani strain (LdCen−/−) was shown to

induce a protective immunity against L. infantum in beagles (Fiuza et al., 2015).

2.1 FIRST-GENERATION VACCINES

The injection of live Leishmania parasites is still the most effective vaccination method, but they do have

inadequate ability to produce long-lasting immunity and often cause intolerable toxicity (Jain and Jain,

2015). Attenuated live vaccines can be obtained by in vitro culturing in different media (Mitchell et al.,

1984), using temperature sensitivity (Gorczynski, 1985), chemical mutagenesis (Kimsey et al., 1993) or

gamma radiation (Rivier et al., 1993). Transgenic parasites that secrete host immune mediators to boost

anti-parasite responses and facilitate parasite clearance have also been used (Beattie et al., 2008).

2.2 SECOND-GENERATION VACCINES

It has been shown that immunization of dogs with diverse strains of L. infantum provided protection.

Second-generation vaccines contain whole crude antigen of cultured parasite, fraction purified parasites

and recombinant Leishmania antigens (Coler and Reed, 2005). Several proteins can be used, such as

surface glycoproteins and receptors (e.g. GP63, GP46), proteinases (e.g. cysteine proteinase A and B),

histones (e.g. H1), translation factors [e.g. elongation and initiation factor (LeIF)], anti-oxidants [e.g. thiol-

specific-antioxidant (TSA)], stress response proteins [L. major homologue of the eukaryotic stress

inducible protein 1 (LmSTI1)] and sand fly saliva factors (e.g. SP15) (Coler and Reed, 2005). TSA,

LmSTI1 and LeIF have been used for the development of the recombinant polyprotein vaccine Leish-111f.

16

These antigens provide a protective response both in mice as in non-human primates (Coler and Reed,

2005). The combination of these three antigens together provides a protective immune response in dogs

(Fujiwara et al., 2005). Second-generation vaccines currently seem the better choice for CVL vaccination

because of the lack of long lasting immunity by first-generation vaccines and because of the unclear

impact of DNA vaccines (Foroughi-Parvar and Hatam, 2014).

2.3 THIRD-GENERATION VACCINES

Since there is no need for a cold chain for DNA vaccines, a lot of studies are ongoing for developing DNA

vaccines against CVL (Palatnik-de-Sousa, 2012). In this strategy, a vaccine antigen is encoded on

plasmid DNA which upon expression in the host can lead to strong antibody production and complete cell

mediated immune responses. It has been shown that both CD4+ and CD8+ T cell mediated immune

responses are evoked and long lasting immunity can be achieved (Gurunathan et al., 2000).

2.4 COMMERCIAL CVL VACCINES

Leishmune® is a second-generation vaccine that was first licensed in Brazil in 2003 and consists of

L. donovani glycoprotein (fucose mannose ligand) with saponin as adjuvant. The Leish-Tec® vaccine,

consisting of A2 antigen (a recombinant protein from the amastigote stage of different Leishmania

species) and saponin, was registered by the Brazilian Ministry of Agriculture in 2007. Research showed

that animals immunized with Leishmune® presented higher IgG, IgG1 and IgG2 humoral reactivity by

ELISA. Dogs that were immunized with Leish-Tec® showed higher frequency and intensity of adverse

reactions. There were no significant differences in the rates of seroconversion, clinical signs, parasitism,

and transmission to the vector by xenodiagnosis among the dogs immunized with the Leishmune® or

Leish-Tec® during natural exposure to infection for 11 months in a CVL endemic area. Both vaccines elicit

a potent immune response but do not provide 100% protection against infection and clinical signs. In this

study, 32.5% of the dogs vaccinated with Leishmune®

and 30.9% of the dogs vaccinated with the Leish-

Tec® vaccine exhibited seroconversion (Fernandes et al., 2014).

There is yet another vaccine on the market, CaniLeish® (LiESP/QA-21), which is used in Europe. It

consists of purified excreted-secreted proteins of L. infantum (LiESP) and QA-21 (a highly purified fraction

of Quilaja saponaria saponin) as adjuvant (Moreno et al., 2014). CaniLeish® must be administered in 3

doses, with 3 week intervals (Martin et al., 2014; Moreno et al., 2012). No adverse effects have been

reported and it also suffices to give annual booster vaccinations (Moreno et al., 2014). This vaccine elicits

a Th1 response that persists for at least a year and has been licensed for prophylaxis against CVL (Jain

and Jain, 2015). Dogs vaccinated with CaniLeish® develop an igG2 response against LiESP and parasite

surface antigen (PSA), associated with a Th1 response (Moreno et al., 2012). After vaccination with the

LiESP/QA-21, the risk of progression to an active infection is significantly reduced, as is the bone marrow

parasite load (Martin et al., 2014).

17

3. HUMAN VACCINES AGAINST ZOONOTIC VL?

There is yet no vaccine available for use in man against leishmaniasis. The recombinant polyprotein

vaccine antigen Leish-111f (also known as LEISH-F1) formulated with monophosphoryl lipid A in stable

emulsion (MPL-SE adjuvant) is the first subunit vaccine to be evaluated in humans. The antigen

component of the vaccine includes three proteins derived from L. major and conserved across various

Leishmania species, including L. donovani; L. chagasi and L. braziliensis. The three proteins are

Leishmania elongation initiation factor (LeIF), thiol-specific antioxidant (TSA), and Leishmania major

stress-inducible protein 1 (LmSTI1) (Chakravarty et al., 2011). This vaccine is protective against both

cutaneous and visceral leishmaniasis in mice (Coler et al., 2002). Previous studies have already indicated

that the Leish-111f vaccine is safe and well-tolerated in humans (Velez et al., 2009) and is also

immunogenic in healthy people (Chakravarty et al., 2011). This vaccine is now in Phase-II clinical testing

(Jain and Jain, 2015).

18

DISCUSSION

Even though there has already been a lot of progress in vaccine studies over the last couple of years,

there are still a lot of challenges for vaccine improvement in the future. There are still several issues with

the effectiveness of current commercially available vaccines.

The genetic differences between the Leishmania strains make it difficult to make an adequate vaccine that

provides sufficient protection against the many circulating strains. In addition, most immunological studies

have been performed on L. major, which causes CL. These findings cannot necessarily be extrapolated to

VL.

The animal models that are used for testing and the route of infection also differ significantly from a natural

infection by an infective sand fly bite (Gradoni, 2015). Intravenous or intracardial injections with

amastigotes could result in a different outcome than a sand fly bite which leads to the intradermal

inoculation of metacyclic promastigotes together with physiologically and immunologically active saliva

proteins. Many experiments have been performed without the use of sand fly saliva. This can change the

outcome of the vaccination/challenge experiments because it has been proven that saliva can exacerbate

the infection and that anti-saliva immunity can protect against a fatal outcome (Gomes et al., 2008).

The pleiotropy of clinical outcomes makes vaccine testing also very difficult. Some dogs are, just as

humans, naturally resistant against a leishmanial infection, while other individuals are more susceptible.

Individuals with inherently enhanced resistance are therefore less suitable for vaccine testing studies.

Because none of the available vaccines fully protects against infection, there is also a chance that

vaccinated dogs will continue to function as asymptomatic carrier or reservoir, imposing a potential threat

to human health. These available vaccines can also give a false sense of safety to the dog owners,

because even when vaccinated, dogs can still be infected and even become symptomatic.

In the progress of developing an effective antileishmania vaccine for both humans and dogs, it is important

to take all these obstacles into consideration. Therefore, more research on the immunology of the different

Leishmania strains, most importantly those that cause VL, will have to be undertaken. The vaccination and

challenge experiments performed in the available animal models should resemble the natural route of

infection as diligently as possible, including the important role of the sand fly vector. It is also very

important to use adequate numbers of animals, for example dogs, in vaccine trials. However, the high

costs of conducting such trials and the increasing ethical constraints in using large cohorts makes vaccine

development even more difficult (Gradoni, 2015).

19

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