Use of a LiESP/QA-21 Vaccine (CaniLeish) Stimulates an Appropriate Th1-Dominated Cell-Mediated Immune Response in Dogs
[['Canine leishmaniasis , a vector-borne disease of dogs , is caused by Leishmania infantum in the Mediterranean basin and is a significant problem for the canine population of endemic areas [1] .', 'It is transmitted in the Mediterranean area by the bite of certain species of sand flies of the Phlebotomus ( Larroussius ) subgenus , and so natural transmission can only occur in areas where competent vector species are present [2] .', 'Currently it is estimated that at least 2 . 5 million dogs are infected in southwestern Europe alone [3] and recent publications have reported a northward spread of the endemic area [4] .', 'Given that canine leishmaniasis is a potentially severe and fatal disease , it represents a source of suffering for affected dogs and many dog owners are highly concerned about how best they can protect their animals .', 'Moreover , the dog is the principle reservoir for human infection; thus a high prevalence of this canine disease also represents a zoonotic risk [5] .', 'In recent years , several topical repellent and insecticide preparations with good trial data have become available and these are able to decrease the intensity of parasite challenge received by the dog by decreasing the number of infectious sandfly bites received .', 'However although these products have good short-term efficacy data when used on an individual dog basis , some evidence exists to suggest that this may not be maintained over the longer term [6] .', 'Even with correct use , these products cannot prevent all infectious bites and there is still a need for further control measures [7] .', 'The outcome of the infection in individual dogs is highly variable and not all dogs which are infected will develop the disease [8] .', 'Some dogs will completely clear the infection , some will remain subclinically infected , while some will develop clinical disease of varying severity from mild papular skin disease to severe generalised disease characterised by renal failure and eventually death [9] , [10] .', 'Studies on canine leishmaniasis have somewhat clarified the risk factors determining the likelihood of disease developing after infection [1] , [3] .', "However it has become clear that the final outcome of infection depends mainly on the efficiency of the dog's immune system [3] , [11] .", 'In murine cutaneous leishmaniasis , it has been demonstrated that there is a clear dichotomous immune response: T helper 1 ( Th1 ) responses are associated with protection while T helper 2 ( Th2 ) responses are associated with the development of disease [12] .', 'In contrast , data from human visceral leishmaniasis cases and the murine model of visceral leishmaniasis have shown that this Th1/Th2 dichotomy is lacking , and a mixed response is essential for protection [13]–[15] .', 'Although dogs which remain asymptomatic after infection with L . chagasi ( =\u200aL . infantum ) develop a predominantly Th1 profile response , whereas oligosymptomatic and symptomatic dogs present a Th2 profile [16] , a clear dichotomous Th1/Th2 pattern is also lacking in this species [17] .', 'However , despite the fact that the situation is extremely complex , it is now widely accepted that the protective canine immune response is mediated by a dominant CD4+ Th1 influence in an overall mixed cellular response [18] , [19] .', 'This protective response is believed to be mediated by the induction of inducible nitric oxide synthase ( iNOS ) in macrophages upon stimulation by Th1 cytokines such as interferon gamma ( IFN-γ ) .', 'This results in a decreased production of arginase , and consequently of the polyamides that are essential for parasite growth and survival in the macrophage , and an increased production of leishmanicidal nitric oxide ( NO ) derivatives [20] , [21] .', 'In addition , in vitro studies propose that while iNOS activity can be considered as an essential effector mechanism to prevent multiplication of Leishmania amastigotes , the NO derivative produced may have additional roles including immunoregulatory functions [22] .', 'Because of this pivotal role for the immune system , several authors have expressed the opinion that an effective vaccine against canine leishmaniasis would be the best control strategy for both canine and human disease [7] , [23] .', 'Two canine vaccines have been available for some time now in Brazil [11] .', 'However , until the recent launch of the LiESP/QA-21 vaccine ( CaniLeish , Virbac , France ) , there were no vaccines against Leishmania available in Europe .', "With any new vaccine , and especially one that is the first of its kind , it is important to understand as much as possible about the mechanism of its action on the dog's immune response and to study known markers of resistance to disease .", 'Indeed investigation of such parameters has recently been proposed as representing an important supplementary data set when assessing any candidate vaccine for canine leishmaniasis [11] .', 'The aim of the study presented here was to follow selected humoral and cellular markers of the immune response in dogs vaccinated with LiESP/QA-21 vaccine during the establishment of the immune response , and specifically to assess if an effective Th1-dominated profile could be generated .'], ['The Virbac Ethical Committee approval confirms that this study was carried out in accordance with the G . R . I . C . E . “Ethical Committee Regulation applied to animal experimentation” guidelines ( implemented in France in 2007 ) .', '20 conventional Beagle dogs ( 10 male and 10 female ) aged 6 months +/−1week on the day of the first vaccination were randomly assigned to two groups ( vaccinated and control ) according to their weight , sex and litter of birth .', 'There were 5 males and 5 females per group .', 'All animals were previously vaccinated with conventional vaccinations against Distemper virus , Adenovirus , Parvovirus , Parainfluenza virus and Leptospira .', 'They were housed in controlled conditions , and dewormed with nitroscanate ( Troscan , Virbac , France ) 1 week prior to the date of the first administration of the LiESP/QA-21 vaccine .', 'The LiESP/QA-21 vaccine is authorised in the European Union under the trade name CaniLeish ( Virbac , France ) .', 'It is composed of purified excreted-secreted proteins of Leishmania infantum ( LiESP ) , produced by means of a patented cell-free , serum-free culture system invented by the IRD ( Institut de Recherche pour le Développement ) [24] , and adjuvanted with QA-21 , a highly purified fraction of the Quilaja saponaria saponin .', 'The doses used in this study were formulated at 100 µgESP and 60 µg QA-21 .', 'This is consistent with the minimum accepted levels in commercially available doses .', 'Dogs in the vaccinated group were given one dose of the LiESP/QA-21 vaccine every 21 days for a total of three doses .', 'Dogs in the control group did not receive any vaccination .', 'All statistical tests were performed using the SAS v9 . 1 software , and for all analyses the significance threshold was set at p\u200a=\u200a0 . 05 .', 'Intergroup comparisons of the results of the CMLA on D0 and the CMLA , LTT and ELISpot assays on D62 were performed using a Wilcoxon test .'], ['Over the course of the study , all LiESP/QA-21 vaccinated dogs developed an IgG2 response to both ESP ( range 1/1350 to 1/4050 ) and , in particular , to PSA ( range 1/450 to 1/4050 ) .', 'See Figures 1 and 2 .', 'All vaccinated dogs also developed an IgG1 response to ESP by the end of the study ( range 1/450 to 1/4050 ) whereas only four of the ten vaccinated dogs developed positive IgG1 titres to PSA by day 56 ( range 1/450 to 1/1350 ) .', 'See Figures 1 and 2 .', 'The serological response induced as a result of vaccination with LiESP/QA-21 is therefore biased towards an IgG2 profile .', 'No dogs in the control group developed positive titres at any point throughout the course of this study .'], ['The results presented in this study confirm that vaccination of dogs with LiESP/QA-21 vaccine is capable of inducing a Th1 profile cellular response , within 3 weeks of the primary vaccine course , which in turn is effective in vitro at reducing the parasite load in pre-infected autologous macrophages .', 'Until in vivo challenge studies are reported , this data provides an understanding of the mechanism and onset of immunity induced by use of LiESP/QA-21 vaccine , which is the first of its type commercially available in Europe .']]
['Introduction', 'Materials and Methods', 'Results', 'Discussion']
['Canine leishmaniasis is an important zoonotic disease of dogs .', 'The clinical outcome of infection is variable , with the efficiency of the immune response being the key determining factor .', 'There is now a general consensus that a predominant Th1 immune profile in an overall mixed Th1/Th2 response is associated with resistance in dogs , and the absence of a strong Th1 influence is associated with a progression to clinical disease .', 'As a result , there has been a growing demand for vaccines that can induce a specific , strong Th1 response .', 'In this study , we measured the impact of a primary course of a newly available LiESP/QA-21 vaccine on selected humoral and cellular markers of the canine immune response during the onset of immunity .', 'All vaccinated dogs developed a humoral response characterised by IgG2 production .', 'More importantly , vaccinated dogs developed significantly stronger cell-mediated immunity responses than did control dogs .', 'Vaccination induced specific cellular reactivity to soluble Leishmania antigens , with a Leishmania-specific lymphoproliferation ( p\u200a=\u200a0 . 0072 ) , characterised by an increased population of T lymphocytes producing IFN-γ ( p\u200a=\u200a0 . 0021 ) and a significant ability of macrophages to reduce intracellular parasite burdens in vitro after co-culture with autologous lymphocytes ( p\u200a=\u200a0 . 0014 ) .', 'These responses were correlated with induction of the NOS pathway and production of NO derivatives , which has been shown to be an important leishmanicidal mechanism .', 'These results confirm that vaccination with LiESP/QA-21 induces an appropriate Th1-profile cell-mediated response within three weeks of completing the primary course , and that this response effectively reduces the parasite load in pre-infected macrophages in vitro .']
['The dog is the principle reservoir of Leishmania infantum , a parasite spread from dog to dog by a sandfly vector .', 'The reduction of canine leishmaniasis is therefore a key factor in the overall management of the epidemiology of this parasite .', 'There is also a need for effective prevention on welfare grounds because of the clinical severity of this potentially fatal disease in dogs .', 'The key factor determining the outcome of infection in dogs is the ability to mount a Th1-dominated immune response , because this is more effective against intracellular pathogens such as L . infantum .', 'Until now , in Europe , only measures to reduce sandfly bites have been available , and for many years there has been a strong demand for a vaccine that provides specific and effective immunity .', "The recent launch of the first European canine leishmaniasis vaccine ( CaniLeish ) has provided a means to achieve this , but data on its impact on the dog's immune system are required .", 'The data presented here demonstrate the specific stimulation of an effective Th1-dominated anti-L .', 'infantum response within three weeks of the administration of the vaccine , and provides a basis for the understanding of the mode of action of this new tool .']
['veterinary immunology', 'veterinary diseases', 'veterinary parasitology', 'zoonotic diseases', 'leishmaniasis', 'immunology', 'veterinary science', 'veterinary medicine']