Chapter 1 : General Introduction
1.6 Vaccine development
Vaccination against gastrointestinal nematodes would be a desirable alternative control strategy to anthelmintic treatments. To date, there have been few commercially available helminth parasite vaccines produced. The only one available for use in ruminants is Bovilis® Huskvac (MSD Animal Health), which was developed for the
Chapter 1: General Introduction 33 control of lungworm in cattle and is based on oral administration of live attenuated L3
(Jarrett et al., 1955; Vercruysse et al., 2004).
Vaccination of sheep against T. circumcincta is a possible option as a protective response can be acquired in the host following repeated exposure to the parasite either through natural field exposure or experimental infection (Smith et al., 1983; Stear et al., 1999). A vaccination programme could potentially be used in conjunction with existing strategies to prolong anthelmintic effectiveness, reduce pasture contamination and to protect the most susceptible sub-group of animals within a flock (Emery, McClure and Wagland, 1993). Successful vaccination could allow lambs to develop immunity before factors such as stress, inadequate nutrition and pasture contamination puts them at high risk of infection (McClure, 2009). The development of a vaccine against parasitic nematodes requires a detailed knowledge of the biology of the parasite, the nature of interactions with the host and the effective immune responses that are generated during infection (Knox, 2010). The first published set of T. circumcincta vaccination experiments, in the late 1970’s, used a mixture of L3 and L4 ES products from ex-vivo worms as antigen and induced 69% reduction in worm burden in vaccinated animals compared to identically-challenged adjuvant-only, recipients (Rose, 1976; 1978).
1.6.1 Approaches used in nematode vaccine development
Development of vaccines against parasitic nematodes has involved targeting molecules from different stages of the parasite life cycle, including products excreted/secreted from larvae or exposed on the parasite surface, using local antibody probes (Murphy et al., 2010; Nisbet et al., 2010a; Redmond et al., 2006; Smith et al., 2009) or the identification of molecules with homology to other known promising vaccine candidates (Nisbet et al., 2009; 2010b; 2011). A successful anti-nematode vaccine is likely to be one that will target several antigens expressed by various developmental stages of the parasite (Knox, 2010).
Chapter 1: General Introduction 34 1.6.1.1 L3 antigens
The L3 represents the first stage to come into contact with the host and its immune system, and therefore is critical in the life cycle as it could determine if the parasite is established within the host or excluded from the gastrointestinal system. One of the first immunogenic antigens to be identified from T. circumcincta L3 was a 31 kDa molecule located to the secretory organelles (McGillivery et al., 1989; 1992). This molecule displayed promise as a vaccine candidate in an immunisation trial where the 31 kDa antigen was purified from a detergent extract of T. circumcincta L3, 400µg of the antigen administered with Quil A adjuvant to 6 month-old sheep and, following immunisation, the sheep subjected to a single bolus challenge of 42,000 T. circumcincta L3 and antibody responses and parasitological parameters monitored (McGillivery et al., 1992). Comparison of vaccinated sheep to adjuvant-only controls showed reductions in both FWEC and worm burdens (42%) alongside the presence of antigen-specific serum IgG as early as 4 days after administration of the first vaccine dose (McGillivery et al., 1992). However, attempts to replicate this result have been unsuccessful as cell- mediated and antibody responses were not induced (Morton et al., 1995). One of the reasons behind this is that challenge dose given was a non-natural large dose and the immunity afforded by the vaccination may not have been adequate to control the parasite infection. Another potential vaccine candidate from T. circumcincta L3 was identified through the bioinformatic analysis of a stage-specific expressed sequence tag (EST) dataset from exsheathed T. circumcincta L3 (Nisbet et al., 2008; 2009). This surface associated antigen, Tci-SAA-1, shared over 77% amino acid identity with Ac-SAA-1, a vaccine candidate from the canine hookworm, Ancylostoma caninum, and was localised to the sub-hypodermal layer below the cuticle of exsheathed T. circumcincta L3 (Nisbet et al., 2009). Abomasal mucus IgA obtained from 5 month-old lambs which had been subjected to an experimental trickle infection (2,000 T. circumcincta L3 three times per week for a 10-week period) bound to the bacterial-expressed recombinant version of Tci-SAA-1, and anti-Tci-SAA-1 antibodies recognised a 12-14 kDa antigen present only in somatic and surface extracts of L3 (Nisbet et al., 2009). Tci-SAA-1 is therefore a
Chapter 1: General Introduction 35 promising vaccine candidate, primarily due to the high level of homology with A.
caninum vaccine candidate, localisation on the surface of exsheathed T. circumcincta L3 and the potential of the antigen to be a target for abomasal mucus IgA. Surface antigens therefore possess promise as vaccine candidates; however the external surface of parasitic nematodes is complex and dynamic. The outer covering of the nematode, termed the cuticle, is a multilayered structure with a unique structural and chemical composition (Blaxter et al., 1992). A carbohydrate-rich surface coat, termed the ‘glycocalyx’ is associated with the epicuticle and has been implicated in immune evasion (Maizels et al., 1993). In addition, following ingestion of infective T. cirucmcincta L3, the L3 exsheath, develop and moult, and the profile of the surface antigens displayed alters (Keith et al., 1990).
Surface antigens of T. circumcincta L3, have been stripped from the epicuticle using detergent and subsequently used in an immunisation trial to investigate their ability to induce a protective response in sheep against challenge (Wedrychowicz et al., 1992; 1995). Sheep vaccinated with L3 surface extracts in conjunction with beryllium hydroxide adjuvant and then challenged with a single bolus of 50,000 L3 had a 72% reduction in worm burden compared to adjuvant-only recipients (Wedrychowicz et al., 1992; 1995). Vaccination with L3 surface extracts induced a humoral response, with vaccinated sheep having a higher level of L3 surface antigen-specific serum IgA and IgG compared to control sheep (Wedrychowicz et al., 1992). However, when repeated in 1995, the same level of protection was not seen as there were no differences in serum IgA responses between vaccinated and control sheep (Wedrychowicz et al., 1995).
An L3-specific carbohydrate-larval antigen (CarLA), was found to be present on the cuticular surface of numerous nematode species, including Trichostrongylus colubriformis, T. circumcincta, H. contortus, Nematodirus americanus and Cooperia curticei (Harrison et al., 2003a; 2003b). Studies with T. colubriformis found that antibodies specific to CarLA were strongly associated with immune-mediated rejection of incoming L3, suggesting that CarLA is implicated in L3 establishment in the tissue niche (Harrison et al., 2003a; 2008). However, further research has revealed that there
Chapter 1: General Introduction 36 is variation in the versions of CarLA antigens displayed on individual worms, i.e.
individual larvae have different versions of CarLA (Maas et al., 2009). The implication of CarLA in L3 establishment and the finding that anti-CarLA antibodies mediate rejection of incoming L3 into the abomasum highlight that carbohydrates displayed on the surface of nematode parasites have the potential to be recognised by the host immune system.
1.6.1.2 L4 antigens
At the L4 stage, the parasite has already established in the abomasum and a vaccine that targets this stage could inhibit or prevent mechanisms by which larvae survive and grow. In the abomasal gland, L4 parasites release ES products and these could be a critical area to target to interrupt interactions between the host and parasite. Nematode ES products have been identified as important sources of protective antigens in other ruminant species (Meyvis et al., 2007). In an attempt to identify some of the components of T. circumcincta L4 ES products, and hence possible vaccine candidates, a proteomic analysis of L4 ES products collected at 8 dpi was conducted (Redmond et al., 2006). Analysis revealed that the most abundant protein present in L4 ES products was the proteolytic enzyme cathepsin-F (Tci-CF-1) (Redmond et al., 2006). This was a proposed vaccine candidate due to other members of the cathepsin family being validated vaccination targets in other helminth species (Dalton et al., 2003; Tort et al., 1999) and because of the essential roles proteinases play in nematode survival and development, including moulting, tissue invasion, digestion and evasion of host immune responses. Tci-CF-1 was a proven target of the local immune response in sheep subjected to a trickle infection and bolus challenge regime, as abomasal mucus IgA levels specific to Tci-CF-1 corresponded to the observed peak in total IgA in efferent gastric lymph (Redmond et al., 2006). A later study also used a proteomics approach to identify key molecules in T. circumcincta L3 and L4 ES products (Smith et al., 2009) from worms collected at 1, 3 and 5 dpi. Tci-CF-1 was identified in L4 ES products
Chapter 1: General Introduction 37 collected from worms harvested at 3 dpi (Smith et al., 2009). Another potential vaccine
candidate identified by the proteomic analysis of L4 ES products was an activation- associated-secreted protein (ASP), Tci-ASP-1 (Nisbet et al., 2010; Smith et al., 2009). ASPs are nematode-specific members of a diverse protein family, called SCP/Tpx- 1/Ag5/PR-1/Sct (SCP/TAPS), present in eukaryotes and a range of parasites (Cantacessi et al., 2009; 2012). The first report of their existence in parasites was in the ES products of serum-activated L3 of the canine hookworm, A. caninum (Hawdon et al., 1996; 1999). They are key vaccine candidates against hookworm species and are thought to be important in the establishment of parasites in the host (Hawdon et al., 1999; Tawe et al., 2000). Tci-ASP-1 was found to be specific to the parasitic stages of T. circumcincta, and a bacterial-expressed recombinant version was shown to be the target of abomasal mucus IgA obtained from sheep rendered immune to challenge through a prolonged experimental trickle infection/challenge regime (Nisbet et al., 2010).