Biological Processes of Designing Candidate Anti-Tick Vaccines: Comparison
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Ticks are obligate hematophagous arthropod ectoparasites distributed worldwide, and belong to two families; Ixodidae (hard-bodied ticks) and Argasidae (soft-bodied ticks). They affect 80% of the world’s cattle population and are associated with numerous health and economic effects. In developing tropical countries, tick-borne diseases (TBDs) constitute a major constraint to the livestock production, especially among smallholder farms of East, Central and Southern Africa.

  • ticks
  • enzyme
  • vaccine

1. Origin of Anti-Tick Vaccines and Immunological Control of Tick Infestations

Control of tick parasitism using immunological strategies has been studied for more than 40 years [1][2]. Earlier studies have revealed that some bovines acquired an immunological response to ticks from the inoculation of various antigens and molecules of tick salivary gland origin [3][4]. These observations form the basis for ATV research and development [5].
Anti-tick vaccines became commercially available in the early 1990s to control tick infestations in cattle [6][7]. Despite how long they have existed, alternative and more effective anti-tick vaccines (ATVs) are still not available. Identification of molecules essential for both tick survival and for host–vector–pathogen interactions have been hypothesized as strategies for the development of novel vaccines, and for simultaneous control of ticks and tick-borne pathogens [8][9]. In the recent years, a number of tick proteins (candidate antigens) have been identified and assessed in controlled pen trials, yielding variable results [10][11].
Elvin and Kemp, 1994 [12] proposed a candidate antigen for the development of an anti-tick vaccine as one for which the host antibodies can sufficiently gain access to the target protein. 

2. Exploring Tick Biology for Antigen Identification and Vaccine Development

Knowledge of specific physiological processes of ticks has been utilized to rationally develop promising vaccine candidates that can impair tick biological processes. These targeted physiological processes effect tick attachment to the host, uninterrupted feeding on the host, intracellular digestion of large blood volumes and metabolism of ingested blood into massive clutches of eggs laid by the engorged female ticks [13]. 
(a)
 Tick Attachment and Feeding to Repletion
Interfering with tick attachment to the host would be the ideal intervention for preventing both tick feeding and pathogen transmission to the host [13]. During tick attachment, an Ixodid tick secretes glycine-rich proteinaceous cement-like substances in the saliva that harden around the inserted mouthparts [14]. This cement cone enables the tick to remain attached to the host during the long duration of feeding (up to 14 days) and prevents host immune mediators from accessing the tick’s proboscis. Thus, a vaccine targeting components of the cement cone could ideally interfere with tick attachment and pathogen transmission [3].
The tick bite stimulates host defenses such as itch, homeostasis, inflammation and immune response. Homeostasis (blood coagulation, vasoconstriction and platelet aggregation), complement activation and inflammation constitute the early protection against tick infestation [15]. The processes leading to acute inflammatory response begin when host tissue is first damaged, but it is the subsequent migration and degranulation of white blood cells, particularly the granulocytes, at the bite site that mark the beginning of inflammation. This may occur within three hours and can last several hours. While pathogens such as the Powassan virus require a short transmission time (15 min) and may elude the inflammatory response, most bacterial and protozoan pathogens require several hours of tick feeding before transmission [7][16]. It has been demonstrated that the cellular response attracting inflammatory cells to the feeding site of Phlebotomus papatasi is sufficient to block transmission of Leishmania [17]. Upon tick attachment and feeding, both cellular and humoral mediators of vertebrate adaptive immunity are activated, with T and B memory cells amplifying the host inflammatory response to subsequent tick infestation through cytokine and antibody production [15][18].
Uninterrupted feeding, therefore, requires ticks to counter the complex host immune responses mounted against them. Tick saliva is a complex mixture of bioactive molecules that are used by the tick to modulate, deviate or inhibit various cellular and molecular functions of the vertebrate’s defense mechanisms, creating an immune-privileged environment at the bite site, which facilitates transmission of pathogens to the vertebrate host [15][19]. This phenomenon is termed saliva-assisted transmission (SAT) [20]. Ticks have evolutionarily developed a range of molecules that counteract almost all the vertebrate’s immune defenses. Gene expression and production of saliva molecules is upregulated soon after tick attachment. Correlation of real-time tick salivary gland transcript and protein expression with corresponding changes in host skin and regional lymph node gene expression elucidates the complex interaction between the tick and host responses [19][21][22], which can guide identification of anti-tick antigens for vaccine development. Although some saliva molecules have been identified and their functions described [19][20], there is extensive redundancy at the molecular, cellular and functional level, and the proteins generally exhibit low immunogenicity [15][19].
A 29-kDa salivary protein (p29) was identified by Mulenga et al. (1999) [23] while screening the cDNA library of Haemophysalis longicornis with rabbit immune serum raised against tick saliva proteins. Due to its structural homology to collagen, the protein was presumed to be a constituent of the extra-cellular matrix that forms the cement cone during tick attachment. When recombinant p29 was used to immunize rabbits against H, longicornis reduced female tick engorgement weights and caused mortalities of 40–55% among larvae and nymphs.
Salivary proteins HL34 and HL35 were identified by Tsuda et al. (2001) [24], by immune-screening of a cDNA library of an adult H. longicornis combined with amplification and cloning of the genes. Since expression of the HL34 and HL35 genes is induced during the slow feeding phase, both in the tick salivary glands and in other organs, the proteins are suspected to play a role in tick feeding. The presence of proline and tyrosine repeat amino acid domains, which characterize adhesive molecules, also suggest the proteins to be components of the cement cone. Vaccination of rabbits with recombinant HL34 protein affected nymphs and reduced oviposition in adults due to impaired blood digestion.
Another cement cone component protein (36kDa) designated Rhipicephalus Immuno-dominant Molecule 36 (RIM36) was identified from the R. appendiculatus cDNA library. The protein is principally localized in the e cells of the type III salivary gland acinus, in which Theileria parva sporozoites also develop. During tick feeding, RIM36 induces a strong host antibody response, to which some studies partly attribute R. appendiculatus resistance among experimental guinea pigs [25]. Recombinant RIM36 also reacted with immune sera from cattle either experimentally infested with ticks or obtained from field infestations [3].
Tick protein 64P, identified in R. appendiculatus, is a putative secreted component of the cement cone. Its compositional similarity to the host skin proteins suggests evolutionary mimicry to avoid rejection of the tick by the host’s immune response. For better exposure of epitopes to the host’s immune system, recombinant truncated constructs of 64P (64TRPs) were fused with GST and used to immunize rabbits. These elicited both humoral and cell-mediated immune responses among the vaccinated animals, which was amplified following tick infestation. This amnestic immune response and the observed local cutaneous inflammatory response are prerequisites to development of naturally acquired resistance to tick infestation, and are desirable as a candidate for an anti-tick vaccine. Cross-reactivity of anti-64TRPs antibodies with salivary, midgut and hemolymph epitopes of both adult and nymph R. appendiculatus subsequently caused mortalities after tick detachment. Similar protective effects of R. appendiculatus 64TRPs were observed against adult and nymphal Rhipicephallus sanguineus and Ixodes ricinus [26], indicating the potential of 64TRPs as a broad-spectrum anti-tick vaccine. In addition, R. appendiculatus 64TRPs successfully protected mice against the tick-borne encephalitis virus (TBEV) transmitted by infected I. ricinus ticks to a level comparable to that of a dose of commercial TBEV vaccine [27].
(b) 
Immunomodulation and regulation of enzyme activity
Mammalian hosts can acquire immunity (resistance) against ticks as a result of prolonged infestation or vaccination with tick antigenic proteins, which affects tick physiological processes such as feeding, reproduction and viability [18]. As noted in (a) above, a tick bite induces host homeostatic and immune regulatory responses, which interfere with the tick’s attachment and feeding [28][29]. A variety of proteases, notably serine proteases, are involved in the mediation of mammalian homeostatic and immune processes. The activity of these enzymes is regulated by a group of proteins collectively known as protease inhibitors [30][31].
In arthropods, serine protease inhibitors (serpins) are presumed to regulate endogenous homeostatic processes and to protect against infection by inhibiting pathogen-derived proteases. Similarly, ticks are likely to deploy serpins to counter host homeostatic and immune responses to facilitate uninterrupted feeding or to maintain their own physiology. This, therefore, makes serpins a potential target as an anti-tick candidate antigen [32]. Serpins are produced in different tick organs, such as the salivary glands [33], the gut [34] and the hemolymph [32], and different tick species target different serine proteases. For example, the serpin HLS2 has only been demonstrated in the tick H. longicornis, and regulates tick endogenous proteases during feeding. A candidate recombinant anti-tick vaccine based on HLS2 (r HLS2) yielded about 40% mortality for tick nymphs and adults fed on vaccinated rabbits. It was, however, not suitable for use as a single vaccine antigen [32].
Other serpins which have been evaluated as possible anti-tick vaccines include Rhipicephallus appendiculatus-derived RAS-1 to -4 [35], among which a combination of rRAS-1 and -2 reduced nymph engorgement by 60% and increased adult tick mortality by 28–43% in cattle immunization trials [36]. Other combinations of these serpins mostly affected parasite-infected ticks.
Cystatins constitute a different superfamily of protease inhibitors targeting papain-like cysteine proteases and legumains, and are present in vertebrates and invertebrates. In soft and hard ticks, type 1 (also called stefins) and type 2 cystatins have been identified, and these only share limited amino acid sequence similarity with cystatins in other organisms (<40%). They have been mostly described in tick salivary glands and the midgut, where they may possibly affect blood digestion. The Bmcystatin in R. microplus midgut plays a role in the tick’s embryogenesis, since it inhibits the vitellin-degrading cysteine endopeptidase (VTDCE) [37]. Similar to Bmcystatin is the midgut stefin, Hlcyst-1 of H. longicornis, which regulates host blood digestion by inhibiting the hemoglobinolytic activity of a cathepsin L-like cysteine protease, HlCPL-A. Type-2 cystatins are secretory in nature and have been studied extensively. Among these are sialostatins L (SL) and L2 (SL2) from I. scapularis, which are capable of inhibiting cathepsin L, with SL additionally inhibiting cathepsin S. It is noteworthy that cathepsins S, L and V are vital for mammalian immunity due to their involvement in antigen presentation processes of dendritic cells and macrophages [38].
Experiments silencing sialostatins L and L2 prevented tick feeding on rabbits by 40%, demonstrating their role in tick blood feeding. Sialostatins L2 was particularly upregulated in salivary glands of feeding ticks, and when used as a recombinant vaccine in animal experiments, early rejection of ticks at feeding sites or prolonged feeding times were observed. A strong immunosuppressive effect of sialostatins in the host was also observed [39][40]. Other type 2 cystatins described in H. longicornis include Hlcyst-2 and -3 (midgut) and HLSC-1 (salivary glands). These are capable of inhibiting papain and cathepsin L, with Hlcyst-2 additionally inhibiting Cathepsin H [41][42]. Hlcyst -2 also interacts with HlCPL-A to affect host blood digestion in a way similar to Hlcyst-1 [43][44]. Hlcyst -2 is additionally implicated in the tick’s innate immunity, as shown by in vitro experiments with Babesia bovis [42]. Among the soft ticks, type 2 cystatins identified in O. moubata include om-cystatin 1 and 2. These strongly inhibit papain and cathepsin B and H, but om-cyastatin 2 also binds cathepsins C, L and S [45][46]. Due to the immunosuppressive action of tick salivary components, such as cystatins, their neutralization by host antibodies or gene silencing in ticks can significantly reduce tick feeding and, possibly, control tick infestation [43].
(c)
 Osmoregulation (water balance)
Unlike insects, which use malpighian tubules, hard ticks rely on salivary glands for osmoregulation [47]. In order to attain full engorgement, an ixodid female tick can suck 200–300 times its own weight in blood. Using the salivary glands, the tick actively excretes up to 70% of the excessive fluid and ions back into the feeding lesion, to concentrate blood components for efficient digestion and to allow further intake of blood [47][48]. Part of this large salivary flow constitutes the transmission route for pathogens and bioactive molecules which modulate the host’s immunity [49]. On the other hand, during long spells when an unfed tick is off the host, salivary glands can produce a hyperosmotic secretion which facilitates the absorption of atmospheric moisture [47]. Given the critical importance of osmoregulation and water balance in tick physiology and survival, water channels (aquaporins) constitute a suitable target for designing an anti-tick vaccine. Aquaporins (AQPs) are protein structures that render the lipid bilayer of cell membranes permeable to water [50], and their role in the salivary glands of I. ricinus has been demonstrated [47]. Three aquaporins have been identified in cattle tick R. microplus, and are designated RmAQP1, RmAQP2 and RmAQP3. Cattle vaccination with recombinant RmAQP1 yielded an efficacy ranging between 68–75% [51].
(d)
 Blood digestion (Hemoglobinolysis)
Besides survival, the major importance of a blood meal for an adult female tick is maintenance of the reproductive vigor, which is measured by the massive egg production [5]. Tick blood digestion occurs intracellularly in the gut epithelial cells, where it is carried out by a network of acidic peptidases. These hemoglobinolytic enzymes mainly comprise aspartic endopeptidases (cathepsin D-like), cysteine endo- and exopeptidases (cathepsin L, B and C type) of the papain family, asparaginyl endopeptidases (legumain peptidases) and monopeptidases [52]. These hemoglobinases have also been functionally characterized in various tick species, mainly based on gene-specific RNAi silencing [53][54], and their gene expression has been shown to be induced and upregulated by tick feeding [44][54][55]. Since their molar concentration and activity increases with feeding, with most of them peaking at full engorgement, a vaccine targeting hemoglobinolytic enzymes may not block pathogen transmission. Moreover, whereas vaccination using recombinant antigens of these enzymes stimulates high antibody titers, only limited efficacy was detected, possibly due to the high redundancy of their coding genes [56].
(e)
 Heme and iron transport and storage
Whereas other blood-sucking arthropods excrete excess heme and iron through feces or polymerize it into insoluble hemozoin, the hemoglobin ingested by ticks is phagocytosed by specialized epithelial cells of the midgut, and is digested intracellularly inside large acidic vesicles [57][58]. A network of acidic lysosomal peptidases is involved in the hydrolytic process [59], generating great amounts of heme inside the cells. Consequently, ticks always have to contend with excessive amounts of toxic heme and iron-derived metabolites from their blood meal [60]. Heme is capable of catalyzing the formation of reactive oxygen radicals, which can, in turn, cause oxidative damage and disrupt the cellular lipid bilayer [61].
As an adaptation to heme toxicity, ticks have evolved heme detoxification mechanisms. During the first days following a blood meal, heme is mainly absorbed from the midgut and transferred into the hemocoel, where it is bound by hemolymph hemelipoprotein (HeLp), which transports and delivers it to peripheral tissues, particularly the ovaries. Since HeLp forms the bulk of hemolymph proteins, heme noticeably accumulates into the oocytes and ovaries (making 80% of ovarian proteins), which facilitates vitellogenesis [60][62][63]. In ticks, vitellin, which is the main yolk protein, is a hemoprotein [64]. Towards the end of blood digestion, most of the heme generated (>90%) aggregates into specialized organelles called hemosomes, and only a limited amount is used by the tick’s own metabolic demands [60][62]. Heme in the tick’s body is always bound to a protein to prevent toxicity [60], and for HeLp, the lipoproteic component serves as an antioxidant [65]. In addition, various peptide products of hemoglobin digestion perform an antimicrobial role (hemocidins), augmenting ticks’ immunity [66]. Due to the critical role played by heme-binding and trafficking proteins (detoxification), targeting them could enable the development of new tick control strategies [60].
Although iron is essential for various biochemical processes, such as oxygen transport, energy metabolism and DNA synthesis, it can catalyze the generation of reactive oxygen species, which can cause oxidative damage to cells and tissues [67]. Different organisms, therefore, use various proteins for the safe metabolism of iron. The protein ferritin, present in most organisms, is important for iron metabolism. Depending on the animal species, it is involved in iron storage, homeostasis, protection against oxidative damage and iron transport in insects [68]. In ticks, ferritin is important for blood feeding and reproduction, and two ferritins have been characterized. Intracellular ferritin 1 (FER1), produced in midgut cells, stores iron within the cell, while a secretory ferritin, FER 2, transports iron to peripheral tissues such as the ovaries and oocytes. Ferritin 2 is, therefore, important for oviposition and embryonic development. Silencing ferritin-coding genes reduces feeding, body weight and fecundity while increasing mortality and morphological defects [68].
Rabbit immunization studies with recombinant ferritins have shown that the proteins are immunogenic and that they induce the production of high levels of antibodies, with anti-FER 2 antibodies being present even in eggs. Since FER 2 is abundant in hemolymphs and circulates in the tick’s body, ingested host antibodies gain significant access to it. The protein (FER2) also exists exclusively in ticks. Thus, with a vaccine efficacy of approximately 50% against H. longicornis in rabbits [69], and over 60% and 70% against R. microplus and R. annulatus, respectively, in cattle vaccination trials [70], it is believed that FER 2 (recombinant), if formulated in combination with other antigens, may improve the efficacy and cross-protectivity of either antigen [69].
(f)
 Detoxification (elimination of toxic substances)
Glutathione S-transferases (GSTs) are a family of enzymes present in various tissues of eukaryotic organisms, and are involved in the metabolic detoxification of xenobiotics, reactive oxygen species, heme and other endogenous compounds [71]. In the presence of glutathione (GSH), GSTs’ catalytic reactions result in less harmful products, which are easily excreted by the cell [72]. The increased activity of GSTs in some pesticide- and drug-resistant parasite strains compared to susceptible ones demonstrates the detoxification function of the enzymes [73][74]. In addition, several acaricides have been shown to target GSTs activity, supporting the possible use of GSTs as a candidate anti-tick antigen [75][76], and since the antigen is relatively conserved across tick species, the induced immune response may be cross-protective [71]. Cattle vaccination trials with H. longicornis recombinant GST antigens yielded a 50% vaccine efficacy [13]. Similar studies by Parizi et al. (2011) [71] recommended the use of GSTs in combination with other characterized antigens to boost efficacy.
(g)
 Embryogenesis (yolk accumulation and degradation)
The tick population in the environment is partly maintained by the tick’s capacity to lay large volumes of eggs that give rise to greater numbers of offspring per subsequent generation. It is, therefore, logical to control tick populations by interfering with their reproductive processes, such as vitellogenesis, embryogenesis and fertility. It is also considered possible to target internal tissues, since studies have shown host antibodies to circulate in the hemolymphs of ticks feeding on immunized hosts [77]. Vitellogenesis or yolk accumulation, which is a process during which extraovarian and ovarian tissues produce protein precursors that are conveyed to and selectively accumulate in the oocytes [78][79], is critical to the tick’s reproductive success. The major protein sequestered from the hemolymph by developing oocytes is vitellogenin, which crystallizes to be stored as vitellin [78] in structures called yolk spheres or yolk granules. Vitellin forms the source of amino acids for tick embryonic development [80]. It is, therefore, imperative to understand the composition of yolk proteins, the tissues where they are produced, how they are transported and sequester in the oocytes, and how they are enzymatically mobilized during embryogenesis. Known among these are two Aspartic endopeptidases, Boophilus yolk pro-cathepsin (BYC) and Tick heme-binding aspartic peptidase (THAP), as well as Cathepsin-L-like vitellin degrading cysteine endopeptidase (VTDCE).
Produced in the gut and fat body, BYC is secreted into the hemolymph and sequesters in the oocytes [81], constituting up to 6% of egg protein [82]. The enzyme is involved in the hydrolysis of vitellin in tick eggs [81] and possibly hemoglobinolysis in the larvae [83]. Cattle immunization with native and recombinant BYC stimulated specific IgG responses with protective efficacies of up to 36% and 25%, respectively, the vaccine effects being notable in the number and weight of engorged females, as well as in egg fertility [84]. Tick heme-binding aspartic peptidase (THAP) is a VT-degrading enzyme present in tick eggs, whose activity can be inhibited by heme at a site remote to the catalytic site [80]. Thus, by binding heme, THAP plays a role in maintenance of the redox balance, preventing oxidative damage [85]. Vitellin-degrading cysteine endopeptidase (VTDCE), is cathepsin-L-like, which refers to a group of enzymes present in all tick developmental stages. Exhibiting higher vitellin-hydrolytic activity than BYC and THAP, VTDCE is distributed in the gut, ovary and hemolymph [79][86][87]. Animal vaccination trials with native VTDCE yielded a vaccine efficacy rate of 21%, the major effects being on egg weights and number of engorging females [86]. The Boophilus microplus Cathepsin-L 1 (BmCL1-recombinant) or RmLCE (native form) is a cysteine endopaptidase found in tick larvae where it hydrolyses hemoglobin and VT subunits generated by previous activity of maternal enzymes (VTDCE, BYC and THAP).
(h)
 Enzymatic disruption and remodeling of host tissues
Naturally, metalloproteases (MPs) are multipurpose proteins involved in many biological functions, and are present in various organisms [88]. In ticks, salivary MPs are used in the remodeling or disruption of host tissues’ structural constituents, as well as interfering with homeostasis [89][90][91]. They are implicated in the degradation of fibrin and fibrinogen at the bite site [92], the inhibition of microvascular endothelial regeneration and the breakdown of cell integrins [20][93]. These enzymatic activities collectively impair natural wound healing at the tick bite site [94], and facilitate pathogen transmission to the host, e.g., Borrelia spirochetes [92].
Gene expression of metalloproteases is induced by tick feeding. A vaccine targeting these proteases could, thus, result in early tick rejection. Using a cDNA library, Decrem et al. (2008) [95] identified and designated two homologous MPs as Metis 1 and 2 in the tick I. ricinus. Characterization of their function by RNAi revealed reduced capacity of salivary gland extracts (SGEs) to disrupt fibrinolysis, while gene knock-down by the same method caused incomplete engorgement and mortalities. Recombinant Metis 1 reduced feeding and oviposition among immunized rabbits, but caused no effect on tick nymphs. Further, three other blood meal-induced MPs were described, and these showed limited genetic similarity to the former [94][95].
The tick H. longicornis metalloprotease (HLMP1) was demonstrated in salivary glands of all instars, and when its recombinant form (rHLMP1) was used to vaccinate rabbits, mortalities of 15.6% and 14.6% in nymphs and adults, respectively, were observed [96]. On the other hand, a 60% protection rate was obtained when recombinant MPs from R. microplus were used in bovine immunization trials. The vaccine mostly affected tick numbers, oviposition and egg hatching [88]. However, these and other studies show that although a vaccine based on MPs can advantageously produce an amnestic immune response, it does not sufficiently suppress tick infestation. This could possibly be due to the extreme redundancy of the antigen as envisaged by presence of MPs in various tissues, and its occurrence as multiple isoenzymes [95][96].
(i)
Tick engorgement and development of reproductive structures
For most Ixodid ticks, there is a transition weight between the slow and rapid phases of tick feeding, termed the critical weight. When this is not attained by a female tick before detachment, no eggs can be laid. On the other hand, most unmated female ticks do not feed beyond critical weight no matter how long they remain on the host. It is believed that a testicular engorgement factor (EF) is introduced into the feeding female during copulation, and stimulates rapid engorgement. This protein was identified from a cDNA library of fed A. haebraeum testicular tissue, and was designated voraxin [97]. It is a combination of two synergistically bioactive peptides, designated rAhEFα and AhEFβ, whose production is upregulated during feeding. The protein also induces salivary gland degeneration and partial development of the tick ovaries. It is believed that a vaccine based on voraxin would reduce oocyte development and pathogen transmission between the tick and the host. This is supported by the observation that the voraxin-based candidate vaccine yielded a reduction in the mean weight of 72% among surviving ticks, compared to 37% by BM86 based vaccines. Other physiologically vital mating factors considered in ticks include the sperm capacitation factor, which stimulates the final phase of sperm cell maturation within the female tick after copulation, and the Vitellogenesis-stimulating factor of the soft tick O. moubata, which is important for oviposition [98].

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