Recent Advances in Tick Antigen Discovery And Anti-Tick Vaccine Development Part 1

Ticks can seriously affect human and animal health around the globe, causing significant economic losses each year. Chemical acaricides are widely used to control ticks, which negatively impact the environment and result in the emergence of acaricide-resistant tick populations. A vaccine is considered as one of the best alternative approaches to control ticks and tick-borne diseases, as it is less expensive and more effective than chemical controls. Many antigen-based vaccines have been developed as a result of current advances in transcriptomics, genomics, and proteomic techniques. 

A few of these (e.g., Gavac® and TickGARD®) are commercially available and are commonly used in different countries. Furthermore, a significant number of novel antigens are being investigated with the perspective of developing new anti-tick vaccines. 

However, more research is required to develop new and more efficient antigen-based vaccines, including assessing the efficiency of various epitopes against different tick species to confirm their cross-reactivity and their high immunogenicity. In this review, we discuss the recent advancements in the development of antigen-based vaccines (traditional and RNA-based) and provide a brief overview of recent discoveries of novel antigens, along with their sources, characteristics, and the methods used to test their efficiency.

vaccinomics; antigen candidates; anti-tick vaccine; tick control.

1. Introduction

Ticks are ectoparasites that infest humans and animals and are responsible for significant economic losses. They are the second most important vectors for the transmission of diseases in humans after mosquitoes [1,2]. They are also one of the most important vectors for the transmission of diseases that impact the global cattle industry and pets [3–5]. Ticks have few natural enemies, making it challenging to control tick infections. Chemical acaricides have been only partially effective, with several nontarget disadvantages, including the selection of acaricide-resistant ticks and contamination of the environment and animal products with chemical residues [6]. 

In addition, to control tick-borne diseases, some antigen-based vaccines are used in various countries; however, new and more effective approaches are needed, including the development of new vaccines that target tick infestations and pathogen infections [7,8].

Traditionally, the "isolate–inactivate–inject" principle has played a crucial role in designing and developing a vaccine for the control of parasites/pathogens. First-generation vaccines were composed of pathogens that were alive, attenuated, or killed. Second-generation vaccines consisted of purified parasite/pathogen components and were developed as a result of advances in cell culture, polysaccharide chemistry, recombinant DNA technology, and immunology [9,10]. The advancement of genomics and other "omics" over the last two decades has resulted in the development of a "third generation" of vaccines, based on technologies such as functional omics, reverse vaccinology, and the systems biology approach.

To overcome the limitations of the conventional vaccine development approaches, vaccine development has become more tailored, with a focus on the antigen moieties that are targeted by the protective immune responses [11,12], with a broad perspective of the pathogen and its interaction with the host immune system [13]. Hence, modern vaccinology relies increasingly on novel omics approaches utilizing high-throughput cutting-edge technologies, such as genomics, transcriptomics, and proteomics, along with advances in basic immunology, host–pathogen biology, immunomics, advanced bioinformatics, computational modeling, and improved understanding and technological innovations.

Compared to using chemicals, vaccination is a wise option because it is environmentally safe and cost-effective to control tick infestation [12,14]. Although vaccination is a rational strategy for controlling tick infestation, only a few vaccines have been commercialized so far, with minimal concern given to the induction of cross-reactive immunity against tick species [15]. 

To develop new vaccines, it is crucial to identify and characterize novel antigen candidates that would be more conserved and have the ability to induce cross-reactive immunity in the host species. The goal of this review is to provide an overview of traditional and RNA-based vaccines and the possibility of their application and novel antigens that have the potential to be exploited as promising antigen candidates for vaccine development.

2. Identification of Antigens: A Road Map to Develop an Anti-Tick Vaccine

The identification of antigens is paramount for the development of an anti-tick vaccine. It is crucial to understand the molecular mechanisms associated with the host–parasite–pathogen interactions to identify antigen candidates that are likely to serve as candidates/targets for the development of a vaccine. The ideal antigen candidate induces long-lasting and effective immune responses in the host [16,17]. Many studies have been carried out since Allen and Humphreys published their findings in 1979, employing a range of antigens, including whole tick homogenates and internal organs, to induce varying levels of immunity against ticks [16].

Several new possibilities have emerged for predicting, screening, and identifying antigens protective against tick infestations since Ixodes scapularis, the first tick species to be sequenced [18]. There are now many nucleotide and protein databases available from different tick tissues and developmental stages, and a wide variety of stimuli that affect ticks, such as tick feeding or infection with pathogens [17,19], are known. 

The probability of selecting protective antigen candidates derived from ticks for the control of tick infestation and pathogen infection has also increased as a result of recent advances in omics technologies (i.e., transcriptomics, proteomics, and metabolomics) [20]. In addition, the use of reverse vaccinology (RV), or vaccinomics, has allowed the discovery of new vaccine antigen candidates [20]. 

As a result of this, synthetic and recombinant proteins have been evaluated and demonstrated to be able to induce some level of protective immunity. The purpose of this section is to discuss antigen candidates originating from different tissues which have been identified, assessed for their efficacy, and are being considered as potential candidates for the development of an anti-tick vaccine, based on the available literature (Table 1 and Figure 1).

Figure 1. An overview of the distribution and efficacy evaluating scheme of tick vaccine antigens targets for the prevention of tick infestations and tick-borne diseases.

2.1. Egg-Associated Antigen Candidates

Egg yolk is an essential component for the development of ticks since it serves as a reservoir of various proteins that play a crucial role during the embryonic development of these arthropods [21,22]. As in insects, yolk proteins are synthesized in the fat body of ticks [21,23]. The degradation of the yolk is carried out by various types of enzymes that are found in eggs. Boophilus Yolk pro-Cathepsin (BYC) is an example of a yolk proteinase that has been isolated from R. microplus eggs and has been reported to be involved in the embryogenesis process of the tick. 

In particular, these enzymes play a key role in the degradation of vitelline, a major proteinaceous component of egg yolk [21]. BYC was first isolated by da Silva Vaz Jr et al. [24] from R. microplus eggs and was then inoculated into cattle to determine its role in the induction of host immunity. This enzyme was found to provide partial protection against ticks and trigger a protective immune response in cattle, but its efficacy was between 14 percent and 36 percent . A subsequent study expressed recombinant BYC protein in a prokaryotic expression system (E. coli). 

Interestingly, the recombinant protein showed an overall higher efficacy (25.24 percent ) compared to the enzyme directly isolated from egg yolk [25,26]. It appeared that various factors may affect the efficacy of this protein, for example, the method of preparation of BYC protein can influence the protein structure and ultimately its functions. Furthermore, this variation may be also associated with the tick strain or other experimental conditions [24].

Vitellin, a lipoglycoprotein also occurring in the egg yolk similar to other yolk proteins, is synthesized in the fat bodies of arthropods [27,28]. In ticks, vitellin or vitellogenins be crucial for egg development and oviposition as demonstrated by the silencing of three vitellogenin genes in H. longicornis [29]. Vitellin protein was purified from tick eggs as a non-covalent complex of six polypeptides of high molecular weight (44–107 kDa). Parallel to this study, an 80 kDa glycoprotein (GP80) was isolated and purified from R. microplus larvae. 

Both proteins were then inoculated to investigate their efficacy. Vitellin and GP80 vaccination showed an overall 68 percent efficacy, suggesting that a vaccine containing both antigens can induce an immune response and also provide partial protection against R. microplus in sheep hosts [28]. 

Remarkably, when recombinant hexahis-GP80 (HH-GP80), which was incorrectly folded and not glycosylated, was injected into the host under the same experimental conditions, it displayed no efficacy [28]. Based on the findings of the above study, it appears that vaccination of vitellin and GP80 can elicit immune responses in sheep and may partially protect sheep against the tick B. micro plus. The correct folding of HH-GP80 is crucial for its activity, since protective epitopes are associated with the folding of the protein and/or the oligosaccharides attached to it, and these epitopes are essential for its activity.

Vitellin degrading cysteine endopeptidase (VTDCE) is another egg-associated enzyme that was identified and isolated by Seixas et al. [30]. Similar to BYC, this enzymatic protein is not synthesized in the ovary of R. microplus and is implicated in vitellin hydrolysis, thereby providing nutrients to developing embryos. 

However, both enzymes were found to regulate vitellin hydrolysis differently [30]. The same research group later analyzed purified VTDCE protein as an antigen and found that this protein also provides partial protection against ticks, as the immunization of livestock resulted in 21 percent efficacy and a 17.6 percent reduction in the weight of fertile eggs [31]. The egg-associated proteins BYC and VTDCE provided limited protection to the host against tick infestation, and therefore seem to be not suitable antigen candidates when used alone in a vaccine.

2.2. Salivary Gland-Associated Antigen Candidates

Ticks contain an angiotensin-converting enzyme-like protein that can control blood pressure by regulating fluid volume, similar to the angiotensin-converting enzyme in mammals [32,33]. This control allows the tick to feed continuously on the host's blood. The salivary glands and midgut of tick B. micro plus contain a low abundance glycoprotein, which is named Bm91 [32]. Bm91 is currently not included in commercial anti-tick vaccines, but it is considered to be a candidate for controlling ticks [34]. When the recombinant Bm91 protein was assessed alone under field conditions with natural tick infestation, the results were disappointing, as this protein showed only 6 percent efficacy, which seems inappropriate for the development of a vaccine for tick control [35]. 

However, when recombinant Bm91 protein which was produced in E. coli was combined with Bm86 (an antigen candidate that is used in commercial vaccines) and then this protein combination was used as a vaccine, the results were much more promising, since the Bm91 addition enhanced the efficacy of the Bm86 antigen [33], suggesting that the combination of these two proteins (Bm91 and Bm86) seems to be an effective strategy to develop a new anti-tick vaccine.

Transcriptomic and differential gene expression analyses of salivary glands have shown that the genome of tick (e.g., R. microplus and Dermacentor andersoni) species comprises a protein sequence named flagelliform silk protein [36,37]. The characterization of differential gene expression in the salivary glands of R. microplus in response to A. marginale infection highlighted the molecular mechanisms of how the tick interacts with the pathogen. Subsequent functional studies have shown that flagelliform silk protein (SILK) may play a crucial role in the infection and multiplication of A. marginale in ticks. An interaction between tick- and pathogen-derived molecules are involved in the multiplication of A. marginale in salivary gland cells [36,38]. 

Following this study, it was proposed that flagelliform silk protein could be a suitable antigen candidate to develop a vaccine. For this purpose, Merino et al. [14] produced recombinant flagelliform silk protein and analyzed its antigenic activity by injecting it into a cattle host. The recombinant protein was found to be an excellent antigenic candidate, as it provided 62 percent protection against tick infestation and tick-borne infection (e.g., babesiosis) in cattle. Vaccination with flagelliform silk protein reduced the multiplication of A. marginale in cattle. Theantigen-specific antibody titers correlated with reduced tick infestations and pathogen infection, indicating that the effect of the vaccine is a result of the antibody response. 

Furthermore, the expression of gene-encoding vaccine antigens in ticks feeding on cattle was also affected by vaccination and co-infection with A. marginale and B. bigemina. Thus, it appears that vaccines using tick proteins that are involved in vector–pathogen interactions can be effective in both controlling tick infestation and preventing pathogen infection at the same time [14].

Salp15 is an immune suppressive salivary protein of I. scapularis with a molecular weight of 15 kDa that inhibits the activation of CD4 plus T cells, the complement activity, cytokine production, and the dendritic cell function in the host [39–41]. Subsequent studies investigated the molecular mechanism of Salp15. The outer surface protein, OspC, is produced by B. burgeri on the outer surface of the cell. The producție de spirochete (B. burgdorferi spirochete) în midgut de infectate căpușe este inițiat când it feed on blood from the host, which is then transported to the host. 

During the exit from the salivary glands and transmission of the B. burgdorferi spirochetes to the host, Salp15 physically interacts with OspC on the surface of B. burgdorferi spirochetes, which facilitates the survival of spirochetes, pathogen transmission, and host infection [38,42]. Salp15–OspC interaction may thus potentially obscure OspC from the host immune response so that the spirochete is protected from the immune response [38]. Recently, the Escherichia coli expression system was used to synthesize Salp15 recombinant protein, and the system was found to be efficient in producing this protein in a considerable yield with good solubility. These characteristics of Salp15 recombinant protein indicate that this has practical application and can be used to generate anti-tick vaccines [41,43,44].

Metalloproteases (MPs) are multifunctional proteins that participate in a wide variety of complex physiological and pathologic processes in living organisms [45]. Several MPs have been identified in different tick species and are considered to be crucial for the maintenance of blood meal-associated functions in ticks [46–49]. For example, the salivary glands of ixodid ticks contain MPs that are recognized as key bioactive components in vital physiological functions and are therefore considered for use as potential targets in control strategies to combat these ectoparasites [49]. To evaluate the antigenic potential of MPs, Ali et al. (2015) [50] amplified a fragment of the sequence encoding an R. micro plus MP, expressed it as a recombinant protein, and used the purified form of this protein as a vaccine antigen against R. microplus in cattle [50]. The recombinant R. micro plus MP protein demonstrated an overall efficacy of 60 percent . 

In addition, it reduced the number of feeding ticks, the number of eggs produced, and the number of eggs hatched, making [41] it an ideal candidate for anti-tick vaccine development [50]. To further explore suitable antigen candidates from R. microplus, Maruyama et al. [51] performed an RNA-seq study on salivary glands at all feeding stages of R. microplus, and they detected a fragment from the transcriptome which was similar to MP (Rm239) along with three other genes, including Rm39, Rm76, and Rm180. Application of these proteins as vaccines showed that all of them can inhibit hemostatic responses, suppress the host's antibody responses, and reduce the tick's ability to bind to the host using a glycine-rich cement protein. 

Therefore, the authors developed a multicomponent anti-tick vaccine using these four different types of proteins [51]. The immunization of cattle with this multicomponent vaccine resulted in a reduction in the infestation of R. microplus by 73.2 percent , indicating that the formulation of a multi-antigen anti-tick vaccine may be more effective than monocomponent vaccines [51].

Ribosomes, also called protein factories, are components of all living organisms. It has been shown that the ribosomal protein P{{0}} plays a pivotal role in regulating the translational activity of ribosomes and assisting an organism to adjust its metabolism to various environmental conditions. It belongs to a group of acidic proteins that form a stalk-like structure in the largest ribosome subunit of the ribosome [52]. There is evidence that shows that tick saliva contains ribosomal proteins that play a role in evading the defensive mechanisms of the host [53–55]. It was recently reported that rabbits vaccinated with recombinant ribosomal protein P0 exhibited strong humoral responses that primarily reduced nymph molting and female reproduction. The protein demonstrated a 57.5 percent protection against infestations of O. erraticus but did not provide cross-protection against infestations of the African tick Ornithodoros moubata [56]. 

În un alt studiu, cercetători chimic sintetizat a peptidă de 2{0 amino acizi, care a fost derivat din the ribosomal P0 protein of Rhipicephalus ticks, and successfully conjugated it to the Keyhole Limpet Hemocyanin (KLH) protein of Megathura crenulate to serve as an antigen against R. microplus, showing 96 percent efficacy in cattle [57]. In this study, the results suggested that P0 conjugated la KLH is an excellent vaccine. However, the production of such a vaccine will be expensive and may therefore not be cost-effective for livestock. It is therefore essential to conduct further research on the recombinant production of an antigenic vaccine to evaluate its effectiveness and to make its production more economic viable.

Serina protează inhibitori: Încercări izolare antigene de la căpușă specie au identificat some serina proteinază inhibitori (serpine), care au apărut să aibă antigen abilități. Serpine sunt implicate în diverse activități fiziologice in animale 2c în particular in bovine, where they influence blood clotting, altering protrorombin time and partial activating thromboplastin time [58–60]. Serpins interfere with the immune system of ticks and thus facilitate the initial feeding process of these parasites [61]. Andreotti et al. [62] isolated and identified R. micro plus trypsin inhibitors (BmTI) from larval extracte. 

To evaluate its antigenic activity, crossbred cattle were vaccinated with BmTI, which was found to interfere with leukocyte migration at the site of larvae fixation [63,64]. Vaccination of calves with BmTI antigens remarkably reduced engorged female tick numbers and their weight, resulting in a 72.8 percent efficacy against R. microplus. This data suggested that BmTI immunization may act in the early phase of larval development [65]. To investigate whether truncated BmTI can also induce immunization, the N-terminal fragment of BmTI was synthesized and showed a lower efficacy (18.4 percent ) in cattle compared to the full-length protein. 

Thus, immunization with the N-terminal domain is not sufficient to improve the effect of BmTIs on host–parasite interactions [64]. Similarly, when the recombinant R. microplus larvae trypsin inhibitors (rRmLTIs) were employed as a vaccine trial, the efficacy (32 percent ) was again low, suggesting that both the truncated or whole recombinant protein are less effective, probably due to a lack of precise folding of the protein in vitro. Overall, these results indicate that trypsin inhibitors seem suitable candidates to produce an effective vaccine; however, the method to produce them on a large scale needs to be improved to enhance their efficacy [66]. Various other serpins have been evaluated as possible anti-tick vaccine candidates from different tick species, including Amblyomma americanum (AAS19), Haemaphysalis longicornis (HLS2), Rhipicephalus (Boophilus) microplus, and so on. All of these serpins demonstrated partial protection to the host; however, the level of protection may vary with tick species and the type of serpin [67–69].

Combinarea proteinelor de la unu sau mai mult căpușe în a singur polipeptidă lanț reprezintă an atractiv anti-căpușe vaccinare strategie. Prin urmare, tripsină inhibitori/or serpins combinat cu imunogen fragmente de alte căpușe proteine poate fi utilizat ca multi-antigen constructs. De exemplu, a himerică proteină conținând recombinant Bm86-Campo Grande antigen (BmCG), rRmLTI, și the heat-labile enterotoxin B subunitate from Escherichia coli (LTB) as a molecular adjuvant was synthesized. This chimeric RmLTI–BmCG–LTB antigen had a 55.6 percent efficacy against R. microplus in cattle.

2.3. Midgut-Associated Antigen Candidați

Feritina proteine sunt importante pentru fiziologic depozitare de fier in a nontoxic dar biologic disponibil forma. Sunt importante pentru metabolismul de fier de fier de la ingerat sange during tick hranire [70,71]. Pana far, doi ferritina molecule (Feritina 1 si Ferritina 2) au been identificat si caracterizat. Feritina 1 (FER1) este localizat in celule, unde este 0aimplved in fiziologic depozitare de fier. 

Pentru Ferritin 2 (FER2), there are no functional orthologs in vertebrate. It is mainly expressed in the gut and plays a crucial biological role in iron transport to the salivary glands and ovare [71]. The FER2 protein has been reported in various tick species including D. variabilis, R. microplus, I. ricinus, Haemaphysalis longicornis, and I. scapularis [71,72]. Pe baza pierderii funcției studiilor de FER2, it is a promising vaccine candidate, because suppression of this gene impairs tick-feeding ability, lowers oviposition, and reduces larval hatching [71]. 

Besides FER2, FER1 has been shown as a suitable antigen candidate to control a variety of tick species. Hajdusek et al. tested the recombinant FER2 protein of R. microplus (RmFER2) to immunize cattle and found that the FER2-based vaccine showed an overall efficacy of 64 percent [73]. Similarly, the recombinant proteins FER1 and FER2 of H. longicornis have been used to immunize rabbits. Both proteins are highly immunogenic and induced host antibody production. Immunizing the host significantly reduced the engorged weight of the infested ticks and reduced the number of eggs and the number of ticks with completely hatched eggs. However, recombinant FER2 caused a greater reduction with a higher efficacy (49 percent ) than recombinant FER1 (34 percent ) [72]. 

Mai mult recent, Manjunathachar și co-lucrători [74] raportat că a vițel vaccinat cu H. anatolicum FER2, a vector de Crimeea–Congo hemoragie febră, a fost puternic protected de la larval (51,7 procent ) și adult (51,2 procent ) căpușă infestări, as well as as against ticks with FER2 knocked down by RNAi. Multe alte studii recente au confirmat confirmat că FER2 oferă semnificativ protecție to the host against tick infestation by using a recombinant protein of FER2 [75–77]. Mecanismul molecular de protecție implică în principal the producție de anti-FER2 anticorpi în gazdă corp, care este transferat în căpușă specii în timpul furajării procesului, și anti-FER2 anticorpi bind to FER2 inside the tick gut cells or hemolymph, thus preventing FER2 assembly and/or function. 

It has been recently discovered that predicted antigenic regions on the FER2 protein are conserved across different tick species. This protein can therefore be used to produce a vaccine for cross-species protection [77]. In a recent study, FER2 orthologues in O. moubata (OMFER2) and O. erraticus (OEFer2) were characterized, and the researchers found that they have high sequence similarity (85.3 percent ). The recombinant form of O. moubata Fer2 (tOMFER2) can elicit strong humoral responses in rabbits. However, in O. erraticus, this protein does not exhibit any protective effect, despite the high sequence similarity, which suggests that a slight difference in their sequences may determine whether or not they have a protective effect. Despite this, the results of this study confirm that OMFER2 has the potential to serve as an antigen candidate for vaccines [78].

TROSPA is a tick receptor that is required for spirochete colonization in I. scapularis. The B. burgdorferi outer surface protein A (OspA) is abundantly produced on these spirochetes and is critical for adhesion to the vector via specific binding to TROSPA [79,80]. In different tick species, including R. microplus, I. scapulars, and R. annulatus, TROSPA may play a role in infection mechanisms and the multiplication of Babesia pathogens. In addition, the outer surface proteins OspA and OspB are expressed when the spirochetes enter and reside in ticks [81]. However, their expression is suppressed during transmission to the host, whereas the expressions of OspC and bba52 are upregulated. BBA52, along with the OspC protein of borrelial, has complementary but non-essential roles in the transmission process, as these antigens are all localized in the outer membrane and co-expressed in feeding ticks [82–84]. 

The biological function of the receptor is unknown, but binding of OspA to TROSPA is necessary for ticks for the bacterium B. burgdorferi to colonize the tick gut, which supports bacterial infection in the vector [79]. Infection of B. burgdorferi induces the production of particular tick genes (TROSPA and salp15), which can be targeted to inhibit the transmission of Borrelia spirochetes and other tick-borne microbes [80,85]. Blocking TROSPA with TROSPA antisera or via RNAi reduces the adherence of B. burgdorferi to the gut of I. scapulars, and thus reduces the bacterial colonization of the vector and potential pathogen transmission to the host [79]. As a result of this interaction, recombinant TROSPA was analyzed in cattle as an antigen vaccine to control tick infestation and pathogen transmission, but it did not affect tick feeding or fecundity [14].

Aquaporins (AQPs) sau transmembrane apă canale play a major rol in apă homeostasis și crioprotecție [86,87]. Sunt evolutiv foarte conservate membri of a mai mare familie de major intrinsec proteine. Ei form pori in membrana celulară că transport apă sau alte solutes [{86,88,88,89]. In addition to transporting water and small neutral solutes, AQPs are involved in numerous fiziologice procese [90]. 

In ticks, AQPs have been reported in the digestive tract, Malpighian tubules, and also in salivary glands [91]. AQPs reduce the host blood volume in tick guts, an important physiological function since ticks ingest large volumes of a blood relative to their size and weight [62]. A fragment of an aquaporin from R. micro plus engorged females has been isolated and subsequently recombinantly produced and designated as a RmAQP1 vaccine [62]. This vaccine was tested in two cattle pen trials for efficacy against R. microplus, demonstrating 68 percent and 75 percent efficacy. This suggests that RmAQP1 may be a potential vaccine antigen [62] and that aquaporins can be used in anti-tick vaccines [62]. In a recent study on the RmAQP2 of the same species, it has been demonstrated that cattle vaccinated with the synthetic peptide of the extracellular domains of the RmAQP2 were able to reduce the number of ticks feeding to repletion by 25 percent overall, suggesting that this target (RmAQP2) may be a useful component of a vaccine cocktail against tick bites [92]. 

Another study on I. ricinus confirmed the efficacy of the tick AQP antigens for the control of tick infestations by showing the effect of IrAQP and CoAQP vaccination on I. ricinus tick larvae in rabbits. The efficacy of the vaccine containing the AQP conserved region present in the CoAQP antigen was higher than that of the IrAQP vaccine [93]. Furthermore, vaccination with synthetic immunogenic peptides derived from Ornithodoros erraticus AQPs (OeAQP and OeAQP1) provided significant protection to cattle against the homologous species O. erraticus, but the cross-species protection against Ornithodoros moubata was lower [94]. Besides, some other studies have also identified AQPs from different species, including O. moubata and Ixodid ticks, with bioinformatics analyses suggesting that these AQPs have a good potential to be used as a vaccine. Therefore, further experimental evidence is required to confirm the antigen potential of these AQPs [95,96].

I. ricinus is one of the tick species responsible for the growing prevalence of tick-borne diseases in companion animals in Europe [4]. The effect of the AQP-based vaccines on I. ricinus larvae infestation and molting could result in a reduction in tick infestations in vaccinated animals and supports that CoAQP might be a candidate protective antigen for the control of different tick species feeding on the same host.

A studiu using expression library imunization against a mouse model of tick infestations showed that the 4D8 protein, later named subleasing (SUB), is a potential antigen that could be used as a vaccine against I. scapularis [97]. It was found that the the sequences of the gene and protein of SUB are conserved across invertebrates and vertebrates. În plus, this genă a fost identificat și caracterizat în diferit căpușă specie și a fost găsit la be exprimat la diferit dezvoltare etape și în diferite țesuturi de adult căpușe [98]. Due to sub's broad distribution, it was proposed to be a good antigen vaccine candidate. The antigen potential of sub has previously been investigated in cattle using recombinant proteins, and it was found that sub can protect (51 procent eficacitate) contra căpușe. 

Furthermore, a combination of SUB vaccination and tick autocidal control following SUB gene knockdown in ticks feeding on cattle to control R. microplus, attained 75 percent efficacy after treatment [99,100]. Furthermore, Shakya and co-workers produced recombinant SUB of R. microplus and used this recombinant protein to immunize bovine. These ruminants were then challenged with R. microplus larvae. In addition, the efficacy of this protein against another geographically different tick strain was assessed. The efficacy of recombinant SUB ranged from 32.7 percent to 44.1 percent and indicated a high sequence homology between tick strains from Mexico and India [101]. In another study, recombinant SUB was synthesized as a chimeric protein with MSP1a and subsequently applied to cattle to control R. microplus. 

Surprinzător%, 2c this chimeric protein demonstrated an 81 percent efficacy [102]. As a result of the successful and promising results of SUB application, the combination of this antigen with Bm86 7d was tested and assumed to give better results, but the overall efficacy did not support the use of this combination as a vaccine. Deși it has been shown that high levels of specific antibodies are activated for each antigen when two antigens are administered simultaneously, they are separate into different formulations and used at iferent inoculation sites in the the animal [103,104].

Anterior cunoscute ca ligandine, glutation S-transferases (GSTs) form a family of multifuncțional protein distributed widely in the animal kingdom. These enzymatic proteins play a role in intracelular transport, digestion, production of prostaglandins, detoxification of both endogenous and exogenous substances, and defense against oxidative stress. GST expression levels are increased in organisms when exposed to insecticides% 0aand acaricide [105]. A iepure ser conținând policlonal anticorpi împotriva GST de la R. microplus reacționat cu recombinant GST de H. longicornis și R. apendice, suggesting that the tick GSTs could be a constituent of a universal vaccine that protects against more than one tick species [106]. 

Based on this preliminary study, Parizi and coworkers (2011) isolated GST from H. longicornis and produced recombinant GST, and used this to vaccinate cattle against R. microplus [107]. This protected cattle against R. micro plus with an efficacy of 57 percent . The recombinant GST protein provided partial cross-protective immunity in the host, suggesting that the protein protective capacity of GST is not sufficient, and thus the use of this protein in a single-antigen vaccine would appear not to be effective in preventing tick infestation [107].

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