Recent Advances in Tick Antigen Discovery And Anti-Tick Vaccine Development Part 2

2.4. Malpighian-Associated Antigen Candidates

In living organisms, 50 -nucleotidases are a widely distributed group of enzymes in various tick species. There are considerable similarities between the 50 -nucleotidase of ticks and the enzymes that are present in vertebrates, and a range of putative functions are carried out by these enzymes, including involvement in the purine salvage pathways [108]. Among ticks, this group of enzymes is found in many different tissues, such as the gut, salivary glands, and ovaries. 

However, they are most abundant in the Malpighian tubules, particularly on the surface of the Malpighian tubules and ovarian cells. The features of 50 -nucleotidases indicate that they are a potential target for antibodies [109]. Hope et al. investigated 50 -nucleotidases for their possible involvement in host immunization, and they found that injections of recombinant 50 -nucleotidase alone in sheep caused a significant upregulation in anti-nucleosidase antibodies, suggesting that they may be good antigen for the development of a vaccine [104]. 

However, when the same group of researchers analyzed their functions as antigens in cattle, there was no rise in antibody levels. Therefore, 50 -nucleotidases were not investigated further as antigens for vaccine development. However, a recent study conducted by another group of researchers has found that the level of 50 -nucleotidase/apyrase production increases in O. erraticus after feeding [110]. Furthermore, they suggested that blocking the apyrase function by host immunization with a recombinant apyrase protein can strongly reduce feeding in O. moubata ticks, demonstrating that 50 -nucleotidases/apyrases are potentially promising candidate antigens for the development of an anti-tick vaccine [110,111].

Nucleotidase is an enzyme that can catalyze the hydrolysis reaction of nucleotides, breaking down nucleotides into nucleosides and inorganic phosphates. Nucleotides have important biological functions in organisms, including the construction, storage, and transmission of DNA and RNA, energy transfer, and cell signal transduction. Therefore, nucleotidases play an important role in living organisms. Immunity is the body's ability to resist pathogens in the external environment, involving a variety of biological processes, including the expansion, differentiation, and regulation of immune cells. Several studies have shown that nucleotidases can affect the function of the immune system and thus immunity. 

For example, some variants of nucleotidases have been linked to the development of autoimmune diseases. In addition, nucleotidase can also affect the proliferation and activation of immune cells, and promote the function of immune cells. Therefore, there is a certain relationship between nucleotidase and immunity. From this point of view, we must pay great attention to the improvement of immunity. Cistanche has a significant effect on improving immunity. Meat ash contains a variety of biologically active ingredients, such as polysaccharides, two mushrooms, Huang Li, etc. These ingredients can stimulate the immune system. Various types of cells in the system, increase their immune activity.

Click cistanche tubulosa benefits

2.5. Tick-Cement-Associated Antigen Candidates

Tick cement is a mixture of glycol and lipoproteins secreted into the host via tick saliva shortly after attachment to the host, and is a valuable source of tick-derived antigens for vaccine development [112]. In addition to adhering tick mouthparts to the host skin [113], tick cement has been shown to act as a depot for B. burgdorferi sensu lato (s.l.) and the tick-borne encephalitis virus [114,115]. So far, various antigens have been identified and characterized from tick cement which has also been shown to be effective in controlling tick infestation and tick-borne diseases.

Truncated constructs of 64P (64TRPs), a 15 kDa cement protein secreted by the salivary glands of Rhipicephalus appendiculatus, showed cross-protection against Rhipicephalus sanguineus and Ixodes ricinus by targeting antigens in the midgut and salivary glands, causing mortality in adults ticks and nymphs. The vaccination of tick-naïve hosts with recombinant 64P significantly reduced the number of nymphal and adult tick infestations, resulting in 48% nymphal and up to 70% adult mortality, with some effects on engorgement weight and egg masses as well [116]. 

From these results, it appears that this protein is a broad-spectrum vaccine antigen and is effective against adult and immature stages of different tick species, including I. ricinus [100]. This cement antigen performed a dual function (i) as a vaccine in hamster, guinea pig, and rabbit models by impairing attachment and feeding and (ii) by cross-reacting with the “concealed” midgut antigens, ultimately causing the death of engorged ticks [100,117]. This antigen not only boosts antibody titers in response to tick infestation but also has cross-reactivity with different tick tissues; therefore, it combines the benefits of both “concealed” and “exposed” antigens [118].

3. The Types of Anti-Tick Vaccines

Ticks are the most prevalent arthropod parasites that feed on humans and livestock and transmit diseases [19]. Zoonotic diseases account for more than 60% of all infectious diseases affecting humans, and it is estimated that 22.8% of these infections are transmitted by tick vectors [131]. There are also massive economic losses for livestock farmers worldwide due to the diseases vectored by ticks that affect their livestock [132,133].

Therefore, it is relevant to control ticks to reduce the socioeconomic burden. The control of ticks has become challenging, as ticks can develop resistance to commercially available acaricides [134–136]. Innovative environmentally sound control technologies are needed due to concerns about the safety of acaricides for workers, food, and the environment and the rising costs associated with acaricide discovery, development, and marketing. Vaccines have many advantages over acaricides since they are generally non-toxic, nonpolluting, and less expensive compared to chemicals. 

However, they tend to be very species-specific. The pharmaceutical industry can play a crucial role in supporting research to develop a vaccine that can provide maximum protection to the host and is effective against multiple tick species [133,137]. Vaccine research programs are underway in various countries and the remainder of this article will focus on the progress of new vaccine technologies and on those that are already available (Figure 2). 

As with chemical applications, vaccine resistance cannot be ignored, and existing vaccines can be modified using sequencing and cloning procedures to isolate new antigens or change existing antigens to restore their efficacy. It is also possible to include two or more unrelated antigens in a single vaccine product to reduce the risk of resistance developing to any single antigen or antigenic determinant.

3.1. DNA-Based Anti-Tick Vaccines

The invention of the DNA vaccine and its use has raised safety concerns; in particular the possibility of stable transfection of genetic material (DNA) into somatic or even germ cells, which may result in altered gene expression and mutations. An extrachromosomal plasmid-encoding luciferase vector was detectable in skeletal muscle for more than 19 months following intramuscular treatment (138). Furthermore, intramuscular injection after electroporation greatly increased the overall transfection rate. The chromosomal integration of vector DNA at random sites is related to any increase in the transfection rate (139]. According to these studies, the integration frequency was well below the number of spontaneous gene mutations. 

However, Manam et al. found that the majority of plasmid DNA administered into the skeletal muscles of different rodents remained at the injection site. Minor fractions were also detected in the gonads but were not integrated into the genome (140). Repeated intramuscular application of a luciferase-encoding reporter vector in primates resulted in long-term reporter expression but induced no anti-DNA antibodies (141,142). Despite this, it should be noted that the aforementioned and additional safety concerns relating to DNA vaccines should be considered regarding their translation into clinical practice (143].

Over the last two decades, various groups of researchers have focused on developing a DNA vaccine to control tick infestation, and so far, several DNA vaccines have been introduced to immunize hosts (130,144,145). DNA vaccines, which differ from traditional protein-based vaccines in that they are based on bacterial plasmids that encode antigenic proteins, and the transcription is controlled by efficient eukaryotic promoters, have the advantages of a simple design, high stability, and safe administration (143]. In DNA vaccination, the injected plasmid DNA molecules are thought to actively enter the nucleus and remain there lifelong as episomal DNA, generating the protective antigens continuously for as long as the cell is alive (146). The problem of repeated boosting to maintain a high antibody titer could be solved by the continuous synthesis, processing, and presentation of antigens to T cells in vivo in DNA-vaccinated animals. 

Furthermore, since a DNA vaccine only contains plasmid DNA and has no contaminating proteins, it seems plausible that receiving multiple or repeated vaccinations would not result in an immune reaction to the vector DNA (147). The expressed antigen can be presented by MHC class I and complexes, which can induce CD4+ and CD8+ T cells, stimulating cellular and humoral immune responses, respectively (148].

The evidence came from the study of De Rose et al. [144], in which Merino crossbred sheep were immunized against B. micro plus using a DNA vaccine. When a plasmid containing a full-length gene sequence of Bm86 was administered either alone or with a plasmid carrying the ovine genes for the cytokines, granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin (IL)-1beta induced a relatively low level of protection against subsequent tick infestation. In addition, co-vaccination with Bm86 and GM-CSF plasmids resulted in a statistically significant reduction in the fertility of ticks. In all groups injected with the Bm86 DNA vaccine, antibody titers against Bm86 were low. In addition, a low level of antigen-specific stimulation of peripheral blood lymphocytes occurred in these groups. DNA vaccination, however, resulted in a strong subsequent antibody response following a single injection of recombinant Bm86 protein in adjuvant. The production of antibodies, however, did appear to be slightly less effective than following two vaccinations with recombinant proteins.

Furthermore, many other researchers investigated the efficacy of DNA-based antigens to immunize hosts against tick infestations. For example, Sayed et al. [149] extracted DNA from Argas persicus eggs and used it to immunize chickens with doses of 200–800 µg DNA/kg chicken body weight. The outcome was supportive, as the feeding success of ticks was reduced by 74.64% (50 µg DNA/kg chick body weight) and 89.39% (100 µg DNA/kg chick body weight) when they were exposed to DNA-immunized chicken. In addition, the authors reported that the serum of chickens immunized with DNA has activity against the gut proteins of A. precious; however, further analysis using other tick species indicated that the serum activity is species-specific. The electrophoretic pattern of the immunized chicken serum showed three new protein bands, which were assumed to be involved in the development of the immune defense of the chicken against ticks [149]. Afterward, many studies focused on antigen-specific DNA vaccination; however, their protection level varied with different types of antigens. It has been shown that BALB/c mice injected with Plasmid pBMC2-encoding antigen Bm86 showed resistance against Boophilus microplus. 

A higher dose of vaccination-induced Anti-Bm86 antibody production and higher interleukin (IL) levels (IL-4, IL-5, and IL-12 (p40)) and interferon-gamma (IFN-γ) levels in the sera of mice immunized with pBMC2. Mice immunized with pBMC2 showed antigen-specific stimulation of splenocytes according to the incorporation of bromodeoxyuridine and IFN g secretion. Application of this vaccine in livestock caused antibody production, suggesting that Bm86 DNA vaccination induces a strong immune response against B. micro plus [150]. Another study evaluated the immune protection elicited by recombinant plasmids encoding Paramyosin (Pmy) of H. longicornis (pcDNA3.1(+)-Pmy) in rabbits. The rabbits developed a high level of IgG, suggesting that a humoral immune response is induced by vaccination. Some ticks (27.31%) that fed on the vaccinated rabbits died, whereas the remaining ticks’ average engorgement weight and the oviposition of female adults were reduced by 36 and 39%, respectively. 

Thus, it seems that a Pmy DNA vaccine can induce an effective humoral immune response however it is provided and partially protects rabbits against H. longicornis infection [151]. Interestingly, a multi-epitope DNA vaccine incorporating both CD4+ and CD8+ cytotoxic T lymphocyte epitopes provided 100% protection to sheep under laboratory conditions against Ehrlichia ruminantium. However, the results were not repeated under field conditions. In this study, pLamp co-administration with MPL via the intramuscular route, in addition to topical application, protected the sheep by up to 60% against ticks by inducing activation of memory T cell responses [152].

Several other antigens, such as Salp14 and lipocalins, either alone or in combination with other antigens have recently been evaluated as DNA vaccines, which further provides hope of developing a DNA vaccine against ticks [130,153]. It has been shown that the salp14 DNA vaccine-elicited erythema at the tick bite site after the tick challenge [153]. Similarly, a lipocalins (LIP) vaccine comprising the recombinant plasmid pcDNA3.1-HlLIP of the LIP homolog from H. longicornis (HlLIP) was applied to immunize a rabbit host. Although this application induced humoral immunity of the host and also influenced the engorgement weight, oviposition, and hatchability of H. longicornis, the efficacy was too low, suggesting that this antigen is not suitable for vaccines as it provides partial protection to the host [130].

Finally, the main objective of developing a DNA vaccine should be to design the vaccine in such a way that it polarizes the immune response of the host towards the Th2 response since the humoral immune response plays a major role in tick immunity. In cattle that had been vaccinated with B. micro plus midgut antigens, the levels of specific IgG1, which are modulated by Th2 cells, were found to correlate with the protection. There is a possibility that if the secretory signal sequence is placed appropriately downstream of the target gene, then the target antigen could be secreted out to the extracellular compartment and induce a greater humoral immune response. It is also likely that the selection of Th2 cells would be favored if they are coinfected with other immunomodulatory genes such as IL4 and IL10.

3.2. mRNA Vaccine

In recent years, many attempts have been made to discover new protective antigens that can be used in the development of an anti-tick vaccine, with numerous improvements. To test the efficacy of the vaccine candidate, recombinant proteins, regardless of whether or not they are associated with other proteins or adjuvants, have been the platform of choice for testing their efficacy through the use of model organisms. 

Due to the ease with which DNA and mRNA vaccine platforms can be generated, there have been significant developments in the use of genetic (DNA and mRNA) vaccine platforms in recent years [130,145]. An mRNA vaccine encoding a cocktail of tick salivary proteins induced “tick immunity” in guinea pigs, thereby remarkably reducing the transmission of tick-borne Borrelia burgdorferi, the causative agent of Lyme disease (borreliosis). Even though many tick antigen candidates have been demonstrated to elicit immune responses in a host, it has not yet been possible to replicate robust tick immunity using vaccination. There is a possibility that this may be because, at different stages of feeding, the composition of salivary proteins in ticks may alter dynamically, possibly to manage host internal changes. This information was provided in a recent study, in which a group of researchers identified and rationally selected 19 salivary proteins of the black-legged tick I. scapularis, which is a common vector for Lyme disease in humans. They engineered nucleoside-modified mRNAs that encode these proteins. 

To produce the mRNA–LNP vaccine 19ISP (19 Ixodes salivary proteins), these were encapsulated in equal amounts in lipid nanoparticles (LNPs). To evaluate the impact of the vaccine on the feeding behavior of I. scapularis, guinea pigs were immunized intradermally three times in 4-week intervals, which resulted in robust antibody responses to at least ten of the encoded antigens. In the next step of the experiment, the animals were challenged with uninfected I. scapularis nymphs. The animals that had been vaccinated developed considerable erythema within 24 h. 

Furthermore, ticks on animals that had been vaccinated fed poorly and began to detach by 48 h, with 80% of ticks detached from vaccinated animals after 96 h, compared with 20% on animals that had not been vaccinated. To further examine whether the altered feeding behavior affects the transmission of pathogens, B. burgdorferi-infected I. scapularis nymphs were placed on guinea pigs that were vaccinated either with 19ISP or with an mRNA vaccine encoding firefly luciferase. Each of these animals received three ticks that were infected. Considering that humans are likely to remove a tick that causes erythema-related itching, the ticks were detached in a double-blind manner as soon as redness appeared. 

A total of 46% of the control animals were infected with B. burgdorferi three weeks after the challenge, whereas none of the vaccinated animals were infected with this pathogen. A gene expression analysis has shown that the vaccine activated several immune pathways, including T and B cell receptors, chemokine, FcεRI, and IL-17 signaling, as well as natural killer cell-mediated toxicity. Moreover, bite site analyses also showed that the vaccine had induced T-cell responses [154].

Concurrently, the same group of researchers used Salp14 as a model antigen to examine tick immunity using mRNA lipid nanoparticles (LNPs), plasmid DNA, or recombinant protein platforms [153]. In this study, vaccination including the nucleoside-modified mRNA lipid nanoparticles encoding (mRNA-LNPs) Salp14 was delivered intradermally, with two boosts every 4 weeks. The development of Salp14-specific antibodies was compared among the different immunization strategies. Salp14 mRNA immunization was the platform that induced the strongest humoral response compared to DNA and protein vaccination. Guinea pigs immunized with the salp14 mRNA elicited the most robust, and intense erythema observed at the bite site in all the immunization groups; however, it did not affect the rate of tick detachment and did not alter engorgement weights [153]. A tick vaccine should induce erythema to be effective, and one approach to change later aspects of tick feeding, including attachment and engorgement, is to use a vaccine that contains several salivary tick antigens [154]. 

Therefore, it seems that immunization with nucleoside-modified mRNALNP salp14, which can be used as a potential vaccine candidate, can lead to higher antibody titers and an earlier and higher degree of redness than immunization with either DNA or protein, which suggests that Salp14 could be a good candidate for a vaccine, either alone with optimizations or in combination with other candidate antigens [154].

On the whole, it appears that a multivalent mRNA vaccine may have the ability to elicit tick resistance in laboratory animals such as guinea pigs and to prevent tick infestation and tick-borne infection, probably by limiting the time duration of tick feeding on their host. It has also been suggested that a mRNA–LNP formulation which enables slow, continuous antigen delivery, may mimic natural tick bites. If this strategy can be translated to humans, it would be the first vaccine that does not directly target a pathogen or microbial target, but instead its vector. Moreover, since anti-tick vaccines are still being developed to assist humans in the prevention of the transmission of tick-borne diseases, the strategy of immunization and the selection of antigens for immunization need to be taken into consideration.

3.3. Protein-Based Vaccines

Some protein-based vaccines are commercially available and effective. A vaccine program was first started in the 1970s when researchers began to experiment with two different types of vaccine formulations to immunize the host against the tick (D. Anderson) at the time. The first included antigens obtained from the gut and ovary, while the second included all the internal organs extracted from semi-engorged D. Anderson females. 

This study discovered that antibody-mediated immune responses are activated against tick intestinal tissue when the cattle host is inoculated with extracts obtained from adult R. microplus females [155]. This initial study to evaluate the effectiveness of vaccine formulation encouraged researchers around the globe to focus on the development of a vaccine for the control of ticks and tick-borne diseases. Thus, in the 1980s, two separate groups of researchers carried out the first scientific investigations using vaccine formulations to analyze the immune response of bovines against R. microplus [156].

Following the above studies, it was found that a tick gut-associated glycoprotein can induce immunoprotection in the host [157]. In a subsequent study, the same research group isolated a glycoprotein with a molecular weight of 89 kDa, which was named Bm86 and reported to be associated with the gut cells of R. microplus [158,159]. The Bm86 recombinant protein was produced on a large scale using a yeast expression system. 

So far, only the Bm86-based vaccine is commercialized with different brand names, for example, it is sold in Australia with the TickGARD® brand name and in Cuba under Gavac® [160,161]. These vaccines are largely used in different countries to reduce the tick pressure on cattle. It has been shown that the use of these vaccines can reduce the tick population by up to 74% and their overall efficacy ranges from 51% to 91%, which varies with the tick population and nutritional condition of the cattle [160–163]. There is evidence that some Columbian, Mexican, and Brazilian R. micro plus tick strains exhibit lower overall efficacy compared to Cuban and Australian R. micro plus tick strains, and even the Argentinian R. micro plus strain A seems to be resistant to vaccination with Bm86 [34,164]. Further analysis was conducted on the variation in the efficacy of the Bm86 vaccine on populations of the same tick species in different parts of the world, and it was concluded that different populations of ticks most likely have a polymorphism in Bm86 antigen genes in terms of the amino acid sequence of the gene, and this is the main reason that existing Bm86-based vaccines are not so efficacious. 

For example, there was a polymorphism in the gene homologous to Bm86 (designated as Bm95) identified in tick populations in Argentina, resulting in differences in the sequence of the Bm86 between tick populations such as those found in Cuba and Australia, which may explain why the Bm86 vaccine was not as effective against the Argentine tick [34]. Considering the resistance problems of the Bm86 vaccine, researchers produced a recombinant Bm95 vaccine that has proven to be highly effective, with an overall efficacy of 89% in Cuba and Argentina and 81% in India in terms of reducing tick infestation [34,102,120,165].

Besides the above-mentioned commercial vaccine and its efficacy trial, many other studies have focused on further improving the efficacy of the Bm86-based vaccine. Some recent studies have synthesized peptides, including SBm4912, SBm7462®, and SBm19733, which were obtained from Bm86, and also produced an rSBm7462® recombinant peptide, and analyzed their efficacy. The percentage efficacy of these peptides ranged from 35.87% to 81.05%, suggesting that these peptides, in particular, SBm7462® and rSBm7462®, play a crucial role in inducing host immunity and can be commercialized as they are highly effective in terms of reducing tick infestation [121,122]. Furthermore, to improve the Bm86 recombinant protein vaccine effectiveness, recently, Lapisa S.A. has introduced a Bm86-based Bovimune Ixovac® vaccine in Mexico. However, this vaccine has not been studied in terms of its effectiveness against ticks; therefore, studies are needed to determine its effects on different tick populations to determine its efficacy.

A Bm86 homolog-based vaccine (TickGard) appears to be suitable, as this vaccine has a broad application and can trigger cross-reactive antibodies in different tick species, such as Rhipicephalus sanguineus, Hyalomma anatolicum anatolicum, Rhipicephalus (Boophilus) decoloratus, Rhipicephalus (Boophilus) annulatus, and Hyalomma dromedary [166–168]. However, this vaccine is unable to induce cross-reactive protection in some other tick species (e.g., Rhipicephalus appendiculatus, Amblyomma variegatum, and Amblyomma cajennense) [169,170]. It is interesting to note that the Bm86 vaccine has a 100% efficacy against R. annulatus, resulting in greater efficacy than the reported efficacy of the homologous vaccine with R. microplus. The reason for this might be due to physiological factors (e.g., less blood engorgement and lower levels of protease activity in the body) or tick genetic factors. These factors may influence BM86 protein levels or tick physiological processes such as feeding and protein degradation, leading to more efficient antibody–antigen interactions [171]. Bm86 vaccination provides excellent protection against R. microplus ticks, but it is challenging to extrapolate these experiences to an Ixodes tick vaccine. 

In contrast to I. ricinus and I. scapularis, R. microplus is a single host tick that feeds exclusively on cattle [168]. It also has a brief life cycle, does not molt, and finds a new host when the blood meal is finished. The efficacy of the Bm86 vaccine was investigated in cows that had been exposed to R. microplus tick larvae, and measurements were made of the parameters relating to tick immunity on the engorged adult females that dropped off following vaccination. Thus, the measured protection is the sum of the influence on two molting periods and three tick stages. It has been shown for R. microplus that Bm86 vaccination causes damage and subsequently reduces the engorgement weight in adult female ticks [172]; nevertheless, the relative influence of Bm86 vaccination on the immature life stages of R. microplus is not precisely known. Bm86 homologs have also been isolated and identified from Ixodes ticks. I. ricinus contains two homologs of Bm86, Ir86-1, and Ir-86-2, and I. scapularis also has two homologs of Is86-1 and Is86-2 [173]. A subsequent study explored that vaccination of recombinant Ir86 proteins enhanced the serum IgG titers against recombinant Ir86 proteins; however, the antibodies were not able to protect rabbits against the I. ricinus challenge; neither the number of attached ticks nor tick weights were reduced [174]. 

Therefore, vaccination against Bm86 homologs in Ixodes is not considered to be an effective approach to control Ixodes ricinus populations,  even though Bm86 vaccination has a clear effect against R. microplus. Even though the Bm86 vaccine has shown considerable success, it is critical to understand that the vaccine is unlikely to replace acaricides because it lacks the “knock-out effect” associated with acaricides. Despite this, field experience has shown that the use of Bm86 considerably reduces the requirement of applying acaricide treatments in the field. For example, Cuba’s tight regulation of its tick control program led to a reduction in the amount of acaricide used in the country by 87%, which is comparable to the results of a recent study conducted in Venezuela [161,175,176]. 

Furthermore, the use of the Bm86 vaccine has also considerably decreased tickborne diseases, including Bovine anaplasmosis and Bovine babesiosis. The application of this vaccine has also enhanced the productivity of livestock, e.g., cattle, and consequently reduced the economic losses of farmers [160]. The above aspects of Bm86 vaccination show that it is a highly cost-effective method compared to chemical applications in dealing with tick infestations. As such, this vaccine may prove to be highly useful in reducing tick-borne diseases as well as in improving the management of tick outbreaks on livestock farms to decrease tick-borne diseases.

4. Concluding Remarks

Ticks feed on blood for development, growth, and reproduction, and they are responsible for the transmission of various tick-borne diseases. Ixodes ticks, for example, transmit a large number of pathogens, including bacteria, protozoa, and viruses. It is important to note that anti-tick vaccines reduce tick infestation and prevent pathogen transmission, and in addition are safer than chemical control, which may have negative side effects. Several novel antigens from a variety of tissues/organs have been identified and their efficacy has been examined in laboratory animals, which has led to important progress towards developing a vaccine against ticks with no reported side effects. Vaccines based on the Bm86 protein have been commercialized in various countries, and their use against R. microplus has shown that vaccination against ticks can be efficiently used. However, Bm86-based vaccines are not equally effective against other tick species, such as Ixodes ticks. It has been shown that vaccination with the Bm86 homolog of I. ricinus does not have any effect on tick feeding [174]. 

Additionally, because R. micro plus only feeds on one host, larvae were used to challenge cows, which resulted in fully engorged adult female ticks, which not only reduced the number of ticks but also had a significant impact on all three life stages of the tick. In contrast to R. microplus, Ixodes ticks change hosts throughout their life cycle and therefore Ixodes tick vaccination requires the effective prevention of attachment and/or feeding of ticks during one blood meal on one host. Nonetheless, vaccination against Ixodes ticks seems possible and realistic. It is thus important to note that cross-protection of the host can be a challenging task with this type of vaccine. There are some other proteins, besides Bm86, that might also have therapeutic potential as immunosuppressive or anticoagulant agents [39,61]. 

Recent advances in genomics and proteomics have enabled us to discover novel antigens and employ molecular techniques to manipulate identified proteins and test new vaccines considerably more quickly and cost-effectively than in the past. DNA vaccination is also an excellent option; however, in general, this type of vaccination can only lead to low levels of antigen expression and limits the non-professional antigen-presenting cell activating CD4+ T helper cells via the MHC class II pathway [177]. However, DNA vaccination not only provides significant protection to the host but is also considered to provide cross-protection against ticks if it is followed by a chimeric vaccine or recombinant protein vaccination [178]. 

Furthermore, mRNA-LNPs may help in the elicitation of erythema at the tick bite site, which is one of the most important early indicators of acquired tick resistance. mRNA-LNPs containing tick genes are a useful platform for the development of vaccines that can potentially prevent selected tick-borne diseases [153,154]. Both DNA and mRNA vaccination also seem to be effective strategies and there is future hope that a mRNA or DNA vaccination for the control of tick infestation and tick-borne diseases may be developed and also provide cross-protection. However, so far no vaccine has been commercialized, indicating that further studies are needed to determine the efficacy of increasingly more antigens for their use to develop a DNA or mRNA vaccine. The identification and characterization of novel tick vaccine candidates can prevent tick feeding and pathogen transmission. Using these antigens in vaccines for domesticated animals and wildlife, let alone humans, remain a challenge.

Author Contributions:

M.N.A., M.A.J., and M.K. designed the structure of the article. All authors performed the literature search and wrote parts of the manuscript/assembled the data. M.N.A. and I.M. created and edited the figures. I.D. and M.K. made critical revisions and proofread the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding:

MK received funding from the Grant Agency of the Czech Republic (grant 19-382 07247S) and ERD Funds, project CePaVip OPVVV (no. 384 CZ.02.1.01/0.0/0.0/16_019/0000759). The funders had no role in the design of the study, data collection, and analysis, the decision to publish, or the preparation of the manuscript.

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Data Availability Statement: Not applicable

Conflicts of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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