CathL, cathepsin L-like cysteine proteinase; GST, glutathione S-transferase; P0, 60S acidic ribosomal protein. L, larva; N, nymph; AF, adult female; AM, adult male. ^a protection for specific parameters. ^b For Bos indicus (Linnaeus, 1758) and B. indicus × Bos taurus (Linnaeus, 1758) breeds, respectively. ^c B. indicus × B. taurus breed. ^d peptide conjugates. ^e cocktail vaccine. Previously, an overall 50% reduction in the engorgement weight of H. a. anatolicum nymphs fed on Bm86-vaccinated calves was obtained [52]. Furthermore, Bm86 and its orthologue Haa86 from H. a. anatolicum were used in a vaccination trial against R. (B.) microplus and H. a. anatolicum infestations [53]. Although both Bm86 and Haa86 presented cross-protection, these antigens showed a higher species-specific efficacy, similar to other H. a. anatolicum subolesin, calreticulin, and cathepsin L-like cysteine proteinase antigens [38]. Additionally, Bm86 was effective against H. dromedarii infestations in cattle and camels. On the other hand, the same antigen failed to protect cattle against Amblyomma sculptum (Berlese, 1888) [26]. In another cross-vaccination trial against Hyalomma spp., Hd86 (Hyalomma scupense (Schulze, 1919) Bm86 orthologue), immunized calves presented a decrease in the number of H. scupense larva, an effect not observed in calves that were immunized with Bm86 [54]. Intriguingly, Hd86 and Bm86 immunization increased H. scupense and Hyalomma excavatum (Koch, 1844) adult female weight, respectively, showing that Hd86 and Bm86 efficacy varies according to the tick life stage. It was suggested that Hd86 and Bm86 protection rates variation could result from differences in amino acid sequences of these antigens among tick species [55] and tick populations [56], as well as different levels of protein expression in immature and adult life stages [57]. Distinct Bm86 vaccine efficacies against Rhipicephalus (Boophilus) annulatus (Say, 1821) and R. (B.) microplus could also be related to the activity of tick endogenous protein degradation machinery (proteases) [58]. Since Bm86 protein expression levels in both tick species are similar, distinct physiologic factors could have caused an increased R. (B.) annulatus susceptibility to Bm86 vaccine. Proteomic analysis showed R. (B.) annulatus has reduced protein degradation machinery compared to R. (B.) microplus, which leads to a higher susceptibility to host immune response factors such as antibodies and the complement system [58]. It was proposed that Bm86 could be useful in controlling R. (B.) annulatus and R. (B.) microplus in endemic regions, e.g., North America, using vaccination and standard eradication practices simultaneously ^[59][60][59,60].

2.2. Subolesin

Subolesin and their orthologs (akirins) are transcription factors that regulate the expression of signal transduction and innate immune response genes in ticks and other parasites ^{[61][62][63][64]}[61,62,63,64]. Cross-protection experiments showed high vaccine efficacies using subolesin and akirins against different parasite species, making these antigens very promising for a broad universal vaccine [29]. These trials supported further investigations analyzing the efficacy of subolesin antigens against different parasite and host species using alternative vaccination protocols. Mono- and multi-antigenic formulations of R. appendiculatus, R. (B.) decoloratus and A. variegatum subolesins were used in B. indicus and B. indicus × B. taurus crossbred cattle, with an efficacy range of 47–94% [39]. However, the protection developed by bovines immunized with this multi-antigenic formulation was not improved when compared to mono-antigenic formulations. A different vaccine strategy, using an oral vaccine combining R. (B.) decoloratus subolesin and heat-inactivated Mycobacterium bovis, induced protection against an R. (B.) decoloratus and R. appendiculatus infestation in B. indicus × B. taurus crossbred cattle [41]. In light of evidence showing that subolesin and akirin induce partial protection against Hyalomma spp. and Rhipicephalus spp. infestations in red and white-tailed deer [65], field vaccination trials were performed using roe deer, another cervid that is highly important as a host for ticks and tick-borne pathogens in Europe [66]. For this trial, a chimera of Ixodes scapularis (Say, 1821) subolesin and Aedes albopictus (Skuse, 1894) mosquito akirin (Q38) was used, since its protective epitopes are conserved among distinct parasites [67], which is a necessary feature for a universal vaccine. Although the tick species collected in the field were not definitely identified, the roe deer showed a smaller number of ticks (likely Hyalomma marginatum (Koch, 1844), Hyalomma lusitanicum (Koch, 1844), Rhipicephalus bursa (Canestrini and Fanzago, 1878), and Ixodes ricinus (Linnaeus, 1758) compared with non-vaccinated animals. Noteworthy, Q38 was able to induce immune protection in rabbits against larvae of I. ricinus and Dermacentor reticulatus (Fabricius, 1794) [68], showing Q38’s potential to protect against different tick life stages. Vaccinations trials using subolesin against the soft ticks O. erraticus and O. moubata showed a low degree of protection [69], probably due to the induction of non-protective antibodies for immunodominant subolesin epitopes in highly structured regions [37]. Subsequently, the prediction of subolesin linear B-cell epitopes, using peptide arrays, epitope mapping (pepscan), and modelling, was used to design synthetic peptides that increased the protection rates against these ticks [37]. Besides the use of the complete protein as an antigen, immunization with synthetic subolesin peptides stimulated the host immune system to develop antibodies against subolesin unstructured regions. Therefore, the prediction of B-cell and T-cell epitopes represents a promising tool to improve the immunogenicity of other tick antigen candidates that show low degree of protection [70], as well as to design chimeras containing subolesin or other protective antigenic epitopes [71]. Recently, an antigenic B-cell epitope peptide from subolesin was tested as a potential vaccine candidate using artificial capillary feeding experiments [72] and vaccination under stable [73] and field [74] conditions, showing partial protection against tick infestation. Moreover, an alternative approach using a priming immunization with a DNA vaccine expressing R. haemaphysaloides subolesin, followed by boosters of R. haemaphysaloides recombinant subolesin or R. haemaphysaloides subolesin and H. longicornis P0 chimeric polypeptide provided cross-protection against H. longicornis of 79% and 86%, respectively [40]. The combination of a DNA vaccine with mono- or multi-protein immunizations is a promising strategy to increase the efficacy of anti-tick vaccines.

2.3. Glutathione S-Transferases

Glutathione S-transferases (GSTs) are enzymes involved in the detoxification of xenobiotics and endogenous compounds [75]. The potential of H. longicornis GST (GST-Hl) to protect bovines against R. (B.) microplus parasitism has been analyzed [76]. A vaccination trial showed that GST-Hl combined with R. (B.) microplus Vitellin-Degrading Cysteine Endopeptidase and Boophilus Yolk pro-Cathepsin decreased R. (B.) microplus infestation in cattle, conferring an overall protection ranging from 35% to 61% for 3 months [76]. Moreover, R. appendiculatus adult females fed on rabbits immunized with GST-Hl are negatively affected, leading to an overall vaccine efficacy of 67% [42]. However, the same antigen was ineffective in protecting rabbits against R. sanguineus s.l. [42]. Furthermore, immunogenicity and cross-recognition of GSTs from R. appendiculatus, R. (B.) decoloratus, R. (B.) microplus, A. variegatum, and H. longicornis were analyzed [43]. Rhipicephalus (B.) decoloratus and A. variegatum GST presented high cross-protection potential and were selected to compose an experimental cocktail vaccine, which was able to reduce 35% of R. sanguineus s.l. adult female numbers in immunized rabbit. All together, these data demonstrate that GSTs are potentially useful antigens to compose vaccines against Rhipicephalus spp.

3. New Cross-Protection Antigen Candidates

Protein P0, one of the eukaryotic 60S ribosomal proteins, has a high identity among ticks of different genera but a low sequence similarity with their hosts’ P0 [77], which is an important feature for candidate antigens. Rhipicephalus sanguineus s.l. and R. (B.) microplus P0 have identical sequences, from which a peptide composed of 20 amino acids within a highly immunogenic region was synthetized and used in vaccination trials [77]. The overall efficacy of this peptide as a protective antigen was 90% for R. sanguineus s.l. [77] and 54% for A. mixtum [45] in rabbits, and 96% against R. (B.) microplus in cattle [44]. Surprisingly, when the same peptide was tested as a conjugate with Bm86 to immunize dogs and cattle, the efficacies were 95% (peptide alone) and 85% (conjugated) against R. sanguineus s.l., and 89% (peptide alone) and 84% (conjugated) against R. (B.) microplus [78], indicating that the effect is not additive. However, O. erraticus P0 showed no cross-protection against O. moubata infestation [50]. Thus, the antigen P0 may be useful to compose a broad-spectrum vaccine against hard ticks, as well as against pathogens transmitted during tick feeding, as demonstrated for Babesia spp. [79]. Using a vaccinomic approach, new tick proteins with high efficacy in controlling I. ricinus and D. reticulatus in rabbits and dogs [80] were successfully identified. These trials showed efficacies up to 80% in animals individually inoculated with the following proteins: (1) heme lipoprotein from I. ricinus, (2) glypican-like protein, (3) secreted protein involved in homophilic cell adhesion, (4) sulfate/anion exchanger, or (5) signal peptidase complex subunit 3 from D. reticulatus. Similarly, through omics and in silico prediction algorithms, peptides from O. moubata midgut membrane antigens were selected for rabbit vaccination against O. moubata and O. erraticus [51]. Interestingly, the levels of protection were higher against O. erraticus than O. moubata. Other proteins in the Ixodidae and Argasidae families, such as R. (B.) microplus [46] and O. erraticus [47] aquaporins, O. erraticus ABC transporter, selenoproteins T, chitinase, secreted protein PK-4 ^[47][50][47,50], Ixodes persulcatus ferritin 2 [48] and R. (B.) microplus cystatins [49], showed limited cross-protection rates. Further studies are necessary to improve the protection levels achieved with these antigens.

4. Vaccines Blocking Pathogen Transmission

An alternative strategy to control tick-borne pathogens is through a vaccine that can block the transmission of these organisms to the host [81]. Some pathogens take advantage of tick saliva molecules for their transmission at an early stage of tick attachment, which increases their dissemination to the host [82]. If a vaccine is able to interfere in key tick physiology processes, pathogen transmission could be blocked. Examples of such processes and potential antigens include: (1) disruption of tick defense mechanisms targeting the microbiota biofilm modulator PIXR [83], (2) reduction in tick attachment time on the host by targeting the cement cone protein 64TRP ^[84][85][84,85], and (3) impairment of tick molecules that favor pathogen development and invasion into the tick, such as the antimicrobial protein IAFGP [86]. In this sense, a proposal for a universal anti-tick vaccine with a transmission-blocking potential is a promising tool to control ticks and pathogens, as suggested for Bm86 homologues to control Borrelia burgdorferi (Burgdorfer, 1982) host infection [87]. Another example of a candidate antigen to control pathogens includes a R. (B.) microplus subolesin fused with Anaplasma marginale (Theiler, 1910) Major Surface Protein 1a (SUB-MSP1a), which showed cross-protection potential against R. (B.) annulatus [88]. SUB-MSP1a was further used in bovine and sheep vaccination trials against R. (B.) microplus [89]. In this trial, the prevalence of Babesia bigemina (Wenyon, 1926) was reduced, showing the potential of SUB-MSP1a to control tick infestations and pathogen infection/transmission. Indeed, subolesin was also shown to be important for tick transmission of Anaplasma phagocytophilum (Foggie 1949), A. marginale, and B. burgdorferi [90]. Nevertheless, the multi-antigenic vaccine containing R. (B.) microplus subolesin, R. appendiculatus TRP64, and 3 histamine binding proteins, as well as Theileria parva (Theiler, 1904) sporozoite antigen p67C, did not affect R. appendiculatus survival and transmission of T. parva to cattle [91]. Using a different methodology, I. ricinus fed by artificial membranes with blood containing antibodies against an I. scapularis lipocalin or against an I. scapularis lectin, impaired tick engorgement, as well as A. phagocytophilum infection [92]. These two antigens identified using a vaccinomic approach are good examples of how this methodology can be useful for antigen discovery. In addition to that, a live anti-tick vaccine was developed using attenuated Babesia bovis as a carrier to express tick proteins in calves [93]. The calves immunized with B. bovis carrying the H. longicornis GST gene generated anti-HlGST antibodies and developed mild babesiosis. Rhipicephalus (B.) microplus reared in these calves were smaller, and their fecundity was affected. This dual vaccine approach opens the perspective to develop universal vaccines not only against several tick species but also against tick-borne pathogens.

5. Advances and Remaining Challenges to Universal Tick Vaccines

This essay reviews studies showing tick antigens being evaluated for vaccine protection levels, including different stages of method development: from in silico or in vitro antigenicity, antibody cross-binding, and artificial feeding analysis, to vaccination trials using target hosts such as cattle, deer, and dogs. However, it is difficult at this point to compare them directly in order to find the best options to be included in a multi-antigenic vaccine. Even when limiting the comparison to antigens that were already analyzed in natural hosts, there are many biological variables to consider in differentiating vaccine efficacies, including host and tick diversity ^[39][94][95][39,94,95]. Moreover, although Canales’ method [33] for calculating the overall efficacy of the vaccine has become a standard and has been widely used in vaccination trials, alternative considerations have also been employed to evaluate levels of vaccine protection. These differences make it challenging to compare the level of protection across different studies ^[96][97][96,97]. The use of standard protocols would be helpful to allow the estimation of vaccine efficacy for ticks with similar biology and for similar experimental approaches. Another important point in cross-vaccination studies is to verify if a given tick species is not actually two or more related but distinct species. Accurate species identification is fundamental to analyze homologous or heterologous vaccine efficacies [98]. Some examples of ticks that were the subject of vaccination trials and were under-speciated groups at the time include: (1) the formerly Amblyomma cajenense (Fabricius, 1787), now known as a complex of six valid species, including A. sculptum [99], (2) R. sanguineus s.l., which is contested to comprise multiple distinct species [100], and (3) R. (B.) microplus, which was also split into two species, giving rise to the new species Rhipicephalus (Boophilus) australis (Fuller, 1899) [101]. Thus, for example, vaccine antigen candidates such as protease inhibitors used against A. sculptum [102] must be considered as heterologous protection against other A. cajennense complex species. Importantly, the low protection rates obtained in some vaccination trials performed on R. (B.) microplus using TickGard and R. australis using Gavac could be the result of physiologic and/or Bm86 amino acid sequence variations between these two species ^{[36][103][104]}[36,103,104]. Nevertheless, the selection of vaccine antigens from conserved proteins among tick species or isolates from different geographic areas will contribute to avoid vaccine inefficacy due to tick species misidentification ^[105][106][105,106]. In this context, the development of universal vaccines is highly desirable.

5.1. Rational Strategies for Improving Tick Vaccines

Different protocols for improving vaccines against multiple tick species were developed recently, thanks to advances in molecular biology, bioinformatics, and new material for vaccine components, such as adjuvants. Furthermore, the use of multiple antigens in a universal vaccine has been proposed, offering advantages such as protection against different tick species and different stages of the tick life cycle, in addition to the pathogens carried by them [107]. Multi-antigenic vaccines prove particularly advantageous for ticks with a single host, such as R. (B.) microplus, as they remain on the same host throughout their larval, nymphal, and adult stages. During this parasitic cycle, the tick produces distinct molecules to evade the host’s immune response ^{[108][109][110][111]}[108,109,110,111]. Moreover, for multi-antigenic vaccines to be effective against ticks with three hosts, they must incorporate proteins specific to each life stage of the parasite that infests each host. Therefore, regardless of the number of hosts affected by tick infestations, a multi-antigenic vaccine should encompass protective antigens targeting various life stages of different tick species, as well as different secreted molecules by the same tick species. The use of multiple antigens in single vaccines or chimeric constructs has shown promising levels of protection in some cases, although some antigen combinations did not increase vaccine efficacy [107]. An interesting strategy proposed was the use of oral vaccines for the control of tick infestations, which, despite being incipient, affected tick survival and fertility, justifying further experiments ^[41][112][41,112]. In addition to in vivo trials, the immunogenicity of a number of tick molecules was analyzed in silico, revealing the potential of cross-protective tick antigens to be used for other tick species, such as aquaporin [113] and GSTs [114]. Similarly, potential proteins to be used for vaccines were discovered by reverse vaccinology analysis ^[97][115][97,115]. The use of systems biology is also a promising technique for the development of vaccines against ticks and their vectored pathogens [116]. Using this tool, it is possible to propose antigens for new generation vaccines that make use of transcriptomes, proteomes, and immunogenomics. Through “omics” analyses, it is possible to find not only antigens but also adjuvants for use in tick vaccines [107]. New generation adjuvants are under development to increase the immune response level, quality, and duration against infection and parasitic diseases ^[117][118][117,118]. For ticks, the performance of adjuvant formulations is currently being analyzed to improve the efficacy of known antigens such as Bm86, showing increased protections levels, although adverse reactions such as cutaneous side effects remain an issue [119]. A self-adjuvancing antigen carrier study indicated the immunogenicity, biodistribution, and safety of Bm86 carried by silica nanoparticles, demonstrating the potential of this delivery platform in anti-tick vaccine applications [120]. New vaccine delivery systems are being developed to protect against ticks and tick-borne diseases, including the use of lipid nanoparticle (LNP)-mRNA vaccines ^[121][122][121,122]. This technology offers an alternative to live attenuated or subunit-based vaccines [123]. An Ixodes scapularis salivary gland protein encoded by mRNA using this system decreased tick engorgement and B. burgdorferi transmission [122]. Interestingly, immunization with mRNA encapsulated into lipid nanoparticles induced not only a humoral immune response against Powassan virus, another pathogen transmitted by I. scapularis, but also against other tick-borne flaviviruses [121]. Other promising delivery systems include viral vectors [124] and live parasite vaccines [125].

5.2. Impact of Tick Expansion into New Territories for Vaccine Development

Tick geographic expansion is speeding up worldwide, including for species that affect human wealth or economic activities, such as I. scapularis [126], H. longicornis [127] and R. (B.) microplus ^[128][129][128,129]. The losses caused by tick parasitism worldwide are estimated to be around USD 20 to USD 30 billion annually [130], a figure that will likely increase as ticks move to new areas due to global climate change. The presence of compatible hosts, climate, physical, and biotic factors is essential for tick establishment into new territories ^{[104][129][131][132]}[104,129,131,132], where tick-borne pathogens can eventually also be introduced. For long-distance dispersion, human action as well as mammal and bird migration play a main role in such expansions ^[133][134][133,134]. Otherwise, for shorter distances, factors such as proximity to a tick-parasitized area, forest cover, and rivers are predicted to be favorable for tick spread [126]. Appropriate climate is one major restriction for tick establishment into new areas [131]. Consequently, the ability to exploit new geographic areas relies on the tick ecological plasticity. Species Distribution Modeling (SDM) is an in silico methodology used to analyze climatic niche expansion of I. ricinus, the most important tick vector of pathogens in Europe [131]. This work predicted that I. ricinus is expanding to about two times the current area due to an increase in human demographics and CO₂ production. Otherwise, changes in temperature, rainfall, and moisture can shrink or increase tick geographic range, as a model predicted for the eastern paralysis tick Ixodes holocyclus (Neumann, 1899) in Australia [132] or for the bont tick Amblyomma hebraeum (Koch, 1884) in Zimbabwe [135]. Similarly, I. ricinus expansion would be affected by the balance between rising temperatures that extends the questing season and favor survival, and droughts that promote tick mortality [136]. Model predictions were used to forecast suitable habitat for R. (B.) microplus distribution in West Africa [137] and South America [129], where temperature was a key determinant for potential tick invasion. The presence of ticks in new geographic areas imposes challenges to control strategies since new hosts will be susceptible to ticks and tick-borne pathogens [138]. This is a concern, especially for highly generalist ticks such as H. longicornis, already spread in North America and with the potential for expansion to new areas in Russia, equatorial Papua New Guinea, and Pacific Islands [129]. Another example includes the wild host reservoir for pathogens, e.g., B. burgdorferi transmitted by I. scapularis, which could increase the difficulty to eliminate Lyme disease in North America [126]. In these cases, the success of vaccination programs could be hindered by the presence of wild animals living near humans and livestock. Additionally, the invasion of R. (B.) microplus into new areas can disturb not only hosts but also native tick species, as is the case of R. (B.) decoloratus in Angola [128], Benin [138], and South Africa [139]. The recent expansion and establishment of R. (B.) microplus in these countries has already resulted in the partial displacement of R. (B.) decoloratus, affecting the stability of native and non-native pathogens. Monitoring new emergence, as well as predicting the invasion of ticks into specific areas, is essential to guide strategies to develop a vaccine protecting against several tick species. For instance, hosts such as bovines or white-tailed deer would be immunized, respectively, against R. (B.) microplus and R. appendiculatus in Africa ^[128][139][128,139], and R. (B.) annulatus and R. (B.) microplus in America [140]. Universal vaccines would be extremely valuable for regions where the economy is heavily dependent on livestock activity.