EJBR2022v12i1art77


ISSN 2449-8955 
European Journal  

of Biological Research 
Review Article 

 

European Journal of Biological Research 2022; 12(1): 77-101 

DOI: http://dx.doi.org/10.5281/zenodo.6386931  

Tick saliva antigen-based vaccines, disease protection 

and prophylaxis 

Nidhi Yadav, Ravi Kant Upadhyay * 

Department of Zoology, Deen Dayal Upadhyaya Gorakhpur University, Gorakhpur 273009, U.P. India 

* Corresponding author: E-mail: rkupadhya@yahoo.com 

Received: 04 December 2021; Revised submission: 01 March 2022; Accepted: 23 March 2022 

 

https://jbrodka.com/index.php/ejbr 
Copyright: © The Author(s) 2022. Licensee Joanna Bródka, Poland. This article is an open-access article distributed under the 

terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/) 

ABSTRACT: This review emphasizes the immune responses to tick infestation and the administration of 

vaccine to save the life of man and his livestock. There are so many vaccines in operation in various parts of 

the world. These vaccines have been developed by using tick saliva toxins or recombinant antigens 

synthesized. This article explains the use of modern molecular tools such as genomics and proteomics in 

identification and search of new potent antigens which could prepare sizable defense against tick-borne 

pathogens. The present article also highlights explorations on salivary gland secreted molecules, genes and 

their expression for preparation of the highly efficacious targeted anti-tick vaccine. There is a need to search 

feeding inhibitors of ticks so that pathogen transmission can be blocked and easy disruption of enzootic cycle 

become possible. In addition, protein antigens from tick midgut must be searched to have a new multi-target 

vaccine to counter-attack tick infestation in various animal and human hosts. 

Keywords: Tick; Vaccine; Tick-borne. 

1. INTRODUCTION 

 Ticks are ubiquitous blood-sucking (hematophagous) arthropod external parasites. These suck blood 

and feed on various classes of terrestrial animal hosts mainly livestock and wild mammals. These are major 

vectors of various human pathogens and animals worldwide.  Tick parasitism is maintained by its various life 

stages and completes among different hosts and mainly domestic, wild and zoo and dairy farm animals. The 

main factors of tick parasitism are blood-sucking and saliva secretion, host immune status, age, breed and 

local ecology. All these factors play important role in the tick-borne pathogen harbor, release and morbidity in 

man and other animals [1]. For feeding ticks grasps the skin, cut the area of attachment and insert their 

feeding tube and cut into the surface [2]. Ticks transmit various disease pathogens and cause morbidity and 

pathogenesis in humans and animals around the world. Tick infestation is noted almost in America, Europe, 

Africa, Australia and Asia, R. microplus, Rhipicephalus (Boophilus) microplus, Amblyomma and Ixodes ticks 

are the medical tick species which transmit so many tick-borne pathogens in humans. Ticks use hypostome to 

puncture the skin, during feeding ticks discharge saliva in the blood of host, through which easily transmit 

disease pathogens. Moreover, anticoagulatory molecules found in tick saliva interfere with the host defense 

mechanisms. These pathogens evoke immune responses after an interaction to cells and tissues of immune 

function. Thus, ticks salivary secretions transmit a variety of disease pathogens bacteria, viruses, and 



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European Journal of Biological Research 2022; 12(1): 77-101 

protozoans in man, livestock and wild animals [3]. Ticks are the major cause of morbidity and mortality; 

they generate significant economic losses to dairy owners [4]. Hard ticks feed very slowly but establish long-

term blood-feeding and much extended parasitic morbidity and show specific host responses [5].  

 There is one unique property of ticks that they are resistant to saliva pathogens and have evolved 

molecules of immune defense. These provide tolerance to pathogens; and assisted in the development of 

immune protection. The main factor which establishes the ticks on hosts is salivary secretion and the 

multistage life cycle of ticks. Saliva upon secretion acts like a carrier for diverse group pathogens and easily 

transfers them into the bloodstream of human and animal hosts. Due to their adaptability to adjust always to 

new conditions makes ticks more perfect transmitters of pathogens of several human diseases and other 

animals. Ticks transmit pathogens during the normal course of feeding, from an infected host to an uninfected 

host. After blood-feeding on infected host, pathogens easily transfer into the gut, from where they reach to 

salivary glands via hemolymph. Again during feeding, ticks transfer these pathogens to another subsequent 

host [2]. Many tick species secrete a cement-like substance that keeps them firmly attached during the meal. It 

is a complex protein that assists ticks in blood-feeding the transfer of disease pathogens. More specifically 

both tick salivary gland secretions and host-derived compounds modulate long-term tick feeding [6]. In 

addition, some pathogens live inside the ovarian tissue of the tick and transmit pathogens directly to their 

progeny. In ticks, saliva plays important role in feeding, the establishment of hemostasis and defying host 

immune responses. It also assists in the formation of a favorable niche for the survival and inoculation of 

various disease pathogens. 

 Tick bite causes severe inflammation on biting sites, swelling and pain. After 2 days it converts into 

red patches on the skin and marks as allergic signs on the skin. This allergic dermatitis develops due to contact 

of saliva components during its release on the skin, and due to expression of IL-6, CXCL-8 and CCL-2 genes 

and synthesis of pro-inflammatory chemokines. More often, low expression of these genes affects the 

production of salivary gland proteins that also decrease host immunity, inflammation and coagulation [7]. 

This shows a delayed-type response in susceptible hosts. To combat allergy granulocytes and T lymphocytes 

are recruited [7]. However, vaccines prepared gut proteins or antigens obstruct both feeding and transfer of 

pathogen at the tick feeding site [8].  

 Though, for the protection of livestock so many countermeasures have been developed. But repeated 

tick infestations result in the development of natural immunity in livestock. It protects from tick bites and 

blocks the transfer of disease pathogens. But it does not seem satisfactory in controlling wider tick attacks. 

However, for control of ticks various acaricides are used but these were not proved sufficient in successful 

control of ticks. Its repetitive use and exposure cause problems to host skin as these absorb through the skin 

and reach in the blood. It has become a major cause of drug resistance in ticks; contamination of dairy 

products and fouling of surroundings. It is true that dairy products obtained from treated dairy animals 

contain acaricide residues which persist for a longer duration. These also impose adverse effects on the 

environment and human beings. Therefore, to avoid undesirable physiological effects of acaricides and 

environmental contamination vaccines are proved much better and appropriate solution.  

 However, before the development of an appropriate vaccine all related factors which essentially target 

tick saliva feeding must be known. These major factors are host-parasite interaction, its participatory 

molecules, pathways, gaps and interconnections, tick immunity mainly cellular and molecular components of 

humoral and cellular immunity must be known. In addition, adverse effects of tick antimicrobial compounds 

on signaling pathways, metabolism and physiology of the host body must be known [9]. In addition, various 



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European Journal of Biological Research 2022; 12(1): 77-101 

antigens from tick saliva or gut proteins released by ticks during feeding must be identified. These could be 

used as appropriate molecules for generating potential vaccines to stop blood-sucking by ticks. Feeding 

inhibition by administration of vaccine will also help in obstruction pathogen transmission from the tick to 

hosts. There is a need to purify and obtain new novel and highly effective vaccine antigens to control tick-

borne pathogen attacks [10]. However, for the preparation of effective and appropriate vaccine tick tissue 

extracts or saliva toxins could be used as antigens.  

2. TICK-BORNE DISEASES AND ECONOMIC LOSS 

 Ticks are ectoparasites and major disease transmission vectors among all arthropods. Tick menace is 

the biggest challenge for livestock and dairy owners in tropical and subtropical regions of the world. Though 

for control of tick menace so many traditional control methods have been applied to kill this dreadful parasite, 

but it has gained resistance to acaricides and made heavy to the cattle industry [11]. There are so many species 

of soft and hard ticks which parasitize different hosts and doing annual losses of billions of dollars. Tick 

menace is severely affecting dairy farming units as the annual loss has been enhanced up to ~ $22-30 billion 

annually around the globe. Around 80% of the global loss is only due to Rhipicephalus microplus transmitted 

infection [12]. Rhipicephalus microplus, is a monoxenous tick that causes massive damage to livestock in 

India and the world. There was obtained variability in tick infestation Cattle breeds mainly taurine breeds 

display higher loads of the tick parasite while indicine breeds are somewhat lesser [13]. 

3. HOW TO COUNTER-ATTACK TICK INFESTATION 

 For tick control, various strategies and methods have been applied so far. All these are based on 

controlling the spread of tick-borne pathogens which after transmission affect human and animal health. Tick-

borne diseases spread with the close interactions of human contact with livestock and wild animals. Therefore, 

interaction and exposure of wild animals to humans must be reduced [14]. Hence, there is a need to 

understand the complex cycle of ticks, its associating hosts and various pathogens transmitted in the human 

population. There is a need of disruption of tick life cycle among various mammalian hosts by using 

alternative approaches. It could become possible by applying cultural, acaricidal rotation, use of synergists, 

mixed pesticide formulations, use of biopesticides, safe animal care methods, and selection for host resistance, 

nutritional and environmental management. For providing protection cover regular vaccination of farm 

animals is highly important with dip bathing by use of plant origin acaricides, plant extracts and essential oils. 

However, the systematic use of different control methods will help to eliminate the acaricide-resistant tick 

population [15]. 

Tick saliva contains pathogens which after release into the host bloodstream show immune interactions 

and the host body starts preparing both innate and acquired immune defenses.  In response to control agents 

ticks have developed resistance to them, ticks also subvert immune defense prepared by various hosts [16]. 

Ticks also have evolved adaptations to chalk out immune functions of crossbreed livestock and challenged its 

genetic constitution [17]. This leads to a lower efficacy of recombinant vaccines. Hence, there is a need to 

find certain uniqueness in modules and gaps to conquer and finish the tick menace. There is a need to focus on 

the search of new noble antigens, biologicals, signaling molecules, complement molecules, cytokines, death 

programmers, feeding inhibitors, antimicrobials, antibodies and vaccines [2]. Lastly, new molecules could 

generate strong innate immune responses and break the adaptational cover made by various tick species in 

form of immune tolerance and efficiency to resistance to their own pathogens [2]. 



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European Journal of Biological Research 2022; 12(1): 77-101 

 Vaccines are more effective against ticks as they easily disrupt feeding in ticks, thereby blocking 

pathogen transmission among hosts and much able to break enzootic cycles [3]. It should reduce exposure of 

infected ticks to humans, domestic and wild animals [3]. Recombinant vaccines provide long-term cover as 

they generate enough immune protection against diverse tick-borne pathogens. These are cost-effective and 

do not put any harm to the surrounding environment. These are the best alternative to replace chemical 

acaricides. Hence, there is an immense need to search for new protective antigens for vaccine development.   

For this purpose, host-pathogen interaction, transmission methods, mechanisms of acquisition, and persistence 

of parasitism must be known. More often, all concerned attack points and elements of the tick life cycle must 

be identified [18]. It will not only help in the success of vaccine programs but also prove a sharp-edged 

weapon to kill acaricide-resistant tick populations [19]. There is a need to search a series of noble and unique 

feeding inhibitory devices, molecules, genes, biochemicals to repel ticks from taking a blood meal and target 

the adaptational shield of ticks [12]. However, combined methods and combined efforts are essentially needed 

to finish the biggest challenge given by ticks to medical and veterinary scientists and public health institutions 

to control tick-transmitted pathogens [17]. 

 There is a need to develop commercial and low-cost vaccines, but till date only two vaccines have been 

prepared which are in use and available in the international market. A single vaccine has been developed by 

using tick midgut protein Bm86 as an antigen. It protects from tick bites and the transfer of disease pathogens. 

It shows limited efficacy to only some tick species and is currently used for field applications [20]. A midgut 

glycoprotein Bm86 was used as an antigen to prepare TickGARD and Gavac vaccines. It was found highly 

effective against R. microplus tick found in different geographical areas [21]. 

4. HOST ANTIBODIES 

 Sera from tick-susceptible Holsteins contain antibodies, which can neutralize tick salivary proteins 

involved in parasitism. Alternatively, antibodies can prohibit tick feeding and thereby control tick infestations 

[22]. Antibodies synthesized against tick-protective antigens can successfully control tick-borne pathogens [9] 

and blood-feeding by nymphs [23]. However, administration of a live vaccine having Enterobacteriaceae 

bacterium Escherichia coli strain BL21 produces anti-E. coli and anti-α-Gal IgM and IgG. These antibodies 

efficiently kill I. ricinus nymphs by inhibition of feeding in them [24]. Aquaporin antigen was used to make a 

vaccine that targets infestations of R. microplus. Being an active ingredient in cattle vaccines it has increased 

the effectiveness of vaccines and reduced the numbers of adult female ticks from blood-feeding [25]. 

5. HEMOSTASIS 

For establishing parasitism in various hosts, ticks synthesize and secrete many anti-haemostatic 

molecules such as vasodilators and a group of peptides which inhibit blood coagulation. These act as platelet 

aggregation inhibitors and increase fibrin breakdown in the host [26]. These anticoagulants disrupt elements 

of both the intrinsic and extrinsic pathways. These stay at the injury spot or cut mark, and assist in providing 

uncoagulated uninterrupted supply of blood to the ticks from the blood pool. Thus, host hemostasis is 

challenged due to the release of antagonistic molecules or vasodilators at ticks biting sites on host skin. Most 

of these vasodilators are lipid derivatives, mainly prostacyclin and prostaglandins [27]. Ticks also secrete 

histamine release factor (tHRF) protein molecules, which stimulate histamine, interleukin 4 and IL-3 

production from IgE-sensitized basophils and mast cells inside the host body. Ticks also synthesize four 

categories of serine protease inhibitors i.e. Kunitz domain inhibitors, Kazal domain inhibitors, trypsin 



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European Journal of Biological Research 2022; 12(1): 77-101 

inhibitor-like cysteine-rich domain (TIL) inhibitors, and serpins. Serine proteases or serpins secreted in tick 

saliva stop the action of a hemostatic system [28]. These also inhibit the activity of trypsin and thrombin [26] 

and obstruct platelet aggregation and blood clotting [29,30]. Tick salivary serpins do indirect inhibition of 

mainly factor X and thrombin (IIa). Few tick species also synthesize Salp14 and tissue factor pathway 

inhibitors. These inhibit the action of factor X and factor VIIa [26,31]. In addition, there are numerous 

salivary proteins i.e TIX-5 which act as an anticoagulant and halt the action of factor V and Xa. Few of them 

are reported in I. scapularis saliva, which accelerates fibrinolysis, degrades fibrin clots and acts as 

metalloproteases [32]. 

 Due to multiple tick bites and mixing of salivary molecular repertoire mainly anticoagulants hosts due 

to invasion and tick multiple bites severely destroy and damage blood vessels and capillary endothelium. In 

response to tick bites, several agonists such as ADP, thrombin and collagen are secreted from the host body. 

These bind to specific platelet membrane receptors and activate platelets. Tick saliva proteases inhibit platelet 

activation and aggregation at different stages. More often these substances block the binding of fibrinogen to 

activated platelets [26]. Ticks I. scapularis transmit B. burgdorferi, secrete proteins that directly or indirectly 

support the multiplication of B. burgdorferi. After blood-feeding both tick pathogens secreted molecules and 

tick components interact with each other that support pathogen persistence and transmission [33]. Lipocalin 

(ISCW005600) is an inhibitor of lectin pathway inhibitor is secreted by I. scapularis [34]. Thus, ticks 

maintain continuous blood-feeding throughout life. Similarly, in response to repetitive tick bites host body 

also develop a multi-layered defense system against foreign pathogens and ectoparasites, coagulants try to 

check blood loss and maintain homeostasis [34]. 

6. SEARCH OF ANTIGENS AND VACCINE PRODUCTION 

6.1. Identification of  elements related to tick pathogens’ life-cycle 

 Various recent molecular and genetic methods have been used to find novel antigens (Figure 1) for 

the generation of vaccines for tick control. For this purpose, salivary gland transcriptome analysis is 

performed to find functional genes which are responsible for the synthesis of toxins [35]. By using genomics, 

and proteomic methods to novel antigens can be identified for the preparation of a vaccine against ticks. The 

main focus is required on saliva secreted antigens associated with tick gut. Tick salivary secretions are quite 

important for their reproduction and survival. Once the action of saliva proteins will be inhibited pathogen 

transmission is also inhibited. By using a similar concept anti-tick vaccines were made by using inhibitory 

molecules as antigens. The administration of these vaccines in livestock prevents tick feeding and efficiently 

prevents infections [36] finally controlling blood-feeding nymphs and adults [6,8]. 

Tick saliva is a natural source of toxins which could be used as an antigen. It interplays between ticks 

and host's immune system is affected by various antigens, these evoke sizable immunity in host’s body and 

mostly used for the production of vaccine. For the generation of natural protection tick salivary extracts from 

ticks are administered as immunogen. It was found successful when salivary gland extracts of Rh. (B.) 

annulatus tick were injected in cattle. It leads to the synthesis and secretion of protective antibodies against 

saliva antigens. It is a safer non-chemical method of tick control [37]. Thus, immunization with anti-tick 

vaccines inhibits feeding efficiency in ticks that also stop the spread of various disease pathogens. Therefore, 

the production of an effective and appropriate vaccine production, identification, purification and 

characterization is highly important to have a noble antigen. Few cell-surface proteins are expressed which are 

also known as exposed antigens are released in tick saliva during blood feeding. The second type of antigens 



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European Journal of Biological Research 2022; 12(1): 77-101 

is hidden antigens which are very hard to collect and concealed type. The third type of antigens is very hard to 

identify and possess properties of both exposed and concealed type vaccine. This third category of antigens is 

widely used for the production of vaccines with broad-spectrum anti-pathogen efficacy. These were found 

effective against both immature and adult stages of ticks [36]. In comparison to acaricides, vaccines are the 

environmentally safer method to control ticks. This is true that appropriate anti-tick vaccines are required to 

combat tick-borne diseases [3]. 

 

 

Figure 1. Important tick vaccine antigen targets for control of pathogenicity and tick infestation. 

  

 Genes which are responsible for the synthesis of toxins could be used to make the highly efficacious 

vaccine. These are based on protective antigens which provide protection cover against tick bites and tick-

borne diseases. By using genetic and DNA sequencing methods new genes can be identified and their 

expression libraries can be used for searching new antigens for immunization. Therefore, by using expression 

sequence tags of various genes and their annotations, and comparison with available antigens can accelerate 

the antigen screening. It will also assist in the opening of the new comprehensive way for having an 

appropriate antigen/s for generation of highly effective against tick-borne pathogens [38]. Differential 

expression analysis is also being done to identify differentially expressed genes (DEGs). Many of these genes 

found in hosts are related to pathogen susceptibility or resistance to tick bites and feeding. Besides this, 

certain genes might control stress response during blood feeding [35]. 

 The dilemma of tick-host and pathogen interaction can be resolved by identifying and encountering 

tick-origin infectious agents and antigens [21]. These unique molecules could control tick feeding and 

pathogen transmission by so many species of ticks not only Rhipicephalus (Boophilus) microplus [35]. In 

addition, there is a need to find molecules which inhibit gene expression of saliva proteins which are 

responsible for hemostasis, inflammation and inhibit coagulation and platelet aggregation anticoagulation and 

suppress host immunity [7].  

 For searching novel, tick-protective antigens for the formulation of anti-tick vaccines various 

molecular and immunological methods are used [39]. For identification of functional gene transcripts of 

salivary proteins and for detection of parasite antigen encoding genes high-throughput microfluidic RT-PCR is 

used [40]. Immunogenetic and molecular biology tools are also used for epidemiological surveys and improve 

TBD prevention and control of ticks [40].  



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European Journal of Biological Research 2022; 12(1): 77-101 

 Further, exploration of molecules can also help in the analysis of cattle cross-breeding tolerance and 

tick infestation [42] and may prove highly beneficial for control programs to combat tick-borne diseases [10]. 

These novel candidates can serve as tick protective antigens for making potential vaccine prevention and 

control of many tick species [34]. Further, for control of ticks inhibitors of microRNAs (miRNAs) should be 

searched, because these are important regulators of gene expression. Once action microRNAs is blocked, the 

synthesis of saliva proteins is inhibited that will result in blood-feeding inhibition in ticks. Further by using, 

genetic methods manipulation of vector microbiome is also possible that can assist in tick control at a large 

scale. Genetically produced tick salivary glycoproteins are highly specific and viable vaccine targets to inhibit 

tick feeding and are found as anti-tick vaccine candidates [43].   

For better control, tick feeding and its parasitism must be explored. All different tick species transmit 

pathogens through blood-feeding; it is also responsible for inter-host transmission or migration of pathogens 

from the gut lumen to the hemocoel. Through blood-feeding ticks release large numbers of pathogens salivary 

secretions which mix in host blood. Thus both saliva and its derived proteins transfer pathogens in the number 

of vertebrate hosts. These modulate responses at the site of attachment and biting sites and frame conditions, 

to make blood-feeding essays, uninterrupted. Besides, pathogens (spirochaetes Lyme borreliosis) tick saliva 

also contains diverse molecular components i.e. Iris, Salp15 which modulate T cells while complement 

proteins i.e. ISAC, Salp20, TSLPI activate B cells. Similarly, a close interaction has been observed between 

outer surface proteins of unfed ticks spirochaetes and OspA receptors found in gut region of ticks [45,46]. 

Spirochetes synthesize OspC a lipoprotein during the tick blood-feeding; this protein is down-regulated after 

transfer of B. burgdorferi to the mammalian hosts. While after feeding is upregulated [47]. OspC is very 

essential for B. burgdorferi infestation and transfer of this pathogen from gut to tick salivary glands for 

initiation of infection [48,47]. More specifically, OspC binds to Salp15 and saves spirochaetes from antibody-

mediated killing. g. It is also responsible for their transmission into the host [49]. Tick antigens useful for 

vaccine production are presented in Table 1. 

 

Table 1. Various tick antigens used for vaccine production with their efficacy and protection.  

Vaccine 

candidate   
Function  Method applied  

Tick stage during 

discovery 

Antibody/ 

screening used 

during discovery 

References 

                                                                                 Salivary proteins 

Sialostatin 
L2 

Specifically inhibits 
cathepsin L activity 

in cytotoxic T 
lymphocytes 

I. scapularis salivary gland 
cDNA expression library 

and sialome 

I. scapularis salivary 
glands from fed nymphs 

and (un)fed adults 

Inhibition of tick 
feeding by anti-
sialantibodies 

[64] 

TSLP 
Tick Salivary Lectin 

Path inhibitor 

Gel filtration, salivary 
protein facilitating B. 

burgdorferi 

Inhibiting the host lectin 
complement pathway 

Inhibition of feeding in 
I. scapularis 

[65] 

BPTI-Kunitz 
thrombin 
inhibitors 

ixolaris, pethalaris, 
Salp14 and 

BmTIs/RsTIs  

Gel filtration, tick anti-
haemostatic antigens 

Block binding of 
fibrinogen to activated 

platelets 

Inhibition of feeding in 
I. scapularis due to 

blood clotting 
[26] 

TIX-5  
An anticoagulant 
salivary protein 

Gel filtration, TIX-5 in 
salivary gland extract of I. 

scapularis adults 
Inhibition of tick feeding Render tick immunity [75] 

Tick cement 
cone protein 

64P 
Structural protein 

Gel filtration escape host 
rejection during tick 

infestation 

Escape host rejection 
during tick infestation 

Render tick immunity [77] 

Ferritins FER1 and FER2 
Gel filtration iron during 

blood feeding and  

Reduce tick infestations 
and the transmission of 
tick-borne pathogens 

Anti-ferritin vaccine 
reduce tick feeding in 

R. microplus, R. 
annulatus and H. 

[20] 



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European Journal of Biological Research 2022; 12(1): 77-101 

Vaccine 

candidate   
Function  Method applied  

Tick stage during 

discovery 

Antibody/ 

screening used 

during discovery 

References 

longicornis 

HlFER and  
HlFER2 

Iron-binding protein 
ferritin (HlFER), an 
intracellular 1 and a  
secretory HlFER2 

Gel filtration, iron during 
blood-feeding 

Inhibition of blood-
feeding in ticks  

HlFER2 anti-tick 
vaccine effective 

against multiple tick 
species 

[20] 

AAS19 
Immunogenic  tick 

saliva protein 

Gel filtration, serine 
protease inhibitor serpin, 

AAS 

Anti-haemostatic 
functions 

Similar to trypsin-like 
proteases 

[80,117, 
123] 

Concealed 
antigens 

A heterogeneous 
group of proteins 

 Host immune system  [10,78] 

                                                                                         Gut proteins 

TROSPA 

Gut-protein, binding 
partner of the 

Borrelia protein 
OspA 

Screening of I. scapularis 
cDNA library 

I. scapularis protein 
involved in pathogen 

colonization is TROSPA 

Borrelia protein OspA, 
impaired B. burgdorferi 

acquisition 
[9,46] 

ISDLP and 
ixofin3D 

Two new Ixodes gut 
proteins 

Gel filtration   
Reduced Borrelia loads 
in the skin 7 days after 

tick challenge 

Antibody/vaccine 
obstruct tick feeding 

[86,87] 

Regulatory proteins 

Subolesin 
Signal transduction, 

impaired tick 
feeding 

cDNA expression library 
immunization and analysis 

of expressed sequenced 
tags 

I. scapularis cell line 
(IDE8) derived from tick 

embryos 

Tick-immunised mouse 
sera, Subolesin based 
vaccine obstruct tick 

feeding 

[90,91] 

OspA a Cyclin-
dependent 

kinases CDKs 

Participate in cell 
cycle control 

cDNA expression 
library of Outer surface 

proteins 

Recombinant CDK10 
followed by tick 

challenge 

Anti-cdk based vaccine 
obstruct tick feeding 

[95] 

                                                                       Recombinant antigens 

B. annulatus 
Bm86 ortholog, 

Ba86 

Midgut membrane 
glycoprotein 

rDNA technology 
Expressed in yeast Pichia 

pastoris 
Bm86-based vaccine 
obstruct tick feeding 

[12] 

IrSPI and 
IrLip1 

Recombinant protein, 
Kunitz elastase 

inhibitor 
cDNA expression library 

Enhance tick 
engorgement and molting 

and decrease tick  

immunomodulator 
implicated in I. ricinus 

feeding 
[5,19,126] 

Salp14 
Factor Xa inhibitor I. 

scapularis 

Salivary gland cDNA 
expression library 

I. scapularis salivary 
gland 

Tick immune rabbit 
sera 

 [74] 

Salp15  
 

Impair B. burgdorferi 
transmission and 

inhibits activation of 
murine CD4 positive 

T cells and human 
dendritic cells  

Salivary gland cDNA 
expression library 

Expression library 
engorged Ixodes ricinus 

Tick-immune rabbit 
serum 

 [49,59,84] 

Salp20 
I scapularis 

complement inhibitor 
Salivary gland cDNA 

expression library 
Engorged I. scapularis 

nymphs 
Tick-immune rabbit 

serum 
[45,46] 

Salp25D Anti-oxidant protein  
Salivary gland cDNA 

expression library 

Engorged I. scapularis 
nymphs, impaired B. 

burgdorferi transmission  

Tick-immune rabbit 
serum 

[33] 

64P/TRP 

Tick cement cone, 
reduce tick mortality, 

and TBEV 
transmission, Ixodes 
ricinus tick feeding 

Immunoblot analysis adult 
Rhipicephalus 

appendiculatus ticks 
Salivary glands extracts 

Antibody, tick-immune 
guinea pig serum 

  
[77] 

 

FER2 
Binding and transport 
of iron, impaired tick 

feeding 

Isolated and cloned from 
cDNA libraries 

Unfed adult I. ricinus 
ticks and larvae of 

Ornithodoros moubata 

cDNAs encoding 
ferritin 

[84] 

tHRF 
Tick histamine 

release factor (tHRF) 
in I. scapularis saliva  

2-dimensional fluorescence 
difference gel 

electrophoresis (DIGE) 

B. burgdorferi-infected 
and uninfected I. 

scapularis 

Salivary glands extracts 
none 

[29] 

Isac-1 
I. scapularis 

complement 
inhibitor 

Random screening of a 
salivary gland cDNA 
library by sequencing 

Partially engorged female 
I. scapularis ticks feeding 

for 3-4 days on a rabbit 

None, impaired B. 
burgdorferi 

transmission 
[66] 



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European Journal of Biological Research 2022; 12(1): 77-101 

Vaccine 

candidate   
Function  Method applied  

Tick stage during 

discovery 

Antibody/ 

screening used 

during discovery 

References 

IRAC-I 
I. ricinus complement 

inhibitor   

Isolation from 
transcriptome of 

I. ricinus salivary glands 

I. ricinus salivary gland 
of engorged adult female 

ticks 

Antibody response, 
marginally impaired 

tick feeding 
[29] 

IRAC-II 
I. ricinus complement 

inhibitor   
Transcriptome of I. ricinus 

salivary glands 

I. ricinus salivary gland 
of engorged adult female 

ticks 
None [29] 

TSLPI 
Tick salivary lectin 
pathway inhibitor  

Immunoscreening of I. 
scapularis salivary gland 

yeast surface display 
library  

I. scapularis salivary 
gland 

Tick immune rabbit 
sera, impairs neutrophil 

phagocytosis and 
chemotaxis   

[75] 

Iris 

I. ricinus suppress T 
cell proliferation, 

induces a Th2 
immune response, 

inhibits production of 
IL-6 and TNFα  

Analysis subtractive library 
from salivary glands 

Unfed and 5 day fed 
female I. ricinus ticks 

None, impaired tick 
feeding 

 [58] 

Tix-5 Anticoagulant 

Immunoscreening I. 
scapularis salivary gland 

yeast surface display 
library 

Adult I. scapularis 
salivary glands 

Tick immune rabbit 
sera 

 [62] 

CDK10 
Cyclin-dependent 

kinases participate in 
cell cycle control 

Bioinformatic search for 
CDK homologues 

R. microplus 
None, impaired Ixodes 

feeding 
[95] 

Ir86-1 and 
Ir86-2 

Bm86 homologue, 
gut protein I. 

scapularis 

Bioinformatic search for 
Bm86 homologues 

I. ricinus 
None, no effect on 
weight/attachment 

[108] 

ISDLP 
Gut-protein, binds to 
Borrelia and involved 
in Borrelia migration 

Probing of I. scapularis 
midgut yeast surface 

display library 
I. scapularis midgut Borrelia proteins [65] 

Ixofin3D  
Gut-protein, binds to 
Borrelia and involved 
in Borrelia migration  

Probing of I. scapularis 
midgut yeast surface 

display library 
I. scapularis midgut 

Borrelia proteins 
vaccination, no effect 

on tick feeding 
[87]  

6.2. Salivary gland proteins 

 Tick saliva is important for pathogen transmission, as it takes place through saliva secretions during 

tick feeding. Tick salivary gland proteins are the main operators of blood-feeding and establishment of 

parasitic life on the host’s body surface [50]. Repetitive tick feeding and secretion of various salivary proteins 

also partially raised immunity in the cattle herds. It plays a protective role against tick inflammation and 

inhibits feeding. These saliva proteins have pharmacological and immunological significance. These are the 

most appropriate candidate molecules for making a potential anti-tick vaccine which can work against various 

tick species and their associated pathogens [51]. More often, multiple tick saliva toxins and multiple antigens 

could be used to make a cocktail of tick antigens or multivalent vaccine [52]. Vertebrate host proteins have 

been detected post blood-feeding in tick midguts. Host blood is the main source of nutrients for ticks. For easy 

intake of blood meals ticks synthesize anticoagulants and other inhibitory proteins from their salivary glands 

during tick feeding and release pathogens which are responsible for pathogenesis. Thus both tick saliva toxins 

and pathogen secreted molecules interact to host immune molecules, interfere in signaling pathways and 

affect synthesis and secretion of protective metabolites and invade the host body. These molecules which are 

responsible for host-pathogen-interaction must be identified to explore new targets for the development of 

potent vaccines eradication of ticks and tick-borne diseases [53]. Few of them have been identified in 

Rhipicephalus bursa salivary glands genes which show differential expression in response to blood-feeding. 

These genes synthesize saliva secreted toxins which also raise adaptive immunity that is possibly induced 

upon TBEV infection and vaccination [54]. Few other protective antigens such as lachesin, vitellogenin-3, and 



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cement proteins have been identified which can be used as suitable antigens development of novel tick 

vaccines [55]. However, functional characterization of these tick saliva toxins is an important issue regarding 

vaccine and antibody generation [56]. These proteins could be used to develop anti-paralysis vaccines. Thus, 

both vector and/or pathogen secreted molecules could be used as protective antigens which might be cross-

reactive to different tick species and most appropriate for the development of novel control strategies [55-57].  

Till date, so many salivary proteins have been isolated and characterized tick saliva which have an 

essential role in tick-host and pathogen interaction. Certainly, these will prove the best candidates for the 

potential vaccine. Iris is a protein secreted from ticks from female I. ricinus salivary glands both by nymphs 

and adults. It causes impairment of tick feeding in vaccinated experimental rabbits [58]. Similarly, another 

protein Iric-1 a homolog of Salp15 is secreted by I. ricinus, that is essential for the transmission of B. 

burgdorferis. B. burgdorferi sensu stricto, B. garinii and B. afzelii and save these pathogens from antibody-

mediated killing in vitro [62].  

Ticks transfer so many pharmacologically active chemical components during blood feeding inside 

hosts. Normally hosts are repeatedly bitten by ticks. These releases a few immunomodulatory proteins like 

Sialostatin L2 which also generate adaptive immune responses after repeated tick bites. This protein acts as an 

antigen and is used in the active immunization of animals [64]. 

A tick salivary protein tHRF is secreted to do multiple functions. It induces the release of histamine 

from basophils and mast cells of mammalian hosts. It participates both in IgE-dependent and IgE-independent 

mechanisms. It leads to an increase in the recruitment of pro-inflammatory cells and increase vascular 

permeability and produces an itching response. This protein increases vascular permeability and blood flow 

just after the tick bite site [29].  Immunization with the recombinant protein blocks Borrelia transmission and 

tick feeding. This tHRF blocking could become the best strategy to develop vaccines which might inhibit the 

feeding of blood and transfer of tick-borne pathogens [29]. Similarly, a protein TSLPI, from I. scapularis 

inhibits the host lectin complement pathway and produces antibody response to infectious toxin antigens from 

various ticks species when mixed in host blood [65]. 

There are thousands of compounds isolated from tick saliva and most of them have been identified by 

transcriptomic gene sequencing methods [66-69]. Besides this, there are dozens of genes which are expressed 

in tick salivary glands [69]. The expression of these genes is responsible for homeostasis and pathogen 

transfer. A few important proteins are Salp15, lipocalins, metalloproteases whose function is still unknown 

[70,71]. Normally these proteins were detected post 24 hours of feeding; these assist in blood feeding and 

pathogen transmission [68]. Ticks and tick-borne pathogens need a suitable environment for pathogen 

establishment [72]. 

6.3. Tick anti-haemostatics 

Tick saliva is a reservoir of pathogen-induced inhibitory proteins which interfere in host immune 

response and signaling. More specifically, tick Ixodes ricinus, secrete tick salivary glands. Iripin-3 protein 

shows anti-haemostatic activity in vitro and immunologically active [50]. Iripin-3 impairs proliferation of 

CD4+ T lymphocytes, cut down T helper type 1 immune response, and induction of regulatory T cell 

differentiation. It does proteolytic inhibition of serine proteases kallikrein and matriptase. Iripin-3 also inhibits 

the blood coagulation pathway and cut down the production of pro-inflammatory cytokine interleukin-6 by 

lipopolysaccharide-stimulated bone marrow-derived macrophages [52]. It modulates the adaptive immune 

response and decreases the survival of mouse splenocytes [50]. These generate make a favorable environment 



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for pathogen transmission. Other important proteins are salivary cystatins, these target two host cysteine 

proteases, cathepsin S and cathepsin C . Cathepsin S is required for antigen- and invariant chain-processing, 

while cathepsin C plays a critical role in processing and activation of the granule serine proteases [73]. 

Cystatin OmC2 found in the saliva of ticks inhibits the activity of several lysosomal cysteines [73]. 

Tick saliva also contains BPTI-Kunitz thrombin inhibitors, ixolaris, pethalaris, Salp14 and 

BmTIs/RsTIs, all these act as anti-hemostatic antigens [26]. Both BmTIs and Salp14 are injected in the host 

body to trigger immune response and obstruction of tick feeding through skin surface [26, 74]. Similarly, a 

recombinant anticoagulant salivary protein TIX-5 is also administered for immunization of experimental 

rabbits. Its homologs are also injected which show very high tick feeding inhibition and prepare a sizable 

immune defense in hosts [75].  

For stabilization of parasitic life, survival and continuity on host body ticks essentially need blood 

feeding. Blood feeding is highly required for normal development and reproduction in ticks. In blood-feeding 

ticks, saliva and its derived molecules work as anti-hemostatic and immunomodulatory candidates. Among 

these proteins are lipocalins, metalloproteases, protease inhibitors including the Kunitz/BPTI-family, proteins 

with phospholipase A2 activity, acidic and basic tail proteins, and vitellogenins. These proteins also prepare 

tick immune protection [76].  

6.4. Structural components 

Tick saliva contains a large number of various non-proteinaceous substances and proteins are 

differentially produced and secreted during tick feeding on the blood of hosts. Ticks have also a cement-like 

substance that holds them during feeding. This is a complex protein polymerization substance secreted 

by ticks. A protein 64P assists in host attachment formation of cone-like structure [77]. It shows similarity 

mammalian skin proteins, and play role in host rejection during tick infestation if not secreted [78]. Truncated 

versions of the protein (64TRPs) showed significant adult and nymphal mortality in guinea pigs. It generates 

potent adaptive immune response [79]. 

Tick saliva and salivary glands contain exosomes which could play important role in therapeutics [80]. 

These can be isolated from the saliva of blood unfed ixodid ticks. Exosomes inhibit wound 

healing via downregulation of C-X-C motif chemokine ligand 12 (CXCL12) in vivo and upregulation of 

interleukin-8 (IL-8) at the tick–human skin interface [80]. Further, inhibition of IL-8 or CXCL12 delays 

exosome-mediated cell migration, wound healing, and repair process. In contrast, exogenous treatment of 

CXCL12 protein completely restored this delay and enhanced the repair process [80]. 

Ticks release various Kunitz inhibitors, serpins, and cystatins in saliva during feeding. Among Kunitz 

inhibitors serve as anti-hemostatic agents and obstruct blood coagulation and platelet aggregation. Serpins and 

cystatins work as anti-hemostatic effectors proteins. All these are potent modulators of the host immune 

system and manage tick-host-pathogen interaction.  

Besides this, enzyme inhibitors mainly serine protease inhibitors (SPIs), have their involvement in 

various tick biological processes. SPIs are also secreted during tick feeding, and inhibit blood clotting but 

assist in blood digestion and nutrient extraction. SPIs secreted from tick hemocytes make innate immune 

defenses in ticks but severely affect host defense mechanisms.   

6.5. Concealed antigens 

'Concealed' antigens are those which do not stimulate an immune response during a natural parasite 

infection, typically because of their physical location. More specifically, these are heterogeneous proteins 



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European Journal of Biological Research 2022; 12(1): 77-101 

which are excreted in intracellular space; these are not limited to a particular tick species. Among them are 

intrinsic membrane glycoproteins Bm86, which function as antigen synthesized inside tick gut. Livestock 

synthesizes antibodies against Bm86 but it remains unidentified from the host's immune system during a 

natural infestation. It may be due to carbohydrate determinants on many tick glycoproteins are cross-reactive 

immunologically and show non-specific reaction with carbohydrate determinants on tick glycoprotein [10] 

[78]. But its recombinant forms evoke immunogenicity when administered vertebrate host [78]. Antibodies 

generated against these antigens were effective and obstruct the transmission of pathogens by feeding 

inhibition. These could be generated in the required amount to raise sufficient levels of antibodies.  

Bm86 is a gut protein secreted by tick B. annulatus, but its multiple orthologs are produced. This is an 

appropriate target of an anti-tick vaccine. Similarly, an Is86 antigen contains EGF-1, EGF-2, and EGF-3 

domains. It is upregulated during B. burgdorferi infection but does not assist for tick engorgement during 

feeding. It only cutdown spirochete loads in the host skin. It may be useful in making anti-tick measures and 

fighting against tick-borne illnesses. Contrary to this, a cocktail of various tick antigens may be more 

appropriate to generate a multivalent vaccine to inhibit feeding in nymphs of R. appendiculatus and ably 

break the natural cycle of tick pathogen transmission [52]. Besides this, genes which encode tick gut proteins 

show differential expression to susceptible and resistant hosts. Possible inhibition of synthesis of these 

proteins mainly sialoproteins could help to achieve the target of complete feeding inhibition in ticks [81]. In 

addition, the level of immune functions may differ from host to host; hence, hosts with strong immune 

defense are resistant and they affect gene expression of proteins in tick salivary glands. Alum-adjuvanted 

antigens generate strong immunogenicity, by having more T-cell epitopes and preparing much better antibody 

responses against ticks [82, 83]. Alum-adjuvanted vaccines based on recombinant proteins have shown much 

better efficacy against tick feeding.  

6.6. Ferritins 

Ticks also generate iron-binding proteins i.e. ferritins during blood feeding [20,84]. FER1 is an 

intracellular form (FER1) while FER2 acts as an iron transporter in the tick hemolymph and is expressed in all 

life stages of ticks. By employing gene silencing and interference of RNA blood feeding and reproduction in 

ticks can be controlled [84]. Moreover, a vaccine generated by using recombinant FER2 protein mainly I. 

ricinus (IrFER2) found 98% efficacious against ticks bites [84]. It successfully inhibits feeding in R. 

microplus, R. annulatus and H. longicornis [20,84]. Ferritins are secreted almost in all tick species; hence a 

cross-reactive vaccine also becomes possible. Similar proteins HlFER and HlFER2 have been identified and 

characterized in hard tick Haemaphysalis longicornis. These are also highly effective antigen targets to make 

the anti-tick vaccine effective against multiple tick species [20]. Similarly, RNAi silencing of the fer1, fer2, 

and irp1 genes impose an adverse impact on the hatching rate and decrease post-blood meal weight in tick 

females [85]. Salp15 is a protein that is secreted by Ixodes persulcatus and Ixodes pacificus salivary glands. It 

is responsible for the transmission of Lyme disease spirochetes in hosts [85].  

6.7. Gut proteins 

Tick survival depends on gut synthesized proteins play an important role. These proteins assist 

pathogens multiplication, transfer to various hosts and generation of immune responses. TROSPA is a gut 

protein delivered in intercellular spaces and the luminal surface of the gut [48]. Its expression is seen in 

almost every life stage of tick. It is an important candidate for the generation of antibodies which significantly 

inhibited feeding in ticks [9]. Similarly, dystroglycan-like membrane-bound proteins ISDLP and ixofin3D are 



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European Journal of Biological Research 2022; 12(1): 77-101 

synthesized by Ixodes gut [86,87]. ISDLP protein is upregulated upon both feeding and infection. Silencing 

by RNA interference (RNAi) of ISLDP reduced Borrelia in the salivary glands of the tick low down tick 

infestation. Vaccination against ixofin3D did not inhibit tick feeding and cutdown Borrelia loads significantly 

1 week after tick feeding [87]. 

Tick Hyalomma (H.) dromedarii gut synthesizes their glycoproteins (range 40-97 kDa) [88]. Vaccines 

generated against these proteins successfully cut down the reproductive index and egg hatchability. GLPs are 

good immunogens and can be useful in the vaccination of cattle against tick infestation. An RNA-mediated 

interference does silencing of pixr in mice, PIXR impairs the ability of B. burgdorferi to colonize the tick gut 

[89]. 

Few other vaccine types were also generated by using a synthetic peptide. These were specially 

designed to target the neuropeptides which innervate Ixodes Ricinus salivary glands and hindgut. 

Administration of this vaccine makes a sizable immune defense against nymphs or larvae and of Anaplasma 

phagocytophilum. These myoinhibitory synthetic peptides (MIP) have been used to make multiple antigenic 

peptide constructs (MAPs) and used for immunization.  These generate robust IgG antibody response and 

gave sizable protection against tick borne pathogens [19]. 

6.8. Regulatory proteins 

Subolesins are gene regulatory proteins isolated from ticks; these are responsible for pathogen 

transmission to the hosts during blood-feeding. These proteins remain involved in signal transduction 

pathways. Subolesins are used to generate vaccines which work against diseases vectors, mosquitos, and 

sandflies other than ticks [90]. Effect of Subolesins was tested in cDNA and/or recombinant protein 

immunization experiments; it has provided protection against all tick developmental stages [91]. SUB-based 

vaccines are mainly used to provide cross-vector protection and could be used for the control of important tick 

species by inhibiting the transmission of pathogens [92,93]. In another method ticks essentially need blood 

hemoglobin for embryonic development, if the hemoglobin-depleted serum is, ticks embryogenesis could be 

stopped. More specifically, the acquisition of exogenous heme is essential for tick reproduction [94]. If heme 

and iron metabolism is interrupted it will help to manage anti-tick interventions [94].   

6.9. Cyclin-dependent kinases 

Cyclin-dependent kinases (CDKs) control both cell division and modulate transcription after receiving 

extracellular and intracellular signals in eukaryotes [95]. Cyclin-dependent kinases enzymes phosphorylate 

specific target proteins. This attached phosphate group during phosphorylation functions like a switch, making 

the target protein more or less active. But cyclins are proteins, associated to a Cdk, activate the Cdks.  These 

are thought to be appropriate antigens and are used for the generation of the anti-tick vaccine. Upon 

immunization, these vaccines display inhibition in tick feeding and reproduction. Besides, protein antigen 

vaccines, Pmy DNA (Paramyosin DNA) vaccine generated a sizable immune response and show protection 

against Haemaphysalis longicornis blood feeding. It induces effective cellular and humoral immune responses 

in rabbits and protect against H. longicornis infection [96]. DNA vaccines show lesser side effects than the 

vaccines generated against attenuated pathogens [97]. Similar immune protection was obtained after 

immunization with Toxoplasma gondii 14–3-3/pSAG1 protein. It has generated high levels of IgG antibody 

responses in experimental animals [98]. Besides, the phylogeny of Haemaphysalis doenitzi was decided based 

on mitochondrial 16S rDNA [99]. A similar vaccine generated by using Plasmodium berghei 

circumsporozoite protein provided protection against malarial sporozoite infection as it cut down mosquito 



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bites [100]. Pmy vector series is a batch of expression plasmids that are used for recombinant production of 

single proteins and protein complexes in bacterial cells. These plasmids could be used to recombine the most 

appropriate molecular candidate for the production of an anti-tick vaccine [101]. In addition, pcDNA3.1 (+)-

Pmy plasmid was also constructed that also display immune protection against ticks infections [102]. 

7. TARGETING BOTH TICKS AND TICK-BORNE PATHOGENS 

Host blood proteins are essential for body metabolism and reproduction of ticks; therefore, blood 

feeding is highly important for ticks. However, obstruction of tick feeding and killing of pathogens released 

into the blood of hosts must be prohibited by adopting any method. Tick saliva proteins induce innate and 

acquired immune responses in host animals. As ticks rely on blood, these are established ecto-parasites, and 

no human protective vaccines is available against them. There are protein vaccines prepared from outer 

surface protein, OspA from B. burgdorferi . This was found active against both Ixodes species and its 

pathogen. This Borrelia antigen is approved by FDA in 1998 and a vaccine against it is available [103]. 

Though, these autoantigens are not of broad immunological use [104]. TROSPA and Salp15 interact are 

parasite proteins which were also found suitable for tick-pathogen combined vaccination [75,105]. Many 

antigens have been isolated from Borrelia, and the use of this multiantigen vaccine has much greater efficacy 

protection against tick-borne diseases [71]. 

Production of anti-Salp15 antibodies has shown much broader protective efficacy than OspA and OspC 

antibodies [29]. Hence, both tick and pathogen antigens could be used for making a more perfect vaccine that 

may be found active against many species of ticks [75]. More specifically, the structurally stable antigen will 

provide high efficacy against ticks [10]. In addition, anti-vector vaccines provide better protection cover 

against lethal vector-borne pathogens [106]. However, an anti-tick vaccine derived from a tick cement protein 

(64TRP) of Rhipicephalus appendiculatus. It shows greater protection in experimental mice against tick-

borne encephalitis virus (TBEV) transmitted by infected Ixodes ricinus ticks [106]. The 64TRP vaccine also 

interrupts pathogen transmission but shows a local cutaneous inflammatory immune response at the tick-

feeding site [106]. 

8. BM86-BASED VACCINES 

 Tick population shows polymorphism in Bm86 antigen genes, it is the main reason that existing Bm86 

based vaccines are not so much efficacious. Hence, there is a need to make hybrid recombinant Bm86 

antigens for having more effective vaccines against R. microplus and R. annulatus. BM86 is a midgut protein 

that is used for production of vaccine to immune cattle for tick control [107]. RNA interference of these gut 

proteins target feeding in ticks, and such vaccines provide wiser control against R. microplus infestations 

[108]. Bm86 gene is the most stable and found expressed in all life phases of many tick species. More 

specifically, Bm86 is expressed in lifelong in R. appendiculatus and R. microplus. This expression differs in 

one-host life cycle and in three-host tick R. appendiculatus [109]. Vaccination with Ba86 recombinant B. 

microplus gut antigen significantly cut down spread of tick borne pathogens. Ba86 blocks oviposition and egg 

fertility in two major species of ticks B. annulatus and B. microplus, respectively [109]. There is a need of 

induction of vaccine-mediated enhancement tick control by checking blood feeding from host skin [19]. Two 

more antigens subolesin and TROSPA are used to prepare vaccines which could inhibit feeding in ticks. These 

are specially used to control cattle tick Anaplasma marginale and its transmitted pathogen Babesia bigemina 

[90]. 



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 A recombinant Bm86 was prepared by using Gavac and Mozambique strains (99.6%) [91]. Gavac 

shows 55-100% efficacy against B. microplus infestations in grazing cattle 4-9 months after the first 

vaccination [11]. Bm86-based vaccine provides also shows high efficacy against some tick species having 

lower Bm86 sequence homology [109]. It shows 83.0% homology with Ba86 from B. annulatus [91]. This 

vaccine is used in Cuba, Colombia, Brazil and Mexico for veterinary uses. A similar vaccine prepared by 

using recombinant Ba86 antigen from Israel strain is administered to livestock from B. annulatus infection. 

Besides this, combined antigens are also used to increase the protection cover of tick vaccines [12]. For better 

efficacy of a vaccine, interactions among ticks and their resident pathogens will help to combat tick-borne 

illnesses [110]. EGF antigens show a partial reduction in spirochete loads in the skin [110]. 

 Tick aquaporins (AQPs) are intrinsic membrane proteins which assist in the transport of water, 

glycerol and small solutes such as urea across the cell membrane. By using transcriptomic studies a cDNA 

that codes for aquaporin was characterized in cattle tick, Rhipicephalus microplus [25]. Both genetic and 

DNA sequencing methods were used to identify new genes of tick antigen proteins for immunization. From 

cDNA expression libraries annotations, and comparison with available antigens, novel antigen candidates can 

be identified. This most appropriate antigen/s can be used for the generation of highly effective against tick-

borne pathogens [38]. More specifically, microRNAs (miRNAs) regulate gene expression in ticks. After 

infection rickettsial Anaplasma phagocytophilum infection, starts down-regulation of tick microRNA-133 

(miR-133). It induces organic anion transporting polypeptide (isoatp4056) gene expression in Ixodes 

scapularis. Inhibition of microRNAs activity may stop bacterial survival in the ticks and its transfer to the 

vertebrate host [41]. 

8.1. Bm95 recombinant tick antigen-based vaccines 

Bm95 recombinant antigen is used for immunization of calves, this antibodies generate against this 

antigen showed protective efficacy against Rhipicephalus (Boophilus) microplus ticks. These also cut down 

oviposition and affected egg hatchability in ticks parasitize over immunized calves [111]. The overall 

protective efficacy noted in Bm95 recombinant cattle tick antigen was 81.27% [111]. BM95 is a glycoprotein 

that shows more than 90% homology to Bm86 antigen protein.  For achieving better efficacy the recombinant 

chimeric protein comprising tick BM95 immunogenic peptide is fused to the A. marginale MSP1a N-terminal 

found on the Escherichia coli membrane. This shows significant elevation in protective efficacy against R. 

microplus infestations in rabbits. Chimeric vaccines protection against multiple cattle tick infestations [112]. 

8.2. SUB-based vaccines 

 Subolesins are antigenic proteins which manage blood-feeding in ticks. These were first identified in 

Ixodes scapularis as gene regulatory proteins. These are responsible for the transfer of pathogens from ticks to 

the hosts during blood feeding. SUB-based vaccines after injection generate high levels of immune responses 

in cattle ticks [90] and stop feeding in ticks and inhibit the transfer of infectious agents in dairy farm animals 

[113]. These are used for the preparation of potential vaccines to control Rhipicephalus appendiculatus, R. 

decoloratus and Amblyomma ariegatum ticks species. These are major vectors of farmyard animals which 

impose multiple morbidities in ticks and affect milk production in them [114]. Similarly, the SUB-based 

vaccine control tick infestations and inhibits pathogen infection/transmission in cattle and sheep [113]. SUB 

vaccine antigens physically interact with histone. These are real actors of interaction between tick-host and 

pathogen and regulate all these important activities. In all the ways, subolesin suppress tick blood-feeding and 

reproduction using RNAi treatment. Moreover, importin-α interaction with subolesin reduces tick weight in 



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sub RNAi-treated ticks as it obstructs feeding [115]. H. longicornis Subolesin (HlSu) was identified and 

expressed as a recombinant protein using E. coli [115]. 

8.3. Disruption by RNAi silencing 

Disruption of mRNA activity by RNAi silencing is an important molecular method for control ticks by 

inhibition of feeding on blood meals. Moreover, RNase III- causes degradation of dsRNA into small 

interfering RNAs (siRNAs), which form an RNA-induced silencing complex.  Disruption of RNAi silencing 

is a unique way to control ticks. AAS19 is an immunogenic tick saliva protein that assists in blood meal 

feeding in Amblyomma americanum. This is an inhibitor of a serine protease (serpin, AAS) and trypsin-like 

proteases. It acts as a blood clotting cascade that shows anti-hemostatic functions. More specifically, if its 

mRNA is disrupted by RNAi silencing, blood-feeding could inhibit in experimental animals. In experiments 

rAAS19 was found highly immunogenic and as immunized rabbits show lesser infestation egg-laying. Tick 

engorged females which feed on AAS19 immunized rabbits were fully stopped from egg-laying. It also results 

in the termination of infestation. This is protein shows homology in its functional domain, which is 100% 

conserved across so many tick species [7]. No doubt rAAS19 can be used generation of cocktail tick vaccine 

[116]. Unfortunately, RNAi silencing was not found successful for Is86 genes. It could not influence tick 

engorgement or B. burgdorferi sensu stricte persistence from blood-feeding [110].   

 AP-1 is a transcriptional activator protein that is responsible for the transmission of pathogens by 

infected Ixodes scapularis ticks during feeding. This acts as a molecular switch and regulates expression of 

iafgp gene. RNAi-mediated silencing of ap-1 expression affects the synthesis of saliva proteins that inhibits 

feeding in A. phagocytophilum nymphal ticks [117].  

 Similarly, knockdown of the expression of kat mRNA alone or in combination with isoatp4056 mRNA 

significantly affected A. phagocytophilum survival and isoatp4056 expression in tick cells [118]. A. 

phagocytophilum specifically up-regulates I. scapularis organic anion transporting polypeptide, isoatp4056 

and kynurenine aminotransferase (kat). This kat gene is involved in the production of tryptophan metabolite 

xanthurenic acid (XA), for its survival in ticks [118]. Non-coding RNAs or microRNAs regulate gene 

expression. These miRNAs are transcribed from DNA sequences into primary miRNAs and processed into 

precursor miRNAs, and finally mature miRNAs. If the function of these miRNAs is obstructed, survival of 

various bacterial pathogens in ticks in vertebrate hosts can be finished.  Inhibition of miRNA activity by its 

homologs miR-133 mimic lower down isoatp4056 expression and bacterial burden in ticks Anaplasma 

phagocytophilum [119]. 

8.4. IrSPI as a Kunitz elastase inhibitor 

 Ixodes ricinus secretes serine protease inhibitor (IrSPI) which showed much similar activity to Kunitz 

elastase inhibitor. Its inhibition increases molting and mortality.  It also acts as an immunomodulator and 

obstructs feeding in ticks [120]. After passing into host blood through the salivary gland secretions during 

blood-feeding find their way into the host body. It represses the proliferation of CD4+ T lymphocytes and 

proinflammatory cytokine secretion from both splenocytes and macrophages. It also acts work as an 

immunomodulator in ticks [120]. In addition, ticks midgut synthesizes thousands of proteins and secretes 

them during a blood meal. These modulate various host defense mechanisms [120]. Unfortunately, IrSPI and 

IrLip1 antigen-based vaccines failed to check pathogenicity against I. ricinus nymph transmitted pathogens.   

 Ticks salivary glands infected with A. phagocytophilum show differentiated regulation of apoptosis 

pathways. These were identified in I. scapularis nymphs and adult female midguts. The bacterial infection 



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was found enhanced after A. phagocytophilum infection. Bacterial infection inhibited the intrinsic apoptosis 

pathway in tick salivary glands. This was due to RNA interference-mediated porin knockdown that 

significantly increases tick colonization by A. phagocytophilum. It resulted in the inhibition of Cytochrome c 

release as the anti-apoptotic mechanism to facilitate bacterial infection [121]. These tick-host interactions can 

be used to make anti-tick vaccines targeting this immunomodulator implicated in I. ricinus feeding [122]. 

8.6. Serine protease inhibitors (serpins) 

 Serine protease inhibitors or (serpins) are responsible for tick’s invasion of the host’s serine protease-

mediated defense pathways.  Serpins in general inhibit multiple enzymes and control serine proteases activity, 

these are responsible for inflammation and blood clotting [122]. Serpins also block the action of digestive 

enzymes. Moreover, these also inhibit tick salivary molecules which are responsible for pathogen 

transmission and do tick-mediated skin immunomodulation and affect immune defense in many unique ways. 

These could be used for designing and production of new potential anti-tick vaccines [123]. Similarly, genes 

related to tick parasitism are differentially expressed and have a wide concern with the level and stage of host 

immunity. These could be used for the development of new sustainable molecular and immunological 

methods for tick control [13]. 

8.7. Inhibition of extracellular vesicles mediated pathogen transmission 

 Unfed ixodid tick saliva contains exosomes are small membrane-bound extracellular signaling vesicles 

which are secreted in tick saliva and salivary glands of partially fed or unfed ixodid ticks [124]. These vesicles 

assist in pathogen transmission from arthropods to humans and other animals diseases [80]. These are rich in 

CD63 ortholog protein and heat shock protein 70 (HSP70) [80]. If the formation of extracellular vesicles is 

stopped, lethal pathogens can be finished by using targeted therapeutic molecules. It has been experimentally 

proved as lethal pathogen Francisella tularensis transmitted by Dermacentor andersoni was stopped by using 

this method [125]. It was also found effective against Blacklegged tick, Ixodes scapularis [126]. These also 

block transmission of pathogens of some other tick-borne pathogen diseases like Lyme disease, anaplasmosis,   

babesiosis, Borrelia miyamotoi disease, Powassan virus disease, and ehrlichiosis associated with Ehrlichia 

muris eauclarensis [127].  

9. CONCLUSION 

Blood feeding is an important and highly essential step for the establishment of the tick life cycle. 

Blood meals are the sole source of metabolically required components which are also essential for the 

reproduction and survival of ticks. Once tick feeding is inhibited parasitic life of ticks will finish, it will not 

only control ticks but also tick-borne pathogenesis. Certainly knowledge about molecular, physiological and 

genetic pathways and functioning of antigen and other biomolecules will make real and needful assessments 

about achieving optimal protection after inducing or administration of antigens. However, for searching new 

novel antigens for single and multi-target vaccines genomic and proteomic tools will be more helpful. 

Identification of new protective antigens and their functional transcripts can revolutionize the vaccination and 

prophylaxis for the management of ticks. Further, there is an immense need to find out gaps among host and 

tick immunity, pathogen interaction to host and resistance and immune tolerance. There will require a 

complete discourse on ticks saliva toxins and gut secreted anti-hemostatic molecules. Inhibition of gut protein 

synthesis and their secretion is a highly important step that can solve the problem of tick menace. In this 

work, both proteomic and transcriptomic studies can assist in finding new alternatives related to vaccine 



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European Journal of Biological Research 2022; 12(1): 77-101 

development. These will also provide direction and production of saliva-focused vaccines and vaccine 

strategies. For this purpose, besides the study of parasitology of ticks, a study of genetics, cell biology, 

immunology, and pharmacology will be more helpful to understand ticks and host acquired resistance. There 

is a need for more clarifications on digestion physiology and tick salivary secretions to end tick-borne 

diseases.  More often, a combination of immunological, ecological, and veterinary methods and microbiology 

tools could lead resolve the complexity of tick–host and pathogen interaction and achieve more successful 

vaccine strategies. 

Authors' Contributions: RKU and NY were responsible for conception, literature review, writing and revising the 

manuscript. Both authors read and approved the final manuscript. 

Conflict of Interest: The authors declare no conflict of interest. 

Acknowledgments: Authors are thankful to H.O.D., Department of Zoology for research facilities. 

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