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. Author manuscript; available in PMC: 2022 Dec 20.
Published in final edited form as: Vaccine. 2021 Nov 30;39(52):7661–7668. doi: 10.1016/j.vaccine.2021.11.003

Tick immunity using mRNA, DNA and protein-based Salp14 delivery strategies

Jaqueline Matias 1,#, Cheyne Kurokawa 1,#, Andaleeb Sajid 1,#, Sukanya Narasimhan 1, Gunjan Arora 1, Husrev Diktas 1, Kathleen DePonte 1, Norbert Pardi 2, Jesus G Valenzuela 3, Drew Weissman 2, Erol Fikrig 1,*
PMCID: PMC8671329  NIHMSID: NIHMS1760981  PMID: 34862075

Abstract

Guinea pigs exposed to multiple infestations with Ixodes scapularis ticks develop acquired resistance to ticks, which is also known as tick immunity. The I. scapularis salivary components that contribute to tick immunity are likely multifactorial. An anticoagulant that inhibits factor Xa, named Salp14, is present in tick saliva and is associated with partial tick immunity. A tick bite naturally releases tick saliva proteins into the vertebrate host for several days, which suggests that the mode of antigen delivery may influence the genesis of tick immunity. We therefore utilized Salp14 as a model antigen to examine tick immunity using mRNA lipid nanoparticles (LNPs), plasmid DNA, or recombinant protein platforms. salp14 containing mRNA-LNPs vaccination elicited erythema at the tick bite site after tick challenge that occurred earlier, and that was more pronounced, compared with DNA or protein immunizations. Humoral and cellular responses associated with tick immunity were directed towards a 25 amino acid region of Salp14 at the carboxy terminus of the protein, as determined by antibody responses and skin-testing assays. This study demonstrates that the model of antigen delivery, also known as the vaccine platform, can influence the genesis of tick immunity in guinea pigs. mRNA-LNPs may be useful in helping to elicit erythema at the tick bite site, one of the most important early hallmarks of acquired tick resistance. mRNA-LNPs containing tick genes is a useful platform for the development of vaccines that can potentially prevent selected tick-borne diseases.

Introduction

Ticks are ectoparasites of medical and veterinary importance [1, 2]. They are responsible for transmitting a wide variety of infectious agents, including diverse bacteria, protozoa and viruses [3]. They are naturally found parasitizing numerous vertebrate animals in the completion of their natural life cycle, and sometimes inadvertently feed on humans. In North America and some other parts of the world, ticks remain one of the main vectors of infectious diseases to animals and humans [2]. Ixodes scapularis is the tick species in the United States that is most commonly associated with the transmission of human diseases, including Lyme borreliosis, babesiosis, human granulocytic anaplasmosis, Powassan virus, and other infectious agents [4-6].

From the initiation of the tick bite to the detachment from the host, I. scapularis employ sophisticated strategies to successfully obtain a blood meal. I. scapularis secrete cement-like substances capable of keeping the tick firmly attached to the host [7]. During blood-feeding, the tick also deposits saliva, which is composed of numerous molecules with pharmacological properties, and together act to prevent blood coagulation, inhibit pain, compromise the complement system and modulate host immune responses, among other activities [8-10].

All these mechanisms acting together allow I. scapularis to go unnoticed when it engorges on animals that are important for the completion of its natural life cycle, such as the white-footed mouse, Peromyscus leucopus [11]. However, some animals such as guinea pigs, which are non-natural hosts, after being exposed to successive tick infestations, develop an immune response capable of causing early detachment of the tick. This mechanism of acquired resistance to ticks is also known as tick immunity [12, 13] and is mainly characterized by reducing the number of engorged ticks, and decreasing the weight and viability of eggs [14]. At the site of the tick bite, it is also possible to observe the presence of erythema and the recruitment of inflammatory cells, such as macrophages, basophils, neutrophils and eosinophils, as a measure of host immune response [11]. Erythema is the most important early visible hallmark of natural tick immunity, but does not necessarily result in the later aspects of tick resistance. It should be noted that tick-bite erythema is different from erythema migrans, which is the cutaneous rash that is commonly associated with Lyme disease and usually occurs several days to a week after infection.

Tick immunity was first defined by Trager (1939) [13] and has previously been observed against different species of ticks [15-17]. Narasimhan et al. (2007) observed tick immunity against I. scapularis in guinea pigs repeatedly exposed to I. scapularis and demonstrated that tick immunity could inhibit transmission of tick-borne Borrelia burgdorferi infection to guinea pigs [18, 19]. Analysis of tick immune animals indicates that the adaptive immune response targets salivary proteins [20]. Studies have also shown that animals immunized with salivary gland extract or saliva develop partial tick immunity [20]. These data suggest that a successful tick vaccine can induce tick immunity that can inhibit transmission of some pathogens, particularly those that are not immediately transmitted to the host following a tick bite, such as B. burgdorferi [18] and some other tick-borne pathogens. Previous studies have demonstrated that immunization with a tick recombinant salivary protein 14 (Salp14) can confer partial tick protection [20]. Salp14 has a critical role in maintaining the flow of blood during tick feeding by inhibiting the coagulation factor Xa and thereby preventing the activation of the blood coagulation cascade by the host [21]. These results demonstrate that inhibiting essential salivary proteins can be a potential mechanism to prevent the incidence and/or duration of tick bites and potential to spread disease.

Efforts to discover new protective antigens in order to develop a tick vaccine have been reported over the past few decades. The use of recombinant proteins, whether associated or not with other proteins or adjuvants, has been the platform of choice to test the efficacy of the vaccine candidate. Although in recent years, there is a significant development in the use of genetic (DNA and mRNA) vaccine platforms due to ease of generation and optimization. In this study, we compare DNA, nucleoside-modified mRNA and different protein-based immunization platforms to induce tick immunity. For this comparison, we utilized Salp14 since immunization with this antigen can confer partial protection, thus allowing for the potential to observe an enhancement in protection by different vaccine platforms. The information and protocols identified in this study will allow potential vaccine candidates to be screened using the platform that will maximize protection.

Results

Immunization with plasmid DNA, mRNA, or protein

Guinea pigs exposed to multiple tick infestations develop an adaptive immune response towards tick salivary proteins deposited in the skin during the feeding process. Upon tick challenge in an immune animal, the deposited tick antigens are rapidly recognized, resulting in epicutaneous erythema, severe epidermal hyperplasia, edema and hyperkeratosis at the tick bite site, leading to decreased tick engorgement and rejection [11]. However, the array of protective antigens has yet to be identified. Here, we sought to compare DNA, nucleoside-modified mRNA and protein-based immunization strategies for tick salivary protein 14 (Salp14), an antigen that has been previously demonstrated to be associated with partial tick immunity [20].

For DNA vaccination, the gene encoding salp14 was cloned into the VR2010 plasmid. Guinea pigs were immunized intradermally with 80 μg of plasmid DNA encoding salp14 or empty vector. Immunized guinea pigs received 2 booster vaccinations every 4 weeks. Similarly, nucleoside-modified mRNA lipid nanoparticles encoding (mRNA-LNPs) salp14 or murineIL-21 (muIL-21, control) were delivered intradermally, with two boosts every 4 weeks (20 μg). muIL-21 has no impact on tick feeding and was therefore used as a negative control. For protein immunizations, guinea pigs received recombinant Salp14 (20 μg), followed by 2 boosts (Fig. 1A). Additionally, we performed a slow-delivery immunization [22], in order to more closely mimic a tick bite. For sustained immunization, a total dose of recombinant salp14 (20 μg) was immunized intradermally over the course of one week. Guinea pigs received one booster (single dose) immunization two weeks after the primary immunization.

Figure 1. Salp 14 immunization strategies elicit Salp14 IgG responses in guinea pigs.

Figure 1.

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(A) Guinea pigs were immunized intradermally with salp14 mRNA-LNPs or murine (mu)IL-21 mRNA-LNP (control), salp14 DNA or empty plasmid VR2010 (control), Salp14 protein (bolus and sustained) or Ovalbumin (OVA control). Two weeks after the last immunization, sera were collected to assess the humoral immune response and challenged with 25 nymphal I. scapularis ticks. Illustration generated using Biorender.com. (B) Salp14-specific IgG antibodies were detected by ELISA. The error bars represent mean ± SEM of at least 3 values. Statistical significance was determined by two-way ANOVA; P value = 0.0003.

DNA, mRNA and protein immunization elicit a comparable humoral response

The development of Salp14-specific antibodies was compared among the different immunization strategies. Sera were obtained from each group prior to tick challenge and assessed by ELISA. Salp14 mRNA immunization was the platform that induced the strongest humoral response with Salp14-specific antibodies detected at all dilutions tested (1:500, 1:5000, 1:50,000 and 1:500,000). Salp14 protein immunization resulted in antibody titers detected up to a dilution of 1: 50,000) and salp14 DNA vaccination produced antibody titers evident up to a dilution of (1: 5000). These results indicate that all immunization strategies elicited a humoral response; however, mRNA encoding salp14 generated the most robust response (Fig. 1B).

Erythema at the bite site upon tick challenge

Immunized guinea pigs were challenged with approximately 25 I. scapularis nymphs two weeks after the final boost. Erythema at the bite site is a strong indicator of an immune response to ticks, and one of the initial hallmarks of acquired tick resistance [20, 23]. Guinea pigs were monitored for signs of erythema at the bite site following the tick challenge. Erythema was detected in all immunization groups by 24 h after tick attachment (Fig. 2 and 3A). Guinea pigs immunized with the salp14 mRNA elicited the most robust, and intense erythema at the bite site among all the immunization groups (Fig. 3B and C). Additionally, significant erythema was observed by as early as 18 h in this experimental group, relative to control-immunized guinea pigs and the other platforms (Fig. 2 B and 3A).

Figure 2. Comparison of erythematic response generation:

Figure 2.

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Guinea pigs were challenged with I. scapularis nymphs and monitored for erythema at the bite sites. Graphs show erythema after tick challenge in the animals immunized with (A) salp14 DNA (n=3), empty plasmid VR2010 (control) (n=3); (B) salp14 mRNA (n=3), murine (mu)IL-21 mRNA-LNP (control) (n=3); (C) Salp14 protein bolus (n=3), sustained (n=3) and Ovalbumin (OVA control) (n=3). Immunization with salp14-mRNA elicits robust erythema upon tick challenge. The error bars represent mean ± SEM. Statistical significance was determined by two-way ANOVA; (A-B) P value < 0.0001 and (C) P value = 0.0068.

Figure 3. Rapid erythema induced at the bite site of mRNA-salp14 vaccinated animals.

Figure 3.

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(A) Guinea pigs were monitored following the tick challenge and redness at the bite site was photographed. Representative animals are shown at 16, 24 and 48 hours post-tick challenge. (B-C) Visual inspection was performed to determine the degree of erythema at the site of the tick bite at 24 and 48 hours. Erythema was scored as: No erythema (−), Light (+), Moderate (++), Strong (+++), and the percentage of the degree of erythema was generated from these data.

Tick rejection among vaccinated guinea pigs

After the tick challenge, guinea pigs were monitored daily for evidence of tick rejection, including the rate of tick detachment and engorgement (tick weight) at the bite site. Immunization using a salp14 DNA plasmid resulted in modest tick rejection at day 4 after tick attachment (Fig. 4 A). Immunization with the salp14 mRNA did not impact the rate of tick detachment (Fig. 4 B). Similar to previous reports, immunization with Salp14 protein resulted in modest tick rejection at days 4-5 post-tick attachment for both sustained immunizations, as well as bolus immunization (Fig. 4 C). Also, sustained immunization resulted in an increase in tick detachment at day 4 (Fig. 4 C). Although immunization with mRNA-LNPs did not reject the ticks, immunization resulted in the greatest erythema and dermal inflammation at the bite site, possibly from infiltrating T cells.

Figure 4. Tick feeding kinetics on immunized guinea pigs.

Figure 4.

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Guinea pigs (n=3 per group) were immunized with salp14 DNA (A and D), salp14-mRNA (B and E), and Salp14 protein (bolus and sustained) (C and F) and challenged with I. scapularis. Guinea pigs were monitored for evidence of tick rejection and the tick feeding kinetics were monitored for the duration of the experiment (A-C). The success of tick feeding was determined by examining engorgement weights of the recovered ticks (D-F). Statistical significance was determined (A) by two-tailed student t-test, P value = 0.0013 and (C) Mann Whitney test, P value = 0.0059.

As an additional indicator of tick feeding, we assessed the engorgement weights of the collected ticks. Immunization with the DNA plasmid encoding salp14 did not impair engorgement weights relative to control (2.9 mg versus 2.0 mg) (Fig. 4 D). Similarly, immunization with the salp14 mRNA did not alter engorgement weights (2.2 mg versus 2.1 mg), nor did immunization with recombinant protein (2.75 mg versus 2.1 mg) (Fig. 4 E, F). Sustained immunization also did not significantly alter engorgement weights (1.2 mg versus 1.7 mg).

Antigenic regions of Salp14

After establishing the antigenicity of Salp14, we aimed to characterize the dominant antigenic epitopes within the salp14 sequence. The soluble portion of the Salp14 protein (without signal peptide) was divided into six peptides, each approximately 20 amino acids long, with an overlapping sequence of 5 amino acids (fragments, Fr 1-6, Fig. 5 A).

Figure 5. Identification of the immunogenic domains of Salp14.

Figure 5.

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A) Small peptides (approximately 20-25 amino acids) were synthesized that covered the entire Salp14 protein sequence (without signal peptide). ELISAs were performed to identify the fragment recognized by sera obtained from animals immunized with B) salp14 mRNA-LNPs, C) salp14 DNA, D) Salp14 protein.

Next, we tested which of the six peptides were recognized by antibodies in the sera from guinea pigs immunized with Salp14 protein, salp14 DNA or salp14 mRNA. Interestingly, Fr 2 and Fr 6 were recognized by antibodies in the sera of salp14-mRNA-immunized guinea pigs with the highest titers (Fig. 5 B). In comparison, sera from Salp14 protein-immunized animals and salp14-DNA-immunized guinea pigs showed weaker responses to these peptides, consistent with their lower level of general antibodies against salp14 antibodies (Fig. 5 B, C, D). No other peptides were readily recognized by these sera. This indicates that Fr 2 and Fr 6 are the predominant regions of salp14 recognized by antibodies that recognize linear epitopes using all of the regimens examined. We did not map conformational epitopes bound by antibodies, which could involve other regions of salp14.

To further test the immunoreactivity of salp14 peptides in vivo, we utilized tick immune guinea pigs, which had been repeatedly infested with I. scapularis. Skin testing was performed by injecting 2 μg of peptide intradermally on the shaved backs of guinea pigs, in order to evaluate immune reactivity that is associated with local erythema. Salp14 protein was taken as a positive control and BSA was taken as a negative control. As shown in Figure 6, Salp14 and Fr 6 showed evidence of an inflammatory dermal response, lasting for at least 72 hours. No other peptide or BSA showed any skin response. These results further demonstrate that the C-terminal region of Salp14 is important in the genesis of immunoreactivity associated with early skin immune response to tick bites, a signature for the initiation of acquired tick resistance in guinea pigs.

Figure 6. Skin testing with Salp14 peptides.

Figure 6.

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Skin tests identify the immunoreactive domain of Salp14 in tick immune animals. The synthetic peptides were injected intradermally into the skin of tick immune animals and monitored for erythema. The degree of erythema was scored as: No erythema (−), Light (+), Moderate (++), Strong (+++). The figure represents the skin reaction caused by Salp14 (+), fragment 6 (++) and bovine serum albumin -BSA (−) 48 hours after the injection. All other fragments showed no erythema and are not represented in the figure.

Discussion

Evidence supporting the development of an effective anti-tick vaccine is derived from experiments demonstrating that laboratory animals, such as rabbits and guinea pigs, can acquire resistance to tick feeding following multiple tick infestations, referred to as “tick immunity” [24]. Importantly, guinea pigs with tick immunity to I. scapularis can be protected from infection by tick-borne B. burgdorferi [19]. Guinea pigs exposed to multiple tick infestations develop an adaptive immune response towards tick salivary proteins deposited in the skin during the feeding process. Upon tick challenge in an immune animal, the deposited tick antigens are rapidly recognized, resulting in epicutaneous erythema, epidermal hyperplasia, edema and hyperkeratosis at the bite site, leading to decreased tick engorgement and rejection [11]. Importantly, immunity to I. scapularis can also influence the ability of Amblyoma americanum and Dermacentor andersoni to successfully feed on a host [25].

While antigen discovery is important, vaccine delivery is also critical in eliciting protective immunity. To date, immunization with a single tick salivary proteins has not yet reproduced tick immunity equivalent to naturally acquired tick resistance. Identifying vaccine delivery protocols could be an important factor in how vaccine candidates are screened. The goal of this study is to address limitations in our understanding of vaccine delivery against tick salivary proteins. We used Salp14 to compare different immunization platforms, DNA, mRNA and protein-based (sustained and bolus), in eliciting some of the markers of tick immunity, including erythema, decreased tick attachment and engorgement. Antigens recognized by the immune system are required to be presented as proteins. In the DNA immunizations, the DNA construct containing the gene of interest has to cross the cell and reach the nucleus before it can be transcribed and translated. In the RNA immunization, mRNAs must cross the cell membrane and then become translated to proteins. Expression efficiencies of individual genes, and other factors can alter the amount of protein that is ultimately produced, but the required dosage of these antigens can be defined as DNA>mRNA> protein. Consequently, the dosage was chosen based on our previous studies with optimization for guinea pigs and published information about the different platforms [19, 20, 26-28]. No adjuvant was used with protein immunizations, as a follow-up from our previous studies, where the addition of incomplete Freund’s adjuvant showed no effect in the overall immune response to saliva-based immunizations [20].

This study demonstrates that guinea pigs immunized with salp14 nucleoside-modified mRNA-LNP developed substantial erythema at the tick bite site within the first 18 hours. Guinea pigs immunized with salp14 DNA or Salp14 protein only started to develop erythema after 24 h. Since Salp14 has been shown to provide only partial immunity to ticks, it was an ideal candidate for our study, for comparison of different platforms and to allow a window for improvement in generating an immune response. In view of this, the erythema after salp14 mRNA-LNP vaccination was more pronounced that after Salp14 DNA or protein immunization. The capacity for the different delivery strategies to elicit different degrees of redness following tick challenge may be associated with the degree and type of humoral and cellular immune response generated following vaccination. Eliciting erythema is an important component for an effective tick vaccine, and a vaccine containing numerous salivary tick antigens affords another way to alter later aspects of tick feeding, including attachment and engorgement [29]. Nevertheless, erythema is one of the parameters associated with tick immunity, with the potential to generate itching and pain [30], and it is possible that early recognition and self removal of a tick bite may be sufficient to prevent the transmission of some pathogens. In previous studies, histological examination of the tick bite-sites on tick-immune animals reveals that erythema is the result of degranulation of immune cells such as eosinophils, mast cells and basophils migrating to the bite site [11].

While identifying the antigenic region of Salp14, we designed six overlapping peptides within the cytosolic region of Salp14. The peptides were tested with guinea pigs immunized with salp14 mRNA, salp14 DNA or recombinant protein, to be recognized by the immunogenic region. All three groups of sera elicited antibody responses primarily by fragments 2 and 6, with 6 comprising part of C-terminus of Salp14. Furthermore, skin testing of the various Salp14 epitopes demonstrated that only fragment 6 elicited erythema, when injected into the dermis of guinea pigs that were naturally made tick immune by repeated tick infestation. Collectively, these studies suggest that the C-terminal region of Salp14 is associated with responses, likely humoral and/or cellular, linked to tick immunity. The C-terminal of Salp14 is highly basic, which is important for the anticoagulant properties of Salp14 and the inhibition of the lectin complement pathway, as Salp14 has similarity to the TSLPI protein [21, 31]. In our previous studies, adding an adjuvant to tick saliva did not markedly alter the elicited immune response [20], but a direct comparison using diverse adjuvant would provide additional data that may be useful for future human studies.

Although evidence for tick immunity in humans has been difficult to study in large controlled studies, there are reported cases of individuals developing hypersensitivity reactions at the tick bite site, similar to the observation in tick immune animals [32]. Additionally, individuals with frequent exposures to tick bites have been shown to develop antibodies to tick proteins, confirming observations in laboratory tick immunity models [33, 34]. Importantly, individuals that report itching at the tick bite site have a decreased probability of acquiring B. burgdorferi [30]. These results suggest that previous exposure to ticks can induce protective immunity, resulting in erythema at the bite site upon future bites. This can be a promising strategy to prevent transmission of B. burgdorferi since transmission does not occur during the first 24 hours of tick attachment [35-37]. Indeed, it is most likely that if a human noticed a tick bite due to increased redness and/or itching, the immediate response would be to remove the tick, and this would likely result in protection from infection with the Lyme disease agent. In contrast, later phenomeno that are associated with tick immunity, such as decreased attachment and altered engorgement may not be as critical as early recognition and removal of the tick for the prevention of human Lyme disease. Certainly, erythema would only be evident on areas of skin that are observable by an individual, but this comprises a substantial portion of the body. In addition, if pruritis occurred in unobserved area of the skin, this would still result in identification of the tick, and early removal.

Overall, the results of this study compare immunization strategies that are best able to elicit the primary components of tick immunity – early erythema at the tick bite site and generation of robust humoral responses. Nucleoside-modified mRNA-LNP salp14 immunization elicits an earlier and greater degree of redness and higher antibody titers, than either DNA or protein immunization, implying the use of Salp14 as a potential vaccine candidate, either alone with optimizations or in combination with other candidate antigens [29]. Thus, this study elaborates that the method of immunization, as well as the selection of antigens, must both be considered, as anti-tick vaccines continue to be developed to help aid humans in the prevention of Lyme disease, and possibly other tick-borne infections.

Methods

Ethics statement

This study was conducted according to the instructions in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, USA. All protocols have been approved by Yale University Institutional Animal Care and Use Committee (YUIACUC - protocol number 2020-07941).

Ticks and experimental animals

I. scapn laris nymphs were obtained from Oklahoma State University (Stillwater, OK, USA). Ticks were kept in the incubator at 23°C and 85% relative humidity under a 14 h light, 10 h dark photoperiod at the Infectious Diseases Laboratory, Department of Internal Medicine, Yale University School of Medicine. 4-5-weeks old female Hartley guinea pigs (Charles River laboratory, MA) were used for immunizations and tick challenge experiments.

Formulation of Salp14 mRNA-LNP, plasmid DNA and recombinant protein

mRNA-LNP-

Salp 14 (accession number AF209921) and muIL-21 mRNA-LNPs were generated as previously described[38]. Briefly, mRNAs were transcribed to contain 101 nucleotide-long poly(A) tails. N-1-methylpseudouridine (m1Ψ-5’)-triphosphate (TriLink) instead of UTP was used to generate modified nucleoside-containing mRNA. Capping of the in vitro transcribed mRNAs was performed co-transcriptionally using the trinucleotide cap1 analog, CleanCap (TriLink). mRNA was purified by cellulose purification, as described [39]. All mRNAs were analyzed by agarose gel electrophoresis and were stored frozen at −20°C. The mRNA was then encapsulated in LNPs using an aqueous solution of mRNA at acidic pH 4.0 and mixed with a solution of lipids [40, 41], consisting of an ionizable cationic lipid/phosphatidylcholine/cholesterol/ PEG-lipid (proprietary of Acuitas, Vancouver, Canada) (50:10:38.5:1.5 mol/mol). For encapsulation, RNA was mixed with the lipids at a ratio of ~0.05 (wt/wt). The LNP had a diameter of ~80 nm as measured by dynamic light scattering using a Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, UK) instrument, and stored at −80°C.

DNA-

salp14 gene was PCR amplified using I. scapularis salivary gland cDNA (described below) and cloned into the VR2010 vector derived from VR1020 plasmid (Vical, Inc.) [42]. The cloning was performed under the restriction sites BamHI and BglII using Gibson assembly cloning kit (New England Biolabs) and the sequence of clone was validated by DNA sequencing (Keck sequencing facility, Yale University). For immunization, plasmids were purified using Endo-free plasmid purification kit (Qiagen), as previously described [42].

Recombinant protein-

Total RNA was isolated from the salivary glands of fed I. scapularis ticks using Trizol (Life Technologies, Inc) and cDNA was synthesized according to the manufacture’s protocol (iScript cDNA synthesis kit, Bio-RAD). Gene-specific primers were used to amplify the salp14 and the amplicon was cloned into pMT-Bip-V5-HisA vector. To validate the clones, the recombinant DNA was sequenced at the Keck sequencing facility, Yale University. Recombinant Salp 14 protein was generated using the Drosophila expression system according to the manufacturer’s protocol (Invitrogen, CA) and as already described for tick salivary proteins [20, 43, 44]. The purity of the recombinant protein was assessed by SDS-PAGE using 4–20% gradient precast gels (Biorad, CA) and quantified using the BCA protein assay kit (Thermo Scientific, MA).

Guinea pig immunization

Female guinea pigs (4-5-weeks old) were immunized intradermally with 20 μg of Salp14 mRNA-LNPs, or human (hu)IL-21 mRNA-LNP (control), 80 μg of plasmid DNA encoding Salp14 or empty plasmid constructions VR2010, 20 μg of recombinant Salp14 or Ovalbumin (OVA control) and sustained immunization with 20 μg of recombinant Salp14 over the course of one week.

The animals were boosted twice at 4-week intervals. Two weeks after the last boost, the animals were bled retro-orbitally to obtain 500μl of blood and the serum separated for use in ELISA experiments. At least 3 animals were used in each group.

Tick challenge

Prior to the tick challenge, the backs of guinea pigs were carefully shaved. Guinea pigs were anesthetized with a mixture of 40 mg/kg ketamine/xylazine and 25-30 I. sccipttlaris nymphs were applied to the shaved backs. Guinea pigs were housed individually in cages with wire racks above water to recover engorged and/or rejected ticks. Animals were monitored multiple times throughout the day for evidence of tick rejection.

Visual inspection was performed to determine the degree of erythema at the site of the tick bite at 24 and 48 hours. Erythema was scored as: No erythema (−), Light (+), Moderate (++) and Strong (+++). The percentage of the degree of erythema was generated from these data and is represented in figures 3B and C. All the experiments were performed in a blinded manner for scoring the erythema.

Generation of Salp14 peptides and skin testing

The sequence of Salp14 protein (104 aa without signal peptide) was divided into 6 overlapping peptides, each ~20 aa long, as follows: HNCQNGTRPASEQDREGCDYY (Fr1), GCDYYCWNAETKSWDQFFFG (Fr2), QFFFGNGEKCFYNSGDHGTC (Fr3), DHGTCQNGECHLTNNSGGPNETDD (Fr4), PNETDDYTPAPTEKPKQKKK (Fr5) and KQKKKKTKKTKKPKRKSKKDQEKNL (Fr6). The peptides were synthesized by GenScript (Piscataway, NJ).

Guinea pigs were made tick-immune by being fed upon with I. scapularis ticks for four days, twice, at an interval of two weeks, as described previously [23]. To study the host response against each peptide fragment, skin testing [45] was performed by intradermal injection of 2 μg of peptides, recombinant Salp14 protein (positive control) or bovine serum albumin- BSA (negative control), on the shaved backs of tick-immune guinea pigs. Two guinea pigs were utilized for skin testing and all the injected sites were on each guinea pig for accurate comparison of redness as a measure of the host response. The animals were monitored for the generation of skin redness at the site of injection for 96 hours.

ELISA assessment

Sera were obtained from the blood of guinea pigs collected by retro-orbital bleeds. To determine antigen-specific antibody responses, ELISAs were performed as previously describe [20]. Briefly, 96-well plates were coated overnight with the indicated proteins and peptides (250 ng) diluted in carbonate-bicarbonate buffer, pH 9.6. The wells were then washed with PBST (PBS containing 0.05% Tween-20) and blocked using PBS supplemented with 3% BSA. Sera were serially diluted and incubated for 2 hours at 37 °C. The wells were then washed and incubated with the secondary goat anti-guinea pig IgG-HRP antibody (ThermoFisher, Waltham, A, USA), After washing, TMB HRP substrate solution was added and incubated for 30 min, followed by addition of TMB stop solution. The plates were read at 450 nm.

Statistical analysis

Statistical analysis was performed using Prism 8.0 software (GraphPad Software, CA). The numbers of animals used in each experiment are indicated in the figure legend. Statistical significance was determined using two-way ANOVA, two-tailed student t-test and Mann Whitney test.

Acknowledgments

This work was supported in part by the NIH grant AI138949PO1 and the Steven and Alexandra Cohen Foundation. This research was supported by the Intramural Research Program of the NIH, National Institute of Allergy and Infectious Diseases (JGV). CK was funded by NIH Immuno-Hematopathology Research Training Grant (T32HL007974).

Footnotes

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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