Ixodes scapularis saliva components that elicit responses associated with acquired tick-resistance

Journal Pre-proof Ixodes scapularis saliva components that elicit responses associated with acquired tick-resistance Sukanya Narasimhan, Cheyne Kurokawa, Husrev Diktas, Norma ˇ Olivares Strank, Jiˇr´ı Cern´ y, Kristen Murfin, Yongguo Cao, Geoff Lynn, Jos Trentleman, Ming-Jie Wu, Kathy DePonte, Fred Kantor, Juan Anguita, Joppe Hovius, Erol Fikrig

PII:

S1877-959X(19)30406-6

DOI:

https://doi.org/10.1016/j.ttbdis.2019.101369

Reference:

TTBDIS 101369

To appear in:

Ticks and Tick-borne Diseases

Received Date:

21 September 2019

Revised Date:

20 December 2019

Accepted Date:

23 December 2019

ˇ Please cite this article as: Narasimhan S, Kurokawa C, Diktas H, Olivares Strank N, Cern´ y J, Murfin K, Cao Y, Lynn G, Trentleman J, Wu M-Jie, DePonte K, Kantor F, Anguita J, Hovius J, Fikrig E, Ixodes scapularis saliva components that elicit responses associated with acquired tick-resistance, Ticks and Tick-borne Diseases (2019), doi: https://doi.org/10.1016/j.ttbdis.2019.101369

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Ixodes scapularis saliva components that elicit responses associated with acquired tickresistance

Sukanya Narasimhan 1,*, Cheyne Kurokawa 1, Husrev Diktas 1, Norma Olivares Strank 1, Jiří Černý 1

, Kristen Murfin 1, Yongguo Cao

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, Geoff Lynn 1, Jos Trentleman 3, Ming-Jie Wu 1, Kathy

Section of Infectious Diseases, Department of Internal Medicine, Yale University School of

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DePonte 1, Fred Kantor 4, Juan Anguita 5,6, Joppe Hovius 3, Erol Fikrig 1,7,*

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Medicine, New Haven, CT 06420.

Key Laboratory for Zoonosis Research, Ministry of Education, College of Veterinary Medicine,

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Jilin University, Changchun, 130062, People's Republic of China Amsterdam UMC, University of Amsterdam, Center for Experimental and Molecular Medicine,

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Amsterdam Infection and Immunity, Amsterdam, Netherlands. Section of Allergy and Clinical Immunology, Department of Internal Medicine, New Haven, CT,

06520, USA.

Center for Cooperative Research in Biosciences (CIC bioGUNE), Derio, Spain.

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Ikerbasque, Basque Foundation for Science, Bilbao, Spain.

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Howard Hughes Medical Institute. Chevy Chase, MD-20815.

* To whom correspondence should be addressed: Sukanya Narasimhan Ph.D.(Email: [email protected]) or Erol Fikrig, M.D (Email: [email protected])

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ABSTRACT

Ticks and tick-borne diseases are on the rise world-wide and vaccines to prevent transmission of tick-borne diseases is an urgent public health need. Tick transmission of pathogens to the mammalian host occurs during tick feeding. Therefore, it is reasoned that vaccine targeting of tick proteins essential for feeding would thwart tick feeding and consequently prevent pathogen

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transmission. The phenomenon of acquired tick-immunity, wherein, repeated tick infestations of

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non-natural hosts results in the development of host immune responses detrimental to the tick feeding has served as a robust paradigm in the pursuit of tick salivary antigens that may be vaccine

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targeted. While several salivary antigens have been identified, immunity elicited against these

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antigens have only provided modest tick rejection. This has raised the possibility that acquired tick-immunity is directed against tick components other than tick salivary antigens. Using Ixodes

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scapularis, the blacklegged tick, that vectors several human pathogens, we demonstrate that immunity directed against tick salivary glycoproteins is indeed sufficient to recapitulate the These observations emphasize the utility of tick salivary

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phenomenon of tick-resistance.

glycoproteins as viable vaccine targets to thwart tick feeding and direct our search for anti-tick vaccine candidates.

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Keywords: Ixodes scapularis, acquired tick-immunity, salivary proteins, salivary glycoproteins

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Introduction

Ixodes scapularis, the blacklegged tick, vectors many human pathogens in North America including Borrelia burgdorferi sensu stricto, the agent of Lyme disease (Barbour and Fish, 1993). Incidences of diseases transmitted by Ixodes ticks are on the rise and vaccines to control the vast majority of the tick-borne human diseases are currently not available (Paules et al. , 2018). While

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vaccines targeting specific pathogens is the traditional approach (Plotkin and Plotkin, 2011), it also The growing

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requires the development of multiple tick-borne pathogen-specific vaccines.

molecular understanding of the critical role of tick saliva in facilitating tick feeding (Hovius et al.

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, 2008, Kotal et al. , 2015, Ribeiro and Francischetti, 2003) and in assisting pathogen transmission

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(Nuttall and Labuda, 2004, Simo et al. , 2017) has directed a paradigm shift in our approach to vaccines against tick-transmitted diseases (Embers and Narasimhan, 2013). Hard ticks such as I.

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scapularis, remain attached to the host for several days in order to feed to repletion, a process essential for the tick to complete its life cycle (Anderson and Magnarelli, 2008).

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pharmacologically active salivary components introduced into the feeding lesion modulate diverse host responses and are central for the tick to engorge successfully (Kazimirova and Stibraniova, 2013, Ribeiro et al. , 2006, Wikel, 2013). Microbes transmitted by an infected tick are deposited into the bite-site along with tick saliva (Wikel, 2013). These microorganisms sometimes exploit

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components of tick saliva to enhance entry and survival in the host (Ramamoorthi et al. , 2005, Schuijt et al. , 2011a). Therefore, salivary proteins that facilitate tick feeding and pathogen transmission have emerged as attractive vaccine targets (Murfin and Fikrig, 2017) and offer a parsimonious approach to control multiple tick-transmitted diseases.

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Over the last two decades, mining of the tick sialome by biochemical and by in-silico methods has revealed that the tick salivary proteome is complex (Francischetti et al. , 2009, Kim et al. , 2016, Narasimhan et al. , 2007a, Perner et al. , 2018) and is composed of a dynamic array of proteins whose expressions are modulated by physiological changes that occur in the tick during feeding, and by concomitant host immune responses. Hence, identifying critical tick salivary proteins that can serve as vaccine targets has posed a challenge (Mulenga et al. , 2000b). Seminal

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observations made by William Trager 80 years ago showed that guinea pigs or rabbits repeatedly

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infested with Dermacentor variabilis, the dog tick, develop robust acquired immunity to D. variabilis resulting in rapid tick rejection (Trager, 1939). Interestingly, repeated infestations of

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mice, which are natural hosts of I. scapularis, does not provoke tick-resistance by mechanisms

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that remain to be deciphered (Anderson et al. , 2017, Narasimhan et al. , 2019). The phenomenon of acquired tick resistance has since been observed on other tick-non-natural host models (Brown

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et al. , 1984, Wada et al. , 2010) including in the I. scapularis-guinea pig model (Nazario et al. , 1998). More importantly, acquired resistance to nymphal I. scapularis also resulted in decreased

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transmission of B. burgdorferi to the host (Narasimhan et al., 2007a, Nazario et al., 1998). Acquired tick-resistance is characterized by recruitment of inflammatory cells, predominantly basophils, to the tick bite-site followed by degranulation of basophils that is suggested to be detrimental to tick feeding (Askenase, 1977, Brown and Askenase, 1981, Tabakawa et al. , 2018).

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While a molecular and mechanistic understanding of the phenomenon of acquired tick-resistance is not available, initial studies invoke both cellular and humoral immune responses directed, presumably, at salivary antigens critical for tick feeding (Brossard and Wikel, 2004, Brown and Askenase, 1983). These insights have bolstered the assumption that critical salivary antigens may be targeted to block tick feeding and pathogen transmission, and has facilitated the identification

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of immuno-reactive salivary antigens (Das et al. , 2001, Lewis et al. , 2015, Radulovic et al. , 2014, Schuijt et al. , 2011b). However, immunity to these antigens or antigen subsets has only modestly recapitulated the tick-resistance phenotype. This raises the possibility that the robust tickresistance phenotype observed upon repeated tick infestations is likely driven by multiple factors other than, or in addition to, tick salivary components. In this study we provide experimental proof that tick saliva is indeed sufficient to elicit a robust tick-resistance phenotype. We also assess the

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relative contributions of protein and non-protein components of tick saliva in eliciting the tick-

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resistance phenotype and provide a new thrust to tick salivary antigen-based tick vaccine efforts.

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Materials and Methods

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Ethics statement

Animal care and housing followed the rules described in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, USA. The protocols described below

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for the use of mice and guinea pigs were reviewed and approved by the Yale University Institutional Animal Care and Use Committee (YUIACUC) and the approved Protocol number is 2018-07941. All animal experiments were conducted in a Biosafety Level 2 animal facility

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according to YUIACUC rules. All data generated in this work will be readily shared and available upon request.

Ticks and animals

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I. scapularis adults, nymphs and larvae were obtained from a tick colony at the Oklahoma State University, Stillwater, OK, and maintained in an incubator at 23°C and 85% relative humidity under a 14-hour light, 10-hour dark photoperiod. 4-5-weeks old female Hartley guinea pigs (Charles River, MA) were used to feed nymphal ticks. Female New Zealand white rabbits (Charles River, MA) were used to feed female adult ticks essentially as described earlier (Schuijt et al., 2011b). Adult tick saliva was collected from engorged adult female ticks using Pilocarpine as

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described by Patton et al. (Patton et al. , 2012). Approximately 10 l saliva/adult tick was obtained

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Immunization of guinea pigs against tick saliva

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and saliva from 40-50 fed adults was pooled, aliquoted and stored at -80 C prior to use.

4-5-weeks old female Hartley guinea pigs (Charles River, MA) were immunized l (~ 4

g of total protein as judged by BCA protein assay kit,

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subcutaneously with 20

(ThermoFisher Scientific)) of tick saliva in the absence of added adjuvant. The animals were l of tick saliva in the absence of adjuvant. Control

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boosted twice at 3-week intervals with 20 animals were immunized with 4

g of Ovalbumin (Ova) and boosted twice at 3-week intervals in

the absence of adjuvant. The animals were bled retro-orbitally 2 weeks after the last boost to obtain 500

l of blood and the serum separated for use in ELISA experiments to assess saliva-

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specific antibody titers. At least 2 animals were used in each group and experiments repeated three times. Immunizations with saliva or Ova was also performed in incomplete Freund’s adjuvant following the same immunization regimen as described above to determine if the oil-water emulsion-based delivery of saliva would enhance immunity elicited by saliva.

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To assess the dose-dependent impact of saliva immunization on tick-resistance, guinea pigs were immunized subcutaneously with 1

l (~200 ng), or 0.1 l (~ 20 ng) 0r 0.01

l (~ 2 ng) of

tick saliva without added adjuvant following the same regimen as described above using at least two animals /group.

l of tick saliva was incubated with a cocktail of

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To remove glycosylations, 20

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Glycosidase, protease, phosphorylase or lipase treatment of tick saliva

glycosidases that removes both N and O-glycosylations and provided in the EDGLY deglycosidase

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kit (Sigma, MO). Deglycosylation reaction was conducted under denaturing conditions as

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recommended by the manufacturer. For protease treatment, saliva was digested with 2.5 Units of proteinase K (Sigma-Alrich) in CutSmart buffer (NEB) and 1% SDS for 1 hour at 37°C. For l calf intestine alkaline

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dephosphorylation saliva was incubated in 1x CutSmart buffer, 5

phosphatase and PBS at 37°C for one hour. For lipase treatment saliva was mixed with 5 l porcine l of PBS and incubated at 37°C for one hour. Enzymatically-treated

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lipase A (1mg / ml) and 265

saliva was frozen at -80 C overnight, and thawed prior to immunization of guinea pigs using immunization regimens described above in the absence of adjuvant. Control animals were

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similarly immunized with untreated saliva in respective buffers.

Generation of recombinant salivary proteins (Salps)

RNA was isolated from salivary glands dissected from I. scapularis ticks fed to repletion and cDNA synthesized according to the manufacture’s protocol (iScript cDNA synthesis kit, Bio-

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RAD). Gene specific primers was used to amplify the mRNA region encoding the mature protein of each Salp listed in Suppl Tables 1 and 2. Purified amplicons were then cloned into pMT-BipV5-HisA vector and recombinant DNA sequenced at the Keck sequencing facility, Yale University, to validate the clones. Recombinant proteins of each Salp was generated using the Drosophila expression system as described earlier for SalpC1, and SalpC24 (Schuijt et al., 2011b), Salp12 (Murfin et al. , 2019), Salp15 (Anguita et al. , 2002), TSLPI (Schuijt et al., 2011a), and

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TIX (Schuijt et al. , 2013) and according to the manufacturer’s protocol (Invitrogen, CA). To

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generate recombinant Salp14 and TSLPI in the mammalian expression system (henceforth referred to as Salp14-m and TSLPI-m), the respective amplicons encoding the mature proteins were

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subcloned into the pEZT-DLUX vector (Addgene,MA) and recombinant DNA sequenced at the

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Keck sequencing facility, Yale University, to validate the clones . Expression and protein purification of Salp14-m and TSLPI-m were performed using the Expi293 expression system

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(Thermo Scientific, MA). Protein purity was assessed by SDS-PAGE using 4-20% gradient precast gels (Biorad, CA) and quantified using the BCA protein estimation kit (Thermo Scientific, MA).

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Recombinant Salp25D was generated using the bacterial Maltose Binding Protein (MBP) expression system (New England Biolabs, MA) as described earlier (Narasimhan et al. , 2007b). Recombinant PDIA3 was generated as a GST-fusion (glutathione -S-transferase) protein in E. coli using the pGEX-4T2 vector and recombinant protein purified according to the manufacturor’s

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protocol (GE Healthcare Life Sciences, PA).

Immunization of guinea pigs against recombinant Salivary proteins (Salps)

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4-5-week-old female Hartley guinea pigs (Charles River, MA) were immunized subcutaneously with two individual cocktails of recombinant Salp proteins (listed in Suppl Tables 1 and 2, Cocktail 1 and Cocktail 2) in IFA. The animals were boosted twice at 3-week intervals. Control animals were immunized with Ovalbumin (Ova) and boosted twice at 3-week intervals in the absence of adjuvant. The animals were bled retro-orbitally 2 weeks after the last boost to obtain 500

l of blood and serum separated for use in ELISA experiments to assess rSalp-specific

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antibody titers.

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ELISA assessment of saliva-specific or recombinant Salp-specific IgG levels

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To assess saliva-specific humoral response 96-well ELISA plates were coated overnight with 500 ng of saliva prepared as described above and incubated with guinea pig anti-saliva sera

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collected 2-weeks post last immunization and prior to tick challenge at 1:500 or 1:5000 dilution. Bound antibody was detected with HRP-conjugated goat anti-guinea pig IgG and TMB substrate

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solution (ThermoFisher Scientific, IL). Guinea pig anti-Ova sera collected 2-weeks post last immunization and prior to tick challenge served as control sera. Salp-specific humoral response was similarly assessed using 500 ng of each of the recombinant Salps (Supplemental Tables 1 and 2) to coat the 96-well ELISA plates and seroreactivity to guinea pig anti-saliva sera or guinea pig

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anti recombinant Salp cocktail sera.

Uninfected tick challenge of guinea pigs

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Immunized or naïve guinea pigs were anesthetized by intramuscular injection of ketamine and xylazine mixture and then challenged with 30 nymphal I. scapularis ticks by placing ticks on their shaved backs. Ticks were allowed to attach prior to housing guinea pigs individually in wirebottom cages with 3 layers of tick containment involving a pan of water below the wire-bottom, a hopper-inclusive lid, and Vaseline grease around the outer edges of the cage. Guinea pigs were monitored daily to monitor the numbers of tick feeding, erythema in skin and to collect any fallen

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ticks from the water pan and the numbers of ticks obtained was used to calculate percent recovery.

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Erythema at the tick bite-sites were assessed by two researchers blinded to the experimental groups and scored based on percentage of erythematous tick bite-sites as follows: redness at < 10 % of

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tick bite sites : 0; redness at 10-20 % of tick bite-sites: 0.5; redness at 20-50 % of tick bite sites: 1;

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redness at 50-80 % of tick bite sites: 2; redness at >80% of tick bite-sites: 3. Repleted ticks were individually weighed using a Sartorius balance to measure engorgement weights as a measure of

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Statistical analysis

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feeding success.

In scoring for seroreactivity to saliva or specific salivary antigens, erythema, and rate of tick detachment, the significance of the difference between the mean values of control and

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experimental groups was analysed by 2-way ANOVA and Tukey’s multiple comparison using with Prism 7.0 software (GraphPad Software, CA).

p ≤ 0.05 was considered statistically

significant. To assess if percent recovery of ticks and engorgement weights were significantly different between control and experimental groups ordinary ANOVA or two-way ANOVA with

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Tukey’s or Holms-Sidak’s multiple comparison or Mann-Whitney test was done using Prism 7.0 software. p ≤ 0.05 was considered statistically significant.

Results

l (~4

g of total protein) of adult tick saliva in the

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Guinea pigs were immunized with 20

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Immunization of guinea pigs against tick saliva provokes robust tick-resistance

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absence of any adjuvant and control animals were immunized in parallel with Ovalbumin (Ova) as described in the Materials and Methods. After the last boost, blood was drawn from each animal

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and serum levels of antibody specific to tick saliva was confirmed by ELISA prior to challenge of

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each animal with ~ 30 I. scapularis nymphs (Fig. 1A). Within 24 h of tick attachment, we observed the hallmark redness at the tick bite sites (Fig. 1B) that significantly increased in intensity by 48 h as judged by visible erythema at all tick bite-sites (Fig. 1C) and was comparable to that seen on

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tick-resistant guinea pigs. Little or no redness was observed in control animals (Fig. 1B-C). Ticks also detached significantly more rapidly on saliva-immunized animals (Fig. 1D) when compared to that on Ova-immunized animals. Although, tick rejection on tick-resistant guinea pigs was

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significantly more rapid than that on saliva-immunized animals (Fig. 1D), the recovery of engorged ticks from saliva-immunized animals was comparable to that on tick-resistant animals and was significantly less than that obtained from control animals (Fig. 1E). The engorgement weights of the small number of ticks that fed to repletion on saliva-immunized animals was decreased compared to that on Ova-immunized animals (Fig. 1F).

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To determine the minimum concentration of saliva that would provide tick resistance, we immunized guinea pigs with decreasing amounts of saliva as described in the Materials and Methods. We observed that immunization of guinea pigs with as low as 1 (~ 200 ng of total protein) and 0. 1 and 0.01

L (~20 ng of total protein) of saliva elicited visible redness at tick bite-sites,

L did not provide any redness at the bite-site (Suppl Fig. 1B-C). Although, tick

recovery was comparable to that observed in control animals (Suppl Fig. 1C), engorgement L saliva-immunized compared to

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weights of ticks were significantly reduced on 1, 0.1 and 0.01

L saliva (Suppl Fig. 1).

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animals immunized with 1

l (~ 2 ng of total protein) of tick saliva when compared to that on

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were immunized with 0.01

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ticks that fed control animals. Tick resistance was however significantly reduced when animals

While elicitation of tick-resistance phenotype was achieved without any adjuvant (Fig. 1), we

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examined if immunization in presence of Incomplete Freund’s (IFA) would enhance the phenotype. Although not a classic adjuvant, by virtue of the oil-water emulsion to form an antigen

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depot at the injection site and enhance the immune responses, we reasoned that IFA could boost immune responses to saliva. Animals were immunized with 10

l (~2

g of total protein) in

presence of IFA and boosted twice as described in the Materials and Methods. After the last boost, antibodies specific to tick saliva on the serum was assessed by ELISA (Fig. 2A) and shown to be

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comparable to that observed in animals immunized with saliva alone (Fig. 2A). Further, upon tick challenge of animals immunized with saliva and IFA we observed the hallmark redness at tick bite-sites, tick rejection and tick recovery that was comparable to that observed in animals immunized with saliva alone (Fig. 2B-E). The engorgement weights of ticks that repleted on the

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immunized animals were also comparably decreased when compared to that on control animals immunized with Ova and IFA (Fig. 2F).

Salivary proteins and glycosylations are critical for eliciting tick-resistance

In an effort to determine the components of saliva that play a critical role in eliciting tick-

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l of saliva (corresponding to ~3-

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glycosylations, phosphorylations and lipidations. About 15 -20

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resistance we focused on the salivary proteins, and their post-translational modifications including

g of total protein) was treated with protease to enzymatically digest proteins in saliva, with a

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cocktail of glycosidases to enzymatically deglycosylate salivary proteins, with lipases to remove

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lipid moieties, or with phosphatase to remove phosphorylations as described in the Materials and Methods. Treated or untreated saliva was used to immunize guinea pigs and 10 days after the final

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boost challenged with ticks as described in the Materials and Methods and the development of tick-resistance monitored. ELISA assessment of IgG antibodies specific to saliva showed that

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glycosidase or protease treatment significantly diminished the reactivity to saliva (Fig. 3A). Protease treatment significantly decreased the development of erythema at the tick bite-sites (Fig. 3B-C) and abolished the development of tick resistance as seen by tick detachment rate (Fig. 3D), percent recovery of ticks (Fig. 3E) and tick engorgement weights that were comparable to that on

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control animals (Fig. 3F). Although, tick bite-sites on glycosidase treated saliva-immunized animals showed the hallmark redness that was significantly greater than that on untreated salivaimmunized animals (Fig. 3C), deglycosylation significantly diminished the development of tick resistance as seen by a slower tick detachment rate and higher percent recovery of ticks compared to untreated saliva-immunized animals (Fig. 3D-E). Engorgement weights of ticks that repleted

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on untreated saliva were significantly decreased compared to ticks that repleted on protease- or glycosidase-treated saliva-immunized animals (Fig. 3F).

ELISA assessment of IgG antibodies specific to saliva showed that lipase treatment, but not phosphatase treatment, significantly diminished the reactivity to saliva (Suppl Fig. 2A). Phosphatase-treated saliva-immunized animals showed all the parameters of tick-resistance

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including redness at the bite-sites (Suppl Fig. 2B-C), rapid tick detachment and decreased tick

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recovery and decreased engorgement weights (Suppl Fig. 2D-F) that was comparable to that on untreated saliva-immunized animals. Lipase treatment prevented the development of erythema at

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the tick bite-sites (Suppl Fig. 2B-C) but did not significantly impact the elicitation of other

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parameters of tick-resistance including tick detachment, tick recovery and engorgement weights

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(Suppl Fig. 2D-F).

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Saliva-immunized animal sera elaborate robust humoral responses to Salp14 and TSLPI

Using various screening approaches we have earlier identified several salivary proteins (Salps) that avidly react with tick-resistant animal sera (Das et al., 2001). Since saliva-immunized animals were significantly protected from tick infestation, seroreactivity of these Salps to saliva-

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immunized guinea pig sera was assessed by ELISA and western blot using recombinant proteins of these Salps generated in the Drosophila expression system.

Recombinant (r) Salp14

(Narasimhan et al. , 2002) and rTSLPI (Schuijt et al., 2011a) showed strong reactivity to antisaliva sera when compared to all other recombinant Salps (Fig. 4A). Therefore, we immunized guinea pigs with a cocktail of rSalp14 and TSLPI as described in the Materials and Methods and

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challenged the animals with I. scapularis nymphs to examine if immunity to rSalp14 and rTSLPI was sufficient to elicit tick-resistance. After the last boost, blood was drawn from each animal and presence of antibodies specific to rSalp14 and rTSLPI in the sera was confirmed by ELISA prior to challenge of each animal with ~ 30 I. scapularis nymphs (Fig. 4B). The tick bite-sites on rSalp14/TSLPI-immunized animals showed erythema by about 24 h of tick attachment and significantly increased by 48 h when compared to that on control animals (Fig. 4C-D). Tick

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attachment was reduced significantly by day 4 (Fig 4E), and tick recovery was diminished (Fig.

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4F). The engorgement weights of the recovered ticks were comparable to that on Ova-immunized

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animals (Fig. 4G).

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Given that glycosylations on proteins played a significant role in tick rejection, we also examined if immunization of guinea pigs with Salp14 and TSLPI generated in a mammalian

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system (rSalp14-m and rTSLPI-m) would impact tick resistance. Guinea pigs were immunized with rSalp14-m/rTSLPI-m and challenged with nymphal ticks as described in the Materials and

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Methods. Presence of antibodies specific to rSalp14m and rTSLPIm in the sera was confirmed by ELISA prior to challenge of each animal with ~ 30 I. scapularis nymphs (Suppl Fig. 3A). In contrast to the results using rSalp14 and rTSLPI made in a Drosophila expression system, no significant redness was observed at tick attachment sites in the first 3-4 days (Suppl Fig. 3B-C).

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However, consistent with the previous results, tick attachment was reduced by day 4 (Suppl Fig. 3D) when compared to Ova-immunized animals and the recovery of repleted ticks from rSalp14m/rTSLPI-m was reduced compared to Ova-immunized animals (Suppl Fig. 3E). Engorgement weights of the recovered ticks were comparable in both groups (Suppl Fig. 3F).

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To determine if inflammation at the tick bite-sites on rSalp14/TSLPI immunized animals was unique to Salp14 and TSLPI or if it simply represented reactivity to the respective Salps in tick saliva, we also immunized animals with a cocktail of salivary antigens that did not show reactivity to anti-saliva sera (Supplemental Tables. 1-2). Indeed, only cocktails that contained rSalp14 and rTSLPI showed redness at the tick bite-sites (Fig. 5 and Supplemental Fig. 4) and none of the cocktails tested provided tick-resistance phenotype as seen by tick detachment rate,

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tick recovery and engorgement weights that were comparable to that on Ova-immunized animals

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(Fig. 5 and Supplemental Fig. 4).

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Discussion

While several proteomic, transcriptomic and functional genomic strategies to develop anti-

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tick vaccines continue to emerge (Artigas-Jeronimo et al. , 2018, Contreras et al. , 2016, de la Fuente et al. , 2010, Nuttall et al. , 2006), Trager’s observations (Trager, 1939) that selected non-

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permissive hosts reject ticks upon multiple infestations remains a robust paradigm to define potential tick vaccine targets to control ticks and prevent tick-transmitted diseases. Over the last several decades, research aimed at understanding the molecular and mechanistic basis of acquired tick-resistance has revealed insights into various host immune components that drive this

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phenomenon (Tabakawa et al., 2018, Wada et al., 2010, Wikel, 1984, 1996, Willadsen, 1980) and also invoked several salivary antigens that likely play a role in eliciting tick-resistance (Das et al., 2001, Mulenga et al. , 2000a, Mulenga et al., 2000b, Schuijt et al., 2011b). However, the paramount goal of exploiting this phenomenon to develop anti-tick vaccines has not been achieved (Mulenga et al., 2000b, Willadsen, 2004). Salivary antigens invoked in acquired tick-resistance

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when tested in vaccine-challenge experiments provided partial protection from tick infestations and pathogen transmission (Brown and Askenase, 1986, Dai et al. , 2009, Das et al., 2001, Mulenga et al., 2000a, Mulenga et al. , 1999, Schuijt et al., 2011b). It was suggested that salivary antigens “exposed” to the host immune responses have likely evolved to counter the immune pressures of the mammalian host (Kotsyfakis et al. , 2008, Nuttall et al., 2006) and dampened enthusiasm for the search for tick salivary antigen-based vaccine targets. Given the complexity of the functional

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genome of ticks (Gulia-Nuss et al. , 2016, Pagel Van Zee et al. , 2007, Ribeiro et al., 2006), it is

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likely that multiple factors need to be taken into consideration to fully harness the vaccine potential of tick salivary antigens. In this study, we utilized the guinea pig model of acquired tick-resistance

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(Allen, 1973) to examine whether immunity directed against I. scapularis tick saliva elicits robust

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tick-resistance and to determine salivary components that are critical for eliciting this phenotype.

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I. scapularis ticks remain attached to the host for several days and feeding progresses in phases of slow to rapid as feeding culminates in repletion (Anderson and Magnarelli, 2008). It is

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now recognized that the tick salivary proteome is dynamic, shifting in composition during the different phases of feeding to counter the defense responses of the host and successfully feed to repletion (Kim et al., 2016, Narasimhan et al., 2007a). While targeting salivary antigens expressed early in feeding is presumed critical to interrupt tick feeding early (Lewis et al., 2015, Narasimhan

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et al., 2007a), it also suffers from the potential disadvantage of a short window of time for a robust anamnestic response to develop. We reasoned that salivary proteins secreted into the host throughout the process of feeding are likely to elicit a robust host response. Therefore, we utilized tick saliva collected from repleted adults that is expected to include secreted salivary antigens expressed throughout the course of tick feeding. Tick salivary and gut proteins have been reported

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to contain diverse post-translational modifications including glycosylations, phosphorylations and lipidations (de la Fuente et al. , 2006, McKenna et al. , 1998, Sauer et al. , 1989, Sonenshine, 1991) and these modifications could provide an adjuvant effect. Given that repeated tick infestations deposit natural saliva into the host and elicit a robust immune response that rejects tick feeding, we examined whether immunization of animals with saliva without added adjuvant was sufficient to provoke host immune responses critical for tick rejection. Indeed, when animals immunized

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with tick saliva were challenged with I. scapularis nymphs we observed the hallmarks of acquired

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tick-resistance (Fig. 1) including significant erythema at the tick bite-sites, impaired tick feeding, and diminished tick recovery when compared to control animals. Animals immunized with as low

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as 20 ng of tick saliva provided partial tick-resistance phenotype as seen by erythema at the bite

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site, but not tick rejection (Suppl Fig. 1), attesting to the potency of tick saliva. The phenotype was not significantly enhanced when animals were immunized with saliva in presence of adjuvant

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such as IFA (Fig. 2). Histologic examination of the tick bite-sites on saliva-immunized animals demonstrated increased inflammation characterized predominantly by neutrophils and

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mononuclear cells and scattered basophils and mast cells. These observations were consistent with that observed by Anderson et al (Anderson et al., 2017) on repeatedly tick-infested guinea pigs. OVA-immunized animals did not show significant inflammation at the tick bite-site.

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To determine the role of different components of tick saliva in eliciting tick- resistance we

enzymatically depleted the saliva of proteins, glycan moieties, phosophorylations or lipid moieties and immunized animals with specific enzyme-treated saliva. The abrogation of the tick resistance phenotype upon depletion of proteins and glycosylations, but not phosphorylations or lipidations suggested that proteins and glycosylations are critical players in eliciting the tick-resistance

18

phenotype (Fig. 3, and Suppl Fig. 3). These findings, especially the role of glycosylations, emphasize earlier observations that recombinant salivary antigens generated in eukaryotic expression systems were more effective antigens than those made in bacterial expression systems (de la Fuente et al., 2006). There is currently no robust tick cell-line-based protein expression system and most studies utilize insect expression systems such as Drosophila (Anguita et al., 2002) or yeast expression systems such as Pichia pastoris (Kumar et al. , 2016). Characterization of tick

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glycosylation patterns and development of tick-expression systems would help refine tick vaccine

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antigen and adjuvant development.

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Both humoral and cellular immunity is invoked in the elicitation of acquired tick-resistance (Wikel

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and Allen, 1976, Willadsen, 1980) and transfer of serum from tick-resistant guinea pigs to naïve guinea pigs was shown to confer partial yet significant tick-resistance phenotype (Askenase et al.

lP

, 1982, Brossard and Girardin, 1979, Brown, 1982). Degranulation of basophils at the tick bitesite, a critical prelude to tick rejection (Brown and Askenase, 1985) is initiated when specific

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salivary antigens engage with antigen-specific IgG bound to cognate receptors on basophils (Brown and Askenase, 1983), emphasizing the role of humoral immunity in acquired tickresistance. Wada et al. (Wada et al., 2010) and Tabakawa et al. (Tabakawa et al., 2018) utilized the Haemaphysalis longicornis tick and murine host model of acquired tick resistance and showed

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that IgE plays an essential role in the development of tick resistance. Immunological reagents for the guinea pig model are limited and using the currently available reagents to assess guinea pig IgE we were unable to discern significant amounts of IgE in tick-immune and in saliva-immunized animal sera. A definitive evaluation of the role of IgE in the guinea pig model will require more sensitive and specific reagents to assess IgE amounts in guinea pig sera.

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Our earlier studies aimed at defining tick salivary antigens that react with tick-resistant animal sera had identified several antigens (Das et al., 2001, Schuijt et al., 2011b). Of these antigens, we observed that Salp14, a putative anticoagulant, and TSLPI, an inhibitor of the lectin pathway of the complement system, reacted avidly with anti-saliva sera from saliva-immunized guinea pigs (Fig. 4). The observation that saliva immunization with IFA increased sero-reactivity

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to several other antigens in addition to Salp14 and TSLPI (Fig. 4A), but did not enhance the tick-

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resistance phenotype (Fig. 2) suggests that Salp14 and TSLPI are likely among the critical elicitors

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of tick-resistance.

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Salp14 and TSLPI are glycosylated proteins and share 93% identity in the N-terminal region (Narasimhan et al., 2002) and belong to a family of structurally related proteins (Valenzuela

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et al. , 2002). Guinea pigs immunized with a cocktail of recombinant Salp14 and TSLPI (rSalp14/rTSLPI) generated in the Drosophila expression system and challenged with I. scapularis

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nymphs provided significant erythema at the tick bite-sites, a notable hall mark of tick-resistance, about 24 h post tick attachment (Fig. 4). Despite the significant erythema at the tick bite-sites reminiscent of acquired tick-resistance, immunity against rSalp14-rTSLPI provided modest tickrejection only around 72-96 hours post tick attachment, and showed a trend towards decreased tick

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repletion. It is likely that antigens in addition to Salp14 and TSLPI might be required to achieve a more robust tick-resistance phenotype.

Interestingly, when we immunized guinea pigs with rSalp14-m/rTSLPI-m generated in a mammalian expression system, we did not observe erythema at the bite site (Suppl Fig. 3),

20

although tick rejection was comparable to that seen on animals immunized with rSalp14/rTSLPI generated in the Drosophila expression system. It is likely that glycosylations on recombinant proteins generated using the mammalian expression system might be less immunogenic in the mammalian host. It is important to note that insect-cell-generated glycosylations by themselves are not contributing to the erythema and that it is a combination of the antigen-glycan epitope. When guinea pigs immunized with cocktails of different subsets of recombinant salivary antigens

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generated in the Drosophila expression system were challenged with ticks, only cocktails that

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included rSalp14/rTSLPI provided erythema at the tick bite-site (Fig. 5 and Supplemental Figs. 4).

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While immunization against rSalp14/rTSLPI did not provide optimal tick-rejection, it did

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provide significant erythema at the tick bite-site. Erythema at the tick bite-site is a result of the congregation of immune cells at the bite site that are thought to initiate responses detrimental to

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tick feeding, including release of histamines from platelets, mast cells and basophils (Wikel, 1996). This would potentially initiate itching of the skin, alert the host to the presence of the tick and

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result in removal of the tick. We reasoned that a vaccine formulation that would alert the host of tick presence would result in rapid tick detection and tick removal that could potentially interrupt tick-transmission of pathogens. We recognize that ticks often attach on parts of the body that are not readily visible, but itching and accompanying redness would promote a more rapid surveillance

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for tick attachment and removal.

We must bear in mind that tick-resistance phenotype observed upon multiple tick

infestations was more effective at rejecting ticks (Fig. 1) compared to that observed on saliva immunized animals. Natural tick infestations might boost the host immune responses additionally

21

by components including the cement cone, and mouth parts directly or indirectly and accelerate tick rejection earlier. Therefore, it is likely that saliva immunizations using higher doses of saliva and using adjuvants might provide more potent tick rejection. Further, saliva obtained from adult ticks is likely not fully reflective of nymphal saliva and could also account, in part, for the differences in the tick-resistance phenotype between saliva-immunized and tick-immune animals.

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The demonstration that immunity against tick saliva is sufficient to elicit the hall marks of

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acquired tick resistance narrows the search to salivary proteins represented in tick saliva and advances in proteomic strategies (Villar et al. , 2017) make this a tractable proposition. Our

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observations, overall, indicate a correlation between humoral responses to specific salivary

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components, as measured by total IgG, and the elicitation of tick rejection. Erythema, a hall mark of tick resistance, appears to be less critical for tick rejection.

It is also evident that the

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immunogenicity of saliva must be assessed in conjunction with adjuvants to further improve the efficiency of tick-rejection. These observations renew our focus on tick saliva and demonstrate

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that salivary antigens are key players in eliciting tick resistance, and expand our understanding of the biochemical coordinates on the salivary antigens to enable a viable vaccine design and development.

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CONCLUSIONS

Trager’s observations (Trager, 1939) that non-permissive hosts reject ticks upon multiple

tick infestations remains a robust paradigm to define potential tick vaccine targets to control ticks and prevent tick-transmitted diseases. In this report we provide evidence that immunity elicited by tick saliva in the absence of added adjuvant is sufficient to recapitulate the parameters of tick-

22

resistance including erythema at the tick bite-sites and tick rejection. We also demonstrate that protein components of tick saliva in conjunction with glycan moieties on these proteins are key elicitors of tick-resistance. These observations redirect our focus on tick salivary proteins as potential anti-tick vaccine targets and emphasize the need to select appropriate recombinant

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protein expression systems to achieve optimal vaccine formulations.

Author contribution statement:

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SN conceptualized, designed the experiments, analyzed the data, acquired funding and

prepared the manuscript; CK designed and conducted the experiments, analyzed data, and

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prepared the manuscript; HD, NOS, JC, GL conducted the experiments, and analyzed data; KD

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and MW conducted the experiments; KM, YC, JT, JA and JH provided input and resources; FK conceptualized, and designed the experiments; EF conceptualized, analyzed data, acquired

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ACKNOWLEDGEMENTS

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funding, and prepared the manuscript.

We wish to thank Dr. Michael Tiemeyer, University of Georgia, for his input on saliva deglycosylation experiments. This work was conducted by funding support from the Steven and

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Alexandra Cohen Foundation to EF. This work was also supported in part by R21 AI128182 award from NIAID/NIH to SN, by the John Monsky and Jennifer Weiss Monsky Lyme Disease Research Fund to EF, and by the multi-PI PO1 AI138949P01 to EF. CIC bioGUNE thanks the Spanish Ministry of Economy and Competitiveness for the Severo Ochoa Center of Excellence Award (SEV2016-0644). EF is an HHMI investigator.

23

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FIGURE LEGENDS

Figure 1. Immunity elicited by tick saliva recapitulates tick-resistance phenotype on guinea pigs. A. Sera from guinea pigs immunized with 20

l of adult saliva with no adjuvant (Saliva) or

Ovalbumin (OVA) or from tick-immune guinea pigs (TIGP) were assessed by ELISA for specific antibodies to tick saliva. About 30 clean I. scapularis nymphs were allowed to engorge on each of l of adult saliva (Saliva) or Ovalbumin (OVA)

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3 Hartley female guinea pigs immunized with 20

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or on tick-resistant (Tick-immune) guinea pigs and the following parameters assessed: B. Visualization of redness at the tick bite sites 24 h post-tick attachment; C. Erythema over the

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course of feeding; D. Rate of tick detachment; E. Percent recovery of repleted ticks; and F.

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Engorgement weights of individual nymphs. Error bars in A and C through F represent means + SEM. Significance of differences assessed in: C, and D by 2-way ANOVA with Tukey’s multiple

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0.05; **p<0.005).

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comparison test; A, E and F by one-way ANOVA with Tukey’s multiple comparison test (*p<

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Figure 2. Tick saliva elicits protective immunity in the absence of adjuvant. A. Sera from guinea pigs immunized with 10

l of adult saliva with no adjuvant (Saliva) or with adjuvant

(Saliva+IFA) or Ovalbumin (OVA) were assessed by ELISA for specific antibodies to tick saliva. About 30 clean I. scapularis nymphs were allowed to engorge on each of 2 Saliva, Saliva+IFA or OVA-immunized female guinea pigs and the following parameters assessed: B. Visualization of

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redness at the tick bite sites 24 h post-tick attachment; C. Erythema over the course of feeding;

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D. Rate of tick detachment; E. Percent recovery of repleted ticks; and F. Engorgement weights of individual nymphs. Error bars in A, and B through D represent means + SEM. Significance of

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differences assessed in: A by one-way ANOVA with Holm-Sidak test; C, and D by 2-way

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ANOVA with Tukey’s multiple comparison test; E and F by one-way ANOVA with Tukey’s

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multiple comparison test. (*p< 0.05; **p<0.005).

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Figure 3. Proteins and glycosylations are critical elicitors of tick-resistance. A. Sera from guinea pigs immunized with 20

l of adult saliva (Saliva) or Ovalbumin (OVA) or saliva treated

with a cocktail of glycosidases (Saliva-deglycosylated) or saliva treated with proteinase K (Saliva-

34

protease) were assessed by ELISA for specific antibodies to tick saliva. About 30 clean I. scapularis nymphs were allowed to engorge on each of 3 Hartley female guinea pigs immunized with Saliva or OVA or Saliva-deglycosylated or Saliva-protease and the following parameters assessed: B. Visualization of redness at the tick bite sites 24 h post-tick attachment; C. Erythema over the course of feeding; D. Rate of tick detachment; E. Percent recovery of repleted ticks; and F. Engorgement weights of individual nymphs. Error bars in A, and C through F represent means

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+ SEM. Significance of differences assessed in: C, and D by 2-way ANOVA with Tukey’s

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multiple comparison test; A and E by one-way ANOVA with Tukey’s multiple comparison test;

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F by one-way ANOVA with Dunn’s multiple comparison test. (*p< 0.05; **p<0.005).

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Fig 4. Salp14 and TSLPI are predominant immunogens in saliva. A. Sera from guinea pigs l of adult saliva (Anti-saliva) or Ovalbumin (Anti-OVA) or saliva with IFA

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immunized with 20

(Anti saliva+IFA) or Ovalbumin with IFA (OVA+IFA) were assessed by ELISA for specific antibodies to tick saliva or to individual recombinant secreted salivary protein antigens listed in Supplemental Table 1. B. Sera from each of 3 guinea pigs immunized with a cocktail of 20

g

each of recombinant Salp14 and TSLPI (Anti-Salp14/TSLPI) or Ovalbumin (Anti-OVA) were assessed by ELISA for specific antibodies to Salp14 or TSLPI. About 30 clean I. scapularis

36

nymphs were allowed to engorge on each of 3 Hartley female guinea pigs immunized with Salp14/TSLPI or OVA and the following parameters assessed: C. Visualization of redness at the tick bite sites 24 h post-tick attachment; D. Erythema over the course of feeding; E. Rate of tick detachment; F. Percent recovery of repleted ticks; and G. Engorgement weights of individual nymphs. Error bars in A, B and D through G represent means + SEM. Significance of differences assessed in: D, and E by 2-way ANOVA and Sidak’s multiple comparison test; F and G by Mann-

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Whitney non-parametric test. (*p< 0.05; **p<0.005).

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Fig 5. Immunity elicited by a cocktail of recombinant salivary proteins including Salp14 and TSLPI elicits erythema at tick bite sites. A. Sera from each of 2 guinea pigs immunized with a cocktail of 20 g each of recombinant salivary proteins (Anti-Salp cocktail 1) or Ovalbumin (AntiOVA) were assessed by ELISA for specific antibodies to each of the salivary proteins. About 30 clean I. scapularis nymphs were allowed to engorge on each of 2 Hartley female guinea pigs immunized with Salp cocktail 1 or OVA and the following parameters assessed: B. Visualization

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of redness at the tick bite sites 24 h post-tick attachment; D. Erythema over the course of feeding;

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E. Rate of tick detachment; F. Percent recovery of repleted ticks; and G. Engorgement weights of individual nymphs. Error bars represent means + SEM. Significance of differences assessed in:

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C, and D by 2-way ANOVA with Sidak’s multiple comparison test; E and F by Mann-Whitney

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test. (*p< 0.05; **p<0.005).

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