Mrna vaccination of rabbits alters the fecundity, but not the attachment, of adult ixodes scapularis

ABSTRACT 19ISP is a nucleoside-modified mRNA-lipid nanoparticle vaccine that targets 19 _Ixodes scapularis_ proteins. We demonstrate that adult _I_. _scapularis_ have impaired fecundity when

allowed to engorge on 19ISP-immunized rabbits. 19ISP, therefore, has the potential to interrupt the tick reproductive cycle, without triggering some of the other effects associated with

acquired tick resistance. This may lead to the development of new strategies to reduce _I. scapularis_ populations in endemic areas. SIMILAR CONTENT BEING VIEWED BY OTHERS IMMUNIZATION

AGAINST ARTHROPOD PROTEIN IMPAIRS TRANSMISSION OF RICKETTSIAL PATHOGEN FROM TICKS TO THE VERTEBRATE HOST Article Open access 30 May 2023 VACCINATION OF CATTLE WITH THE _BABESIA BOVIS_

SEXUAL-STAGE PROTEIN HAP2 ABROGATES PARASITE TRANSMISSION BY _RHIPICEPHALUS MICROPLUS_ TICKS Article Open access 27 September 2023 NEEDLE-FREE, SPIRULINA-PRODUCED _PLASMODIUM FALCIPARUM_

CIRCUMSPOROZOITE VACCINATION PROVIDES STERILE PROTECTION AGAINST PRE-ERYTHROCYTIC MALARIA IN MICE Article Open access 04 October 2022 INTRODUCTION Tick saliva is composed of a diverse set of

bioactive molecules including proteins that facilitate tick feeding1,2. Some of these proteins have been associated with host immune responses that lead to acquired tick resistance (ATR)3.

It was recently shown that a lipid nanoparticle containing the nucleoside-modified mRNAs encoding 19 tick salivary proteins, named 19ISP, can induce ATR. Guinea pigs immunized with 19ISP,

and then exposed to _Ixodes scapularis_ nymphs developed robust _erythema_ at the tick bite sites, diminished tick engorgement, and early tick detachment4. Guinea pigs, however, are not a

good laboratory model for adult _I. scapularis_ feeding, molting and fecundity, which prevents a complete assessment of the influence of 19ISP on this stage of the tick life cycle. ATR has

also been observed in rabbits, dog, and cattle5,6,7, and rabbits are an excellent animal laboratory model for studying feeding by adult _I. scapularis_. We, therefore, tested the ability of

19ISP to induce ATR against adult _Ixodes scapularis_ in rabbits with a focus on the ability of 19ISP to impact fecundity. Rabbits were immunized with 19ISP, or a lipid nanoparticle

containing firefly luciferase mRNA as a control and exposed to adult _I. scapularis_, in order to determine whether vaccination could interfere with the reproductive fitness of the ticks.

The rabbits received 3 doses of 19ISP or the control formulation, given at 4-week intervals. Two weeks after the last boost, sera were obtained from the animals, and examined by ELISA for

antibodies to antigens within 19ISP, at dilutions of 1:500, 1:5000 and 1:50,000. Rabbits elicited antibodies against 11 of the recombinant proteins tested (Salp12, Salp14, Salp15, Salp25A,

Salp25C, Salp25D, Salp26A, IsPDIA3, P11, SG10, SG27) (Fig. 1). The rabbits did not show antibodies against TSLPI and TIX, which differs from the results observed when guinea pigs were

immunized with the same batch of 19ISP vaccine4. The dose administered in rabbits was calculated in a similar way to that administered to guinea pigs4, based on the weight of the animals.

When comparing antibody responses between rabbits and guinea pigs, the rabbits were generally shown to be less responsive to immunization with 19ISP4 . The rabbits also did not show a strong

antibody response to Salp14 compared to guinea pigs immunized with 19ISP4, and this may have been one of the reasons for the absence of _erythema_ in the rabbits. Salp14 was the antigen

most commonly associated with _erythema_ in guinea pigs exposed to _I. scapularis_ nymphs, when all the 19 components within 19ISP were analyzed, either individually or in smaller

combinations8,9. One week after collecting the blood sample, the rabbits were exposed to adult _I. scapularis_. The ticks on the animals were monitored daily for attachment (Fig. 2A), and

the weights of the collected ticks were determined (Fig. 2B). No significant differences were observed in the duration of time that _I. scapularis_ remained attached to I9ISP-immunized or

control rabbits. The engorgement weights of the ticks that fed on both groups of animals was also similar and no difference in _erythema_ at the bite site was observed between the groups.

These results are different from those observed in guinea pigs with nymphal _I. scapularis_4,8. In guinea pigs, early detachment or rejection of the nymphs was observed by 72 h. This

influence of 19ISP mRNA immunization on the feeding time of nymphs, also resulted in a marked reduction of the weights of the recovered ticks. These data underscore the differences observed

in rabbits using adult _I. scapularis_. To assess fecundity, all ticks were evaluated for oviposition regardless of engorgement weight. Ticks were collected, stored individually, and allowed

to complete their reproductive cycle. The weight of the egg mass generated by each female (Fig. 3A) was similar in ticks that fed on 19ISP-immunized or control rabbits. Females from both

groups who did not posture or had a very low weight were also included as a part of Fig. 3A. Interestingly, a significant difference was observed in the percentage of eggs that hatched into

larvae between _I. scapularis_ that fed on 19ISP-immunized rabbits compared to the ticks that fed on control animals (Fig. 3B). These data suggest that the 19ISP vaccination can

significantly impair a major parameter of _I. scapularis_ reproduction, without influencing some of the other parameters associated with ATR. Our study demonstrates that 19ISP immunization

can significantly interfere with the ability of adult _I. scapularis_ to successfully complete their reproductive cycle. In particular, the capacity of adults to produce viable larvae was

substantially reduced. Interestingly, some differences in ATR were evident upon 19ISP immunization in guinea pigs and rabbits. An immune response to Salp14 in guinea pigs is linked to

_erythema_ at the tick bite site. Surprisingly, rabbits do not develop robust antibodies to Salp14 following 19ISP immunization, and these animals did not develop significant _erythema_ at

the tick bite site. These data underscore the importance of the different host immune response to specific tick proteins in the development of selected aspects of ATR. These data also help

expand the principle that not all aspects of ATR, including _erythema_, tick engorgement and attachment and tick fecundity occur via the same mechanisms, or are associated with host

responses to the same antigens. Understanding the specific host responses associated with ATR is complex and requires the combination of different techniques. A detailed analysis of the bite

site, including the examination of specific cellular responses using single cell RNA seq, as well as an examination of local innate, cellular and metabolic responses will help to guide

future research. Adult _I. scapularis_ commonly feed on deer to complete their life cycle, and it will be important to determine if the alterations in fecundity observed in the rabbit model

extend to deer. Overall, these studies suggest that 19ISP can interfere with the fecundity of adult _I. scapularis_, and this finding opens new strategies to reduce tick populations in

endemic areas. METHODS ETHICS All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, USA. The animal

protocols were approved by the Yale University Institutional Animal Care and Use Committee (YUIACUC—protocol number 2023-07941). The authors complied with the ARRIVE guidelines. TICKS,

ANIMALS AND IMMUNIZATION _I. scapularis_ adults were obtained from the Oklahoma State University (Stillwater, OK) and maintained in an incubator at 23 °C and 90% relative humidity under a 14

 h light, 10 h dark photoperiod. Female and male ticks were kept together for at least 48 h before challenge under the conditions mentioned above. New Zealand white rabbits (Charles River)

were used for immunizations and tick challenge experiments. Rabbits were immunized intradermally with 200 μg of 19ISP (n = 2) or luciferase mRNA as a control (n = 2). The animals were

boosted twice at 4-week intervals. Three weeks after the last booster, rabbits were sedated with acepromazine (0.75 mg/kg) and 25 female and 25 male adult ticks were placed on the animal’s

ears. Rabbits were monitored daily for evidence of tick rejection, recovery, and _erythema_ at the bite site. The experiment was repeated three times, totaling 6 animals per group. In

addition, all the rabbits wore Elizabethan collars and the ear that received the tick was isolated with an adapted sock, which prevented the ticks from moving to another location. The

animals were prevented from self-cleaning and received a daily dose of 1.25 mg/kg diazepam as a veterinary recommendation to reduce stress. During tick feeding, rabbits were housed

individually in closed cages with wire grates suspended over water. The ticks that attached to the rabbits were allowed to detach naturally. All recovered fed ticks were kept individually in

plastic tubes sealed and maintained in an incubator under the conditions mentioned above until the end of egg laying (around 6 weeks). The egg mass was weighed and kept in the incubator

until the hatching of the larvae (8 weeks). The hatching success was obtained by the value of visual count of larvae. LABORATORY METHODS To assess antigen-specific antibody responses, ELISA

was performed as described previously4. Briefly, 96-well ELISA plates were coated overnight with 250 ng of recombinant protein diluted in carbonate-bicarbonate buffer pH 9.6, washed with

PBST (PBS with 0.05% Tween 20) and blocked with 3% bovine serum albumin for 1 h at 37° C. Rabbit sera were serially diluted (1:500, 1:5000, or 1:50,000) and incubated for 2 h at 37° C. Wells

were washed, and reactivity was detected using goat anti-rabbit IgG-HRP and TMB HRP substrate solution (Thermo Fisher Scientific). TMB stop solution was added and the plates were read at

450 nm. mRNA-lipid nanoparticles (LNPs) were formulated as previously described10 and this LNP contains 19 mRNAs encoding individual _I. scapularis_ salivary proteins or firefly luciferase

(Luc) mRNA4. mRNAs were transcribed from a linearized plasmid DNA template with a 101 nucleotide-long poly(A) tail and instead of UTP, N-1-methylpseudouridine (m1Ψ) 5’-triphosphate (TriLink)

was used to generate modified nucleoside-containing mRNA. During the in vitro transcription, co-transcriptional capping was performed using the trinucleotide cap1 analog, CleanCap

(TriLink). mRNAs were purified by cellulose purification, as described11, analyzed by gel electrophoresis and frozen at −20 °C. All mRNAs were combined and then encapsulated using an aqueous

solution of mRNA at pH 4.0 and mixed with a solution of lipids dissolved in ethanol12,13. The solution contains an ionizable cationic lipid/ phosphatidylcholine/ cholesterol/ polyethylene

glycol (PEG) lipid (proprietary of Acuitas, Vancouver, Canada) (50:10:38.5:1.5 mol/mol). RNA was mixed with the lipids at a ratio of ~ 0.05 (wt./wt.), 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. STATISTICAL ANALYSES Data were analyzed using Prism

9.5.1 software (GraphPad Software, CA). Data are represented as the mean ± standard error of the mean (SEM) or mean with standard deviation (SD). The significance of the difference between

control and experimental group was determined by Mann Whitney test. P ≤ 0.05 was considered statistically significant. DATA AVAILABILITY The data that support the findings of this study are

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https://doi.org/10.1002/anie.201203263 (2012). Article  CAS  PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS We thank Sukanya Narasimhan for her helpful

suggestions in guiding this research work. This work was support by NIH grants AI165499 and AI138949, the Steven and Alexandra Cohen Foundation, and the Howard Hughes Medical Institute

Emerging Pathogens Initiative. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Section of Infectious Diseases, Department of Internal Medicine, School of Medicine, Yale University, New Haven,

CT, 06520, USA Jaqueline Matias, Yingjun Cui, Geoffrey E. Lynn, Kathleen DePonte, Emily Mesquita & Erol Fikrig * Department of Microbiology, Perelman School of Medicine, University of

Pennsylvania, Philadelphia, PA, 19104, USA Hiromi Muramatsu & Norbert Pardi * Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104,

USA Mohamad G. Alameh, Garima Dwivedi & Drew Weissman * Acuitas Therapeutics, Vancouver, BC, V6T 1Z3, Canada Ying K. Tam Authors * Jaqueline Matias View author publications You can also

search for this author inPubMed Google Scholar * Yingjun Cui View author publications You can also search for this author inPubMed Google Scholar * Geoffrey E. Lynn View author publications

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inPubMed Google Scholar * Drew Weissman View author publications You can also search for this author inPubMed Google Scholar * Erol Fikrig View author publications You can also search for

this author inPubMed Google Scholar CONTRIBUTIONS E.F., and J.M. conceived the study. J.M., Y.C., G.E.L., K.D. performed and interpreted experiments (Rabbits immunization and tick

challenge). J.M., Y.C., and E.M. performed and interpreted tick biological parameters. E.F., N.P., D.W., H.M., M.G.A., and G.D. designed and generated the mRNAs. Y.K.T. prepared the mRNA

nanoparticles. J.M., and Y.C performed ELISAs. E. F., J.M. analyzed and interpreted data. J.M., and E.F. wrote the initial draft, performed statistical analysis, and generated figures. All

authors read, edited, and approved the final manuscript. CORRESPONDING AUTHORS Correspondence to Jaqueline Matias or Erol Fikrig. ETHICS DECLARATIONS COMPETING INTERESTS N.P. and D.W. are

named on a patent describing the use of nucleoside-modified mRNA in lipid nanoparticles as a vaccine platform. We have disclosed those interests fully to the University of Pennsylvania, and

we have in place an approved plan for managing any potential conflicts arising from licensing of our patents. N.P. served on the mRNA strategic advisory board of Sanofi Pasteur in 2022. N.P.

is a member of the scientific advisory board of AldexChem. ADDITIONAL INFORMATION PUBLISHER'S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published

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permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Matias, J., Cui, Y., Lynn, G.E. _et al._ mRNA vaccination of rabbits alters the fecundity, but not the attachment, of adult _Ixodes

scapularis_. _Sci Rep_ 14, 496 (2024). https://doi.org/10.1038/s41598-023-50389-6 Download citation * Received: 29 September 2023 * Accepted: 19 December 2023 * Published: 04 January 2024 *

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