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Sajid et al., Sci. Transl. Med. 13, eabj9827 (2021) 17 November 2021
SCIENCE TRANSLATIONAL MEDICINE | RESEARCH ARTICLE
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INFECTIOUS DISEASES
mRNA vaccination induces tick resistance and prevents
transmission of the Lyme disease agent
Andaleeb Sajid1†, Jaqueline Matias1†, Gunjan Arora1†, Cheyne Kurokawa1, Kathleen DePonte1,
Xiaotian Tang1, Geoffrey Lynn1, Ming-Jie Wu1, Utpal Pal2,3, Norma Olivares Strank1,
Norbert Pardi4, Sukanya Narasimhan1, Drew Weissman4, Erol Fikrig1*
Ixodes scapularis ticks transmit many pathogens that cause human disease, including Borrelia burgdorferi. Acquired
resistance to I. scapularis due to repeated tick exposure has the potential to prevent tick-borne infectious diseases,
and salivary proteins have been postulated to contribute to this process. We examined the ability of lipid nanoparticle–
containing nucleoside-modified mRNAs encoding 19 I. scapularis salivary proteins (19ISP) to enhance the recog-
nition of a tick bite and diminish I. scapularis engorgement on a host and thereby prevent B. burgdorferi infection.
Guinea pigs were immunized with a 19ISP mRNA vaccine and subsequently challenged with I. scapularis. Animals
administered 19ISP developed erythema at the bite site shortly after ticks began to attach, and these ticks fed
poorly, marked by early detachment and decreased engorgement weights. 19ISP immunization also impeded
B. burgdorferi transmission in the guinea pigs. The effective induction of local redness early after I. scapularis
attachment and the inability of the ticks to take a normal blood meal suggest that 19ISP may be used either alone
or in conjunction with traditional pathogen-based vaccines for the prevention of Lyme disease, and potentially
other tick-borne infections.
INTRODUCTION
Tick-borne diseases are currently increasing in North America and
Europe. The black-legged tick, Ixodes scapularis, transmits diverse
pathogens, including Borrelia burgdorferi (the Lyme disease agent),
Babesia microti, Anaplasma phagocytophilum, Borrelia miyamotoi,
and Powassan virus, among other infectious agents (1–3). As one
example, the most common I. scapularis–borne human illness in the
United States, Lyme disease, results in almost 40,000 cases reported
annually, and the Centers for Disease Control and Prevention esti-
mates that the real number of infections may be 10 times greater
(3–5). Given the identification of new types of tick-borne pathogens
along with their diversity and generally unsuccessful efforts toward
the development of single, pathogen-specific vaccines, the develop-
ment of a broad antitick vaccine strategy is highly desirable. A pos-
sible approach to the prevention of one or more I. scapularis–borne
diseases involves developing new methods to detect the ticks early on
and preventing the arthropod from taking a successful blood meal.
The ability of animals to develop acquired resistance to tick bites
after repeated exposure to ticks, so-called “acquired tick resistance”
or “tick immunity,” was first described by Trager in 1939 (6). Tick
immunity is associated with the recruitment of inflammatory cells to
the tick bite site that alter tick feeding, including histamine-secreting
basophils (7–11). The phenomenon of naturally acquired resistance
to I. scapularis has most substantially been observed in animals that
are not important in the natural life cycle of this tick, including
guinea pigs, rabbits, and cows, among other animals (12,13). Dermal
hypersensitivity after repeated tick exposure in humans has been
described, suggesting an association with acquired resistance to
I. scapularis (14,15). Tick immunity in guinea pigs also provides
protection against I. scapularis–transmitted B. burgdorferi infection,
indicating that understanding this process further can lead to new
vaccine strategies to prevent Lyme disease and possibly other tick-
borne infections (16,17).
In contrast to guinea pigs, some animals that serve as a natural
reservoir for I. scapularis, such as mice, do not readily develop robust
resistance upon repeated exposure to I. scapularis (8,9,12,18). Mice,
however, are not a natural reservoir for Haemaphysalis longicornis,
and it has been demonstrated that laboratory mice can acquire re-
sistance to these arthropods, suggesting that evolutionary pressure
may lead, at least in part, to ticks feeding on vertebrate hosts on
which they can readily take a blood meal without the host developing
resistance (8,19,20). These disparities largely influence the choice
of an animal model for studying tick immunity, but also provide a
platform for understanding how diverse host responses contribute
to resistance (9,18,21). Guinea pigs acquire robust tick immunity
after repeated tick infestations at the larval, nymphal, and adult
stages (16,17,22,23).
Naturally acquired tick resistance is generally considered to be
associated with host immune responses to tick antigens that are se-
creted into the bite site and present in saliva and cement (17,24).
Specific I. scapularis salivary proteins have functional properties
that can influence host immune responses, inflammation, and
coagulation, but it is unclear whether these antigens are directly in-
volved in the genesis of tick immunity (25–27). Several tick antigens
have been shown to generate a host response, either after a tick bite
or upon specific immunization, but robust tick immunity has not
been replicated (28–40). For example, vaccination of guinea pigs
with sialostatin L2, an I. scapularis immunomodulator, led to some
degree of diminished tick feeding and detachment by 72 hours (28),
and immunization with a combination of two salivary proteins,
salivary protein of 14 kDa (Salp14) and tick lectin pathway inhibitor
(TSLPI), provided modest tick rejection at 72 to 96 hours after
tick attachment (29). Direct immunization with tick saliva alone
1Section of Infectious Diseases, Department of Internal Medicine, Yale University
School of Medicine, New Haven, CT 06520, USA. 2Department of Veterinary Medicine,
University of Maryland, College Park, MD 20472, USA. 3Virginia-Maryland Regional
College of Veterinary Medicine, College Park, MD 20472, USA. 4Department of
Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
*Corresponding author. Email: erol.fikrig@yale.edu
†These authors contributed equally to this work.
Copyright © 2021
The Authors, some
rights reserved;
exclusive licensee
American Association
for the Advancement
of Science. No claim
to original U.S.
Government Works
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afforded a more substantial degree of tick rejection by 48 to 72 hours,
indicating that multiple salivary components or a specific manner
of immunization are required for eliciting an optimal host response
against tick bites (29). An approach that more closely replicates the
response observed in naturally acquired tick immunity is needed to
definitively interfere with pathogen transmission (16,17).
Tick saliva is a complex blend of several proteins that are expressed
dynamically depending on tick feeding and resulting changes in
host responses (17,29,41–43). Using this information and previous
analysis of the tick sialome (24,29,30,36–38,44–54), we selected 19
salivary proteins to form a cocktail of antigens. These antigens were
carefully selected on the basis of their high immunogenicity and, in
most cases, with known mode of action. Nucleoside-modified
mRNAs encoding these 19 I. scapularis proteins were encapsulated
in lipid nanoparticles (LNPs; I9ISP), which protect mRNA from
degradation and facilitate invivo delivery (55,56). The nucleoside-
modified mRNA-LNP vaccination platform has shown promising
results for infectious diseases in humans, including the recent
coronavirus disease 2019 (COVID-19) vaccine trials (56–60). In the
present study, we demonstrate that 19ISP effectively induces tick
immunity in guinea pigs.
RESULTS
19ISP is an LNP-encapsulated, nucleoside-modified mRNA
vaccine encoding 19 I. scapularis salivary proteins
We selected 19 genes known to be expressed in I. scapularis salivary
glands, many of which are secreted at the bite site (Table1). Most of
Table 1. Genes incorporated into the 19ISP mRNA vaccine.
Gene Gene/protein accession Characteristics*Reference
Salp10 AAK97828/AF278575
Elicits antibodies in host, homology
with Kunitz-type protease inhibitors,
altered expression in response to A.
phagocytophilum
(47, 54)
Salp15 AAK97817/AF209914 Alters CD4+ T cell function, influences
B. burgdorferi infection (37, 44, 47, 51)
Salp25A AAK97825/AF209922 Generates antibodies in host,
putative histamine binding protein (47)
Salp25B AAK97821/AF209918 Produces antibodies in host,
probable histamine binding protein (47)
Salp25C AAK97816/AF209913
Potential histamine binding protein,
altered expression in response to A.
phagocytophilum
(47, 54)
Salp25D AAK97814/AF209911
Peroxiredoxin, role in acquisition of
B. burgdorferi by I. scapularis in the
host
(36, 47)
Salp14 AAK97824/AF209921 Anticoagulant that inhibits factor Xa (29, 50, 53)
TSLPI HQ605983/AEE89466 Lectin complement pathway
inhibition (29, 52)
Salp26A AAK97822/AF209919 Generates antibodies in host (47)
P11 DQ066011/AAY66648 Facilitates A. phagocytophilum
migration to tick salivary glands (48)
Salp16A AAK97818/AF209915 Elicits antibodies in host, homology
with Salp15 (47)
Salp17 AAK97819/AF209916 Expression decreased by A.
phagocytophilum (54)
TIX5 AEE89467/HQ605984 Factor Xa inhibitor (30, 53)
P32 ADO95260/HM802761 Reacts with tick immune serum (53)
Salp12 XP_002433874/XM_002433829 Chemoattractant for B. burgdorferi
and influences acquisition (49)
SG27 XP_002405832/XM_002405788 Putative B. burgdorferi attractant,
protein disulfide-isomerase (45, 49)
IsPDIA3 XP_002406442/XM_002406398
Protein disulfide-isomerase,
contributes to B. burgdorferi
colonization of ticks
(49), (45)
SG10 XP_002411436/XM_002411391 Heme lipoprotein (42, 46, 49)
SG09 XP_002411435/XM_002411390 Hemelipoglyco-carrier protein (42, 49)
*Data from references, Pfam, and Simple Modular Architecture Research Tool (SMART) domain analysis.
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these genes have been characterized in
individual studies, and several have been
tested for their efficacy against tick in-
festation. Salp14, TSLPI, Salp10, Salp15,
Salp16A, Salp17, Salp25A, Salp25B,
Salp25C, Salp25D, Salp26A, tick inhibitor
of factor Xa (TIX5), and a 32-kDa sali-
vary protein (P32) were initially identified
by immunoscreening assays as secreted
salivary proteins that reacted avidly with
tick- resistant animal sera (47,53). Some
of these salivary antigens regulate host
immune responses or influence pathogen
infectivity (30,36,37,44,50–52,54).
P11 is a secreted salivary protein in-
volved in A. phagocytophilum infection
of salivary glands (48). Salp12, SG09,
SG10, SG27, and I. scapularis protein
disulfide isomerase (IsPDIA3) are se-
creted salivary proteins that may influ-
ence B. burgdorferi acquisition (45,49).
SG10 is a heme lipoprotein and SG09 is
a hemelipoglyco-carrier protein present
in I. scapularis saliva with homologs
identified in saliva, hemolymph, and
tissues of a variety of other tick species,
including Ixodes ricinus (42,61,62).
Heme-binding class proteins are abun-
dant in the saliva of I. scapularis, with
known or putative functions in other tick species, including trans-
port and storage of heme, detoxification, and involvement in innate
immunity (42,61, 63). A recent study included an I. ricinus heme
lipoprotein homologous to SG10 among the many potential targets
that may influence tick infestation in rabbits and dogs (46). The
individual nucleoside-modified mRNAs for 19 genes were syn-
thesized invitro and encapsulated in an LNP in equal amounts
to generate 19ISP. The 19ISP mRNA-LNP vaccine was used to im-
munize guinea pigs to test for the generation of immunity against
tick bites.
Immunization with 19ISP generates antibody responses
to specific antigens in guinea pigs
Guinea pigs were immunized intradermally three times at 4-week
intervals with 50 g of 19ISP mRNA-LNP or interleukin-21 (IL-21)
mRNA-LNP as a control. Two weeks after the last dose and before
tick challenge, blood was collected from the immunized guinea
pigs and sera were isolated. Serum immunoglobulin G (IgG) titers
were evaluated by enzyme-linked immunosorbent assay (ELISA)
using recombinant salivary protein antigens. We tested 18 antigens
for the presence of specific antibodies. The primary sequences
of SG09 and SG10 share 75% identity, which precludes conclusive
determination of antibodies specific to these two proteins. There-
fore, recombinant SG09 was not generated for ELISA assays. Anti-
bodies were detected against 10 of the selected proteins: Salp14,
Salp15, Salp25D, Salp26A, TSLPI, IsPDIA3, TIX5, P32, SG10,
and SG27 (Fig.1). Antibodies for these proteins were not detected
in serum obtained from the control animals. These results suggest
that 19ISP vaccination elicits antigen-specific humoral responses
in guinea pigs.
19ISP immunization elicits protective responses against
tick challenge in guinea pigs
Twenty-five uninfected I. scapularis nymphs were placed on 19ISP-
immunized or control (IL-21 immunized) guinea pigs and allowed
to naturally attach. The guinea pigs were monitored for the devel-
opment of erythema at the bite site, which is the earliest hallmark
associated with acquired tick resistance. Substantial erythema was
observed in 19ISP-immunized animals as early as 18 hours after tick
challenge (Fig.2A). The erythema peaked at 24 hours at all the bite
sites and persisted throughout the tick challenge. Tick bite in the
control animals did not show substantial redness (Figs.2B and 3A).
The immunized guinea pigs were also monitored for other
hallmarks of tick immunity that occur after the appearance of
erythema, including tick rejection, feeding, and engorgement weights.
For 19ISP-immunized animals, the ticks fed poorly and started to
detach by 48 hours after tick challenge (Fig.3B). Being small in size
and poorly fed, the recovery of I. scapularis was also reduced with
many of the dead tick shells merely attached to the guinea pigs
(Fig.2, 72 hours; Fig.3C). By 96 hours after tick challenge, 80% of
the ticks detached from the 19ISP-immunized guinea pigs, as com-
pared with 20% detachment in control animals. Because of poor
feeding and early detachment, the engorgement weights of the ticks
fed upon 19ISP-immunized animals were significantly lower (mean
weight,1.02 mg; n=109) as compared with the ticks that fed upon
control animals (mean weight,2.42 mg; n =108) (P< 0.0001;
Fig.3D). These data indicate that 19ISP vaccination induces ac-
quired resistance to tick bites in guinea pigs.
Mice, in contrast to guinea pigs, are important in the natural life
cycle of I. scapularis and do not develop substantial acquired tick
resistance after repeated exposure to I. scapularis (8,9,12,18). It is
Fig. 1. Antibody responses to specific I. scapularis antigens were elicited in guinea pigs after 19ISP vaccina-
tion. ELISA results are shown for guinea pig serum binding to specific recombinant proteins corresponding to the
mRNAs in 19ISP. Serum samples were isolated from animals after vaccination with 19ISP or control mRNAs (19ISP,
n = 6; IL-21, n = 5). ELISA was performed with 19ISP antigens using serum dilutions of 1:500, 1:5000, and 1:50,000.
Control sera were tested at 1:500 and 1:5000 dilutions. Antibodies were detected on the basis of optical density (OD)
450-nm values against 10 of the tested proteins: Salp14, Salp15, Salp25D, Salp26A, TSLPI, IsPDIA3, TIX5, P32, SG10,
and SG27. Data are presented as mean ± SEM of at least six OD values for the 19ISP group and four OD values for
the control group.
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possible that ticks have coevolved with animals important in their
natural life cycle, in part, to optimize their capacity to feed success-
fully. Consistent with this concept, mice immunized with 19ISP did
not demonstrate substantial erythema at the tick bite site, and there
was no impact on tick feeding (fig. S1). As humans are not important
in the natural life cycle of I. scapularis, they are therefore more likely
to respond in a manner similar to guinea pigs.
A subsequent study was performed to determine whether 19ISP
immunization could influence the transmission of B. burgdorferi to
guinea pigs. Although a single B. burgdorferi–infected tick is suffi-
cient to transmit full infection (64), we placed three B. burgdorferi–
infected I. scapularis nymphs on each guinea pig immunized with
either 19ISP or firefly luciferase (Luc)–mRNA control. As most
humans do not commonly get three tick bites at the same time, and be-
cause many I. scapularis in nature are not infected with B. burgdorferi,
we assumed that three ticks on guinea pigs represent a substantial
degree of exposure to the Lyme disease agent. In addition, as humans
are likely to remove a tick that causes a bite with concomitant
erythema or pruritis, we removed the ticks from the experimental
and control guinea pigs, in a double-blind manner, when redness
became evident (fig. S2). At 3 weeks after exposure to ticks, the
guinea pigs were euthanized, and biopsies were taken adjacent to
bite site to evaluate infection. In total, almost half (46%) of the con-
trol guinea pigs were positive (6 of 13) for B. burgdorferi by quanti-
tative polymerase chain reaction (qPCR); in contrast, none (0%) of
the 19ISP-immunized guinea pigs were PCR positive (0 of 16) for
B. burgdorferi (Fig.3E). To further confirm these results, B. burgdorferi
was cultured from skin biopsy specimens from all guinea pigs. All
six PCR-positive guinea pigs were also culture positive (Fig.3F). In
a subsequent experiment, we allowed a single B. burgdorferi–infected
tick to feed without removal on 19ISP-immunized or control guinea
pigs, to mimic an unnoticed human infection from a single tick bite.
We found that 60% (three of five) of control guinea pigs were in-
fected with B. burgdorferi, whereas none of the 19ISP-immunized
animals were infected, as tested by qPCR and culture (fig. S3A). In an
identical follow-up experiment, in which
three B. burgdorferi–infected ticks were
place on guinea pigs, three of five 19ISP-
immunized guinea pigs developed in-
fection, and four of five control guinea
pigs were infected (fig. S3B). Collectively,
these data show that 19ISP immunization
can protect against tick-borne B. burgdorferi
infection when ticks are removed when
redness appears or after short exposure
to the infected tick.
Differential host transcriptional
responses are associated
with 19ISP-mRNA vaccination
To further understand the immune re-
sponses associated with I9ISP vaccination
and host protection, we isolated RNA
from immunized guinea pigs for gene ex-
pression analysis 2 weeks after the final
immunization. We observed differences
in whole-blood gene expression in 19ISP-
immunized animals as compared with
controls. Principal components analysis
(PCA) and cluster dendrogram revealed that the 19ISP-vaccinated
animal group formed a separate cluster from the control animal
groups (Fig.4A and fig. S4A). A total of 125 differentially expressed
genes were identified with a P value less than 0.05 and a fold change
greater than or equal to 2.0. We observed that 113 genes were up-
regulated and 12 genes were down-regulated in the 19ISP-immunized
group as compared with the control group (Fig.4A and data file S1).
To identify signaling pathways involved in response to 19ISP-mRNA
vaccination, we used Kyoto Encyclopedia of Genes and Genomes
(KEGG) pathway enrichment analysis. The top 20 enriched pathways
include many immune pathways (fig. S4B). The top immune pathways
enriched after vaccination were T cell receptor and B cell receptor
signaling pathways, chemokine signaling pathways, IL-17 signaling,
natural killer cell–mediated cytotoxicity, Fc receptor (R) I–mediated
signaling, and C-type lectin receptor signaling pathways (Fig.4B).
In addition, we also compared gene expression at the erythematic
bite site with a nonerythematic site in the same guinea pigs immu-
nized with 19ISP (data file S2). Our data show enrichment of T cell–
related pathways, indicating that T cell responses were elicited by
vaccination (fig. S5).
To further understand some of the cell-mediated immune responses
elicited by 19ISP vaccination, peripheral blood mononuclear cells
(PBMCs) were isolated from 19ISP- and control (Luc)–mRNA immu-
nized guinea pigs 2 weeks after the second boost. PBMCs were stim-
ulated with I. scapularis saliva, and total RNA was extracted. mRNA
expression of selected cytokines and chemokines commonly induced
by activated T and B cells after vaccinations, including interferon-
(IFN-), tumor necrosis factor– (TNF-), CXCL10, IL-2, IL-4, and
IL-8, was examined. The expression of these cytokines was increased
in 19ISP-immunized animals (Fig.5) as compared with controls.
DISCUSSION
In the present study, we attempted to generate acquired tick resistance,
or tick immunity to I. scapularis, using 19 salivary proteins that
Fig. 2. Tick challenge of 19ISP mRNA-LNP immunized guinea pigs induces erythema. Guinea pigs were immu-
nized with 19ISP or control (IL-21) mRNA, and 25 I. scapularis nymphs were allowed to engorge on their shaved backs.
All animals were monitored for the development of erythema as a cardinal initial sign of acquired tick resistance over
a period of 6 days or until all ticks detached. The images show representative (A) 19ISP-immunized or (B) control
animals at the indicated time points.
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have a spectrum of functions in tick feeding, interaction with the
pathogen, or host responses, reflecting a portion of the tick sialome.
To optimize the immune response, we chose a nucleoside-modified
mRNA-LNP platform that allowed for more continuous delivery of
the antigen (65,66) and, therefore, perhaps more closely resembled
a tick bite. Safe and effective nucleoside-modified mRNA-LNP vac-
cines are currently being used in humans as a vaccine platform for
COVID-19; therefore, we believe that this platform represents a safe
and effective strategy to deliver tick antigens to a host.
Guinea pigs were used as the primary animal model because
they are not part of the natural life cycle of I. scapularis and readily
develop tick immunity after repeated exposure to I. scapularis
(9,12,13,67). In addition, guinea pigs can be infected with tick-
borne B. burgdorferi (16) and can therefore be used to determine
whether acquired tick resistance can influence the transmission of
the Lyme disease agent. We show that immunization with 19ISP
provides robust tick immunity in guinea pigs, including early
erythema after tick placement on the animals and rapid tick detach-
ment, along with severely impaired tick feeding and low engorge-
ment weights (66,68,69).
Host responses in the 191SP-immunized guinea pigs were further
assessed using the relatively limited reagents for this species. Robust
antibody responses to a subset of the 19 antigens, Salp14, Salp15,
Salp25D, Salp26A, TSLPI, IsPDIA3, TIX5, P32, SG10, and SG27,
were detected in 19ISP-immunized guinea pigs, suggesting that these
may be the targets most closely associated with tick immunity. Nine
of the proteins did not elicit detectable responses, suggesting that
they are not highly antigenic and may not be directly linked with
tick immunity. PBMCs from 19ISP-immunized guinea pigs stimu-
lated with I. scapularis saliva elicited the production of several T cell–
related inflammatory cytokines, suggesting that a combination of
humoral and cell-mediated immunity accounts for the magnitude
of response by 19ISP mRNA vaccination. Last, RNA sequencing
elucidated the specific genetic signatures associated with 19ISP im-
munization in guinea pigs. The activated pathways included T cell
receptor and B cell receptor signaling pathways, chemokine signaling
pathways, IL-17 signaling, natural killer cell–mediated cytotoxicity,
FcRI-mediated signaling, and C-type lectin receptor signaling path-
ways. FcRI signaling and B cell receptor signaling pathways were
also activated in our previous study to delineate pathways that are
Fig. 3. 19ISP mRNA vaccination elicits protective responses against tick challenge and B. burgdorferi transmission. Immunized animals (19ISP, n = 6; IL-21, n = 5)
were challenged with I. scapularis nymphs and assessed for erythema, tick detachment, recovery, and engorgement. (A) Erythema was calculated as the percent of
nymphs showing redness on each animal, and each symbol represents one animal challenged with about 25 nymphs. (B) The graph measures tick detachment and the
percent of ticks remaining attached at a given time point in all animals of a group. (C) The percent of total ticks recovered from each animal after rejection or detachment
is shown. (D) The weights of ticks recovered from each animal after rejection or detachment are shown, with individual ticks represented by each symbol. The error bars
in (A) to (D) represent mean ± SD, and significance was calculated using Mann-Whitney tests. P values are indicated in the figure. (E) B. burgdorferi transmission was evaluated
by qPCR using skin biopsies from guinea pigs immunized with 19ISP (n = 16) or Luc-mRNA control (n = 13), challenged with B. burgdorferi–infected ticks. Data show relative
expression of B. burgdorferi flaB, normalized with guinea pig Actin. The data from three biological replicates show 6 of 13 B. burgdorferi–infected samples in control versus
0 of 16 infected samples in the 19ISP group. Error bars show mean ± SEM, and significance was calculated using Welch’s t test (95% confidence interval). (F) The table
shows total number of samples that were positive or negative for B. burgdorferi by culture in each group.
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differentially activated at the bite site of guinea pigs with naturally
acquired tick resistance after exposure to multiple tick bites (70).
The activation of common pathways in naturally acquired tick re-
sistance and after 19ISP immunization can help guide future vaccine
development, and studies with mRNA LNPs containing selected
subsets of the genes will help address the contribution of each of the
19 genes in the genesis of acquired tick resistance.
The I9ISP-immunized guinea pigs were protected from tick-borne
B. burgdorferi infection when the ticks were removed when erythema
became pronounced. This time point was chosen because when hu-
mans notice redness or irritation due to a tick bite, the immediate
response is to remove the tick. Such erythema-associated itch is ap-
parent in tick immune guinea pigs and is likely to occur in humans
(18). In addition, when challenged with B. burgdorferi–infected ticks
that were allowed to feed until falling off, protection was more lim-
ited after exposure to multiple ticks but more apparent after a single
tick exposure. Humans, like guinea pigs, are not important in the
natural life cycle of I. scapularis, and it is, therefore, possible that
vaccination of humans with I9ISP can help prevent Lyme disease,
either because the ticks naturally detach early or because humans
more readily remove ticks when erythema is present. Ticks that
attach and feed to repletion on humans are much more likely to
transmit B. burgdorferi than ticks that are removed early in the feed-
ing process (71–74).
Our study has several limitations. We tested the effect of 19ISP
vaccination in two animal models. Immunization with 19ISP shows
significant erythema and early tick detachment in guinea pigs, but
not mice. It will be important to examine 19ISP in other models.
Furthermore, a detailed analysis of the host immune response
will help optimize the response associated with tick resistance. In
addition, the outcome of the B. burgdorferi transmission experiments
was largely dependent on the generation of erythema and removing
the ticks soon after erythema development. Future studies will also
delineate whether a subset of selected antigens within 19ISP, or ad-
ditional tick antigens, can afford protection. Efforts will determine
whether protection extends to other I. scapularis–borne pathogens,
such as B. microti, A. phagocytophilum, and Powassan virus, and also
to the pathogens transmitted by field-collected rather than laboratory-
infected ticks. As some of these infectious agents are transmitted
rapidly by ticks, 19ISP vaccination in humans would need to elicit
almost immediate recognition of a tick bite to prevent infection.
In conclusion, 19ISP immunization can elicit acquired resistance
against I. scapularis and prevent tick-borne B. burgdorferi infection
in guinea pigs. To date, all human vaccines against infectious dis-
ease directly target pathogens or microbial targets. However, there
have been promising studies with anti-sandfly proteins that protect
the hosts against Leishmaniasis (75,76). In addition, commercial
antitick vaccines in animals reduce the tick burden but have not
been generally shown to prevent the transmission of an infectious
disease (77–80). A study in mice, however, demonstrated that vac-
cination with Rhipicephalus appendiculatus cement protein 64TRP
protects against I. ricinus–transmitted tick-borne encephalitis virus
(81). I9ISP may change that paradigm by demonstrating that a human
anti-tick vaccine may prevent infectious diseases and also suggest
that a vaccine that elicits more rapid tick recognition may be suffi-
cient to prevent infection. Studies in additional animal models and
humans will determine whether 19ISP immunization can induce
robust tick immunity and fully determine the degree of protection
that is afforded against the Lyme disease agent. These data demon-
strate that a multivalent mRNA vaccine that targets I. scapularis
Fig. 4. Gene expression analyses by RNA sequencing reveals up-regulation of proinflammatory genes in 19ISP-vaccinated guinea pigs. (A) The heatmap shows
125 differentially expressed genes that were identified by RNA sequencing (P < 0.05) with a fold change greater than or equal to 2.0. Of the 125 genes, 113 were up-regulated
and 12 were down-regulated in the 19ISP-immunized group as compared to the Luc-mRNA control group. (B) Signaling pathways involved in response to 19ISP-mRNA
vaccination were identified by KEGG pathway enrichment analysis. The top immune pathways enriched include T cell receptor and B cell receptor signaling, chemokine
signaling, IL-17 signaling, natural killer cell–mediated cytotoxicity, FcRI-mediated signaling, and C-type lectin receptor signaling.
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antigens has the potential to prevent a common tick-borne infec-
tious disease.
MATERIALS AND METHODS
Study design
The primary objective of this study was to design and evaluate the
effect of an mRNA-LNP–based vaccine composed of 19 tick salivary
proteins as a potential tool to prevent tick-borne diseases. The main
procedures used to achieve this objective were guinea pig and mice
invivo models, generation of LNP-encapsulated mRNAs, immuni-
zation modules, procurement of uninfected and B. burgdorferi–
infected ticks, ELISA, RNA sequencing, cytokine expression
analysis by quantitative reverse transcription PCR (qRT-PCR), and
B. burgdorferi infection analysis by qPCR and culturing. Mice and
guinea pigs were randomized into groups, and vaccination was
initiated when the animals reached a certain age. A minimum of 3
(and up to 10) animals were used in each group, and experimental
replicates were performed as mentioned in the figure legends. No
power analysis was performed for these experiments. Specific atten-
tion was given while handling mRNA-LNPs to maintain the tem-
perature and avoiding multiple freeze-thaw cycles. For uninfected
tick challenge, 25 ticks were attached to each guinea pig. For
B. burgdorferi–infected tick challenge, one to three ticks were attached
to each guinea pig, as mentioned in Results. For postimmunization
studies, two to four biopsies were collected from each guinea pig,
which were used for RNA and DNA isolation and culturing of
B. burgdorferi. Daily monitoring was performed to assess the numbers
of ticks attached, feeding patterns, and skin erythema, and to collect
any detached ticks from the water pan. The numbers of ticks de-
tached and recovered were used to calculate percent recovery and
measure the engorgement weights. Erythema at the tick bite sites
was assessed by two researchers blinded to the experimental groups,
and pictures were taken and scored on the basis of the percentage of
erythematous bite sites on the total attached ticks. The host immune
responses of 19ISP were determined by the presence of specific anti-
bodies in serum by ELISA, RNA sequencing from blood and skin
samples, and cytokine expression analysis in PBMCs. We did not
perform power analysis since the vaccine efficacy was unknown.
Ethics statement
For animal care and housing, the rules were followed as described in
the Guide for the Care and Use of Laboratory Animals of the National
Institutes of Health. The protocols described below for the use of
mice and guinea pigs were reviewed and approved by the Yale Uni-
versity Institutional Animal Care and Use Committee (YUIACUC),
and the approved Animal Protocol number is 2020-07941. All animal
experiments were conducted in a Biosafety Level 2 animal facility
according to YUIACUC rules.
Ticks and animals
I. scapularis ticks were obtained from the Oklahoma State University
and maintained in an incubator at 23°C and 90% relative humidity
under a 14-hour light, 10-hour dark photoperiod. Four- to
Fig. 5. Proinflammatory cytokine expression is enriched in 19ISP-immunized guinea pigs. Cytokine expression was measured using RNA from PBMCs isolated from
19ISP- or Luc-mRNA control–immunized guinea pigs 2 weeks after the second boost and stimulated with I. scapularis saliva. Relative expression of Ifng, IL2, Tnfa, IL4, IL8,
and Cxcl10 is shown normalized to guinea pig Gapdh. The figure shows data from two independent experiments, and each circle represents a guinea pig (n = 6 per group).
The P values were calculated using unpaired t tests between saliva-stimulated 19ISP and mRNA control. Data are presented as mean ± SEM.
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5-week-old female Hartley guinea pigs (Charles River Laboratories)
were used to feed nymphal ticks, as described earlier (29). Six-week-old
female C3H mice (Charles River Laboratories) were used for
tick infection.
Preparation of 19ISP mRNA-LNP
mRNA-LNPs were generated as previously described (82). mRNA
vaccines encoding individual salivary antigens with their own signal
peptide or IL-2 signal peptide, and IL-21 or firefly Luc were codon
optimized, synthesized, and cloned into the mRNA production
plasmid as described (82). mRNA production and LNP encapsulation
were performed as described (82). Briefly, the sequence of mRNAs
was transcribed to contain 101-nucleotide-long poly(A) tailsN1-
methyl-pseudouridine-5′-triphosphate (TriLink) and, instead of
uridine 5′-triphosphate (UTP), was used to generate modified
nucleoside-containing mRNA. Capping of the invitro–transcribed
mRNAs was performed cotranscriptionally using the trinucleotide
cap1 analog, CleanCap (TriLink). mRNAs were purified by cellulose
purification, as previously described (83). All mRNAs were analyzed
by agarose gel electrophoresis and were stored frozen at −20°C. In
the multivalent 19ISP formulation, equal amounts (by weight) from
each of the 19 mRNAs were combined before LNP formulation.
mRNAs were encapsulated in LNPs using a self-assembly process in
which an aqueous solution of mRNA at acidic pH 4.0 was rapidly
mixed with a solution of lipids dissolved in ethanol (84,85), which
contains an ionizable cationic lipid–phosphatidylcholine–cholesterol–
polyethylene glycol (PEG) lipid (50:10:38.5:1.5 mol/mol), and were
encapsulated at an RNA–to–total lipid ratio of about 0.05 (w/w).
LNPs were stored in small aliquots at −80°C at a concentration of
mRNA of about 1 g/l. The LNPs had a diameter of about 80nm
as measured by dynamic light scattering using a Zetasizer Nano ZS
(Malvern Instruments Ltd.) instrument, with a polydispersity index
of 0.02 to 0.06 and an encapsulation efficiency of about 95%. Two or
three batches from each mRNA-LNP formulations were used in
these studies, and we did not observe variability in vaccine efficacy.
LNPs used in this study were prepared by Acuitas Therapeutics.
Immunization of guinea pigs
Female Hartley guinea pigs (5 weeks old) were immunized intra-
dermally with 50 g of 19ISP mRNA-LNPs (about 2.63 g perantigen)
or murine IL-21 or Luc mRNA-LNP (control). Because a combina-
tion of 19 antigens has not previously been used, the approximate
dosage was estimated on the basis of previous studies performed in
mice and guinea pigs (65,68,86). Intradermal immunization is more
efficient when using lower doses of antigens, without compromising
the efficacy (65,68,87). The required amount of frozen mRNA-LNPs
was thawed at room temperature, diluted with sterile phosphate-
buffered saline (PBS), and used within 2 hours of injection. The
animals were boosted twice at 4-week intervals. The animals were
bled retro-orbitally 2 weeks after the last immunization to obtain
blood for RNA sequencing, and the serum was separated for use
in ELISAs.
Generation of recombinant salivary proteins
RNA was isolated from salivary glands dissected from I. scapularis
ticks fed to repletion, and complementary DNA (cDNA) was syn-
thesized according to the manufacturer’s protocol (iScript cDNA
Synthesis Kit, Bio-Rad). Gene-specific primers (table S1) were used
to amplify the mRNA region encoding the mature proteins listed in
Table1. Purified amplicons were then cloned into the pMT-Bip-
V5-HisA cloning vector or the pET28A Escherichia coli expression
vector, and recombinant DNA was sequenced at the Keck sequencing
facility, Yale University, to validate the clones. Recombinant proteins
were generated using a Drosophila expression system as described
earlier for different antigens (36,44). For expression in E. coli, the
clones were transformed in endotoxin-free ClearColi cells BL21(DE3)
(Lucigen) and expressed with an N-terminal His6 tag. The proteins
were purified using Ni2+-NTA-agarose resin according to the man-
ufacturer’s instructions (Qiagen). Protein purity was assessed by
SDS–polyacrylamide gel electrophoresis using 4 to 20% gradient
precast gels (Bio-Rad) and quantified using a bicinchoninic acid (BCA)
protein estimation kit (Thermo Fisher Scientific). Recombinant
IsPDIA3 was generated as a GST-fusion (glutathione-S-transferase)
protein in E. coli using the pGEX-4T2 vector (45), and recombinant
protein was purified using GST resin according to the manufacturer’s
protocol (GE Healthcare Life Sciences).
ELISA assessment of recombinant salivary proteins
To assess the 19ISP specific antibody response against individual
proteins, 96-well Immunosorp ELISA plates were coated overnight
with 250ng of recombinant proteins, blocked with 3% bovine se-
rum albumin for 1 hour at 37°C, and incubated with guinea pig
anti-19ISP sera collected 2 weeks after the last immunization dose
at 1:500, 1:5000, or 1:50,000 dilutions for 2 hours. Each step was
separated by three washes with PBST (PBS with 0.025% Tween 20).
Bound antibody was detected with horseradish peroxidase–
conjugated goat anti-guinea pig IgG secondary antibody and tetra-
methylbenzidine (TMB) substrate solution (Thermo Fisher Scientific).
The reaction was stopped by TMB stop solution, and absorbance
was read at 450nm.
RNA sequencing analysis
Total RNA was extracted from whole blood obtained from guinea
pigs 2 weeks after the final immunization. TRIzol was added to the
whole blood, and RNA was isolated according to the manufacturer’s
instructions (Qiagen). RNA was submitted for library preparation
using TruSeq (Illumina) and sequenced using Illumina HiSeq 2500
by paired-end sequencing at the Yale Center for Genome Analysis.
RNA sequencing analyses including alignment, quantitation, normal-
ization, and differential gene expression analyses were performed
using Partek Genomics Flow software. Specifically, RNA sequencing
data were trimmed and aligned to the guinea pig genome (Cavea porcellus,
Cavpor 3.0 from Ensembl), with associated annotation file using
STAR (v2.7.3a) (88). The aligned reads were quantified to Ensembl
transcripts using the Partek E/M algorithm (89), and the subsequent
steps were performed on gene-level annotation followed by total
count normalization. The gene-level data were normalized by di-
viding the gene counts by the total number of reads followed by the
addition of a small offset (0.0001). PCA was performed using de-
fault parameters for the determination of the component number,
with all components contributing equally in Partek Flow. Hierarchal
clustering was performed on the genes that were differentially
expressed across the conditions (P<0.05, fold change ≥2 for each
comparison). Pathway enrichment was conducted by converting the
guinea pigs Ensembl gene symbol to the Entrez gene ID for mice as
described by Kurokawa etal. (70), since the guinea pig genome is
not annotated in Partek Flow. The top 10 immune pathways were
further plotted on a bubble diagram by ggplot2in R studio.
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Uninfected tick challenge of Guinea pigs
Two weeks after the last immunization dose, guinea pigs were chal-
lenged with uninfected I. scapularis ticks, as described earlier (29).
Briefly, after anesthetizing by intramuscular injection of a ketamine
and xylazine mixture, the guinea pigs were challenged with 25
I. scapularis nymphs. Ticks were allowed to attach to the shaved
backs of guinea pigs. Guinea pigs were housed individually with
three layers of tick containment (a pan of water below the wire
bottom of the cage, a hopper-inclusive lid, and grease around the
outer edges of the cage).
Infected tick challenge and B. burgdorferi transmission
of guinea pigs
To generate B. burgdorferi–infected mice and nymphs, B. burgdorferi
N40 was inoculated in C3H mice as described previously (17).
Approximately, 100 l of 1 × 105 N40 spirochetes/ml was injected
subcutaneously. I. scapularis larvae were placed on B. burgdorferi–
infected C3H mice and fed larvae molted to generate B. burgdorferi–
infected nymphs. For B. burgdorferi transmission to guinea pigs,
three B. burgdorferi N40–infected nymphs were placed on each
guinea pig (at least five animals in each group) and allowed to feed
until the appearance of erythema (up to 120 hours after tick chal-
lenge), after which ticks were pulled off carefully using forceps. All
control and experimental animals were examined for erythema in a
double-blinded manner. Additional experiments were performed
in which one or three B. burgdorferi–infected tick(s) were placed on
each control and 19ISP-immunized guinea pigs, and the ticks were
allowed to take a blood meal until they naturally detached from the
animals. After tick detachment, the transmission was assessed by
culture and by qPCR of skin punches at 3 weeks. In a previous
study, we have examined skin, blood, spleen, and bladder after
B. burgdorferi–infected ticks were allowed to engorge on guinea pigs
and were not able to detect spirochetes in tissues other than skin
(16). In several of the animals in the current study, we looked at
numerous internal sites and, consistent with the previous experi-
ment, were only able to detect B. burgdorferi in the skin of the guinea
pigs, indicating that skin is an ideal site to test transmission.
qPCR to estimate spirochete burden
Guinea pig skin punch biopsies were obtained from sites near and
distal to tick attachment sites at 3 weeks after tick engorgement. The
biopsies were suspended in a DNAeasy suspension buffer (Qiagen)
containing proteinase K and processed for DNA isolation using a
DNAeasy kit (Qiagen) according to the manufacturer’s protocol.
DNA was analyzed by qPCR using the iTaq Sybr Green Supermix
(Bio-Rad) for the presence of B. burgdorferi using flab, and results
were normalized using Actin (table S1).
Cytokine expression analysis from PBMCs
PBMCs were isolated from guinea pig blood 2 weeks after the third
immunization and stimulated with 3 l of tick saliva in a total vol-
ume of 100 l of RPMI-1640, supplemented with 10% fetal bovine
serum (90) for 24 hours at 37°C. RNA was extracted from stimulated
and unstimulated PBMCs using the Qiagen RNeasy kit, and cDNA
was prepared from the purified RNA using an iScript cDNA synthesis
kit (Bio-Rad). cDNA was analyzed by qRT-PCR using the iTaq Sybr
Green Supermix (Bio-Rad) for the expression of guinea pig–specific
cytokines and chemokines, including IFN-, TNF-, CXCL10, IL-2,
IL-4, and IL-8 (table S1). The relative expression was calculated using
the Cq method and normalized to the expression of the guinea pig
gapdh gene.
Statistical analysis
Data for ELISA, skin erythema, engorgement weights, and tick
detachment/recovery were analyzed by Prism 9.0 software (GraphPad
Software). The significance of the difference between controls and
experimental groups was analyzed by ordinary analysis of variance
(ANOVA), two-way ANOVA, or Mann-Whitney/Welch’s test (as
mentioned in respective figure legends). P≤0.05 was considered
statistically significant.
SUPPLEMENTAL MATERIALS
www.science.org/doi/10.1126/scitranslmed.abj9827
Figs. S1 to S5
Table S1
Data files S1 to S3
View/request a protocol for this paper from Bio-protocol.
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Acknowledgments: We thank M. M. H. Sung and Y. K. Tam at Acuitas (Vancouver, Canada) for
preparing the mRNA nanoparticles. Funding: This work was support by grants from the NIH
[AI152206 (E.F.), AI126033 (E.F. and S.N.), and AI138949 (U.P., E.F., and S.N.)] and the Steven and
Alexandra Cohen Foundation (E.F.). Author contributions: This work was conceptualized by
A.S., J.M., G.A., S.N., D.W., and E.F. Guinea pig and mouse immunization, tick challenge, and
B. burgdorferi transmission experiments were performed by A.S., J.M., C.K., K.D., G.L., N.O.S., and
S.N. qPCR and B. burgdorferi culturing were done by A.S., J.M., K.D., S.N., and M.-J.W. Protein
expression and purification and ELISAs were performed by A.S., G.A., M.-J.W., and S.N. RNA
isolation, sequencing, and analysis were performed by G.A. and X.T. PBMC isolation and
cytokine expression were performed by G.A. and A.S. mRNA LNPs were designed and
generated by E.F., N.P., and D.W. Data analysis and interpretation were done by A.S., J.M., G.A.,
X.T., N.P., U.P., S.N., D.W., and E.F. The manuscript was drafted by A.S., J.M., G.A., and E.F. and
reviewed and modified by all the authors. Competing interests: E.F. has an equity interest
and serves as a consultant for L2 Diagnostics. The lipid and LNP composition are described in
U.S. patent US10,221,127. D.W., E.F., and S.N. are coinventors on a patent application
(US 63/234,508) entitled “mRNA vaccines against tick salivary proteins, and methods of using
same.” The other authors declare that they have no competing interests. Data and materials
availability: All data associated with this study are present in the paper or the
Suppleme ntary materials. Reagents, including tick antigens and mRNA vaccines, will be
made available to th e scientific community by contacting E.F. and D.W. and completion of a
material transfer agreement. All RNA sequencing data are deposited at NCBI GEO under
accession number GSE184063.
Submitted 15 June 2021
Accepted 22 September 2021
Published 17 November 2021
10.1126/scitranslmed.abj9827
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mRNA vaccination induces tick resistance and prevents transmission of the Lyme
disease agent
Andaleeb SajidJaqueline MatiasGunjan AroraCheyne KurokawaKathleen DePonteXiaotian TangGeoffrey LynnMing-Jie
WuUtpal PalNorma Olivares StrankNorbert PardiSukanya NarasimhanDrew WeissmanErol Fikrig
Sci. Transl. Med., 13 (620), eabj9827.
An mRNA vaccine for ticks
Repeated exposures to the black-legged tick, Ixodes scapularis, can lead to acquired resistance against ticks or “tick
immunity.” To bypass the need for repeated exposures while still generating tick resistance, Sajid et al. developed
an mRNA vaccine that encoded for 19 I. scapularis salivary proteins (19ISP). Guinea pigs vaccinated with 19ISP
developed erythema at the site of tick attachment, a feature of acquired tick resistance. This led to poor tick feeding
and, in the case of ticks infected with the Lyme disease agent, Borrelia burgdorferi, reduced transmission of the
pathogen. Thus, 19ISP is a promising candidate for antitick vaccines that may also prevent transmission of tick-borne
diseases.
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