Vaccination against Lyme disease: past, present, and future

Division of Bacteriology and Parasitology, Tulane National Primate Research Center Covington, LA, USA.
Frontiers in Cellular and Infection Microbiology (Impact Factor: 3.72). 02/2013; 3:6. DOI: 10.3389/fcimb.2013.00006
Source: PubMed
ABSTRACT
Lyme borreliosis is a zoonotic disease caused by Borrelia burgdorferi sensu lato bacteria transmitted to humans and domestic animals by the bite of an Ixodes spp. tick (deer tick). Despite improvements in diagnostic tests and public awareness of Lyme disease, the reported cases have increased over the past decade to approximately 30,000 per year. Limitations and failed public acceptance of a human vaccine, comprised of the outer surface A (OspA) lipoprotein of B. burgdorferi, led to its demise, yet current research has opened doors to new strategies for protection against Lyme disease. In this review we discuss the enzootic cycle of B. burgdorferi, and the unique opportunities it poses to block infection or transmission at different levels. We present the correlates of protection for this infectious disease, the pros and cons of past vaccination strategies, and new paradigms for future vaccine design that would include elements of both the vector and the pathogen.

Full-text

Available from: Monica E Embers, Sep 18, 2014
REVIEW ARTICLE
published: 12 February 2013
doi: 10.3389/fcimb.2013.00006
Vaccination against Lyme disease: past, present, and future
Monica E. Embers
1
*
and Sukanya Narasimhan
2
1
Division of Bacteriology and Parasitology, Tulane National Primate Research Center, Covington, LA, USA
2
Section of Infectious Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA
Edited by:
Lisa A. Morici, Tulane University
School of Medicine, USA
Reviewed by:
Maria Gomes-Solecki, The
University of Tennessee Health
Sciences Center, USA
Jose Ribeiro, National Institute of
Allergy and Infectious Diseases,
USA
*Correspondence:
Monica E. Embers, Division of
Bacteriology and Parasitology,
Tulane National Primate Research
Center, Tulane University Health
Sciences Center, 18703 Three
Rivers Road, Covington, LA 70433,
USA.
e-mail: members@tulane.edu
Lyme borreliosis is a z oonotic disease caused by Bo rrelia burgdorferi sensu lato bacteria
transmitted to humans and domestic animals by the bite of an Ixodes spp. tick (deer
tick). Despite improvements in diagnostic tests and public awareness of Lyme disease,
the reported cases have increased over the past decade to approximately 30,000 per
year. Limitations and failed public acceptance of a human vaccine, comprised of the outer
surface A (OspA) lipoprotein of B. burgdorferi, led to its demise, y et current research has
opened doors to new strategies for protection against Lyme disease. In this review we
discuss the enzootic cycle of B. burgdorferi, and the unique opportunities it poses to block
infection or transmission at different levels. We present the correlates of protection for this
infectious disease, the pros and cons of past vaccination strategies, and new paradigms
for future vaccine design that would include elements of both the vector and the
pathogen.
Keywords: Lyme disease, vaccine, reservoir, vector, tick, Ixodes scapularis, Borrelia burgdorferi
INTRODUCTION
In the United States, during 2000–2010, a total of 251,720 con-
firmed cases of Lyme disease were reported to the CDC by health
departments in the 50 states, the District of Columbia, and US
territories; the annual count increased 101%, from 9908 cases
in 1992 to 19,931 cases in 2006 and has approached 30,000
more recently . Twelve states account for 95% of cases nation-
ally: Connecticut, Delaware, Maine, New Hampshire, New Jersey,
New York, Pennsylvania, Massachusetts, Rhode Island, Virginia,
Minnesota and Wisconsin. Despite increased public knowledge
of Lyme disease and improvements in diagnosis, the incidence of
Lyme disease in North America has not declined. In fact, evidence
indicates that in Canada and Europe the number of Lyme disease
cases is on the rise (Fülöp and Poggensee, 2008; Koffi et al., 2012)
and may yet increase (Ogden et al., 2008; Mannelli et al., 2012).
In six reporting eastern states of Germany, for example, the inci-
dence rose from 17.8 cases per 100,000 people in 2002 to 37.3
cases per 100,000 in 2006. Across Europe and North America,
the number of reported cases is probably a significant under-
estimate of actual cases (Henry et al., 2011; Scott et al., 2012).
Adding to the public health impact, Lyme disease spirochetes
have also been identified in Asia and Australia, with the actual
disease incidence yet unclear (Mayne, 2011; Stanek and Reiter,
2011).
Vaccination against infection is a high ly effective means to con-
trol the spread of disease in a population. In general, vaccines
in common use protect against highly transmissible diseases and
effectiveness is largely based on the generation of herd immunity.
In this review, we discuss vaccination with regard to protection
against Lyme disease—a disease that is not readily transmitted
from person to person, but one that is both vector-borne and one
whose risk is largely influenced by geography. Despite these limits
of contagion, Lyme disease has become a serious and expensive
public health problem. The impetus for development of a vac-
cine gained momentum in the 1990’s and led to the approval of
the first Lyme disease vaccine for human use. Only on the mar-
ket for 4 years, several factors led to its failure and enthusiasm
for a subsequent product may be founded more in basic science
than in the pharmaceutical industry. Prominent scientists have,
however, called for renewed interest in a Ly me disease vaccine
(Plotkin, 2011; Poland, 2011). Among them, renowned vacci-
nologist Stanley Plotkin published an article calling the removal
oftheLymevaccinea“publichealthfiasco(Plotkin, 2011).
Notwithstanding, the possible avenues to protect against Lyme
disease include interruption of transmission and infection at mul-
tiple points (Figure 1). Current research extends potential well
beyond simple vaccination of humans and here we highlight spe-
cific approaches, with emphasis on vaccination against the tick
vector .
THE ESSENTIAL ANTIBODY RESPONSE
Studies in mice have shown that immunity to reinfection w ith
B. burgdorferi is short-term and declines significantly by 1
year (Piesman et al., 1997). Reports of human or non-human
primate infection with both the Lyme disease spirochete and
Relapsing Fever-causing spirochetes also indicate that incidental
hosts are likewise susceptible to reinfection (Felsenfeld and Wolf,
1975; Nowakowski et al., 1997; Golde et al., 1998). Therefore,
immune responses generated during the natural course of infec-
tion are insufficient for long-term protection. Interestingly, pas-
sive immunization with serum from acute infection in mice
(Barthold et al., 1997) or chronic infection in humans (Fikrig
et al., 1994) has been shown to be protective against challenge in
mice. In fact, the importance of antibody responses in controlling
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FIGURE 1 | Points a t which interruption of B. burgdorfe ri transmission to humans can be achieved through vaccination.
these bacterial infections is well-established (Fikrig et al., 1997;
McKisic and Barthold, 2000). These and other findings indicate
that the B. burgdorferi spirochetes alter antigen expression dur-
ing infection so as to evade the antibody response and do not
elicit effective memory responses to protective antigens (i.e., those
that are expressed by all spirochetes and likely essential for infec-
tivity). Thus, identification of suitable antigens for induction of
protective immunity has been a challenge.
VACCINE DEVELOPMENT: CONSIDERATIONS
Development of protective vaccines requires appraisal of multiple
factors, both common and pathogen-specific. Given the transmis-
sion mode and antigenic variation of B. burgdorferi, qualities that
pertain specifically to this vector-borne infection must be scru-
tinized. As with many pathogens, the use of whole-cell lysates
vs. subunit antigens is a safety concern for human use. In pre-
vious studies, cross-reactivity between anti-Borrelial whole-cell
antibodies and host tissue antigens led to emphasis on a sub-
unit vaccine (Aberer et al., 1989; Sigal, 1993; Garcia-Monco
et al., 1995). Immunodiagnosis and how this may be affected is
another consideration. A whole-cell lysate vaccine would induce
polyclonal antibody responses to multiple antigens that would
make differentiation between vaccination and infection diffi-
cult. Similarly, conserved antigens amongst spirochetes and other
bacteria could confound interpretation of diagnostic tests for
Lyme (Shin et al., 1993). Subunit vaccines, rather, would induce
responses to single or few antigens, easily distinguished from an
infection response. Ancillary to this consideration is the question
of how protection and efficacy can be determined, if a serologi-
cal approach is required. For Borrelia, a pathogen with multiple
species and variants found on several continents, concern about
whether or not the vaccine will protect against other genospecies
or variants should be included. Also, given the ability of the tick
vector to harbor and transmit multiple pathogens concurrently
upon feeding, the protection against possible co-infections must
be taken into account. Lastly, and of significant importance, is the
duration and type of immunity elicited. The generation of long-
lasting B cell memory responses to B. burgdorferi or tick antigens
would be ideal. This would limit the need for multiple booster
injections to retain immunity.
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BACTERINS AND VETERINARY VACCINES
Early studies on the immunogenicity of whole cell, killed (bac-
terin) preparations of the spirochetes demonstrated protection in
hamsters w/formalin-inactivated Borrelia (Johnson et al., 1986a).
Serum from vaccinated animals protected in passive immuniza-
tion studies, indicating that protection is largely, if not com-
pletely, antibody-mediated (Johnson et al., 1986b). Not long
after the discovery of the Lyme disease agent, natural infec-
tion of dogs became apparent (Lissman et al., 1984; Kornblatt
et al., 1985; Magnarelli et al., 1985). As such, so did the inter -
est in a veterinary vaccine. Currently, several licensed canine
vaccines have become available. Bacterin vaccines, manufac-
tured by Fort Dodge Labs (now Pfizer) (Chu et al., 1992;
Levy et al., 1993) and Schering-Plough Animal Health (Galaxy
Lyme) are available. Two subunit vaccines have also progressed
to market—one consisting of just the B. burgdorferi outer sur-
face protein A (OspA) subunit, Recombitek®, manufactured by
Merial (Conlon et al., 2000), and another combining OspA
and OspC subunits (Novibac® Lyme) by Merck Animal Health.
These subunits will be discussed extensively in the next sec-
tion. Though these vaccines appear to exhibit satisfactory effi-
cacy, safety (side effects) and necessity continue to be under
scrutiny (Littman et al., 2006). In certain circumstances serolog-
ical surveillance of dogs can be used as a measure of endemic-
ity for human Lyme disease (Rand et al., 1991; Hamer et al.,
2009). Vaccination with the bacterin may interfere with this t ype
of surveillance unless a properly chosen test (O’Connor et al.,
2004) is used, so in that regard the subunit vaccines may be
preferred.
OspA: THE FDA-APPROVED TRANSMISSION-BLOCKING
VACCINE
Several antigenic subunits of B. burgdorferi hav e been evaluated
for their vaccine potential, many of which are listed in Ta ble 1 .
For one reason or another, all antigens listed, other than OspA,
have not been legitimized as vaccine candidates on their own.
OspA is a lipoprotein whose expression is abundant on in vitro-
cultured spirochetes and spirochetes within the tick midgut.
This lipoprotein is also quite immunogenic, as antibodies are
detect ed in experimentally infected animals. Importantly, due to
the expression of ospA in a natural infection largely confined to
the tick midgut, antibodies are not typically induced following
tick-mediated infection. Compared to the immunodominant B.
burgdorferi antigen, OspC, the OspA lipoprotein is reasonably
well-conserved among North American strains; immunization
with OspA, but not OspC, was shown to provide cross-protection
of mice challenged with North American isolates of Borrelia
burgdorferi (Probert et al., 1997). Sequence analysis revealed that
the ospA genes from these three isolates were >99% homolo-
gous, whereas the ospC genes shared only 81–85% homology.
Western blot analysis suggested antigenic heterogeneity associated
with OspC but not OspA. The production of polyvalent chimeric
OspC molecules may, how ev er, enhance the potential for its use
as an immunogen against Ly me disease (Earnhart and Marconi,
2007a,b; Earnhart et al., 2011).
Initial studies on the protective efficacy of OspA utilized
a mouse model of vaccination with recombinant OspA pro-
tein (Fikrig et al., 1990). Long-term (180 days) protection was
elicited against challenge by intradermal inoculation of cultured
Table 1 | Prospective Lyme vaccine antigens from B. burgdorf eri.
B. burgdorf eri
antigen
Mechanism of protection How tested Result References
OspA Antibody-mediated,
transmission-blocking
Challenge of mice by
injection, tissue transplant
and tick transmission;
challenge of monkeys by tick
transmission
Efficacious, dependent upon
antibody titer
Fikrig et al., 1990, 1992a,b;
Probert and Lefebvre, 1994;
Telford et al., 1995; Philipp et al.,
1997
OspB Antibody-mediated; elicits
bactericidal antibodies
Active and passive
protection against injection
challenge
Potent ial for
strain-dependent efficacy,
due to truncations of OspB
proteins in some strains
Fikrig et al., 1993; Telford et al.,
1993; Colemanx et al., 1994;
Probert and Lefebvre, 1994;
Probert et al., 1997
OspC Antibody-mediated, within
host
Challenge of mice by
injection and tick
transmission
Effective, but with minimal
cross-species protection;
failure to elicit long-term
(anamnestic) response
Probert and Lefebvre, 1994;
Gilmore et al., 1996, 2003;
Probert et al., 1997
DbpA Antibody-mediated, within
host
Challenge of mice by
injection and tick
transmission
Protective against injected,
but not t ick-transmitted
infection
Hanson et al., 1998; Hagman
et al., 2000
Bbk32 (p35) Antibody-mediated, within
tick
Passive immunization
against injection and tick
challenge
Ef ficacy in combination with
DbpA and OspC against
challenge by injection but
not singly
Fikrig et al., 1997, 2000; Brown
et al., 2005
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Embers and Narasimhan Vaccination against Lyme disease
B. burgdorferi (Fikrig et al., 1992a). Immunization in this manner
was shown to be ineffective against challenge with host-adapted
spirochetes, transferred by transplant of skin from an infected
mouse to a naïve mouse (Telford et al., 1995). This finding, we
now know, resulted from the aforementioned down-regulation
of ospA expression once the spirochetes enter and adapt to the
host. This variation in antigen expression compels the testing of
any putative immunogen against B. burgdorferi challenge by tick-
mediated infection. When immunization with OspA was tested
in this manner, protective efficacy was found to be significant,
and the spirochetes were eliminated from the tick vector by the
serum antibodies post-feeding (Fikrig et al., 1992b). Eventually,
the mechanism of protection as it related to expression of ospA
by the spirochetes inside ticks was firmly established (De Silva
et al., 1996). A subsequent study demonstrated that the anti-OspA
antibody titer required to eradicate spirochetes from feeding
ticks (>213 μg/ml) was considerably higher than that required
(>6 μg/ml) for blocking transmission (De Silva et al., 1999).
These groundbreaking studies led to the eventual testing of
safety, immunogenicity, and efficacy in the non-human pri-
mate model of B. burgdorferi infection (Philipp et al., 1997).
Manufacture and distribution of the recombinant OspA vac-
cine, LYMErix, began by SmithKlineBeecham and it became
available to the public in December 1998. LYMErix contained
lipidized OspA adsorbed onto aluminum hydroxide adjuvant
in phosphate-buffered saline with phenoxyethanol as a preser-
vative (pH 6.5–7.1). LYMErix was produced using recombi-
nant DNA technology. The OspA gene from the ZS7 strain of
B. burgdorferi sensu stricto was placed in the pOA15 plasmid
vector and grown in Escherichia coli. The OspA lipoprotein pro-
duced was a single polypeptide chain of 257 amino acids; the
lipid moiety was covalently bonded to the N-terminus after trans-
lation. Each 0.5 ml dose containing 30 μg of lipidated OspA
with adjuvant was administered intramuscularly, and three injec-
tions were recommended. LYMErix was available only through
February 2002 and was not formulated in combination with other
vaccines.
THE OspA VACCINE’S DEMISE
Further evaluation of the immune responses of OspA-vaccinated
animals led to the identification of a target (epitope) of specific
antibodies that was strongly correlated with a protective response
(Golde et al., 1997). The epitope was designated LA-2, after the
monoclonal antibody originally used to identify it. Later, the
importance of antibodies that targeted LA-2 in humans immu-
nized with LYMErix also became apparent (St eere et al., 1998).
Though the OspA vaccine emerged in an environment with Lyme
disease reporting on the rise and heightened awareness of the
risk, both geographically and health-related, it was eventually
withdrawn from the market due to poor sales. Some of the
pros and cons of the OspA vaccine can be found in Ta bl e 2.
Regarding efficacy, the need t o keep serum antibody titers high
with multiple boosts, given the absence of antigen r e-introduction
at tick-transmitted infection, was suboptimal. However, the pub-
lic response to the vaccine is likely what had the most influence
on its diminished use. In particular, the proposed autoimmune
potential for OspA due t o its partial homology with the human
Table 2 | P ositive and negative characteristics of the OspA vaccine.
PROs CONs
Blocks transmission; easier to test
for efficacy
Required maintenance of high
antibody titers for efficacy
(multiple boosts)
Subunit; does not interfere with
immunodiagnosis
Some adverse reactions, potential
for inducing autoimmunity
Targets a reasonably conserved
protein within species
Not effective against other
tick-borne diseases
lymphocyte function associated antigen-1 (hLFA-1) was brought
into question. Patients with treatment-resistant Lyme arthritis
were reported to possess specific HLA alleles, which retain the
ability to present this autoantigen (Gross et al., 1998; Trollmo
et al., 2001). Such HLA alleles, however, were not found more
commonly in persons who developed arthritis after taking the
OspA vaccine, calling its etiology into question (Ball et al., 2009).
Less than 10% of vaccinated individuals reported side effects,
including mild, local reactions and mild arthralg ia (dose inci-
dence 0.2%) from three different formulations of the vaccine;
this result was not significantly different from the placebo group
(Van Hoecke et al., 1996). Nonetheless, reports emerged suggest-
ing that the vaccine could be arthritis-inducing (R o se et al., 2001;
Lathrop et al., 2002). This led to anti-vaccine sentiment and class
action lawsuits, along with reduced support amongst physicians
for the vaccine and eventually enough of a decline in use for
its voluntary removal by the manufacturer. Unfortunately, this
failure in North America led the leading European prospective
Lyme vaccine manufacturer, Past eur Merieux Connaught, to halt
development.
TARGETING THE RESERVOIR (MOUSE) HOST
Another option for interruption of transmission of B. burgdor-
feri to humans is through vaccination of reservoir hosts. In the
North American regions of endemicity, Peromyscus leucopus,or
the white-footed mouse, is considered the primary reservoir host,
especially for larval and nymphal feeding (Levine et al., 1985;
Anderson et al., 1987; Anderson, 1989; Mather et al., 1989). The
majority of human infections are transmitted by Ixodid ticks in
the nymphal stage (Barbour and Fish, 1993)soblockingacqui-
sition at the pre-nymphal (lar val) stage would be most effective
for preventing human infection. Though such a task may seem
daunting, the strategy remains viable for zoonotic infections with
geographic “hot spots. The control of Rabies virus in animal
populations as a method to prevent human infection has proved
effective (Niin et al., 2008; Sterner et al., 2009).
Considerations for a reservoir host vaccine include the anti-
gen type and route of delivery, the type of delivery system, and
the implementation protocol. For generating immunity against
B. burgdorferi infection, antigens that target both the spirochetes
(e.g., OspA) and the tick have been tested. Because the OspA
antigen emerged as the most efficacious vaccine in animals and
also acted by blocking transmission, it was the primary choice for
the first reservoir-targeting vaccine strategies. Also, most of the
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Embers and Narasimhan Vaccination against Lyme disease
studies that characterized OspA as a protective immunogen uti-
lized mice, indicating that the outbred (wild) population would
likely respond to vaccination with OspA. Initial studies were per -
formed on plots with different densities of white-footed mice
(Tsao et al., 2004). Using this catch and release stra tegy to vacci-
nate mice subcutaneously with OspA, the researchers found that
vaccination did have an impact on the percentage of infected ticks
the following year. This reduction in Borrelia-harboring ticks was
positively correlated with the density of both ticks and mice, sug-
gesting that targeting mouse-dense areas can have a significant
impact on carriage, but the contribution of non-mouse species
must also be considered.
Subsequent studies have focused on an approach that may be
more practical on a larger scale, which is the baited oral vacci-
nation strategy (Gomes-Solecki et al., 2006). Here, the antigen
delivery method must be stable and effective by the oral route.
Protection of mice (89%) and reduction of B. burgdorferi in vec-
tor ticks was accomplished by oral vaccination of the mice with
E. coli expressing recombinant OspA (Gomes-Solecki et al., 2006).
Advantages to baited or al vaccination of reservoir (mice) hosts
with E. coli are the efficacy and the absence of safety issues,
wherein the consumed vaccinogen is non-pathogenic and immu-
nity does not wane over time, given a sufficient amount of antigen
(Meir elles Richer et al., 2011). A nother lead group using this strat-
egy has chosen delivery of OspA by Vaccinia virus (VV) for several
reasons: (1) these viruses have a broad host range; (2) VV are sta-
ble under harsh conditions, such as encountered in the digestive
tract; (3) proteins can be expressed at high levels from VV and
only a single dose is required; and (4) ingestion of VV does not
cause disease in wildlife nor is it readily transmissible amongst
infected animals (Bhattacharya et al., 2010). However, the poten-
tial to transmit the virus to unwanted recipients remains (CDC,
2009). Under laboratory conditions, C3H mice vaccinated by oral
gavage with VV-OspA generated readily detectable antibody titers
that peaked at 42 days post-inoculation (Scheckelhoff et al., 2006).
Importantly, where 67% of ticks that fed upon control vacci-
nated mice were B. burgdorferi-positive, only 17% of those fed
upon VV-OspA vaccinated mice harbored B. burgdorferi.TheVV-
OspA was l ater tested in a durable bait formulation with outbred
Peromyscus mice to examine immune responses and the effects
on tick transmission (Bhattacharya et al., 2011). Vaccinated mice
developed antibody titers above the minimum required to pre-
vent transmission, which waned slowly over time (30–40 weeks).
In protection studies, 10/12 vaccinated mice wer e protected from
infection (5 weeks p.i.) and the acquisition of B. burgdorferi by
larval ticks fed upon infected mice was reduced from 85 to 23%
with vaccination.
The tick protein, subolesin, has also been produced in a recom-
binant VV vector for use as a reservoir vaccine (Bensaci et al.,
2012). This strategy holds the potential to prevent human infec-
tion with not just B. burgdorferi, but other pathogens such as
Babesia and Anaplasma species that are often co-transmitted.
The goal in this case is to prevent tick feeding and uptake of
these pathogens from the reservoir hosts. Initial studies with this
vaccine in laboratory mice demonstra ted a significant reduction
(52%) in tick feeding and a modest, albeit significant reduc-
tion (40%) in the transmission of B. burgdorferi by ticks to
vaccinated mice. Perhaps a combination vaccine including OspA
and subolesin would show enhanced efficacy.
The formulation consisting of the OspA antigen expressed by
E. coli was incorporated into bait and tested on outbred P. l eu -
copus mice. These studies showed that the majority of vaccinated
mice generated antibodies to a key epit ope (LA-2), were protected
from tick challenge, and significantly reduced the B. burgdor-
feri prevalence in nymphs when infected ticks were fed upon
vaccinated mice (Meirelles Richer et al., 2011). Using different
vaccine dosing strategies, the authors demonstrated that by offer-
ing repeated dosages from 4 to 16 weeks, antibody titers could
be kept high over a full year. The strategies discussed await field-
testing, with some initial work having been performed (Te lf o rd
et al., 2011). Nonetheless, by combining the knowledge gleaned
from each of these studies, it is conceivable that reservoir-targeted
vaccines could become a reality for implementation in coming
years.
TARGETING THE TICK VECTOR
Historically, vaccines against infectious agents, including
B. burgdorferi, have primarily utilized live attenuated pathogens
or antigens of the pathogen (Plotkin and Plotkin, 2011)toinduce
protective immunity. A potent alternative avenue to protect
against arthropod-borne pathogens is targeting the vector itself,
be it to eliminate the vector by using chemicals toxic to that vector
(Carroll et al., 2009; Rosario-Cruz et al., 2009; Raghavendra et al.,
2011), by para-transgenic approaches that modify the vectors’
ability to transmit pathogens or reproduce (Aksoy et al., 2008;
Hurwitz et al., 2011), or by use of vaccines targeting vector
antigens critical for the vector to feed, reproduce or transmit
pathogens (Wikel, 1988; De La Fuente et al., 2011; Mathias et al.,
2012; P arizi et al., 2012). In this section of the review we will
focus on the utility of vector-based vaccines against Ixodes species
that transmit several human pathogens including B. burgdorferi,
and provide a cohesive ove rview of some of the research efforts
that might lead to a next-generation vaccine against Ixodes ticks
and the pathogens they transmit in North America and Eurasia
(Goodman et al., 2005).
THE HARD LIFE (CYCLE) OF IXODES
A unique feature of the spirochetes of the B. burgdorferi sensu
lato complex when compared with other pathogenic spirochetes
is that it is entirely dependent on the obligate hematophagous
Ixodes tick to infect susceptible vertebrate hosts (Piesman and
Schwan, 2010). B. burgdorferi is transmitted by four species of
Ixodes ticks within the Ixodes ricinus complex, Ixodes scapu-
laris and Ixodes pacificus in the North America, and Ixodes
ricinus and Ixodes persulcatus in Europe and Asia, respectively
(Goodman et al., 2005). Additionally, Ixodes scapularis trans-
mit Anaplasma phagocytophilum, Babesia microti, and Powassan
virus in the North America and I. ricinus and I. persulca-
tus transmit tick-borne encephalitis virus in Europe and Asia
(Kurtenbach et al., 2006). The ticks and the pathogens they
transmit are maintained in a zoonotic cycle involving a diverse
array of vertebrate hosts effectively increasing the breadth of
the transmission cycles of the pathogens (Barbour and Fish,
1993; Keirans et al., 1996; Kurtenbach et al., 2006). We will
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focus on I. scapularis, the predominant vector of B. burgdor-
feri, A. phagocytophilum, B. microti, and Powassan virus in North
America.
Ixodes spp have three developmental stages, the larvae, the
nymph, and adult, and to complete development each stage
requires one blood meal on a vertebrate host. I n North eastern
and North central America Ixodes scapularis larval and nymphal
stages feed on small rodents such as Peromyscus leucopus,thepre-
dominant host (Barbour and Fish, 1993). Larvae hatch clean from
eggs laid by mated female adults, acquire the pathogen/s from
an infected host during feeding, and molt to become infected
nymphs. When infected nymphs feed on mice, the pathogens
are transmitted to the host. The nymphal stage is therefore cen-
tral to pathogen transmission to reservoir hosts. P. leucopus is
the major reservoir host for B. burgdorferi sensu stricto (Barbour
and Fish, 1993). It is not clear if P. leucopus also serves as the
predominant reservoir host for other I. scapularis-transmitted
pathogens. While humans are not natural hosts for Ixodes ticks,
ticks feed on humans upon accidental encounters of humans with
infected nymphs and this often results in transmission of the
pathogen/s (Kurtenbach et al., 2006). The larval acquisition of
B. burgdorferi from the vertebrate host and subsequent molt to
infected nymphs are thus critical determinants of infection preva-
lence, and consequent risk of infection to humans (Brunner et al.,
2011). Infected nymphs molt to become infected adults. Adult
Ixodes feed on white-tailed deer, but deer do not serve as reser-
voir hosts for B. burgdorferi (Nelson et al., 2000). It is suggested
that components of deer serum, especially serum complement
components, are Borrelicidal (Nelson et al., 2000). Further, lar-
vae do not feed on deer, smaller mammals and rodents being
their preferred hosts (James and Oliver, 1990; Schmidt et al.,
1999), additionally precluding the ability of deer to transmit
Borrelia to larvae. Nymphal feeding precedes larval feeding, usu-
ally beginning in early spring and ending in late summer. This
sets the stage for larvae to acquire pathogens that nymphs might
have transmitted to the murine host. Larval feeding begins in
late summer through fall and molt to become nymphs. Nymphs
ov er-winter and begin feeding in the subsequent spring. Nymphs
that fed in spring/summer molt to become adults and female
adults begin feeding in late fall and winter. Fed adults lay eggs
in early spring and larvae hatch in summer ready to acquire
pathogens.
In the western United States, the life cycle of the western
black-legged tick, Ixodes pacificus that is a vector for B. burgdor-
feri is complex and involves another Ixodes species, I. spinipalpis.
I. spinipalpis larvae and nymphs feed on small rodents that
serve as r eservoir hosts for B. burgdorferi. I. pacificus lar vae
and nymphs routinely feed on lizards and lizards are incompe-
tent reservoir hosts for B. burgdorferi (
Lane and Quistad, 1998).
Only occasional feeding of I. pacificus larvae and nymphs on B.
burgdorferi-infected rodents results in infected I. pacificus stages
competent to transmit the spirochete to humans (Peavey and
Lane, 1995; Salkeld and Lane, 2010). Susceptible to changes in
climate, ecology, and host population densities, the zoonotic life
cycle of Ixodes ticks is thus closely entwined with the hosts
it feeds on, and the pathogens it harbors (Kurtenbach et al.,
2006).
VACCINE TARGETING THE TICK—WHERE DO WE BEGIN?
In our pursuit for tick-based vaccines against B. burgdorferi in
particular, and I. scapularis -transmitted pathogens in general,
it is important to bear in mind that vaccines directed against
tick stages that transmit pathogens are vital. Larvae rarely hatch
infected (Patrican, 1997; Richter et al., 2012) and therefore are
unable to transmit the currently known tick-borne pathogens.
Infected nymphs are fully capable of transmitting harbored
pathogens to humans, and domestic animals (Barbour and Fish,
1993). Further, the small size of the ny mphal stage makes it diffi-
cult to notice and remove easily, hence can remain attached to the
human host long enough to promote transmission of pathogen/s
(Piesman et al., 1987). Infected female adults are likely to have
higher pathogen burdens (having had twice the opportunity to
acquire pathogens) and are fully capable of feeding and transmit-
ting pathogens to humans. However the larger size of the female
adult stage makes it easily noticeable and br iskly removed, provid-
ing much less opportunity for transmission to ensue. Therefore,
the general c onsensus is that targeting the nymphal stage might be
most relevant from a human vaccine perspective. Efforts directed
against larvae and larval acquisition of pathogens impact preva-
lence of infected nymphs in endemic areas, and efforts directed
against adult ticks to increase their mortality and decrease fecun-
dity impact tick population densities in endemic areas (Tsao et al.,
2012). The vaccine target search cannot be compartmentalized;
often proteins expressed in the nymphal stage are expressed in
both lar val and adult stages and proteins critical for feeding in one
stage might also be critical for other stages (De La Fuente et al.,
2011; Schuijt et al., 2011a; Bensaci et al., 2012). Tick salivary lectin
pathway inhibitor protein (TSLPI) is critical for transmission of
Borrelia to the murine host and also critical for Borrelia acquisi-
tion from the murine host (Schuijt et al., 2011a). Candidate vac-
cines that specifically target larval feeding or Borrelia/pathogen
acquisition from the murine host naturally have to be deliv-
ered to the reservoir host, P. leucopus. So also vaccine candidates
that specifically target adult tick feeding and fecundity have to
be delivered to the reservoir host, O. v irginianus (white-tailed
deer). We will first focus the review on nymphal antigens, since
vaccine candidates that target nymphal feeding and pathogen
transmission have utility for reservoir host and humans.
LOW-HANGING FRUITS—NYMPHAL SALIVARY PROTEINS CRITICAL
FOR FEEDING
I. scapularis nymphs feed for 3–5 days, gradually engorging to
repletion and falling off the host. During this feeding process,
the nymph inserts its hypostome into the host skin and tears
the host skin with its sharp mouth parts (mandibles) and lodges
itself firmly at the feeding site with a cement cone glue that the
salivary glands secrete in preparation for feeding and pathogen
transmission (Sonenshine, 1991). This event marks the onset of
feeding and is critical, for it brings in close proximity the tick
vect or, its harbored pathogens, and the host dermis with all its
inflammatory arsenals (Nuttall et al., 2000; Nuttall and Labuda,
2004; Hovius et al., 2008b). This event also initiates a series of
molecular signals resulting in physiological changes in the tick
gut and salivary glands critical for successful engorgement (Sauer
and Hair, 1986). Success may be det ermined entirely by the
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Embers and Narasimhan Vaccination against Lyme disease
ability of the tick to surmount host defense responses. The sali-
vary glands secrete pharmacologically active molecules (Ribeiro
and Francischetti, 2003; Nuttall and Labuda, 2004; Hovius et al.,
2008b) that defuse host immune responses including host com-
plement, pro-coagulants, proteases, histamine-binding proteins,
and innate immune cells that are recruited to the feeding site.
This prepares the ground for the exiting pathogens harbored by
the tick, and facilitates pathogen transmission, a phenomenon
described as Saliva Activated Transmission (Nuttall and Labuda,
2004). The Ixodes salivary transcriptome elaborates a variety
of histamine binding proteins, anti-complement proteins, anti-
coagulants, peroxidases, and protease inhibitors (Francischetti
et al., 2005; Ribeiro et al., 2006), a handful of which have been
fully characterized (Paesen et al., 1999; Valenzuela et al., 2000;
Gillespie et al., 2001; Muleng et al., 2001; Francischetti et al., 2002,
2003, 2004; Narasimhan et al., 2002, 2007b; Sangamnatdej et al.,
2002; Daix et al., 2007; Schroeder et al., 2007; Guo et al., 2009;
Juncadella and Anguita, 2009; Schuijt et al., 2011a,b). Further,
the genome encodes structural paralogs of several genes, whose
functions r emain to be determined (Hill and Wikel, 2005; Pagel
Van Zee et al., 2007). Several questions arise: Why this struc-
tural paralog y? Are structural paralogs also functional paralogs?
If so, are all the paralogs expressed simultaneously? Are the struc-
tural paralogs stage-specific? Structural paralogs are thought to
represent a fall-back strategy, but this has not been fully tested.
Because, ticks feed on multiple hosts, the structural and func-
tional paralogs might be preferentially expressed on different
hosts (Narasimhan, Unpublished) and might redirect our search
for vaccine candidates tailored for targeting tick feeding on reser-
voir hosts or humans. The multifaceted strategies of the tick
are the bane of research efforts to identify tick-based vaccine
candidates. Nevertheless, since feeding is central t o tick survival
and pathogen transmission, targeting salivary proteins critical for
feeding presents a logical starting point. Further, salivary proteins
are secreted into the host at the feeding site and have the added
advantage of providing anamnestic responses to boost vaccine
efficacy.
A few salivary proteins have been tested for their vaccine
potential to block nymphal or adult feeding, with some promise
(Hovius et al., 2008b; P arizi et al., 2012). While many provide
impaired feeding, blocking tick feeding has been difficult. Clearly,
ticks encode an impressive array of molecules to defuse host
immune responses and vaccines targeting one or two components
are not sufficient to derail tick feeding effectively. For example,
I. scapularis salivary protein molecules such as ISAC (Valenzuela
et al., 2000), Salp20 (Tyson et al., 2007), Salp15 (Schuijt et al.,
2008), and TSLPI (Schuijt et al., 2011a) inhibit different arms of
host complement pathways and vaccines that target these in com-
bination might be a more viable option; this has yet to be tested.
Similarly, anticoagulant proteins including Ixolar is, Penthalaris,
Salp14 (Francischetti et al., 2002, 2004; Narasimhan et al., 2002),
and P28 (Sc huijt et al., 2011b), identified from I. scapularis adult
or nymphal salivary glands inhibit different components of the
coagulation cascade and might be targeted in combination rather
than individually to inhibit successful feeding. More recently,
I. ricinus salivary glands were shown to elaborate a novel Serpin,
IRS-2, that defused host inflammation and thrombin-induced
platelet aggregation (Chmelar et al., 2011). Homologs of I. scapu-
laris IRS-2 might be added to a vaccine cocktail targeting several
anticoagulants to block tick feeding. Mining the genome and
transcriptome reveals a long list of putative secreted proteins that
might be vaccine targets, but the daunting task of prioritizing a
set of potential targets remains the confounding obstacle.
ACQUIRED RESISTANCE TO TICKS—JUST A MATTER OF TIME
Several decades ago, William Trager obser ved that rabbits infested
repeatedly with Dermacentor ticks develop a robust immune
response against tick components that results in rapid rejec-
tion of ticks (Trager, 1939), and since then this phenomenon
of acquired tick resistance has been noted in various tick-host
models (Wikel and Alarcon-Chaidez, 2001). Iscapularisticks feed
successfully on guinea pigs and rabbits, the laborator y models of
non-reservoir hosts, at first infestation, but subsequent infesta-
tions result in dramatic reduction in feeding and ticks fall off or
die within 12–24 h (Allen, 1989). Interestingly, this phenomenon
does not occur upon repeated infestations of I. scapularis ticks
on the murine host, the chosen reservoir host (Wikel et al.,
1997), for reasons that are not well understood. The hallmark
of tick resistance is the swelling and redness at the tick bite-
site (Figure 2) due to cutaneous basophil hypersensitivity, or the
rapid recruitment of basophils to the tick bite-site (Brossard and
Fivaz, 1982; Wikel and Alarcon-Chaidez, 2001) and is apparently
mediated by the concerted activation of humoral and cellular
responses. Recruitment of basophils to the bite site, followed
by their degranulation, effectively thwarts tick feeding, and pro-
motes tick mortality by mechanisms that are not fully understood
(Brown, 1982; Brown and Askenase, 1983, 1985). It is presumed
that salivary proteins secreted into the bite site provoke the
immune response in the host that recruits basophils to the site
(Bro wn and Askenase, 1985; Wikel and Alarcon-Chaidez, 2001).
Importantly , when B. burgdorferi-infected nymphs were allowed
to feed on tick-immune guinea pigs, B. burgdorferi transmission
was also dramatically impaired (Nazario et al., 1998; Narasimhan
et al., 2007a). Hence, there is an ongoing interest to exploit the
phenomenon of acquired tick resistance to identify tick salivary
proteins that are natural targets of host immunity (Schuijt et al.,
2011b). It is anticipated that this would help define salivary pro-
tein candidates that might serve as vaccine targets to block tick
feeding and Borrelia transmission.
Different molecular approaches have been utilized to identify
proteins in engorged tick salivary glands that react with tick-
immune sera and several I. scapularis nymphal salivary proteins
with interesting biochemical functions have been identified (Das
et al., 2001; Schuijt et al., 2011b). Despite the robust reactivity
of these proteins with tick-immune sera, that include peroxire-
doxins, anticoagulants, anticomplements, and histamine binding
proteins, the vaccine potential of these proteins when teste d in a
cocktail to block nymphal and adult tick feeding remains mod-
est (20–30% reduction in engorgement weights) (Schuijt et al.,
2011b). Further, not all ticks in the immunized group are affected,
and this variation in efficacy also poses a bottleneck, especially
from a human vaccine perspective.
A careful analysis of the tick salivary transcriptome suggested
that the tick salivary proteome might be dynamic, and change
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Embers and Narasimhan Vaccination against Lyme disease
FIGURE 2 | Ixodes scapularis infestations of guinea pigs results in the development of acquired resistance to ticks. Nymphs feeding on: (A) naïve guinea pig
shows no redness at the tick bite-sites and (B) repeatedly tick-infested guinea pig shows increased redness around the tick bite-site within 24 h of tick attachment.
FIGURE 3 | Temporally changing composition of tick saliva spit into
the host skin. Schematic representation of the dynamic tick saliva.
Ixodes scapularis engorge on vertebrate host skin for 3–7 days spitting
saliva into the host dermis at the bite-site. Salivary composition
potentially changes during feeding to confront the different host defense
responses.
during feeding (Narasimhan et al., 2007a). Feeding proceeds not
as one “big gulp nor as a steady “sipping, but proceeds in phases
defined grossly as slow in the first 1–2 days and then rapid in
the l ast 3rd and 4th day (Anderson and Magnarelli, 2008). It is
then plausible that the salivary proteome changes to meet feed-
ing phase-specific requirements (Figure 3). Histopathological
and molecular examination of the dermis at the tick bite site
also showed differences in the composition of the inflammatory
milieu that accumulates in the early and late stages of feeding
(Krause et al., 2009; Heinze et al., 2012). During the final rapid
feeding phase, I. scapularis ticks have been shown to secrete a
protein that facilitates release of histamines from neutrophils,
mast cells and possibly basophils to increase vasodilation and
acceleratetheflowofbloodtothebitesite(Dai et al., 2010).
Understanding the dynamics of the tick proteome reveals a pos-
sible drawback in the approach to identification of tick salivary
proteins targeted by host tick-immunity. Since acquired resis-
tance to tick feeding results in rapid tick rejection within the
first 12–24 h of tick attachment, presumably, host immunity is
directed against salivary proteins expressed in the early phase
and it might be critical to identify this subset of salivary pro-
teins. P erhaps we have to shift the focus away from antigens
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Embers and Narasimhan Vaccination against Lyme disease
expressed later in feeding, to antigens expressed early in tick feed-
ing. Targeting salivary proteins expressed early in feeding has
the advantage of blocking tick feeding early and blocking the
transmission of pathogens such as TBEV, A. phagocytophilum and
B. microti that are transmitted earlier in feeding than B. burgdor-
feri (Goodman et al., 2005). The process to dissect and obtain
sufficient amount of prot eins or RNA from 12 to 24 h fed salivary
glands is tedious. Yet, powerful molecular approaches are now
available to circumvent the limitations of this approach and to
spur the analysis of the early phase transcriptome and proteome
of I. scapularis (Hill and Wikel, 2005).
BLOCKING Borrelia TRANSMISSION—THE REAL DEAL
Blocking tick feeding might just be a monumental task, up against
the powerful evolutionary measures designed to ensure that the
tick saliva is equipped with protein and non-protein biomolecules
critical for feeding (Oliveira et al., 2011). But, from a human vac-
cine perspective, do we really want to block tick feeding? Is it
sufficient that we block pathogen transmission? A vaccine tha t
can effectively block pathogen transmission is undoubtedly the
public health goal and broadly applicable to the murine reser-
voir host and to humans. The first transmission blocking vaccine
against B. burgdorferi,basedontheBorrelia outer surface pro-
tein OspA (Fikrig et al., 1992a,b), was developed in 1998, but
removed from the market in 2001 due to the perceived notion
that anti-OspA antibodies were inherently arthritogenic (Rose
et al., 2001; Lathrop et al., 2002) as detailed in section “OspA: The
FDA-Approved Transmission-Blocking Vaccine. Therefore, a safe
and effective vaccine against Lyme disease remains an unmet
need.
64TRP, a subunit of a secreted salivary cement protein of
Rhipicephalus appendiculatus, was tested in a guinea pig model
of feeding and shown to provide a significant impairment of
adult R. appe ndiculatus feeding (about 23–25%) and a 45%
reduction in egg mass (Trimnell et al., 2005). Further, since
64TRP shared structural homology with proteins from several
tick species, including, I. scapular is, it has the potential util-
ity of being a broad spectrum vaccine to target multiple tick
species and consequently impair the transmission of multiple
tick-transmitted pathogens. Indeed when 64TRP was tested in
murine model of I. r icinus transmitted TBEV, the inflammation
elicited at the feeding site in 64TRP-immunized mice impaired
TBEV transmission from infected mice to nymphs, and from
infected nymphs to mic e (Labuda et al., 2006). This finding
provides new optimism in the search for tick-based vaccines
(Trimnell et al., 2005).
Two I. scapularis salivary cysteine protease inhibitors,
Sialostatin L and Sialostatin L2, were shown to play a role
in ny mphal tick feeding (Schwarz et al., 2012). Inhibition of
Cathepsin S by Sialostatin L decreased inflammation and possibly
facilitated feeding (Kotsyfakis et al., 2008). Sialostatin L2 is specu-
lated to have a role in modulating inflammatory responses, tissue
remodeling and angiogenesis by inhibiting intra or extracellular
cathepsins of innate immune cells (Kotsyfakis et al., 2010). More
importantly, inoculation of mice w ith B. burgdorferi in conjunc-
tion with Sialostatin L2 significantly promoted spirochete survival
and infection in the skin (Kotsyfakis et al., 2010), suggesting that
a vaccine targeting Sialostatin L2 might help thwart B. burgdorferi
transmission.
I. scapularis salivary protein, TSLPI, is an anti-complement
protein that inhibits the lectin pathway of the complement cas-
cade by binding to the mannose binding lectin (MBL) and pre-
venting its ability to activate the downstream signaling (Schuijt
et al., 2011a). When tested as a vac cine to block tick feed-
ing, it was not effective (Schuijt et al., 2011b). B. burgdor-
feri s.l strains are sensitive to human complement (Van Dam
et al., 1997), and in vitro studies on
B. burgdorferi s.l strains
showed that TSLPI offered protection from human comple-
ment, and antibodies against TSLPI abrogated the protective
effect (Schuijt et al., 2011a). Both immunization against TSLPI
and RNA interference-mediated decrease in TSLPI expression
in I. scapularis nymphs impaired B. burgdorferi transmission
by I. scapularis nymphs (Schuijt et al., 2011a). I. ricinus ticks
also elaborate potent anticomplement proteins, IRAC I and
II, that inhibit the alternative pathway of mammalian comple-
ment (Schroeder et al., 2007) and bear structural and functional
homology to ISAC, the I. scapularis anticomplement (Valenzuela
et al., 2000). It is then possible that cocktail vaccines of potent
structurally related anticomplement proteins might serve as a
broad-spectrum vaccine to prevent B. burgdorferi transmission
by I. scapularis and I. ricinus ticks. Various proteins with anti-
inammatory functions have been characterized to-date from
Ixodes species (Hovius et al., 2008b). Theoretically, it should be
possible to test the vaccine potential of these proteins in tick
challenge experiments in the context of Borrelia transmission as
well as in the context of other pathogens transmitted by I. scapu-
laris or I. ricinus. Testing them as a cocktail of functionally
related proteins might be a more tractable approach to accelerate
prioritization.
The discovery that Subolesin, a transcription factor expressed
by several tick species including I. scapularis, can be targeted
to decrease the feeding and fecundity of adult ticks, and feed-
ingoflarvalandnymphaltickshasrevealedanewfacetto
tick-based vaccine development (De La Fuente et al., 2011).
Subolesin is a homolog of vertebrate akirins and is evolution-
arily conserved in insects and in ticks (Galindo et al., 2009). As
discussed in section “The OspA Vaccine’s Demise, oral vaccina-
tion of murine hosts with vaccinia viruses that express Subolesin
provided protection against tick infestations and B. burgdorferi
transmission (Bensaci et al., 2012). This could have utility as
a reservoir-host vaccine. Importantly, like 64TRP, immunity to
Subolesin effectively impaired the feeding ability of several tick
species (De La Fuente et al., 2006; Merino et al., 2011). Since
Subolesin homologs were also expressed in mosquitoes, it has the
potential of being part of a broad-spectrum vaccine formulation
(Canales et al., 2009). Subolesin is an intracellular protein that
functions to tr anscr iptionally regulate NF-kB-dependent genes
(Galindo et al., 2009). The traditional approach is to target
secreted salivary proteins or at least extracellular tick proteins.
The mechanisms by which anti-Subolesin antibodies are able to
enter tick cells to target or neutralize Subolesin is not fully under-
stood, and opens new possibilities that intracellular tick proteins
can also be targets for prote ctive vaccines (De La Fuente et al.,
2011).
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Embers and Narasimhan Vaccination against Lyme disease
SEPARATE AND CONQUER—A NEW PARADIGM
ItwasshownafewyearsagothatTROSPA,atickgutprotein
helps tether B. burgdorferi to the gut by binding to OspA, an
outer surface protein of the spirochete, and that this facilitated
spirochete colonization (Pal et al., 2004). When TROSPA expres-
sion was decreased in I. scapularis nymphs, Borrelia acquisition
was impaired (P al et al., 2004). Similarly, spirochetes that did
not express OspA were unable to colonize the tick gut (Ya ng
et al., 2004) and emphasized that molecular interactions between
the tick and the spirochete were specific, and thus could be tar-
geted to derail the ability of the spirochete to colonize the tick.
Underscoring this understanding of spirochete-tick interactions,
Salp15, a multifunctional secreted I. scapularis salivary protein
has been shown to inhibit activation of CD4
+
T cells (Anguita
et al., 2002), complement activity (Schuijt et al., 2008), and den-
dritic cell function (H ovius et al., 2008a). Salp15 was also shown
to physically bind to OspC on the spirochete surface during
exit from the salivary g lands (Ramamoorthi et al., 2005; Rosa,
2005). Tilly et al. showed that OspC, an outer surface lipopro-
tein that decorates the spirochete surface as it exits the tick, is
critical for Borrelia, early in infection of the vertebrate host (Tilly
et al., 2006). It was suggested that Salp15-OspC interaction poten-
tially cloaked OspC from host immune responses and protecte d
the spirochete from Borrelicidal antibodies (Ramamoorthi et al.,
2005). This provided a significant survival edge upon entry into
the host, escaping the inflammatory host responses that accumu-
late at the tick bite site during feeding. Mice actively immunized
with recombinant Salp15, and challenged with B. burgdorferi-
infected nymphs were significantly protected from infection (Dai
et al., 2009). It is likely that antibodies directed against Salp15
sequester Salp15 away from OspC and leave it exposed to the
immune milieu, or Salp15 antibodies bind to Salp15-coated
spirochetes and deliver the spirochetes more effectively to phago-
cytes (Dai et al., 2009). Salp15 homologs were also identified in I.
ricinus ticks and similarly bound B. garinii and B. afzelii OspC to
facilitate spirochete transmission (Hovius et al., 2008c).
During transmission, the spirochete replicates, and migrates
from the gut to the salivary glands (Rosa et al., 2005), to then
exit the vector and enter the host. Elegant live imaging of ticks
infected with GFP-expressing spirochetes showed that B. burgdor-
feri associate tightly with the tick gut epithelial cells and move
as a meshed network toward the basement membrane of the
gut in preparation for egress from the gut (Dunham-Ems et al.,
2009). Extending the concept of PIP, or Pathogen-Interacting-
Proteins, to the tick gut, it has been shown that the Borrelia outer
surface protein BBE31 binds to a gut protein, TRE31 (Zhang
et al., 2011) to enhance its egress from the gut by mechanisms
that remain to be elucidated. Disrupting the interaction between
BBE31 and TRE31 compromised spirochete egress from the gut.
These findings provide a new understanding of vector–pathogen
co-operation that involves a direct interaction of the vector pro-
tein with the pathogen. These PIPs might be targeted as vaccines
to prevent Lyme disease.
THE TICK GUT-TARGETING ACQUISITION AND TRANSMISSION
Although the search for immunogens to block tick feeding and
pathogen transmission is predominantly saliva-centric, the gut
mustnotbeignored,foritistheentryandexitavenuefortick-
borne pathogens. The blood-meal, critical for tick development,
arrives in the gut and is stored in the gut for several days post-
feeding as digestion proceeds (Sauer and Hair, 1986). The gut
also presents a unique vector–host–pathogen interface wherein
the vector secretes proteases, anticomplements, antibacterial pep-
tides and anticoagulants to protect the epithelial barr ier from
pathogen and host-mediated damage and to keep the blood-
meal fluid during the long feeding period (Rudenko et al., 2005;
Anderson et al., 2008). Targeting gut components critical for the
vector to continue feeding is an equally viable option (Nuttall
et al., 2006). Salp25D, a peroxire doxin expressed both in the
salivary glands and guts was shown to be critical for Borrelia
acquisition (Narasimhan et al., 2007b) by neutralizing reactive
oxygen species at the vector–host interface and facilitating the
viability of spirochetes as they entered the gut. It is conceiv-
able that targeting TROSPA, and Salp25D simultaneously might
help to effectively block Borrelia acquisition from murine hosts,
and such a vaccination strategy could be applicable to reser-
voir host to impact infection prevalence. The gut also presents
a daunting physical and immunocompetent entry-point bar-
rier for tick-borne pathogens (Kopacek et al., 2010). Little is
known about the molecular mechanisms that Borrelia and other
pathogens utilize to surmount the barrier and an understanding
of this should open new ways to block pathogen acquisition and
transmission.
CONCLUSIONS
Several tick molecules with the potential to serve as vaccines
to impair feeding and transmission have been identified in the
last decade. The sequencing of the genome of Ixodes scapularis
(Hill and Wikel, 2005) has contributed largely to this progress
and the time is ripe to put our collective efforts to dev elop
an effective vaccine against Lyme disease. A tick-based vaccine
holds the promise that it might be useful to also simultaneously
block the transmission of other tick-borne pathogens (Wikel,
1996). The application of RNA interference technology to the tick
field has catalyzed our ability to designate physiological functions
to tick genes and to partially remove some of the bottle-necks that
the field had faced a decade ago (De La Fuente et al., 2005; Karim
et al., 2010). Technologies to genetically manipulate I. scapu-
laris are also coming of age (Kurtti et al., 2008) and represent
another milestone that will help increase our understanding of
tick genes in the context of development, feeding, and pathogen
transmission; this will help us prioritize tick antigens for vac-
cine development. Recent work has shown that immunization
of murine hosts with a combination of Salp15 a nd OspA pro-
vided better protection from B. burgdorferi infection than either
alone (Dai et al., 2009). Incorporating antigenic epitopes of crit-
ical tick and Borrelia proteins into a chimeric vaccine might thus
be a viable option for Lyme vaccine development. Vaccination
of the reservoir hosts and/or humans are not mutually exclusive
options, and targeting the reservoir populations to decrease tick
populations and interrupting acquisition or transmission cycles
in conjunction with vaccination of humans should provide the
desired goal of controlling tick-borne pathogens (Tsao et al.,
2012).
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