2NIAID Integrated Research Facility, Ft. Detrick, Frederick MD 21702
*Corresponding author:
Dr. Michael R. Holbrook, PhD
NIAID Integrated Research
Facility
8200 Research Plaza
Ft. Detrick, Frederick, MD 21702 Tel: 301-631-
7265 Fax: 301-619-5029 E-mail: Michael.holbrook@nih.gov
Received July 26, 2010; Accepted August 25, 2011; Published September 25,
2011
Tick-borne encephalitis (TBE) is a disease that is found from western Europe across Asia and into Japan. In recent
years the incidence rate has been increasing as has the endemic range of the virus. Tick-borne encephalitis is caused
by three genetically distinct sutypes of viruses within a single TBE virus (TBEV) serocomplex. These three subtypes
consist of Far-eastern subtype TBEV (TBEV-FE), Siberian subtype (TBEV-Sib) and European subtype (TBEV-Eu).
Each of these subtypes cause clinically distinct diseases with varying degrees of severity. Development of the first
vaccines for TBEV began in the late 1930s shortly after the first isolation of TBEV-FE in Russia. In the 1970s Austria
began large scale vaccine production and a nationalized vaccine campaign that significantly reduced the incidence
rate of TBE. Currently there are four licensed TBE vaccines, two in Europe and two in Russia. These vaccines are
all quite similar formalin-inactivated virus vaccines but the each use a different virus strain for production. Published
studies have shown that European vaccines are cross-protective in rodent studies and elicit cross-reactive neutralizing
antibody responses in human vaccines. European vaccines have been licensed for a rapid vaccine schedule that
could be used in response to a significant outbreak and reasonable neutralizing antibody titers can be achieved after
a single dose although a second dose provides nearly complete and long-lasting protection. This review focuses on
the current status of licensed TBE vaccines and provides a brief summary of technology currently being developed for
new vaccines.
The tick-borne encephalitis (TBE) serocomplex of flaviviruses
(Family Flaviviridae, genus Flavivirus) includes a number of viruses
that cause disease in humans. These include the TBE viruses (TBEV) of
which there are three subtypes, Kyasanur Forest disease virus (KFDV)
and the closely related Alkhurma (ALKV) and Nanjianyin viruses,
Omsk hemorrhagic fever virus (OHFV) and Powassan virus (POWV).
A subtype of POWV is deer tick virus (DTV). Also within the TBE
serocomplex is Langat virus (LGTV), a naturally attenuated virus
that is generally apathogenic in humans following natural infection.
Members of the TBE serocomplex are genetically distinct, but closely
enough related serologically that they are often difficult to distinguish
by antibody-based assays.
The diseases caused by the TBE serocomplex viruses range from
asymptomatic or mild febrile illness to hemorrhagic fever or severe
encephalitis with significant morbidity and mortality. OHFV, KFDV
and ALKV are most frequently associated with hemorrhagic disease
while POWV and TBEV infections can result in encephalitic disease.
The three subtypes of TBEV can be distinguished genetically and
often by clinical presentation. European subtype TBEV (TBEV-Eu)
is generally a biphasic disease, occasionally resulting in neurologic
disease, but with a low case fatality rate. In contrast, Far-eastern subtype
TBEV (TBEV-FE) is more frequently associated with severe neurologic
disease, relatively high case fatality rate and an increased propensity for
neurological sequelae in survivors. The Siberian subtype TBEV (TBEVSib)
is intermediate in disease severity, but has been associated with
chronic infection [1-5].
The incidence of TBE in Europe has increased significantly over the
past 40 years despite active vaccination programs in many European
countries [6,7]. The spread of TBE cases has been attributed to a number
of factors including incomplete vaccine coverage, increased abundance
of ticks, an increased range of the Ixodes ricinus tick, changes in human
life-style, socio-economic conditions and climate change [8]. There are 10,000-12,000 cases of TBE reported annually throughout Europe and
Asia (Tick-borne encephalitis International Scientific Working Group
(TBE-ISW), http://www.tbe-info.com) [9]. KFD accounts for 100-
500 cases per year in India with outbreaks in both humans and nonhuman
primates [10]. The documented incidence of OHFV, ALKV and
POWV infection is very low.
Transmission of TBE serocomplex viruses typically occurs through
the bite of an infected tick although outbreaks sometimes can also be
associated with consumption of un-pasteurized milk products from
infected sheep or goats, a disease termed "biphasic milk-fever" [2].
Percutaneous injury in laboratory or medical settings along with direct
exposure to blood or bodily fluids from infected humans or animals
are also potential routes of exposure. Infection from inhalation of
TBE virus aerosols has been documented after laboratory accidents
[11,12]. Quoting the CDC Special Pathogens Branch internet website:
"Laboratory infections were common before the use of vaccines and
availability of biosafety precautions to prevent exposure to infectious
aerosols".
Flaviviruses are typically transmitted by either mosquitoes or
ticks although there is a small subset of viruses that are not believed
to require an arthropod vector for transmission [13]. The flaviviruses
account for majority significant number of the arthropod-borne
viral diseases worldwide. Tick species associated with transmission
of TBE serocomplex viruses include the hard ticks Ixodes ricinus, Ix. persulcatus, Dermacentor spp. and Hyalomma spp. Soft ticks from
the genus Ornithodoros have also been associated with transmission
of some tick-borne flaviviruses including ALKV [14,15]. There has
been some suggestion that tick-borne flaviviruses are transmitted by
mosquitoes, but this has not been conclusively proven.
The flaviviruses are a family of small, single-stranded RNA viruses
with a positive-sense genome and a host-derived lipid envelope.
The viral genome encodes a single polyprotein that is co- and posttranslationally
cleaved into ten individual proteins, three structural
and seven non-structural. The three structural proteins (C-capsid,
prM/M-premembrane/membrane and E-envelope) organize to
produce the viral particle. The non-structural proteins are principally
associated with viral replication, polyprotein cleavage and may have
additional roles in regulating the host immune response [16,17]. The
viral E protein is the major surface antigen for the flaviviruses and
contains the receptor-binding domain and fusion peptide. The viral
prM/M protein functions as a chaperone for the E protein and also
blocks the fusion peptide during virus assembly to prevent fusion with
exocytic vesicles. The prM protein is cleaved by furin following particle
assembly to produce the mature fusogenic virus. Co-expression of the
prM and E genes in cell culture systems have been used to produce
subviral particles which were used in early characterization of the
membrane fusion process of flavivirus entry and have also have been
tested as potential vaccine candidates. Vaccine candidates for several
flaviviruses, including West Nile and dengue viruses utilize a purified
recombinant E protein that has been produced in insect cell cultures
[18-22].
Tick-borne encephalitis was first described in 1936 as a neurologic
disease in far eastern Russia (then the Soviet Union) that had been
recognized in 1932. In 1937 a large expedition supported by the
Soviet government identified Ix. persulcatus ticks as the likely vector
for the contagion. In the same year separate groups isolated the virus
and were able to demonstrate that this virus was the causative agent
for the encephalitic disease seen in far-eastern Russia. The virus was
subsequently named "Far Eastern encephalitis virus" [23]. The isolation
of the virus also provided the first opportunity to develop a vaccine
to prevent infection. Smorodintsev developed a formalin-inactivated
vaccine that was used to vaccinate forest worker [24]. Subsequent
studies characterizing clinical disease described two similar yet distinct
diseases, one found in the far-east which was more severe and a less
severe disease in western Russia and parts of Eastern Europe. The latter
disease was termed Western encephalitis to distinguish the two diseases
[24]. Western encephalitis was also known as biphasic milk fever given
an apparent relationship with the consumption of unpasteurized milk
from infected animals. Studies during outbreaks in Czechoslovakia
isolated the causative agent of Western encephalitis and identified
the virus as related to Far eastern encephalitis virus [25-27]. Fareastern
encephalitis was found to have a higher case fatality rate and
increased incidence of long-term sequelae than Western encephalitis.
Additional studies identified a third intermediate subtype of what was
now known as tick-borne encephalitis. This third subtype was called
the Siberian subtype and has subsequently been shown to be associated
with chronic infection [1-5,28]. Following the advent of viral genome
sequencing, genetic analysis supported clinical descriptions in clearly
defining three distinct subtypes of TBEV. These are now termed TBEVFE
(Far-eastern), TBEV-Eu (European) and TBEV-Sib (Siberian) [29].
In 1957 a large outbreak of hemorrhagic disease in India was
described in bonnet macaques and humans. This disease was termed Kyasanur Forest disease (KFD), given its locale, and subsequent
studies isolated the causative agent and characterized the virus as
related to TBEV [30,31]. KFDV was clearly distinct from TBEV as
this virus caused a disease that had both hemorrhagic and neurologic
components while TBEV was only associated with encephalitis. For
four decades KFDV was thought to only exist in India. However, in
1996 Alkhurma virus was isolated from cases of hemorrhagic disease
near Jeddah, Saudi Arabia. This virus was characterized and found to
be closely related to KFDV both serologically and genetically. In 2009,
Nanjianyin virus was identified in south central China and found to
virtually identical to KFDV [32].
Omsk hemorrhagic fever was first identified in 1947 in the
Novosibirsk and Omsk Oblast regions of Russia. Few cases of OHF
have been described, but clinical disease frequently has a hemorrhagic
component with evidence of neurologic disease less common [33].
Following isolation of OHFV, serological and genetic characterization
clearly identified this virus as a member of the TBEV serocomplex.
Langat virus was first isolated from rodents in Malaysia in 1956
and was initially thought to be TBEV-FE based on serological results
[34]. Further analysis found that LGTV was a virus within the TBEV
serocomplex that was less virulent that related viruses. Shortly after
isolation and characterization of LGTV, the Elantcev 15-20/3 strain of
the virus was tested in human trials as a potential vaccine for prevention
of more severe disease caused by the more virulent members of the TBE
complex. Unfortunately, LGTV vaccination resulted in unexpectedly
high incidence (~1:10,000) of neurologic disease among vaccinees [35-38].
Powassan virus (POWV) and the closely related DTV are the only
members of the TBEV serocomplex viruses known to be endemic in
North America. POWV was isolated in northeast Canada in 1958 [39]
and has been found in the northern United States, southern Canada and
in far-eastern Asia [2,40]. POWV appears to generally be associated
with subclinical disease, but occasional cases of severe encephalitis do
occur, most recently in June 2011 in Minnesota (ProMED-Mail, June
29, 2011).
Disease
Members of the TBEV serocomplex cause a range of diseases from
subclinical or mild febrile illnesses to severe and lethal encephalitis or
hemorrhagic fever. Although a majority of infections with TBEV-Eu
appear to be subclinical [2], clinical descriptions of European TBE
indicate a primarily biphasic disease with the first phase a relatively
mild flu-like illness that is followed by a symptom-free phase of
about one week. Approximately 65% of TBE-Eu patients recover
after the first phase of disease. Patients who progress to the second
phase of disease generally present with high fevers and evidence of
neurologic involvement. Typical presentations include meningitis,
meningoencephalitis, poliomyelitic or polyradiculoneuritic symptoms
[12]. Neurological symptoms mostly resolve as TBEV-Eu infection has
a case fatality rate of 1-2% with little evidence of long-term sequelae
although the incidence of long-term effects is higher in older (> 60
years) patients [41].
Illness caused by infection with TBEV-FE is generally more severe
than that associated with TBEV-Eu infections. Following a 2-18 day
incubation period, disease onset can be very sudden with symptoms
including headache, high fever, vomiting, myalgia, photophobia and
other indications of neurologic disease including evidence of focal
encephalitis and meningitis. TBE in these cases is often described as mono-phasic with a majority of patients progressing directly to the
severe phase of disease [41]. The disease can be complicated by flaccid
lower motor neuron paralysis and ascending paralysis or hemiparesis
[42]. Neurological sequelae are more common in TBEV-FE cases than
is seen following TBEV-Eu infections. Sequelae can include atrophy
and paresis of the brachial plexus and neck muscles, paresis of the
lower extremities and poliomyelitis-like sequelae. The case fatality rate
following severe TBEV-FE infection can be 20-30%.
Disease caused by infection with TBEV-Sib is generally described
as intermediate between TBEV-FE and TBEV-Eu. However, TBEVSib
has been associated with chronic or persistent infections in
both humans, and primates, [1-5], a phenomenon that has not been
described for either the TBEV-FE or TBEV-Eu subtypes.
Disease seen following infection with KFDV and its subtypes
generally consists of a hemorrhagic fever type of illness and may be
associated with encephalitis whereas infection with OHFV is biphasic
in about 50% of cases with limited neurologic manifestations [33].
Infection with KFDV or ALKV may present as epitaxis, hematemesis,
cutaneous bleeding, melena or bleeding from venipuncture sites
[43,44].
Therapeutics
There are currently no licensed therapeutics for the treatment of
infections by TBEV or related viruses and recommended treatment is
largely supportive. A hyperimmune serum therapy regimen available
previously has been discontinued due to an unfavorable outcome in
disease observed in at least five treated children [45].
Potential as a biothreat agent
Members of the TBEV serocomplex are considered potential
biothreat agents due to their pathogenicity, ability to be aerosolized
[46], limited vaccine coverage and availability in many areas of the
world, a lack of therapeutics and stability in the environment. Reports
in the popular press have also suggested that Russian agents worked
to develop RSSEV as a bioweapon although the extent and purpose
of this effort are not clear. TBEV has been transmitted repeatedly
through consumption of contaminated (unpasteurized milk or milk
products) as was documented in early reports from outbreaks in Russia
and Eastern Europe [1,47-49]. Despite improved safety in production
of milk products, transmission of TBEV in milk continues to be a
problem in rural communities [50]. However, despite these high risk
considerations, the inability of the TBE viruses to be transmitted humanto-
human and the relative inefficiency of tick-to-human transmission
limits the potential impact if used as a weapon. In addition, the relatively
rapid vaccination schedules for licensed European vaccines with high
rates of seroconversion following the second dose would provide some
protection during an outbreak scenario. In non-endemic areas such
as North America, a more significant risk is the introduction of the
virus into the tick populations that could potentially allow the virus to
become established in a new environment. The ability of the virus to
become established requires both competent vectors and susceptible
amplifying hosts. It is not clear if either exists in North America and
if they share the same ecosystem, even though the presence of POWV
suggests that at least certain regions may provide suitable conditions
for permanent establishment.
TBE vaccines
The first effort to derive a TBE vaccine occurred shortly after the
discovery of the virus when Chumakov utilized a formalin-inactivated virus preparation to vaccinate forest workers [24]. In the 1960s TBE
vaccines were developed using cell culture systems [51-55] and clinical
trials demonstrated vaccine efficacy [56]. In 1971 the Institute of
Virology at the University of Vienna and the Microbiological Research
Establishment at Porton Down, UK began a collaboration to develop
a vaccine for use in Europe. This vaccine was based on the Austrian
TBEV-Eu strain Neudörfl. Seed stocks were generated in mouse brain
and the virus was then cultivated in specific pathogen free (SPF)
chicken embryo cells, clarified by centrifugation, inactivated with
formalin and then purified to produce the vaccine virus stock [57].
The purified inactivated virus was stabilized with human albumin and
combined with aluminum hydroxide that functioned as the adjuvant.
In 1976 the Austrian company Immuno took over vaccine production
and began marketing the vaccine as FSME-IMMUN®. This vaccine was
administered to over 400,000 people and studies found a seroconversion
rate of greater than 90% (by hemagglutination inhibition test) following
two doses of the vaccine [58]. An additional booster was indicated
approximately 9-12 months after the second dose after it was found that
antibody titer quickly waned [59]. In 1979 improvements were made to
the vaccine preparations to reduce local and systemic side effects to the
vaccination and to increase antigen purity [60]. The modified vaccine
showed immunogenicity comparable to the original vaccine but with
reduced side effects [61]. Later on, mouse-brain derived seed stocks,
thiomersal and human albumin were eliminated to further improve the
purity of the vaccine and this new formulation was called TicoVac®.
However, the removal of the albumin stabilizer without concurrently
adjusting the antigen content resulted in an increased likelihood of
vaccine-associated fever in infants and young children [62]. In 2001
FSME-IMMUN® containing human serum albumin was reintroduced
in doses for both adult and pediatric applications.
In the late 1980s the German firm Behring-Werke developed a
second product called Encepur that is based on the K-23 strain of TBEVEu
isolated in Germany. Encepur was licensed in 1992 and a pediatric
formulation of Encepur was released in 1995 [63]. The production
processes of Encepur and FSME-IMMUN® are similar with the only
significant differences being in the final formulation. Both vaccines
have been shown to induce significant protective antibody titers and
are considered essentially equivalent. Encepur is now produced by
Novartis while FSME-IMMUN® is produced by Baxter. While FSMEIMMUN
® is now also available in Canada, neither product is licensed
for use in the United States. An additional vaccine is available in China
but little is known about its production or efficacy [9].
In the 1960s and 1970s the LGTV strain Elantcev 15-20/3 was
tested as a potential live-attenuated vaccine for TBEV. Initial studies
were positive with rapid generation of protective responses and safety
in non-human primates and humans [1], but in a large scale clinical
study, a high rate of viral neuroinvasion and evidence of vaccinerelated
neurological illness significantly limited enthusiasm for the use
of LGTV as a vaccine for its more pathogenic cousins. More recently
the LGTV backbone has be used for development of chimeric dengue
vaccines [64,65].
Development of a vaccine for KFDV began in the early 1960s with
the use of a mouse-brain derived formalin inactivated vaccine based on
a TBEV-FE virus that was developed at the Walter Reed Army Institute
of Research (WRAIR) laboratory in Washington, DC. Vaccinees
receiving the vaccine had few vaccine related side effects, but the
vaccine elicited a poor protective immune response as was subsequently
abandoned [66-68]. Subsequent development of both mouse brain and cell culture derived vaccines utilizing inactivated KFDV met with better
success. These vaccines elicited a protective immune response in some
vaccinees, but the response was not consistent and did not appear to
provide complete protection [69,70]. Currently a formalin inactivated
KFD vaccine cultivated in chick embyo fibroblasts has been licensed for
use in India. This vaccine has reasonable efficacy, but is only used on
outbreak situations and requires an annual booster to retain sufficient
protective antibody titers [71].
Two vaccines based on TBEV-FE strains are currently licensed for
use in Russia. The TBE-Moscow vaccine was licensed for adult use in
1982 and for children =3 years old in 1989. TBE-Moscow is based on
the Sofjin strain cultivated in primary chick embryo cells. The virus is
formalin inactivated, purified and stabilized with human albumin. The
EnceVir vaccine was licensed for both adult and pediatric (=3 years)
use in 2001. EnceVir uses the TBEV-FE strain 205 and is similar in
manufacture and preparation to the TBE-Moscow vaccine [9]. Both Russian vaccines have shown safety and efficacy profiles similar to
those seen for the European vaccines. In adults, vaccine associated
reactogenicity was limited to primarily local responses following
vaccination with either Russian vaccine [9,72]. However, an increased
incidence of fever and allergic reactions following vaccination with
particular lots of EnceVir led to it being withdrawn from pediatric
use pending reformulation [9]. The EnceVir vaccine is currently not
recommended for use in children (= 17 years old). Protective antibody
responses were measured in 90-100% of vaccinees, depending upon the
study design and dosing schedule, and surveillance trials following a
large-scale vaccination program indicated antibody persistence for =3
years following a 3 dose vaccination schedule [9].
Given the significant genetic similarity and serological crossreactivity
between the TBEV serocomplex viruses, vaccines developed
for protection against one subtype should be cross protective against
all TBEV subtypes. Cross-protection against multiple subtypes has
been shown in studies in mice [73] and in human cross-neutralization
studies utilizing sera from vaccines [74]. These studies found
equivalent neutralization titers against both TBEV-Eu and TBEV-FE
subtypes and somewhat lower neutralization titers against OHFV [9,73,74]. Preclinical studies in mice also found vaccine dose-dependent
protection against infection with several TBEV-Eu and TBEV-FE
strains in addition to protection against the related Louping Ill virus
[75] and POWV (M. Holbrook-personal observations).
Novel vaccine technologies
The production of inactivated vaccines carries the inherent risk
of utilizing large quantities of potentially highly pathogenic viruses
and the possibility of incomplete inactivation of viruses. In addition,
vaccines based on inactivated viruses as antigens have shown a certain
level of adverse reactions, especially in children, that has to be carefully
balanced with efficacy and durability [76]. These risks, while minimized
by quality control efforts by manufacturers, are real. Subsequently
a number of researchers have evaluated alternative strategies for
development of vaccines that includes development of live-attenuated
viruses [77-79], DNA vaccine technology [80] and the use of subunit
vaccines (Coller et al, in preparation). None of these novel vaccine
strategies have reached clinical trials.
Table 1:
Summary
The members of the tick-borne encephalitis virus serocomplex
present significant health risks in a large proportion of the world,
particularly in Europe and across Asia. With an estimated 10-12,000
annual cases of TBE reported (this figure is believed to significantly
underestimate the actual total), these viruses present a real and specific
risk in their endemic regions. Fortunately, several established vaccines
are very effective for prevention of TBE. Two inactivated virus vaccines
manufactured in Central Europe have been used with very good success
throughout Europe. These vaccines are also available for travelers from
the United Kingdom and Canada. Two vaccines manufactured in
Russia appear to be essentially equivalent to those produced in Europe.
Perhaps the biggest limitation to the effective use of the TBE vaccines
is the ability to maintain complete vaccine coverage within endemic
regions. The vaccination schedule requires three doses to stimulate
the development of a significant and relatively long-lasting protective
antibody response. However, booster vaccinations are required every
3-5 years to maintain protective immunity, especially in an elderly
population.
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