|Vaccine Development for Biothreat Alphaviruses
|Kevin B Spurgers and Pamela J Glass*
|United States Army Medical Research Institute of Infectious Diseases, Frederick, MD 21702, USA
||Dr. Pamela J Glass
United States Army Medical Research
Institute of Infectious Diseases
1425 Porter Street,Fort Detrick
|Received June 14, 2010; Accepted July 13, 2011; Published September 25, 2011
|Citation: Spurgers KB, Glass PJ (2011) Vaccine Development for Biothreat Alpha
viruses. J Bioterr Biodef S1:001. doi:10.4172/2157-2526.S1-001
|Copyright: © 2011 Spurgers KB, et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
|The majority of alphaviruses are non-pathogenic to humans. However, select alphaviruses can cause severe disease
in humans during the course of naturally occurring epizootic outbreaks, or accidental infection of laboratory personnel.
Natural infections occur through the bite of an infected mosquito. However, pathogenic alphaviruses, including
Venezuelan, eastern, and western equine encephalitis viruses, have proven to be highly infectious via the aerosol
route. Given this aerosol infectivity, ease of production of high-titer virus, and low infectious dose, these alphaviruses
are recognized as candidates for use as biological weapons, and are classified as category B pathogens by the Centers
for Disease Control and Prevention and The National Institutes of Health. There are currently no licensed vaccines
to prevent alphavirus infections. Such a vaccine could protect geographically defined human populations during an
epizootic, and enhance national security by serving as a deterrent to the use of these viruses as biological weapons.
To address this critical need, several strategies are being pursued to develop safe, effective, and ultimately licensed
vaccines for use in humans.
|Alphavirus; Vaccine; Attenuated; Venezuelan equine
encephalitis; Eastern equine encephalitis; Western equine encephalitis;
Adenovirus; Furin; Chimeric; Subunit; DNA; Virus-like replicon
|Venezuelan (VEEV), eastern (EEEV), and western equine encephalitis
(WEEV) viruses, members of the genus Alphavirus in the family Togaviridae,
are causative agents of debilitative, acute, and sometimes fatal
encephalitis in North, Central, and South America . These viruses
are maintained in nature in a zoonotic cycle between susceptible nonhuman
vertebrate hosts, and hematophagous mosquito vectors. Natural
human cases are rare, and occur via the bite of an infected mosquito.
Since the discovery of these viruses, several epizootic outbreaks, infecting
human and equid livestock populations, have been recognized. Additionally,
these viruses pose a threat to public health, and military personnel
because of their potential use as bioweapons . This threat is
based on virus characteristics favorable to weaponization, and a known
history of weaponization. First, these viruses have been proven to be
highly infectious by the aerosol route. They are also easy to produce
at high titer, have a low infectious dose, and can be lyophilized. VEEV
was tested as a biowarfare agent during the U.S. offensive program in
the 1950's and 1960's, and may have been weaponized by the former
Soviet Union [3,4]. Because of the potential for weaponization, VEEV,
EEEV, and WEEV are classified as category B pathogens by the Centers
for Disease Control and Prevention (CDC), and the National Institutes
of Health (NIH). Veterinary vaccines utilizing inactivated alphavirus
preparations are available and in routine use to control infection in endemic
areas . Unlicensed, investigational vaccines for VEEV, EEEV,
and WEEV are also in use to protect at-risk laboratory personnel [6-8].
There are currently no vaccines licensed for general use in the U.S. for
prevention or treatment of alphavirus infections.
|Alphavirus virions are small, spherical particles ~ 70 nm in diameter
. The viral nucleocapsid core is surrounded by a host-derived
lipid membrane in which 80 protein spikes composed of trimers of E1/
E2 heterodimers are embedded. The nucleocapsid consists of the capsid
protein (C) surrounding the single-strand, positive sense, ~11 kb
RNA genome. The genomic RNA is capped, has a polyadenylated tail,
and is immediately translated upon entry into the cell cytoplasm. The
5' region of the genome encodes four nonstructural proteins (responsible
for viral transcription and replication), while the 3' region codes for five structural proteins (Figure 1). The structural genes are initially
expressed as a polyprotein from a 26S subgenomic RNA (Figure 1).
Cleavage events (by furin and signalase) produce the mature structural
proteins, including C, E1, and E2, as well as E3 and 6K [1,9,10]. The
E2 glycoprotein is thought to be involved in receptor binding [11,12].
The E1 glycoprotein has a role in endosomal membrane fusion, and
release of the nucleocapsid into the cytoplasm [13,14]. In response to
infection, most neutralizing antibodies are produced targeting the E2
protein. Given that many studies have demonstrated that a neutralizing
antibody response correlates with protection against a subcutaneous
challenge, E2 is the most common antigen used in vaccine efforts
to combat alphavirus infections. Although, neutralizing antibodies
against E1 protein are rare, E1 alone has been successfully used as a
vaccine antigen capable of protecting against lethal challenge [15,16].
|VEEV represents a complex of viruses previously classified as subtypes
I-VI (Table 1). Recent taxonomic changes have classified only the
subtype I viruses as VEEV . VEEV subtype varieties IAB and IC
have been associated with major outbreaks involving hundreds of thousands
of equine and human cases . VEEV subtypes ID, IE, and IF
are enzootic, equine avirulent strains not associated with major epizootics
or epidemics, although they do occasionally cause humans illness,
which can be fatal [19,20]. Subtypes II-VI are now classified as distinct
species within the Alphavirus genus. In many cases, immunity to one
species or subtype/strain does not protect against a heterologous strain.
This is true for the currently available investigational VEEV vaccines
which may not protect against heterologous subtypes/strains
|The EEEV complex consists of two species, North and South American,
which are further divided into four distinct genetic lineages . Lineage
I is found in North America and the Caribbean (EEEV NA); and
lineages II-IV that are found in Central and South America (EEEV SA).
Human disease is associated with the lineage I viruses.
|The WEEV complex consists of six species, WEEV, Sindbis virus,
Highlands J virus, Fort Morgan virus, Aura virus, and Whataroa virus.
The Sindbis group consists of five genotypes found exclusively in Old
World distributions . These viruses cause a relatively mild illness
with symptoms including fever, rash, and arthralgia. The remaining
members of the WEEV complex are New World viruses. There are a
number of WEEV subtypes, some of which are antigenically distinct,
found throughout North and South America [25,26]. A number of
WEEV strains have been associated with disease in humans and horses,
although a majority of the cases are either asymptomatic or present as
a febrile illness.
|Investigational vaccines for VEEV include TC-83 and C-84. TC-83
is a live-attenuated virus generated by serial passage of VEEV Trinidad
(TrD) strain in guinea pig heart cells . This vaccine is immunogenic
and produces a neutralizing antibody response in approximately 80% of human recipients. However, approximately 40% of vaccinated individuals
develop moderate flu-like symptoms, including fever, headache,
and malaise. Although this vaccine strain of VEEV can produce longlasting
immunity, safety concerns remain. In horses, TC-83 vaccination
can produce significant viremia . The virus also causes illness or
death in certain mouse strains after intracranial (i.c.), or subcutaneous
(s.c.) inoculation . C-84 is a formalin-inactivated preparation
of TC-83, which is administered to at-risk individuals who fail to seroconvert
after TC-83 vaccination, and those whose titer wanes over time
. This vaccine strain is safer than TC-83, but produces reduced neutralizing
antibody titers and less durable immune responses. Formalininactivated
virus vaccines for EEEV and WEEV are also in use at the
U.S. Army Special Immunizations Program to protect at-risk laboratory
personnel . The properties of these vaccines are similar to C-84, in
that they are poorly immunogenic, require frequent boosting; and it is
not clear if they would protect individuals from an aerosol challenge.
|A critical need exists to produce safe, effective, and ultimately
licensed vaccines for the prevention of VEEV, EEEV, and WEEV infections.
For such a vaccine to be effective in a biodefense scenario, it
ideally will protect individuals from aerosol exposure to VEEV, EEEV,
or WEEV. As such, most vaccine studies now measure efficacy against
aerosol virus challenge in various animal models of infection. This is
accomplished by testing exposure to artificial aerosols, or infection by
the intranasal (i.n.) route. The most appropriate and predictive correlate
of protection for alphavirus vaccines is still ill-defined. It is unclear if an
antigen specific antibody response (including neutralizing antibodies),
or a cell-mediated response, or a combination of both, is critical for
a successful vaccination against aerosol VEEV, EEEV, or WEEV challenge.
A large number of vaccine development strategies are currently
being employed to produce a vaccine that is more immunogenic, more
efficacious, and safer than current investigational vaccines for alphaviruses
(Figure 2, Table 2). This review summarizes these efforts.
||Figure 1: Alphavirus genome organization. Organization of the alphaviurs genome
is shown. The genomic RNA has a methyl guanine cap (mG), and a polyadenylated
tail (An). The mRNA encoding the structural proteins is transcribed
from a replication intermediate (not shown) and the 26S subgenomic RNA
promoter. Cleavage events (carried out by viral and host proteases) produce
mature, individual structural proteins from the initially translated polyprotein.
||Figure 2: Vaccine platforms used for the development of anti-alphavirus vaccines.
Chimeric virus vaccine candidates typically harbor a recombinant RNA
genome coding for the non-structural proteins of SINV, and the structural proteins
for VEEV, EEEV, or WEEV. Rationally designed, recombinant, attenuated
vaccines feature a deleted furin cleavage site between E3 and E2 coding
sequences. This results in PE2 (E2 precursor protein) being expressed on
the virion surface, along with virus attenuation. Viral replicon particles (VRPs)
are non-replicating, but infectious particles, engineered to express a protein
antigen (ag) of interest upon infection. Genome replication and gene expression
are carried out the by the nonstructural proteins (nsP 1-4). Viruses of
various types, including adenoviruses (Ad) have been engineered to express
a vaccine antigen upon transduction of target cells. DNA vaccines are typically
plasmids that express a vaccine antigen upon entry into target cells. Finally,
peptides from viral proteins or whole viral proteins can be administered to elicit
a protective immune response.
||Table 1: Partial summary of alphavirus strains and subtypes.
||Table 2: Summary of select alphavirus vaccine studies.
|Live attenuated virus vaccines
|Chimeric Vaccines: Construction of virus chimeras often produces
replication-competent, but highly attenuated viruses that are attractive
vaccine candidates. Chimeric viruses have been developed and tested
as vaccine candidates for the prevention of infection with VEEV, EEEV,
and WEEV. The most common strategy uses Sindbis virus (SINV), typically
not pathogenic to humans, as a vector to express the structural
genes of VEEV, EEEV, or WEEV. Such chimeric viruses are attenuated
and can protect mice from lethal alphavirus infection. For example, a
chimeric virus (SIN-83) containing the nonstructural genes of SINV,
and the full structural gene region of VEEV TC-83 protects mice from
lethal VEEV challenge . Although lower than that seen with TC-83
immunization, SIN-83 produced a neutralizing antibody response to
VEEV TC-83. To examine vaccine efficacy, NIH Swiss mice were vaccinated
with one dose of either VEEV TC-83 or SIN-83 by the s.c. route.
Four weeks later, mice were challenged s.c. with VEEV strains ZPC738
or SH3. All mice immunized with either TC-83 or SIN-83 survived
challenge. Although SIN-83 grows well in cell culture, it is highly attenuated.
Intracranial injection of SIN-83 (2x106 plaque forming units,
pfu) produced no mortality in suckling mice. Chimeric viruses using
other SINV and/or VEEV strains have also been examined . These
included SINV chimeras using structural genes from virulent VEEV
strains TrD and ZPC738. Additionally, a more virulent strain of SINV
was utilized with VEEV TrD. These chimeras showed intermediate attenuation
when tested in suckling mice compared to SIN-83, but were
more efficacious than SIN-83 in a mouse challenge model. Efficacy was
also demonstrated in Syrian golden hamsters after s.c. challenge with
VEEV ZPC738. Overall, these SINV/VEEV chimeras were found to be safer than TC-83, and more efficacious than the original SIN-83 chimera
in s.c. challenge models of infection.
|Atasheva et al., reported the use of three recombinant viruses as
vaccines against WEEV infection . These chimeras are based on the
SINV backbone, and express the SINV nonstructural genes required
for virus replication. The recombinant viruses are engineered to express
the structural genes of WEEV (strain CO92-1356 or ON41-McMillan).
SINV/CO92 was safe in adult mice, but was poorly immunogenic, and
provided 100% protection against lethal WEEV infection only at the
highest dose of vaccine tested. Two additional SINV/WEEV chimeras
had greatly improved immunogenicity, as measured by a robust neutralizing
antibody response . One of these proved safe in adult mice,
and provided 100% protection against i.n. challenge with WEEV after
one vaccination dose. Despite these promising results, the chimeras
were highly pathogenic when administered to suckling mice, leaving
some concerns regarding safety. Environmental safety of these chimeras
has been addressed by examining the ability of the viruses to infect,
and be spread by, mosquitoes . By this measure, the viruses appear
to be safe, and unlikely to be reintroduced into a natural transmission
|A similar approach generated chimeras between SINV and EEEV
FL93-939 or EEEV BeAr436087 (termed SIN/NAEEEV and SIN/
SAEEEV, respectively) . Neither chimera produced disease or death
in 8-week old NIH Swiss mice. SINV/NAEEEV was more immunogenic
and produced a good neutralizing antibody response against
homologous EEEV. For both chimeras, the neutralizing antibody response
was slightly cross-reactive to heterologous EEEV. Importantly,
both chimeras provided complete or near complete protection against
intraperitoneal (i.p.) challenge with EEEV FL93-939, at all doses of vaccine
tested. As with the SINV/WEEV chimeras, neurovirulence was
observed in suckling mice. Additionally, dissemination potential of the
chimeric virus was noted in Ae. sollicitans .
|Furin cleavage site mutant vaccines: The E2 protein of alphaviruses
begins its transit through the endoplasmic reticulum and golgi
as PE2, a precursor protein consisting of E3 and E2. In the trans-Golgi
network, or a post-Golgi compartment, E3 is cleaved from PE2 by
furin, producing mature E2 [10,37-40]. Alphaviruses incorporating
PE2 into mature virions can be viable and infectious, but are generally
attenuated in animal models of infection, and grow poorly in mosquito
cells. These observations have led to the rational design of VEEV, EEEV,
and WEEV vaccine candidates with furin cleavage site deletions, and
associated secondary resuscitating mutations [41,42].
|Site-directed mutagenesis was applied to the virulent VEEV TrD
strain, to produce engineered virus with deletion or mutation of the
furin cleavage site of PE2 . Secondary mutations in either E2 or
E1 allow the production of viable, infectious virions incorporating PE2
into the virus membrane. One furin cleavage mutant that has been well
characterized is V3526. This recombinant virus harbors a deletion of
the furin cleavage site and a secondary mutation at codon 253 in E1.
VEEV V3526 demonstrates reduced growth in C6/36 mosquito cells.
Growth of this virus is also slower in mammalian BHK cells, but final
titers of V3526 can equal those of wild type VEEV TrD (clone V3000)
virus. V3526 produces no signs of disease, or death, when administered
to adult CD-1 mice by the i.n. or s.c. routes. These immunized
mice were also completely protected against lethal i.n. challenge with
VEEV TrD V3000 virus . Since this initial characterization, V3526
has proven to be a highly efficacious and safe vaccine in several animal
models, and was transitioned into clinical development [43-48]. However, V3526 vaccination induced unacceptable clinical signs in humans
during phase I clinical trials and the vaccine was placed on hold (Parker
MD, unpublished data). At this point, the decision was made by the
sponsor of these studies not to pursue live virus vaccines for alphaviruses.
An observation that was noted based on the outcome of the clinical
trial was that humans appear to be more sensitive to VEEV infection
than the current nonhuman primate (NHP) models.
|Furin cleavage site mutants also exist for WEEV . Attenuation
was demonstrated for two of these recombinant viruses (WE2102 and
WE2130) by reduced replication in mosquitoes. These vaccine strains
also prevented viremia in chickens after challenge with virulent WEEV
|Second-Generation inactivated vaccines
|Following the cessation
of the live-attenuated vaccine program, studies were conducted to examine
inactivated preparations of V3526 as vaccine candidates. V3526
preparations inactivated with 1,5 iodonaphthyl azide (INA), formalin,
and gamma irradiation, have been investigated for immune response
and efficacy in a mouse model of VEEV infection [49-52]. Methods
for complete inactivation of V3526 virus stocks via formalin treatment
or gamma irradiation were evaluated and optimized . A detailed
dosage and schedule study, with and without adjuvant, was completed
to evaluate the immunogenicity and efficacy of gamma irradiated
V3526 (gV3526) . The adjuvants tested in this study were CpG,
Alhydrogel™ (AlOH), and CpG+AlOH. BALB/c mice immunized s.c.,
or intramuscular (i.m.; low dose), with or without adjuvant, were significantly
protected against s.c. challenge with VEEV TrD. Under these
conditions, protection was absent or poor against aerosol exposure to
VEEV. However, increasing the dose of gV3526 administered i.m. with
adjuvant (CpG alone, or CpG+ AlOH) resulted in 70-90% protection
against aerosolized VEEV TrD.
|A similar study was carried out with formalin-inactivated V3526
(fV3526) . The same adjuvants mentioned above were tested as
well as one additional adjuvant Viprovex. Adjuvant was not required to
achieve 100% serconversion in BALB/c mice after one or two vaccinations,
s.c. or i.m.. Neutralizing antibody titers after fV3526 vaccination
approached those achieved with C84 vaccination. Adjuvant was also
not required for fV3526 vaccination (s.c.) to protect 100% of mice from
s.c. VEEV TrD challenge. However, fV3526 alone offered poor protection
against aerosol challenge. Inclusion of AlOH adjuvant increased
survival to 80% (compared to 70% survival for C84 vaccination).
Similar results were obtained when vaccinating by the i.m. route. One
notable exception was the increased protection against aerosol challenge
afforded by fV3526 + CpG adjuvant administered i.m. compared
to s.c. vaccination. VEEV-specific serum and neutralizing antibodies
were produced to both fV3526 and gV3526 regardless of vaccine dose,
schedule or adjuvant. However, a positive antibody response, total serum
or neutralizing, could not be correlated with protection against
aerosol challenge. In both the gV3526 and fV3526 studies, mice were
vaccinated with extremely low doses. It is likely that further increases
in dose of these vaccine candidates could achieve complete protection
against an aerosol challenge.
|The photoactive compound 1,5 iodonaphthyl azide (INA) sequesters
in lipid membranes and, with ultraviolet irradiation, covalently
binds to lipids and proteins in the lipid bilayer. This reaction inactivates
integral membrane proteins while preserving extracellular epitopes on
those proteins. This compound has been shown to completely inactivate
several viruses including ebolavirus, HIV-1, VEEV (TrD, clone
V3000), and V3526 [52-54]. Interestingly, Sharma et al., observed that RNA isolated from INA-treated VEEV or V3526 was noninfectious,
and did not produce new virions or cell death when transfected into
BHK cells . This raises the possibility that INA inactivates virions
by two independent mechanisms. Vaccination with INA-treated VEEV
offers partial and dose-dependent protection against s.c. challenge with
VEEV TrD. This protection is enhanced with adjuvant. Additional studies
are required to investigate the efficacy of INA-inactivated V3526
against aerosol VEEV challenge.
|While inactivated vaccines were developed as first-generation
vaccines, many advances in techniques and methodologies have
warranted reexamination of this strategy for production of secondgeneration
alphavirus vaccines. Previous studies indicate that
inactivated vaccines are safe and effective. Work is now underway with
inactivated V3526 to examine increased vaccine dose and the use of
adjuvants. Similar strategies for the production of inactivated EEEV
and WEEV vaccines are also being investigated (Glass, P., personal
|Viral replicon particle vaccines
|Viral replicon particles (VRPs),
based on alphavirus vectors, are a promising vaccine platform that has
proven safe and efficacious in a variety of animal models . VRPs
consist of a self-replicating RNA genome (replicon), which expresses
the alphavirus nonstructural genes (producing the proteins required
for viral transcription and genome replication), along with a gene(s) of
interest. The heterologous gene(s) take the place of the viral structural
genes in the replicon RNA. VRPs are generated by transfection of target
cells with the replicon RNA along with helper RNAs which express the
viral structural genes. The helper RNAs do not contain signal sequences
required for packaging into new viral particles, therefore, only the replicon
RNA is packaged into the VRP. The resulting VRPs infect new cells
and express the inserted gene of interest, but do not generate new VRPs.
|VRP-based vaccines have been used to successfully protect animals
from challenge with simian-human immunodeficiency virus (SHIV)
, measles virus , and ebolavirus . A similar vaccination approach
was efficacious in mouse and NHP animal models of alphavirus
infection (Glass, P. and Reed, D., manuscripts in preparation). Specifically,
a replicon containing the VEEV nonstructural proteins was
engineered to express the envelope glycoproteins of VEEV, EEEV, or
WEEV. Mice vaccinated with the VRPs exhibited complete protection
from lethal aerosol challenge with VEEV, EEEV, or WEEV (Glass, P.
manuscript in preparation). Vaccination with the trivalent VRP vaccine
also significantly improved clinical parameters in NHPs challenged
with aerosolized VEEV (Reed, D., manuscript in preparation). In each
of these experiments, vaccine was administered on day 0, with a boost
on day 28. The one shortcoming was that very large doses of VRP were
required to protect NHPs. Ongoing studies are investigating the use of
adjuvants to decrease the requisite dose of VRP needed for protection.
Given successful protection against several viral infections, VRPs remain
an attractive vaccine candidate for advanced development.
|Viral vectors of several types, engineered
to express a transgene/protein antigen of interest upon transduction of
target cells, have been widely studied and utilized as vaccines . In
particular, replication-incompetent adenoviruses (Ad) are being used
as pre-clinical and clinical tools to combat infectious disease, cancer,
and Alzheimer's disease . Adenovirus-based vaccines to prevent
various viral infections, including HIV-1, influenza A, dengue virus,
and Japanese encephalitis virus have been described [61-64]. These vaccines can produce potent
antigen-specific antibody and T-cell immune responses. Adenovirus- vectored vaccines administered i.n. produce mucosal immunity, which
may be an important factor for preventing disease after aerosol exposure
to viral pathogens. Other viruses used as vaccine vectors include
vaccinia virus, Sendai virus, and lentivirus, among others.
|Adenovirus-Vectored vaccines: The adenovirus vaccine platform
has been used successfully to immunize mice against lethal VEEV infection.
Phillpotts et al., utilized an adenovirus vector expressing the
E3-E2-6K portion of the subgenomic region of VEEV TC-83 . This
recombinant adenovirus expresses VEEV E2 protein upon infection of
target cells. Three sequence changes were engineered into the E2 gene
sequence of this construct, to match the sequence of E2 in the VEEV
Trd strain (vaccine designated RAd/VEEV#3). This vaccine candidate
provided significant protection against challenge doses of 640 LD50 or
less; however it did not protect BALB/c mice challenged with a high
dose (8460 LD50) of aerosolized VEEV TrD. This vaccine candidate did
provide protection against heterologous strains of VEEV however. Partial
or complete protection was achieved when mice were challenged
with ~100 LD50 of VEEV strains from five other serogroups. With
this vaccine, an antigen-specific antibody response was achieved. However,
sera from immunized mice were unable to neutralize VEEV TrD.
The antibody response to VEEV antigen after immunization with RAd/
VEEV#3 was not improved with co-administration of CpG oligodeoxynucleotides
as adjuvant . Although more survivors were observed
in the vaccine plus adjuvant group (versus vaccine alone) after challenge
with a high dose of VEEV TrD, this apparent increase in protection
was not statistically significant. Likewise, plasmid or Ad-directed
expression of interferon alpha as an adjuvant to RAd/VEEV#3, did not
improve the immunogenicity of the RAd/VEEV#3 vaccine . Significant
improvements to this adenovirus-vectored VEEV vaccine were
achieved with gene optimization procedures . The RAd/VEEV#3
vaccine construct was altered to optimize codon usage and remove
undesirable RNA motifs. These alterations increased E2 expression in
transduced cells and increased the VEEV-specific antibody response
in mice. The optimized vaccine construct also significantly improved
survival after VEEV challenge when compared to the parental RAd/
VEEV#3 vaccine .
|Adenovirus vectors have also been investigated as a vaccine platform
to prevent WEEV infections. Wu et al., utilized a replication defective
adenovirus vector containing the subgenomic coding region
(E3-E2-6K-E1) of WEEV strain 71V-1658 . Upon infection of cells
with this construct (Ad5-WEEV) WEEV E1 and E2 proteins are expressed.
BALB/c mice administered two doses of Ad5-WEEV i.m. produce
a modest neutralizing antibody response and are protected against
i.n. homologous WEEV challenge . In a follow-up study, the authors
demonstrate both rapid and long-lasting cross-protection against
WEEV challenge with a single dose of vaccine. A single i.m. administration
of Ad5-WEEV protected mice from the 71V-1658, CBA87, and
Fleming strains of WEEV at one week, or 13 weeks, after immunization
. Swayze et al., also constructed and tested an Ad5 vector expressing
only WEEV E1 protein (Ad5-E1) . A single i.m. administration of
this vaccine construct also completely protected mice against intranasal
challenge with WEEV strains 71V-1658 and CBA87. At the time
of WEEV challenge, one week after vaccination, a T-cell response was
detected in the absence of a humoral immune response. Further studies
are necessary to determine if this finding is reflective of a necessary
protective response, or due to assay sensitivity. Regardless, this study
suggests that WEEV E1 protein alone may be a sufficient and effective
vaccine antigen. These studies show that the adenovirus platform is efficacious;
however it is still under debate how pre-existing immunity to adenoviruses will affect the widespread utility of this strategy.
|Equine herpesvirus-vectored vaccine: As an alternative to adenovirus-
based vaccines, a VEEV vaccine has been tested that utilizes
equine herpesvirus type I (EHV-1) as a vector, to express VEEV structural
proteins, and protect against lethal challenge . A possible advantage
of this vaccine platform is the absence of anti-vector immunity
in humans. Like adenoviruses, EHV-1 has broad tissue tropism and
can accommodate large amounts of exogenous DNA. A replicationcompetent
recombinant EHV-1 virus expressing the E3-E2-6K-E1 portion
of the VEEV TC-83 genome was constructed (vaccine designated
rH_VEEV) . NIH Swiss mice received two s.c. vaccinations with
various doses of rH_VEEV, and were then challenged s.c. with 1000
LD50 of VEEV subtype ID strain ZPC738 four weeks after initial vaccination.
The highest dose of rH_VEEV provided complete protection
in this lethal mouse model. A VEEV-specific antibody response was
achieved after the second vaccination, but neutralizing activity was
not detected. Low levels of IgG1 and total IgG antibodies, in the absence
of IgG2a, were detected suggesting possible roles for cytotoxic
T lymphocytes or antibody-dependent cytotoxicity in the protection
against VEEV. A number of studies have reported the lack of correlation
of protection with neutralizing antibody responses [50,51,72,73]
and others have provided evidence of T cell involvement in the immune
response against alphaviruses [74-76]. Further studies are necessary to
determine the protective immune response following vaccination with
this vaccine candidate.
|Vaccinia virus-vectored vaccines: Recombinant vaccinia virus has
also been used as a vaccine vector to express the structural proteins
of VEEV. While these vaccines are efficacious in mice under certain
conditions, they are generally less effective than TC-83, and fail to offer
full protection against aerosol VEEV exposure. VACC/TC-5A is a recombinant
vaccinia virus engineered to express the structural genes of
VEEV TC-83 . A/J mice were immunized by intradermal (i.d.) tail
scarification. Although present, the vaccine-induced antibody response
to VEEV was less than that seen with TC-83 vaccination. Neutralizing
antibodies were present but variable, even at the highest dose of vaccine.
However, this response was durable; neutralizing antibodies could
be detected in some mice up to 16 months after vaccination . Additionally,
TC-5A vaccination produced a T-cell response against both
the vaccinia vector and TC-83 VEEV virus . VACC/TC-5A effectively
protected mice challenged i.p. with VEEV types IAB, IC, ID, and
II . However, this vaccine was not able to protect against i.n. TrD
challenge tested in A/J, C3H, and NIH Swiss mice. TC-83 vaccination
offered complete or near complete protection from aerosol exposure in
each mouse strain.
|Bennett et al., also investigated a vaccinia virus vector expressing
the 26S subgenomic region of VEEV TC-83 . Additionally, the parental
vaccine construct (WR100), was altered in an effort to increase
VEEV protein expression and immunogenicity. WR100 was engineered
to contain a synthetic promoter in front of the VEEV coding sequences,
and to introduce a sequence change in E2 to match the sequence of
VEEV TrD. VEEV protein expression was increased from this construct
(WR103), compared to WR100. Still, the antibody response to WR103
vaccination was low when compared to TC-83, and neutralizing antibodies
were not present. Although an improvement over WR100,
WR103 offered only partial protection from s.c. challenge with VEEV
TrD to BALB/c mice vaccinated by the i.m. route. Vaccinia virus-based
vaccines, expressing only portions of the 26S subgenomic structural
gene region (expressing E2 only, or E1 only) also protect BALB/c mice against peripheral challenge with VEEV TrD, but fail to completely protect
against aerosol challenge .
|As a vaccine platform, DNA offers several advantages
over other strategies. These include the absence of pre-existing vector
immunity, as well as ease and low cost of production. Because of the
inherent stability of DNA, transport and long-term storage are not
problematic. DNA vaccines have proven safe and effective in preclinical
animal models and clinical trials. Yet, immunogenicity is often less than
that produced by other vaccine platforms, including live-attenuated,
inactivated, or viral-vectored vaccines. DNA vaccines are typically
plasmids that express a vaccine antigen of interest when delivered into
target cells. These expressed proteins are then processed and presented
to the immune system to elicit a protective response. Delivery methods
include direct i.m. or i.d. injection of naked DNA, particle-mediated
epidermal delivery (PMED, i.e. gene gun), or i.m. electroporation (i.m. EP).
|DNA vaccination has shown promise in preclinical animal models
as a strategy to prevent VEEV disease. A plasmid DNA vaccine expressing
the 26S sub-genomic region (C-E3-E2-6K-E1) of VEEV TrD was
administered to mice by PMED. This strategy protected 80% of mice
from lethal aerosol VEEV exposure . Partial protection was also
observed with this vaccine and delivery method in NHP . VEEV
DNA vaccination by PMED prevented viremia in two out of three NHP
after aerosol VEEV challenge. Fever, lymphopenia, and clinical signs
of disease were also reduced in vaccinated animals. In efforts to improve
their VEEV DNA vaccine, Dupuy et al., utilized gene-optimization
methods and tested an alternative delivery method . Codonoptimization
was applied to the VEEV DNA vaccine to reflect codon
usage in humans, and the construct was altered to remove unwanted
cis-acting RNA motifs. The resulting vaccine plasmid (VEEVco), containing
the E3-E2-6K-E1 coding region of VEEV TrD, was tested for
increased immunogenicity and/or protective efficacy in several animal
models. BALB/c mice were given three vaccinations with VEEVco or
control VEEVwt (parental, wild type, un-optimized VEEV DNA vaccine),
by i.m. EP. At low doses of vaccine, VEEVco produced a more
robust anti-VEEV antibody response in immunized mice compared
to VEEVwt. VEEVco also elicited a greater neutralizing antibody response
at all doses tested. To assess efficacy, mice were vaccinated twice
with VEEVco by i.m. EP, and challenged by aerosol VEEV TrD (>1000
LD50). All VEEVco vaccinated mice survived challenge and showed no
signs of disease. Efficacy was also investigated with this optimized DNA
vaccine NHPs . Cynomolgus macaques
were vaccinated twice with VEEVco by i.m. EP. After aerosol challenge
with VEEV TrD, no viremia was detected in vaccinated animals. Fever,
lymphopenia, and clinical signs of disease were present, but reduced, in
VEEVco-vaccinated NHPs, compared to controls. Additional experiments
demonstrate a T-cell response to the vaccine in mice, and longlived
(> 6 months) neutralizing antibodies in rabbits.
|Directed molecular evolution (i.e. gene shuffling) has generated
and identified improved vaccine protein antigens with increased immunogenicity
and cross-reactivity. Dupuy el al., applied this technique
toward the generation of a multi-agent DNA vaccine for VEEV, EEEV
and WEEV . In vitro DNA recombination was performed using
cloned E1 and E2 genes from VEEV IAB, VEEV IE, Mucambo virus,
EEEV, and WEEV. Recombination events occurred at regions of sequence
homology, or at sites engineered for forced crossover recombination.
This procedure produced a plasmid library of alphavirus E1
and E2 protein variants, subsequently tested as DNA vaccines in mice.
In vitro and in vivo screening procedures identified DNA vaccines with increased immunogenicity and cross reactivity. Select plasmid DNA
vaccines elicited a greater antibody response to VEEV IAB compared
to the parental, unrecombined VEEV IAB plasmid. Antibodies elicited
by this recombined vaccine also cross-reacted with other alphaviruses.
In efficacy studies, mice were immunized with parental or recombined
DNA vaccines by PMED. After the second and third vaccination, two
of the recombined plasmids produced an anti-VEEV antibody response
comparable to TC-83 vaccination, along with an enhanced neutralizing
antibody response. These plasmids protected 90-100% of mice from
lethal aerosol VEEV infection (compared to 80% protection with parental
DNA vaccine). Given the observed cross-reactivity for VEEV,
EEEV, and WEEV, of the serum antibody from animals vaccinated with
the recombined DNA vaccines, it will be interesting to see if protection
is observed against alphaviruses other than VEEV. Such an approach
could provide an alternative to administering several separate vaccines
to protect against these related viruses.
|Increased performance of VEEV DNA vaccines can be achieved
with a prime-boost immunization strategy . The VEEV gene sequence
from RAd-VEEV#3  was used to generate a plasmid for use
as a DNA vaccine against VEEV. BALB/c mice were vaccinated with
three doses of VEEV DNA vaccine by PMED at two week intervals.
The mice were then boosted with Ad-VEEV#3 or not boosted. The
Ad vaccine boost elicited a greater VEEV-specific antibody response,
and neutralizing antibody response, compared to DNA vaccine alone,
or Ad-VEEV#3 alone. The prime-boost regimen also significantly enhanced
protection against lethal aerosol VEEV TrD challenge. Further
studies are needed to determine the extent of enhanced efficacy that
an Ad vaccine boost might achieve after fewer than three initial DNA
|An expression plasmid (pVHX-6) containing the 26S subgenomic
region of WEEV strain 71V-1658 has been investigated as a DNA vaccine
. This plasmid expresses both E1 and E2 proteins of WEEV.
BALB/c mice were immunized with four doses of pVHX-6, or empty
plasmid control, via Helios Gene Gun i.d. delivery. Mice were given
two doses of pVHX-6 on each of two vaccination days, (total of 4 vaccine
doses), two weeks apart and challenged two weeks after second
set of doses. Immunized mice were 100% protected from i.n. challenge
with homologous WEEV. Significant but incomplete protection was
observed after challenge with WEEV Fleming or CBA87 strains. With
this immunization strategy, an anti-E1 or E2 antibody response was not
detected. However, a T-cell response was generated. Additional WEEV
DNA vaccines, expressing different portions of the 26S subgenomic region,
have been investigated . In comparison to plasmid pVHX-6,
constructs lacking the capsid protein coding region, or constructs expressing
only E1 or only E2 proteins were studied. Mice were administered
three doses of DNA vaccine, two weeks apart, i.d., by gene gun.
Notably, the DNA vaccine expressing E1 (p6K-E1) provided complete
protection against homologous WEEV, while the DNA vaccine expressing
E2 alone provided no protection. However, DNA vaccine p6K-E1
was less effective than pVHX-6 when challenged with a high virulence
heterologous WEEV strain.
|E1 and E2 glycoproteins have been investigated
as subunit vaccines for alphaviruses, and have shown variable efficacy.
The E1 or E2 coding sequence from WEEV strain 71V-1658 were cloned
and expressed in E. coli [87,88]. The recombinant proteins were immunogenic,
producing both antibody and cell-mediated responses in mice,
and were recognized by immune serum from mice immunized with
inactivated WEEV. However, i.m. immunization with recombinant E1 or E2 proteins formulated with TiterMax Gold adjuvant provided only
slight or no protection against homologous or heterologous WEEV virus
challenge. In contrast, one study indicated that the structural proteins
from VEEV are immunogenic and effective vaccine antigens in
mice . Recombinant baculoviruses were utilized to express various
regions of the structural gene region of VEEV in Sf9 insect cells. Lysates
from these cells, containing VEEV structural gene expression products,
were used to immunize BALB/c mice. Lysates containing baculovirusexpressed
E1 and E2, or E1 alone provided 100% protection against i.p.
challenge with VEEV TrD. This study suggests that purified VEEV E1
or E2 proteins would be effective VEEV vaccine immunogens. Differences
in the platforms concerning expression and purity of WEEV and
VEEV immunogens could explain the different outcomes of these studies.
|Peptides derived from VEEV E2 protein are also immunogenic
and protective when administered to mice . BALB/c or NIH Swiss
mice were immunized s.c. with free peptides, with or without adjuvant.
Anti-peptide and anti-VEEV antibodies were elicited in response to
vaccination, although neutralizing antibodies were not detected prior
to challenge. Two peptides provided significant protection against i.p.
challenge with VEEV TrD in BALB/c and NIH Swiss mice.
|Conclusions and Future Directions
|Diverse vaccine platforms have proven efficacious in animal models
for the prevention of VEEV, EEEV, and WEEV infections. Several of
these have been successfully used to develop vaccines for other virus
infections, and have passed important safety hurdles in phase I clinical
trials. DNA vaccines offer many advantages, though they are often
less immunogenic and require more vaccinations or specialized delivery
systems (i.e., electroporation) for effective protection, compared
to other vaccine platforms.VRPs offer great
promise, but are in the early stages of development for alphaviruses.
Live-attenuated virus vaccines are often the most immunogenic and
efficacious, though safety concerns could limit their licensure. Recent
studies of inactivated vaccine candidates indicate that next-generation
inactivation methodologies may provide greater protection than those
utilized in the early 1960s. This strategy could have an advantage given
that a number of FDA-approved vaccines are based on this methodology.
Virus-like particles (VLPs) are also likely to emerge as promising
alphavirus vaccine candidates. A recent report describes the production
and characterization of Chikungunya virus (CHIKV) VLPs .
Immunization with CHIKV VLPs produces a neutralizing antibody
response in mice and NHPs, and prevents viremia in NHP after subsequent
challenge with CHIKV. This strategy will likely be tested in the
future as a vaccine strategy for VEEV, EEEV, and WEEV. To date, many
of the alphavirus vaccine candidates are immunogenic and efficacious
in peripherally challenged mouse models of infection. Additional testing
in inhalational models of infection is necessary to determine which
platform is the most efficacious as well as licensable. A major hurdle for
vaccines against biowarfare agents will be FDA approval. The benefit to
risk ratio is inherently low and licensure will require the use of the animal
rule. One aspect of alphavirus immunity that remains unanswered
is the correlate of protection by which vaccines should be measured for
determination of the best vaccine candidate. It is clear that neutralizing
and nonneutraling antibodies as well as T cells can aid in the protection
against lethal alphavirus infection. Protection in immunocompetent
individuals will likely be provided by a multi-armed immune response
involving both humoral and cell-mediated immunity. One arm of the
immune response may need to dominate depending upon the route of
challenge. For example, neutralizing antibody levels correlate with protection following a subcutaneous challenge; yet do not always correlate
with protection against an aerosol challenge. It is possible that a T cell and/or a mucosal immune
response will be important for protection against lethal disease
following inhalation of alphaviruses. The studies reviewed here suggest
that the correlate of protection will not be a single solution but different
vaccine candidates may have different mechanisms of protection based
on the candidate itself, the formulation, route of vaccination, and route
of infection. Therefore, the best vaccine candidate ultimately may depend
upon the type of infection one is trying to protect against.
|Opinions, interpretations, conclusions, and recommendations are those of the
authors and are not necessarily endorsed by the U.S. Army. KBS is appointed to
the Postgraduate Research Participation Program administered by the Oak Ridge
Institute for Science and Education (ORISE) through an interagency agreement
between the U.S. Department of Energy and USAMRMC. PJG vaccine research
is supported by funding from the Defense Threat Reduction Agency Projects
H.H.0003_07_RD_B and 1.1C0040_09_RD_B.
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