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Infection and Immunity, September 2001, p. 5709-5715, Vol. 69, No. 9
Virology Division1 and
Toxinology Division,2 U.S. Army Medical
Research Institute of Infectious Diseases, Fort Detrick, Frederick,
Maryland 21702-5011
Received 8 January 2001/Returned for modification 23 March
2001/Accepted 11 May 2001
A candidate vaccine against botulinum neurotoxin serotype A
(BoNT/A) was developed by using a Venezuelan equine encephalitis (VEE)
virus replicon vector. This vaccine vector is composed of a
self-replicating RNA containing all of the VEE nonstructural genes and
cis-acting elements and also a heterologous immunogen gene
placed downstream of the subgenomic 26S promoter in place of the viral
structural genes. In this study, the nontoxic 50-kDa carboxy-terminal
fragment (HC) of the BoNT/A heavy chain was cloned into the
replicon vector (HC-replicon). Cotransfection of BHK cells
in vitro with the HC-replicon and two helper RNA molecules, the latter encoding all of the VEE structural proteins, resulted in the
assembly and release of propagation-deficient, HC VEE
replicon particles (HC-VRP). Cells infected with
HC-VRP efficiently expressed this protein when analyzed by
either immunofluorescence or by Western blot. To evaluate the
immunogenicity of HC-VRP, mice were vaccinated with various
doses of HC-VRP at different intervals. Mice inoculated
subcutaneously with HC-VRP were protected from an
intraperitoneal challenge of up to 100,000 50% lethal dose units of
BoNT/A. Protection correlated directly with serum enzyme-linked immunosorbent assay titers to BoNT/A. The duration of the immunity achieved was tested at 6 months and at 1 year postvaccination, and mice
challenged at these times remained refractory to challenge with BoNT/A.
Botulism is a disease resulting from
the activity of botulinum neurotoxins (BoNT) produced by
Clostridium botulinum on the transmission of neuromuscular
stimuli (8, 22, 23). The blockage of stimuli produces
neuromuscular weakness and flaccid paralysis, which can lead to
respiratory failure and death. Food poisoning, infant botulism, and
wound botulism are the three most common BoNT diseases affecting
humans. The BoNT consists of two polypeptides bound by a disulfide
bond, a heavy chain of about 100 kDa and a light chain of about 50 kDa.
Seven different serotypes (A through G) of BoNT have been
characterized. Previous research has shown that polyclonal antibodies
to one serotype can block the effects of the homologous serotype but
not of heterologous serotypes (20). The current human
vaccine, which is administered under Investigational New Drug status to
at-risk laboratory personnel, contains five of the seven serotypes (A
to E) and is formulated as a toxoid. The toxoid vaccine is given as a
primary series of three inoculations given at 0, 2, and 12 weeks,
followed by a booster at 1 year. Since the vaccine is reactogenic in up
to 20% of the recipients and contains only five of the seven
serotypes, an improved vaccine would be preferable.
Venezuelan equine encephalitis (VEE) virus is a member of the
Alphavirus genus in the Togaviridae family. Alphaviruses
contain a 42S single-stranded positive-sense RNA genome encoding four nonstructural proteins (providing the replicase and transcriptase function) and three structural proteins (capsid, E1, and E2). The
nonstructural proteins are translated directly from the 42S genomic
RNA. The structural proteins are translated from a subgenomic, 26S RNA
that is transcribed from the full-length negative strand. The 26S
promoter on the negative strand drives transcription of the 26S RNA to
levels 10 times that of the 42S genomic RNA (20, 21).
Attenuated variants of VEE virus developed initially as live-attenuated
vaccine candidates for VEE have also been configured as vaccine vector
systems for the expression of heterologous genes (15). The
VEE vaccine vector system utilized in the studies described here is
composed of an RNA replicon and a bipartite helper system for packaging
the replicon into propagation-deficient VEE replicon particles (VRPs).
The replicon contains a multiple cloning site immediately downstream of
the 26S promoter, which allows insertion of heterologous genes in place
of the viral structural genes. The bipartite helper system is composed
of two RNAs, one encoding the capsid gene (C) and the other encoding
the glycoprotein genes (E3-E2-6K-E1). The
glycoprotein genes also contain attenuating mutations which
provide an additional level of safety in the unlikely event that
multiple RNA recombination events regenerate replication-competent virus (15). After cotransfection of susceptible cells in
vitro with the replicon RNA and both helper RNAs, VRPs containing
recombinant replicons are produced. Pushko et al. used this system to
evaluate the safety and immunogenicity of replicons expressing either
the Lassa virus nucleocapsid (N) gene or the influenza hemagglutinin gene (15). These researchers observed that cotransfection
did not regenerate replication-competent virus, that
sequential vaccination of mice against Lassa virus and influenza was
possible, and that a protective immune response could be induced in
mice against influenza virus.
Previous studies have also shown that guinea pigs and nonhuman primates
vaccinated with VRP expressing either the glycoprotein (GP)
gene alone or a mixture of VRP expressing either the GP gene or
nucleoprotein genes from Marburg virus (MBGV) were protected from an
otherwise-lethal challenge of MBGV (10). In a different study, mice were protected and guinea pigs were partially protected by
vaccination with a VEE replicon expressing the genes from Ebola virus
(14). Nonhuman primates were also partially protected against simian immunodeficiency virus (SIV) after vaccination with a
mixture of VRP expressing SIV gp160, gp140, and matrix-capsid genes
(6).
Previous research has also shown that the nontoxic 50-kDa
carboxy-terminal fragment (HC) of the BoNT serotype A
(BoNT/A) heavy chain expressed in yeast or in Escherichia
coli can protect mice from a lethal challenge of neurotoxin
(2, 4). In this study, we have cloned the gene encoding
the BoNT/A HC fragment into the VEE replicon vaccine vector
(HC-replicon), assembled the HC-replicon into
VRP (HC-VRP), and assessed the HC-VRP both in
vitro and in vivo. Western blot and immunofluorescence analysis of
whole cells infected with HC-VRP or lysates prepared from
such cells were used to characterize the HC expression
product. To evaluate the immunogenicity and protective efficacy of
HC-VRP, mice were inoculated with various doses of
HC-VRP, at different intervals, and challenged with
increasing amounts of BoNT/A. The results of this study demonstrate that the HC-VRPs are capable of inducing efficient
protection against an otherwise-lethal challenge with BoNT/A and define
the vaccination schedule required for optimal immunogenicity.
Plasmids and production of VRP.
The design and construction
of the VEE replicon vector system and the Lassa virus nucleocapsid
replicon (N-replicon) was described previously (16). The
synthetic BoNT/A HC gene was cloned into a shuttle vector
(5) as a SalI/HindIII fragment
from the pMutAC-1 plasmid previously described by Clayton et al.
(4). The synthetic HC gene was then cloned
from the shuttle vector into the VEE replicon plasmid as an
XbaI/HindIII fragment.
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.9.5709-5715.2001
Candidate Vaccine against Botulinum Neurotoxin
Serotype A Derived from a Venezuelan Equine Encephalitis Virus
Vector System
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
70°C.
Analysis of expression products and titration of VRP. Subconfluent BHK cell monolayers were infected with HC-VRP or, alternatively, cell suspensions were transfected by electroporation with HC-replicon RNA. After incubation for 20 to 24 h at 37°C, cell lysates were prepared for Western blot analysis, and the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). HC protein was detected by using horse anti-BoNT/A HC antisera (obtained from Mark Poli, USAMRIID, Fort Detrick, Frederick, Md.) and a chemiluminescence Western blot assay kit (Amersham Pharmacia Biotech, Inc., Piscataway, N.J.).
Titers of VRPs were determined by infecting subconfluent BHK cell monolayers in eight-well chamber slides (Nunc, Inc.) with serial dilutions of purified VRP. Cells were fixed with methanol, and antigen-positive cells were visualized by indirect immunofluorescence by using a horse anti-BoNT/A HC antisera and fluorescein isothiocyanate (FITC)-conjugated goat anti-horse antibody (Kirkegaard and Perry Laboratories, Inc.). Cells expressing Lassa virus N protein were detected by direct immunofluorescence with FITC-conjugated antibodies obtained from a monkey anti-Lassa serum. Cell nuclei were stained with 1 µg of 4',6'-diamidino-2-phenylindole (DAPI) per ml in VectaShield mounting medium (Vector Labs, Inc., Burlingame, Calif.). The VRP titers are expressed as focus-forming units (FFU), where 1 FFU is equivalent to 1 infectious unit (iu). VRP preparations were monitored for the generation of replication-competent VEE virus by a standard plaque-forming assay in which samples were tested directly and after blind passage of the preparations in BHK cell cultures. No PFU were found in any of the VRP preparations.Vaccination and challenge of mice. Groups of 6- to 8-week-old BALB/c mice were inoculated subcutaneously (s.c.) (200 µl) at 7-, 14-, 21-, or 28-day intervals (as indicated) with 105, 106, or 107 iu of HC-VRP or with 107 iu of N-VRP (negative control replicon) diluted in PBS. Positive control mice were inoculated s.c. with 0.1 or 0.2 ml of botulinum toxoid vaccine at 28-day intervals. Serum for enzyme-linked immunosorbent assay (ELISA) was obtained 1 or 2 days before each inoculation and 3 to 5 days before challenge. Mice were challenged intraperitoneally (i.p.) with 100 µl containing 102, 103, 104, or 105 50% median lethal dose (MLD50) units of BoNT/A (as indicated) diluted in PBS containing 0.2% gelatin 30 or 31 days after the last inoculation.
For duration-of-immunity studies, five groups (I through V) of 6- to 8-week-old NIH Swiss mice were used. Each group consisted of 40 mice. In sets of 10 mice, the sets were inoculated with either 106 or 107 iu of HC-VRP, 107 iu of N-VRP (negative control), or 0.2 ml of botulinum toxoid vaccine (positive control). All of the groups received the appropriate inoculations at days 0 and 28. Group I was then challenged on day 196. Groups II and V were challenged on day 370. Group III animals received booster inoculations on day 168 and were then challenged on day 370. Group IV animals received booster inoculations on day 341 and were challenged on day 370. A minimum of 10 mice were bled at each time point 2 to 3 days before and at 28-day intervals after the second inoculation. The mice were also bled 2 to 7 days before and 28 to 35 days after challenge. Mice were challenged i.p. with 1,000 MLD50 units of BoNT/A on either day 196 or day 370. Note that, in conducting research using animals, we adhered to the Guide for the Care and Use of Laboratory Animals (NIH publication No. 86-23).ELISA.
Microtiter plates were coated with BoNT/A (1 µg/ml)
in 100 µl of PBS and allowed to absorb overnight at 4°C. After the
plates were washed five times with wash buffer (PBS containing 0.1%
Tween 20), fourfold serial dilutions of serum in blocking buffer (100 µl; PBS, 0.1% Tween 20, 5% dried nonfat milk) were applied to the
plates and incubated at 37°C for 1 h. After another washing, 100 µl of horseradish peroxidase-conjugated goat anti-mouse secondary antibody in blocking buffer (diluted 1:1,000; Kirkegaard and Perry Laboratory) was added to the plates and incubated for an additional hour at 37°C. After a washing step, bound antibody was detected colormetrically by using
2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid) (ABTS) as a
substrate (Kirkegaard and Perry Laboratory). Titers were defined as the
reciprocal of the last dilution with an A405 of
0.1. Titers of <2.00 and >5.61 log10 were estimated. Serum samples from individual animals were assayed in duplicate. The
duplicate measurements were then used to calculate a geometric mean
titer for the group.
Statistical analysis.
For ELISA titer data obtained after
BALB/c mice were inoculated at different intervals, an analysis of
variance with a Student-Newman-Keuls multiple-range test was used to
identify differences between the treatment groups. The probability of a
type one error was set at 5% (
= 0.05). The Fisher exact test
was used to determine statistical differences in survival between
groups that received HC-VRP and the negative control group
that received N-VRP.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Packaging and expression of the HC-replicon. The HC-replicon was assembled into VRPs by using the bipartite helper system originally developed by Pushko et al. (15). The amount of packaged HC-replicon obtained in the cell culture supernatants ranged from 1.2 × 107 iu/ml to 4.8 × 107 iu/ml. No replication-competent virus was detected in either the medium from cotransfected cultures or after blind passage of the medium in BHK cell cultures.
Figure 1 shows a photomicrograph of BHK cells infected with HC-VRP. Antigen-positive cells were detected by using a primary polyclonal horse anti-BoNT/A antibody and a secondary fluorescein isothiocyanate-conjugated polyclonal goat anti-horse antibody. The staining pattern was consistent with cytoplasmic expression and retention of the HC polypeptide. Extensive nuclear, Golgi, or plasma membrane staining was not observed in BHK cells infected with HC-VRP. Cell lysates generated from BHK cells infected with HC-VRP contained large amounts of HC polypeptide, as demonstrated by Western blot analysis (Fig. 2). VEE replicons expressing HC produced proteins that comigrated on acrylamide gels with protein expressed in Escherichia coli and reacted with antibodies raised to the same protein.
|
|
Effect of HC-VRP dose on protection against challenge
with BoNT/A.
Figure 3 shows the
ELISA titers and survival for BALB/c mice inoculated with two doses of
105, 106, or 107 iu of
HC-VRP. The amount of HC-VRP (given on days 0 and 28) required to completely protect BALB/c mice from a lethal
challenge of 1,000 MLD50 BoNT/A was between 106
and 107 iu per dose. The prechallenge serum ELISA titers
from BALB/c mice inoculated with 105, 106, or
107 iu of HC-VRP were 1.27, 3.81, and 4.56 log10, respectively, compared to 1.73 log10 for
mice that received N-VRP, the negative control replicon. None of the
animals inoculated with 105 iu of HC-VRP or the
negative control replicon survived challenge, whereas 8 of 10 and 10 of
10 mice that received 106 or 107 iu of
HC-VRP, respectively, survived challenge.
|
, Serum obtained on day 26; 
, prechallenge serum
obtained on day 56; Effect of HC-VRP vaccination schedule on protection
against challenge with BoNT/A.
Results from animal studies
demonstrated that the VEE replicon expressing the 50-kDa HC
fragment of BoNT/A could protect mice from a lethal challenge of BoNT/A
(Table 1). BALB/c mice inoculated with
107 iu of HC-VRP generally produced a primary
antibody response which was maximal at day 19 and remained constant to
day 26 (Table 1). Booster inoculations given 7, 14, 21, or 28 days
after the primary inoculation induced a secondary antibody response
that was 60-to 159-fold greater than the primary response. If both
doses of the HC-VRP were given on the same day (i.e., a
dose of 2 × 107 iu), the primary antibody response 28 days later was 2.96 log10 compared to 1.73 log10 for mice that received two doses of the control N-VRP
at days 0 and 28, and were bled 28 days later. Mice that received the
equivalent of two doses of HC-VRP on day 0 were not
protected from challenge (1,000 MLD50 units of BoNT/A).
However, the time to death was increased from 6 h (for mice that
received the N-VRP) to 33 h. Mice that received a booster
inoculation on day 7 produced a secondary antibody response of 3.23 log10 with 8 of 10 mice surviving challenge (1 mouse showed
symptoms of slight flaccid paralysis). Mice that received booster
inoculations on day 14, 21, or 28 produced higher secondary antibody
responses of 3.87, 4.44, or 4.56 log10, respectively, with
all mice surviving challenge with no apparent symptoms. Thus, at this
dose, the most effective vaccination schedule was two doses of
HC-VRP given at least 14 days apart.
|
Effect of the BoNT/A challenge dose on protection conferred by
HC-VRP.
To determine the level of protection achieved
with the HC-VRP, vaccinated mice were challenged with
increasing amount of BoNT/A from 100 to 100,000 MLD50 units
(Table 2). Two inoculations of 107 iu of HC-VRP given 28 days apart protected
all mice from challenge at 100 and 1,000 LD50 units and 8 of 9 or 9 of 10 mice from a challenge of either 10,000 or 100,000 LD50 units of BoNT/A, respectively. The mice that survived
showed no effects from the neurotoxin, i.e., no flaccid paralysis. The
two mice that failed to survive challenge had ELISA titers of 2.00 and
5.01 log10.
|
Duration of HC-VRP induced immunity against
BoNT/A.
To determine the duration of immunity induced by
HC-VRP vaccinations, Swiss mice were given two inoculations
of 106 or 107 iu of HC-VRP 28 days
apart and then serologically monitored for 13 months (Fig.
4). Two additional groups were inoculated
with a third dose of HC-VRP on either day 168 or day 341. The responses in these mice were compared to control animals that
received the toxoid vaccine or 107 iu of N-VRP. The peak
antibody response in the mice given two doses of 106 iu of
HC-VRP was 4.90 log10, which occurred at day
110 (Fig. 4B). Before the 6-month challenge (day 196), the geometric
mean titer (GMT) of antibody in the mice was 3.99 log10,
which protected all 10 mice from the effects of 1,000 MLD50
units of BoNT/A. The 12-month prechallenge titers in mice were 4.50 log10, which protected 18 of 19 mice challenged with the
same amount of toxin. Mice inoculated with 107 iu of
HC-VRP produced an antibody response of 5.07 log10 at day 110 (Fig. 4C). The antibody response in mice
inoculated with two doses of 107 iu of HC-VRP
tended to be higher than in mice similarly inoculated with
106 iu of HC-VRP. The antibody responses before
the 6- and 12-month challenges were 4.54 and 5.08 log10,
which protected 10 of 10 mice and 18 of 18 mice, respectively. All mice
that survived challenge at either 6 or 12 months remained healthy, with
no signs of BoNT intoxication. Mice inoculated with the toxoid vaccine
produced an antibody response that peaked at 6.28 log10 on
day 63. The antibody response decreased with time such that on day 194 (2 days before the 6-month challenge) the titer was 5.81 log10, and 10 of 10 mice survived the 6-month challenge
(Fig. 4A). Before the 12-month challenge, the titers in the mice had
decreased further to 5.31 log10; but all the mice survived
the 12-month challenge with no morbidity. The antibody response in
control mice inoculated with N-VRP was negligible at 2.17 and 1.30 log10 before the 6- and 12-month challenges, respectively.
As expected, all N-VRP inoculated mice failed to survive challenge.
|
) or 107 iu of
Lassa N-VRP (A, 
) or with 106 iu (B, 
) or
107 iu (C, 
) of HC-VRP. These results are
reproduced in panel D without error bars for comparison. See Materials
and Methods for the inoculation schedule. Different groups of mice were
challenged on either day 196 or day 370 (open arrows) with 1,000 MLD50 units of BoNT/A. Numbers in boxes indicate survivors
versus the total number when challenged at the indicated times. Sera
from individual animals were assayed in duplicate, and the duplicate
measurements were used to calculate the GMT for a minimum of 10 animals
bled in a staggered fashion; titers greater than 5.61 log10
and less than 2.00 log10 were estimated.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Although toxoid preparations for BoNT have proven effective for vaccinating against some serotypes, the production, preparation, and quality control of the required neurotoxins is expensive, labor-intensive, and hazardous. As a result, recent vaccine development efforts have focused on the production of nontoxic recombinant proteins or vectored vaccines. Both E. coli and yeast expression systems have been used in the production of BoNT HC (2, 4).
Vaccines composed of recombinant HC protein have been shown to protect mice from the effects of BoNT/A. Clayton et al. expressed a synthetic HC gene in E. coli (1, 13, 17, 24) and used whole bacterial cell lysates containing the HC polypeptide combined with Freund adjuvant to protect some ICR mice from challenge (4). Subsequently, Byrne et al. developed a method for purification of the HC polypeptide expressed from the synthetic gene in the yeast Pichia pastoris (2) and showed protection in mice vaccinated with purified HC combined with aluminum hydroxide adjuvant (2, 9). Another approach for developing BoNT vaccines has focused on "naked" DNA vaccine vectors. Two research groups have demonstrated that DNA-based vaccines can partially protect animals from challenge with BoNT/A (3, 18).
Alphavirus replicon vectors have the ability to express high amounts of a foreign protein in eukaryotic cells (12, 15), and such vectors show considerable promise as vaccine vectors for the transient expression of foreign genes in animals. By inserting the synthetic HC gene in the VEE replicon vector, we were able to achieve high-level expression of HC polypeptide in eukaryotic cells as visualized by immunofluorescence of cells infected with HC-VRP and also by Western blot analysis of cell lysates generated after cells were infected with the same HC-VRP. Because alphaviruses replicate in the cytoplasm of eukaryotic cells (20), expression of the HC gene in the cytoplasm alleviates the difficulties imposed by conventional nuclear transcription of plasmids, i.e., limiting or incompatible transcription factors, mRNA splicing, and transport of the mRNA out of the nucleus (17).
In the studies reported here, VEE replicon vaccines expressing the HC fragment of BoNT/A (HC-VRP) induced a strong antibody response in BALB/c mice that was both dose and schedule dependent. Mice inoculated with the highest dose of HC-VRP (107 iu) produced the greatest antibody responses and were completely protected from challenge. Vaccinations with doses of less than 107 iu stimulated a weaker immune response, which only partially protected the animals. To define an optimal vaccination schedule, mice were inoculated with two doses of 107 iu of HC-VRP separated by 0, 7, 14, 21, or 28 days. The antibody responses induced varied directly with the length of time between the two doses. Inoculations spread out over several weeks stimulated the strongest immune responses, indicating that anamnestic responses were elicited. Complete protection from challenge was observed in groups of mice vaccinated at an interval of 14 days or more between inoculations. However, 80% of the mice were protected with two doses given only 7 days apart. Mice inoculated with two doses of 107 iu of HC-VRP at an interval of 28 days produced the highest antibody responses and were protected against very high doses of toxin (100,000 MLD50 units).
Previous research utilizing viruses as vaccine vectors has shown that animals vaccinated with such vectors often developed high neutralizing responses against the vector, as well as immune responses against the foreign gene (11). In these studies, the VEE replicon vector induced anti-VEE neutralizing antibodies in the outbred mice but not in the BALB/c mice (data not shown). Nevertheless, the anti-BoNT/A antibody responses induced in the outbred mice were higher than those observed in the BALB/c mice. The VEE neutralizing antibody responses may have been stimulated by the presence of viral glycoprotein in the replicon particles themselves, or from copurified cell membrane debris containing VEE glycoprotein, or from a recombination or copackaging event between the replicon and the glycoprotein helper RNA (7, 15, 16, 25). Additional studies are in progress to define the basis for the induction of VEE neutralizing immune responses in the outbred mice.
The duration of immunity and protection induced by HC-VRP was also evaluated and compared to that achieved with the toxoid vaccine. Outbred Swiss mice were found to produce somewhat higher antibody responses than that seen in BALB/c mice, so the outbred mice were then used to evaluate the duration of immunity induced by HC-VRP. We found that two inoculations of 107 iu of HC-VRP given on days 0 and 28 produced both primary and secondary anamnestic antibody responses that efficiently protected these mice from a BoNT/A challenge at 6 and at 12 months postvaccination. Swiss mice inoculated with the toxoid vaccine initially produced somewhat higher antibody responses than those inoculated with two doses of 107 iu of HC-VRP, but the level of toxoid-induced antibody fell over time such that, at 12 months, the response was similar in both groups. No appreciable fall in antibody titers was noted over a period of 12 months in the mice inoculated with two doses of either 106 or 107 iu of HC-VRP. Swiss mice given three inoculations of 106 or 107 iu of HC-VRP, at days 0 and 28, and then boosted again at day 168 or day 341 were also completely protected from challenge at day 370.
Since seven serotypes of BoNT are known, the ease with which genes may be cloned into the VEE replicon make this vector system attractive as a platform for developing multivalent vaccines. Replicon vaccines expressing the remaining six serotypes have been constructed and are being studied for their immunogenicity both individually and in combination. In addition, single replicons expressing several HC genes are also being evaluated. Our use of the VEE replicon as a vaccine vector for inducing immune responses against BoNT demonstrates that prokaryotic genes can also be accurately and efficiently expressed in eukaryotic cells with this vector system and that such expression can elicit a highly protective immune response of long duration.
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FOOTNOTES |
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* Corresponding author. Mailing address: Virology Division, USAMRIID, Fort Detrick, Frederick, MD 21702. Phone: (301) 619-4912. Fax: (301) 619-2290. E-mail: John.Lee{at}det.amedd.army.mil.
Editor: J. T. Barbieri
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Infection and Immunity, September 2001, p. 5709-5715, Vol. 69, No. 9
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.9.5709-5715.2001
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