مجال
التميز
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تميز دراسي وبحثي
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البحوث المنشورة
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البحث (1):
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عنوان البحث:
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Expression and
Cellular Immunogenicity of a Transgenic Antigen Driven by Endogenous Poxviral
Early Promoters at Their Authentic Loci in MVA
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رابط الوصول:
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Click
here
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تاريخ النشر:
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27/06/2012
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موجز عن البحث:
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CD8+ T cell
responses to vaccinia virus are directed almost exclusively against early
gene products. The attenuated strain modified vaccinia virus Ankara (MVA) is
under evaluation in clinical trials of new vaccines designed to elicit
cellular immune responses against pathogens including Plasmodium spp.,
M. tuberculosis and HIV-1. All of these recombinant MVAs (rMVA)
utilize the well-established method of linking the gene of interest to a
cloned poxviral promoter prior to insertion into the viral genome at a suitable
locus by homologous recombination in infected cells. Using BAC
recombineering, we show that potent early promoters that drive expression of
non-functional or non-essential MVA open reading frames (ORFs) can be
harnessed for immunogenic expression of recombinant antigen. Precise
replacement of the MVA orthologs of C11R, F11L, A44L and B8R
with a model antigen positioned to use the same translation initiation codon
allowed early transgene expression similar to or slightly greater than that
achieved by the commonly-used p7.5 or short synthetic promoters. The
frequency of antigen-specific CD8+ T cells induced in mice by
single shot or adenovirus-prime, rMVA-boost vaccination were similarly equal
or marginally enhanced using endogenous promoters at their authentic genomic
loci compared to the traditional constructs. The enhancement in
immunogenicity observed using the C11R or F11L promoters
compared with p7.5 was similar to that obtained with the mH5 promoter
compared with p7.5. Furthermore, the growth rates of the viruses were
unimpaired and the insertions were genetically stable. Insertion of a
transgenic ORF in place of a viral ORF by BAC recombineering can thus provide
not only a potent promoter, but also, concomitantly, a suitable insertion
site, potentially facilitating development of MVA vaccines expressing
multiple recombinant antigens.
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المؤتمرات العلمية:
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المؤتمر (1):
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عنوان المؤتمر:
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Poxvirus 2012,
The XIX International Poxvirus, Asfarvirus and Iridovirus Conference
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تاريخ الإنعقاد:
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24-28/06/2012
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بلد
ومكان الإنعقاد:
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Salamanca, Spain
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طبيعة المشاركة:
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Poster
presentation
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عنوان
المشاركة:
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ENHANCING TRANSGENE EXPRESSION
AND IMMUNOGENICITY IN MVA
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ملخص المشاركه:
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The
CD8+ T cell response to a transgenic antigen expressed in MVA can be enhanced
by using a promoter with increased early activity. We are pursuing three
approaches to augmenting early transgene expression in recombinant MVA.
First, we are using BAC recombineering to replace MVA open reading frames
with a transgenic reporter construct, placing it under control of viral
promoters at their natural loci. Replacement of B8R, C11R, F11L or
E3L with Renillaluciferase (rLuc) revealed that the endogenous promoters
could drive early transgene expression and elicit murine CD8+ T cell
responses similar to or greater than the conventionally used promoters p7.5
and modified H5 (mH5) (Wyatt et al. 1996 Vaccine 14:1451-8).
Second, we are attempting to boost early activity, starting with the B8R promoter
(pB8R), by replacement of unfavourable nucleotides in the early promoter core
region, as originally described for mH5. Third, we aimed to prolong
translation of early transcripts by inserting an IRES upstream of the rLuc reporter
in order to counteract decapping, but this did not result in any detectable
effect on transgene expression in vitro. In agreement with other
studies,we show that enhancement of the immunogenicity of the transgene
product is associated with reduced immunodominance of viral antigens. A
potential advantage of theendogenous promoter strategy is that each of these
strong early promoters is automaticallyassociated with its own novel
insertion site in MVA. This may find applicationfor development of multivalent
T cell inducing vaccines against diseases such as influenza,tuberculosis and
malaria.
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المؤتمر (2):
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عنوان المؤتمر:
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7th Vaccine
& International Society for Vaccine Congress
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تاريخ الإنعقاد:
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27/10/2013
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مكان
الإنعقاد:
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Barcelona, Spain
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طبيعة المشاركة:
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Poster presentation
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عنوان المشاركة:
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A combination of new insertion loci and
endogenous promoters drives higher transgene immunogenicity in MVA
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ملخص المشاركة:
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Introduction: Modified Vaccinia virus
Ankara has been used as a vaccine vector to elicit strong CD8+ T cell
responses, preferably when strong early promoter is used. We show that F11L,
B8R, and E3L early promoters utilized at their natural loci to drive the
expression of a CD8+ T cell-specific malaria epitope from Plasmodium Berghei
(pb9) showed improved immunogenicity in Balb/c mice. Methods: Recombineering
technology was used to manipulate MVA genome cloned into bacterial artificial
chromosome (MVA-BAC). Then, a helper Fowlpox virus (FP9) was used to derive
recombinant MVAs in BHK cells. Balb/c mice were used for immunization
studies. Results: We demonstrate that using F11L, B8R, and E3L endogenous promoters
at their natural loci in MVAs drive more immunogenicity compared to the
conventionally used mH5 or p7.5 promoter inserted at the thymidine kinase
(TK) locus. Our results suggest that utilizing MVA endogenous promoter while
using their loci as insertion sites gives higher level of immunogenicity.
Furthermore, we show that the improved immunogenicity is partially due to
keeping TK locus intact. Discussion: In conclusion, utilizing F11L, B8R, and
E3L early promoters is important to obtain a better level of immunogenicity
due to (a) the strong activity of these promoters, and (b) keeping TK
undisrupted.
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المؤتمر (3):
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عنوان المؤتمر:
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Saudi Scientific International Conference
2012
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تاريخ الإنعقاد:
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10/10/2012
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مكان
الإنعقاد:
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Brunel, UK
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طبيعة المشاركة:
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Poster presentation
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عنوان المشاركة:
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ENHANCING TRANSGENE EXPRESSION AND
IMMUNOGENICITY IN Modified Vaccinia Ankara Virus
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ملخص المشاركة:
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The CD8+ T cell response to a transgenic
antigen expressed in MVA can be enhanced by using a promoter with increased early
activity. We are pursuing three approaches to augmenting early transgene
expression in recombinant MVA. First, we are using BAC recombineering to
replace MVA open reading frames with a transgenic reporter construct, placing
it under control of viral promoters at their natural loci. Replacement of
B8R, C11R, F11L or E3L with Renilla luciferase (rLuc) revealed that the
endogenous promoters could drive early transgene expression and elicit murine
CD8+ T cell responses similar to or greater than the conventionally used
promoters p7.5 and modified H5 (mH5) (Wyatt et al. 1996 Vaccine 14:1451-8).
Second, we are attempting to boost early activity, starting with the B8R
promoter (pB8R), by replacement of unfavourable nucleotides in the early
promoter core region, as originally described for mH5. Third, we aimed to
prolong translation of early transcripts by inserting an IRES upstream of the
rLuc reporter in order to counteract decapping, but this did not result in
any detectable effect on transgene expression in vitro. In agreement with
other studies, we show that enhancement of the immunogenicity of the
transgene product is associated with reduced immunodominance of viral
antigens. A potential advantage of the endogenous promoter strategy is that
each of these strong early promoters is automatically associated with its own
novel insertion site in MVA. This may find application for development of
multivalent T cell inducing vaccines against diseases such as influenza,
tuberculosis and malaria.
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المؤتمر (4):
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عنوان المؤتمر:
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The 2nd European Seminars in Virology
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تاريخ الإنعقاد:
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13-15/06/2014
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مكان
الإنعقاد:
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Italy
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طبيعة المشاركة:
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Oral presentation
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عنوان المشاركة:
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IMPROVED MVA VACCINE IMMUNOGENICITY
ACHIEVED BY USING ENDOGENOUS EARLY PROMOTERS
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ملخص المشاركة:
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Introduction: Modified vaccinia virus
Ankara has been extensively used as a vaccine vector to elicit strong CD8+ T
cell responses, typically using an early promoter to drive transgene
expression. Our previous published work showed that the early promoter of
F11L or B8R ORF, utilised at their natural loci to drive the expression of a
CD8+ T cell-specific malaria epitope from Plasmodium berghei (pb9), elicited
improved cellular immunogenicity in BALB/c mice, compared to that achieved by
recombinant MVAs (rMVAs) with the conventional p7.5 or mH5 promoters. In the
latter case, those conventional promoters were inserted at the thymidine
kinase (TK) locus. Here, we extend our investigation to determine the
mechanisms underlying this observed enhancement in immunogenicity, which
could be a result of one or many possibilities. These possibilities include
the effect of deleting the natural F11L or B8R ORFs, and the use of these
ORFs as insertion sites instead of the conventional TK insertion locus. The
presence of intact TK gene in rMVAs that have the endogenous promoters as
opposed to the disrupted TK locus in rMVA with the conventional p7.5 or mH5
promoters could also impact on the improved immunogenicity. Finally, the
strong activity of the F11L or B8R endogenous promoter was initially, in our
previous publication, thought to be the driving mechanism behind the enhanced
immunogenicity. We addressed these four possibilities by deriving a range of
recombinant MVAs. Our results suggested that the improved immunogenicity
achieved by utilising the F11L and B8R endogenous promoters in rMVAs is
mainly due to the strong activity of those promoters. However, the
inactivation of the TK gene in the rMVAs with the endogenous promoters
slightly reduced the cellular immunogenicity. Moreover, our results also
showed that the F11L or B8R promoter could drive the same level of
immunogenicity when they were taken out of their natural context and inserted
at the TK locus. This also ruled out the effect of F11L or B8R insertion
sites on the improved immunogenicity. Finally, the deletion of the original
F11L or B8R ORF did not appear to affect the immune responses. In conclusion,
the F11L or B8R endogenous promoters seems strong promoters that could be
used either at their authentic loci or at the TK locus and enhance superior
immunogenicity compared to the use of conventional p7.5 or mH5 promoters.
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المؤتمر (5):
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عنوان المؤتمر:
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EuroSciCon
Vaccine 2014
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تاريخ الإنعقاد:
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20/10/2014
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مكان
الإنعقاد:
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London,
UK
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طبيعة المشاركة:
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Poster
presentation
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عنوان المشاركة:
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IMPROVED
MVA VACCINE IMMUNOGENICITY ACHIEVED BY USING ENDOGENOUS EARLY PROMOTERS
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ملخص المشاركة:
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Modified
vaccinia virus Ankara has been extensively used as a vaccine vector to elicit
strong CD8+ T cell responses, typically using an early promoter to drive
transgene expression. Our previous published work showed that the early
promoter of F11L or B8R ORF, utilised at their natural loci to drive the
expression of a CD8+ T cell-specific malaria epitope from Plasmodium berghei
(pb9), elicited improved cellular immunogenicity in BALB/c mice, compared to
that achieved by recombinant MVAs (rMVAs) with the conventional p7.5 or mH5
promoters. In the latter case, those conventional promoters were inserted at
the thymidine kinase (TK) locus. The observed enhancement in immunogenicity
could be a result of many possibilities: 1) the deletion of the natural F11L
or B8R ORFs, even though those ORFs are fragmented in MVA genome; 2) the use
of F11L or B8R natural loci as insertion sites; 3) the presence of intact TK
gene in rMVAs that have the endogenous promoters as opposed to the disrupted
TK locus in rMVA with p7.5 or mH5 conventional promoter; or 4) the strong
activity of the F11L or B8R endogenous promoter. We addressed these four
possibilities by deriving a range of recombinant MVAs. Our results suggest
that the improved immunogenicity achieved by utilising the F11L and B8R endogenous
promoters in rMVAs is mainly due to the strong activity of those promoters.
However, the inactivation of the TK gene in rMVAs with the endogenous
promoters slightly reduced the cellular immunogenicity. Moreover, our results
also showed that the F11L or B8R promoters could drive the same level of
immunogenicity when they were taken out of their natural context and inserted
at the TK locus. This also ruled out the effect of F11L or B8R insertion
sites on the improved immunogenicity. Finally, the deletion of the original
F11L or B8R ORF did not appear to affect the immune responses. In conclusion,
utilising F11L or B8R endogenous promoters at their natural loci in rMVA
vaccines leads to improved immunogenicity, in comparison to the
conventionally used p7.5 or mH5 promoters inserted at the TK locus. This
improved immunogenicity could be partially due to keeping the TK locus
intact, but is mainly due to the strong activity of these early promoters. In
addition, we did not find the insertion sites, or the deletion of F11L or B8R
ORFs, to have an effect on vaccine immunogenicity.
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