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Two rAd vectors, shown in Figure 1A, were constructed for this study. The test vector, rAd-Bg, carries the human cytomegalovirus (CMV) promoter-driven chimeric EDIII-4/2 antigen
gene (derived by linking the EDIII-encoding cDNAs generated from DEN-4
and DEN-2 viral genomic RNAs) fused to a 3' green fluorescent protein
gene (GFP) tag, inserted into the Ad5 genome, in place of its
E1 region. The control vector, rAd-C, is similar in design, with the
exception that it carries an empty CMV expression cassette. Aliquots of
the rAd viral DNA were subjected to PCR analysis using Ad E1- and insert-flanking primers as described earlier . The results are shown in Figure 1B. The E1-specific
primers produced the predicted ~0.47 Kb amplicon when the template was
DNA from wild type (wt) Ad (lane 2), but not when it was from either
the rAd-C (lane 3), or rAd-Bg (lane 4) viruses. PCR with
insert-flanking primers resulted in the amplification of a ~1.5 Kb cDNA
only from rAd-Bg (lane 8) but not from the wt Ad DNA template (lane 6).
From rAd-C viral DNA as the template, the insert-flanking primers
amplified a ~0.1 Kb vector derived cDNA product (lane 7). Extensive
restriction mapping of the rAd-C and rAd-Bg viral DNAs were found to be
consistent with the construction strategy (data not shown).
Figure 1. The rAds constructed for this study.
(A) Schematic representation of the linear genomes of the rAd-C and
rAd-Bg viruses. The dashed line, close to the left end represents the
position of region E1. The open box located towards the right end
represents a ~2.7 Kb deletion in the non-essential E3 region (ΔE3).
The hatched boxes at either end represent the left (L) and right (R)
inverted terminal repeats (ITRs), and the open circle close to the
L-ITR represents the packaging signal (ψ).
In the rAds, region E1 is replaced either by an empty expression
cassette (rAd-C) or the EDIII-4/2 expression cassette (rAd-Bg).
Components of the expression cassettes are indicated by the following
abbreviations, P: CMV promoter (the arrow indicates its orientation); 4
and 2: DEN-4 and DEN-2 EDIII-encoding regions, respectively, of the
bivalent EDIII-4/2 gene; g: enhanced green fluorescent protein gene (fused in-frame to the EDIII-4/2 gene)
and pA: SV40 polyadenylation signal. (B) Characterization of the rAd
genomes by PCR. The picture depicts agarose gel analysis of PCR
products generated using wt Ad5 (lanes 2, 6), rAd-C (lanes 3, 7) or
rAd-Bg (lanes 4, 8) viral DNA templates. Primers used were either
E1-specific (lanes 2–4) or specific to insert-flanking sequences (lanes
6–8). DNA size markers were run in lanes 1 and 5. Sizes of the lane 1
markers (in Kb) are indicated to the left of the panel. The position of
the 1.6 Kb band in lane 5 is indicated by a white dot for orientation.
The arrows to the right indicate the positions of the amplicons
corresponding to the bivalent insert (top), E1 region (middle) and the
insert-less vector sequences (bottom).
We next examined the ability of the rAd-Bg virus to
express the bivalent antigen by direct fluorescence microscopy to
detect its GFP tag. In the experiment shown in Figure 2A, HEK 293 cells were observed ~24 hours after infection with rAd-Bg (panel ii).
Detection of the C-terminal GFP tag suggests successful rAd-mediated
expression of the bivalent antigen. On the other hand, rAd-C
virus-infected cells (panel i), analyzed in parallel, did not
display any green fluorescence. That the rAd-C virus had efficiently
infected the cells was evident from the cytopathicity observed by
visible light microscopy (data not shown). In addition, we
carried out indirect immunofluorescence assays (IFA) to detect the
EDIIIs corresponding to DEN-2 and DEN-4 in the bivalent antigen. To
this end, we probed rAd-Bg infected cells, separately, with monoclonal
antibodies (mAbs) 3H5 and MAB8704 to identify EDIIIs corresponding to
DEN-2 and DEN-4, respectively. As the bivalent antigen was GFP-tagged,
we used anti-murine IgG-rhodamine conjugate as the secondary antibody
in the IFA experiment. 3H5 is a DEN-2 serotype-specific mAb, which
specifically recognizes an epitope that maps to amino acid (aa)
residues 386–397 of the E protein .
As this epitope falls within the DEN-2 EDIII region, this mAb is
expected to recognize the rEDIII-4/2 bivalent antigen as well. MAB 8704
is a DEN-4 virus-specific mAb. We found that it was able to
specifically recognize E. coli-expressed DEN-4 EDIII (our unpublished data).
Thus, this mAb also is expected to recognize the rEDIII-4/2 bivalent
antigen. Consistent with this, probing rAd-Bg infected cells with mAbs
3H5 (panel iii) and MAB8704 (panel iv) resulted in
red immunofluorescence. We also identified the bivalent antigen in
rAd-Bg virus-infected cells using an immunoprecipitation approach
In this experiment, we used mAb 3H5 to immunoprecipitate the bivalent
antigen expressed in infected HEK 293 cells. Consistent with the direct
fluorescence and IFA data, the 3H5 mAb specifically identified a
protein of the predicted size (~55 kDa) in rAd-Bg infected (lane 4),
but not in rAd-C infected (lane 2) and mock infected (data not shown)
HEK 293 cells. As a positive control, a parallel immunoprecipitation
was performed using cells infected with a previously reported rAd ,
expressing aa 1–395 of the DEN-2 virus E protein (lane 3). Taken
together, these data showed that the rAd-Bg virus is capable of
expressing the intended chimeric bivalent antigen in infected mammalian
Figure 2. rAd-mediated expression of rEDIII-4/2 protein as aGFP fusion.
(A) Detection of rAd-mediated bivalent antigen expression by
fluorescence microscopy. HEK 293 cells were either infected with rAd-C
(panel i), or rAd-Bg (panel ii-iv) for 24 hours and examined either directly for GFP fluorescence (panels i and ii) or analyzed by IFA, using either mAb 3H5 (panel iii) or MAB8704 (panel iv),
as the primary antibody in conjunction with rhodamine-conjugated
anti-murine IgG as the secondary antibody. (B) Immunoprecipitation of
the bivalent protein from infected HEK 293 cells. Cells were infected
with rAd-C (lane 2), a rAd expressing DEN-2 E (lane 3 [ref. 45]) or
rAd-Bg (lane 4) viruses. Infected cells were metabolically labeled with
[35S]-methionine, lysed, immunoprecipitated with 3H5 mAb,
and analyzed on a denaturing 15% polyacrylamide gel. Pre-stained
protein markers were run on the same gel (lane 1); their sizes (in kDa)
are indicated on the left. The arrows on the right show the positions
of the bivalent protein (upper) and the E protein (lower).
A major objective of this study was to investigate if a single rAd
vector encoding an EDIII-based bivalent DEN antigen would elicit
antibodies specific to both the constituent serotypes. To this end, we
undertook mice immunization experiments. As anti-Ad antibodies
resulting from the initial immunization of the mice can potentially
interfere with the efficacy of the rAd vector to function as a vaccine
carrier during booster immunizations, we adopted a heterologous
prime/boost strategy that entailed priming the animals with the rAd
vector, followed by boosting with a plasmid vector encoding the same
bivalent antigen gene. Sera were collected periodically after priming
and after each boost. We performed two series of ELISAs using these
sera. In the first experiment, we determined antibody titers after each
immunization, using the bivalent r-EDIII-4/2 protein as the capture
antigen, as shown in Figure 3A.
It is evident from the data presented that initial antibody titers
which were relatively low, progressively increased after each boost,
with maximal titers being observed after the final plasmid boost. In
the next experiment, we analyzed the capacity of sera obtained after
the final boost, to specifically react with monovalent EDIII proteins
corresponding to each of the four DEN virus serotypes. Accordingly, in
this experiment antibody titers were determined using using rEDIII-1,
rEDIII-2, rEDIII-3 and rEDIII-4 proteins, as capture antigens. The
results of this experiment, depicted in Figure 3B,
showed that serum from the immunized animals manifested high levels of
reactivity towards the rEDIII proteins corresponding to DEN virus
serotypes 2 and 4, but not to serotypes 1 and 3.
Figure 3. Reactivity of immune sera towards monovalent and bivalent rEDIII proteins.
(A) Antibody titers in serial dilutions of murine sera drawn 1 week
after priming with rAd-Bg (blue) and 1 week following the first (red),
second (green) and final (violet) boosts with pVAX-EDIII-4/2 plasmid
were determined in an ELISA using the rEDIII-4/2 bivalent protein as
the capture antigen. The black dashed curve indicates ELISA reactivity
of control sera, drawn from rAd-C primed/pVAX1-boosted mice after the
final boost. (B) Antibody titers in serial dilutions of murine sera
drawn from rAd-Bg primed/pVAX-EDIII-4/2 boosted mice, one week after
the final boost. ELISA was performed using rEDIII-1 (blue), rEDIII-2
(green), rEDIII-3 (red) and rEDIII-4 (violet) proteins as capture
antigens. The black dashed curve indicates ELISA reactivity of control
sera using a mixture of the four rEDIII proteins as capture antigen.
All ELISA values in this experiment represent the average of two
To address the question if these antibodies would
display this serotype preference toward the DEN viruses themselves, we
carried out an immunofluorescence-based assay. BHK cells were
separately infected with each of the four DEN virus serotypes and
probed with the test antiserum (obtained from rAd-Bg primed/bivalent
plasmid boosted animals) in conjunction with a secondary antibody-FITC
conjugate. The prediction was that the test antiserum should light up
cells infected with DEN-2 and DEN-4, but not DEN-1 and DEN-3 viruses.
The results of this experiment which substantiate this are presented in
Figure 4A. Panels in the first (a, e, i and m) and second (b, f, j and n) columns depict the negative and positive controls, respectively. DEN-1 (panel b), DEN-2 (panel f), DEN-3 (panel j) and DEN-4 (panel n) viruses were detected using murine polyclonal antibodies raised against E. coli-expressed rEDIII-1, rEDIII-2, rEDIII-3 and rEDIII-4 proteins, respectively. The detection of immunofluorescence in panels b, f, j and n demonstrates
that DEN-1, 2, 3 and 4 viruses, respectively, had successfully infected
the cells in this experiment. Mock-infected cells did not fluoresce
with any of these antibodies (panels a, e, i and m),
indicating that the antibodies used are virus-specific. The control
serum (obtained from rAd-C primed/empty plasmid boosted animals) also
resulted in no fluorescence in DEN virus-infected cells (panels c, g, k and o). This suggests that the vaccine vectors per se do
not induce a DEN virus-specific immune response. Consistent with the
ELISA data, the use of test serum as the source of primary antibodies
in the assay resulted in the identification of DEN-2 (panel h) and DEN-4 (panel p), but not DEN-1 (panel d) and DEN-3 (panel l)
viruses, as predicted. Taken together, the data strongly suggest that
the bivalent EDIII-4/2 antigen elicits antibodies that specifically
recognize DEN-2 and DEN-4 viruses with little evidence of
cross-reactivity towards DEN-1 and DEN-3 viruses.
Figure 4. Simultaneous induction of antibodies that recognize and neutralize DEN virus serotypes 2 and 4. (A) IFA of DEN virus-infected cells using murine antisera. BHK cells were either mock infected (panels a, e, i and m) or infected separately with DEN-1 (panels b, c, d), DEN-2 (panels f, g, h), DEN-3 (panels j, k, l) and DEN-4 (panels n, o, p)
viruses. One day after infection, cells were fixed and probed
separately with different antisera as the source of primary antibodies.
The antisera were obtained from mice, immunized with purified
recombinant proteins [rEDIII-1 (panels a, b), rEDIII-2 (panels e, f), rEDIII-3 (panels i, j), and rEDIII-4 (panels m, n)] and rAd/plasmid [rAd-C/pVAX1 (panels c, g, k, o) or rAd-Bg/pVAX-EDIII-4/2 (panels d, h, l, p)].
The resultant virus/antibody complexes were visualized using anti-mouse
IgG-FITC conjugate. (B) LLCMK2 monolayers were separately infected with
each of the four DEN viruses (DEN-1: filled circles; DEN-2: filled
squares; DEN-3: open circles; & DEN-4: open squares) that had been
pre-incubated with serial two-fold dilutions of pooled immune serum
drawn from the rAd-Bg primed/plasmid pVAX-EDIII-4/2 boosted animals.
The resultant plaque counts, expressed as percent inhibition of virus
infectivity (with reference to the number of plaques generated in the
absence of antiserum, which was taken to represent 100% infectivity)
are plotted as a function of antiserum dilution. Each data point shown
represents the mean of four replicate assays (the error bars represent
While the data obtained so far indicated that the
EDIII-4/2 antigen induces a bivalent response targeting DEN-2 and DEN-4
viruses, it does not provide any information regarding the capacity of
these antibodies to neutralize their infectivity. To this end, we
performed plaque reduction neutralization tests (PRNTs), by plaqueing
the antibody-incubated DEN viruses on LLCMK2 cells. Figure 4B
presents the percent reduction in virus infectivity as a function of
test serum dilution for each of the four DEN virus serotypes. The data
show that the test serum displayed PRNT50 titers of ~1:80
towards both DEN-2 and DEN-4 viruses, with no significant neutralizing
antibody titers towards DEN-1 and DEN-3 viruses.
Spleen cells in culture, derived from control and test mice, were stimulated in vitro with DEN-2 and DEN-4 viruses, separately, and monitored for proliferation responses and cytokine (IFN-γ and IL-4) production, as depicted in Figure 5. Splenocytes from test mice manifested significant and similar levels of proliferation in response to in vitro stimulation with both DEN-2 and DEN-4 viruses, compared to mock-stimulated splenocytes (Figure 5A).
Control splenocytes did not manifest any proliferative response
regardless of whether they were stimulated or not. The proliferative
response of the test splenocytes was accompanied by IFN-γ secretion (Figure 5B). The elevated levels of IFN-γ,
first evident at 48 hours post stimulation, persisted for the duration
of the experiment. Again, as seen in the thymidine uptake experiment,
both DEN-2 and DEN-4 virus stimulation resulted in comparable levels of
IFN-γ production. At all time points tested, mock-stimulated test cells did not secrete discernible levels of IFN-γ. When control mouse spleen cell cultures were tested, IFN-γ
was barely detectable in mock- and virus-stimulated culture
supernatants. With regard to IL-4, the splenocytes manifested an
increase in the levels of IL-4 secretion in response to virus
stimulation, but it was not dramatic (as seen for IFN-γ),
as basal IL-4 levels in control splenocytes were somewhat elevated. The
magnitude of stimulation of IL-4 secretion in test splenocytes with
respect to mock-stimulated cells, which was about 2 fold higher at 24
hours, increased to about 5 fold at 96 hours (Figure 5C).
Taken together, these experiments demonstrate that the bivalent
antigen, elicits DEN-2 and DEN-4 virus-specific T-cell responses.
Interestingly, the magnitude of these responses was more or less
equivalent for the two serotypes.
Figure 5. Analysis of T cell responses elicited by the bivalent antigen.
Splenocytes were obtained from mock-immunized (open bars) and
test-immunized (gray, black and hatched bars) animals, 7 weeks after
the final immunization and placed in culture. They were either
mock-stimulated (open and gray bars) or stimulated in vitro, either with DEN-2 (black bars) or DEN-4 (hatched bars) for 96 hours for performing the T cell assays. Tritiated [3H]
thymidine uptake was determined in a scintillation counter (A).
Aliquots of the culture supernatant withdrawn at the indicated time
points were assayed for the presence of IFN-γ
(B) and IL-4 (C) by solid phase ELISA. Data depicted represent the mean
value of three separate determinations (the error bars represent SD).
In panel A, two dosage levels of virus were used for in vitro stimulation
[L, low (0.015 PFU/cell); and H, high (0.03 PFU/cell); N, no antigen].
Data in panels B and C were generated using splenocytes stimulated with
the high dose.
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