Immunogenicity of Novel Dengue Virus Epitopes Identified by tic Analysis
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Transcript of Immunogenicity of Novel Dengue Virus Epitopes Identified by tic Analysis
8/8/2019 Immunogenicity of Novel Dengue Virus Epitopes Identified by tic Analysis
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8/8/2019 Immunogenicity of Novel Dengue Virus Epitopes Identified by tic Analysis
http://slidepdf.com/reader/full/immunogenicity-of-novel-dengue-virus-epitopes-identified-by-tic-analysis 2/8
Please cite this article in press as: Sánchez-Burgos,G., et al., Immunogenicity of novel Dengue virus epitopes identified by bioinformatic analysis.
Virus Res. (2010), doi:10.1016/j.virusres.2010.07.014
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VIRUS952101–8
2 G. Sánchez-Burgos et al. / Virus Research xxx (2010) xxx–xxx
et al., 2004) and bacteria (Al-Attiyah and Mustafa, 2004). How-
ever, these approaches have not been fully explored in the case
of DENV. For example, initial studies focused on the prediction
of epitopes from the E, prM or NS1 proteins allowed the identi-
fication of B and T cell epitopes in DENV-2 ( Vázquez et al., 2002;
Leclerc et al., 1993; Jiang et al., 2010). Analyzing proteins C, E and
NS3 for CD4+ T cell epitopes, Wen et al. (2007) identified four new
epitopes which stimulated IFN- production and proliferation of
PBMC isolated from DF convalescent patients infected with DENV-
1. Similarly, Bashyam et al. (2006) identified four epitopes located
in NS4b, NS4a andE proteins from a singleDENV-3 isolate. The role
of MHC class I-restricted CD8+ T cells was investigated with 106
predicted epitopes tested in an IFN- ELISPOT assay, and twelve
peptides derived from DENV proteins (C, M, E, NS2a, NS4b and
NS5) were identified as immunogenic (Yauch et al., 2009). Another
more comprehensive approach focused on the evolutionary anal-
ysis of a large number of DENV genome sequences, allowing the
identification of highly conserved regions among the four DENV
serotypes,but the immunogenicity of the conserved sequences was
notfullyexaminedand only a fewepitopesfrom the non-structural
proteins were identified (Khan et al., 2006). A second more exten-
sive study by the same authors indicated that 34 of the conserved
sequences contained numerous predicted HLA restricted peptide
sequences and 26 of these stimulated T cells in HLA transgenicmice and/or were reported to be immunogenic in humans (Khan et
al., 2008). While these approaches appear promising, it seems that
additional conserved epitopes may be identified more reliably by a
more systematic useof theavailable tools.Therefore, the aimof the
present study was to further explore the usefulness of an extensive
bioinformatic analysis using nine different T cell epitope predic-
tion programs and a representative sample of DENV polyprotein
sequences, to identify novel epitopes conserved among the four
serotypes and to evaluate their immunogenicity in mice. Indeed,
the validation of such strategy would be a key step towards its
application to human HLA for the development of an epitope-based
dengue vaccine.
2. Materials and methods
2.1. Bioinformatic analysis
A multistep immunoinformatic approach was used to identify
new antigens from DENV which consisted on computational anal-
ysis of sequences of whole viral polyprotein (3395 amino acids)
through algorithms for prediction of T cell epitopes presented by
MHC class I or class II molecules, and sequence comparisons, as
described before (Herrera-Najera et al., 2009). First, sequences
from virus prototypes DENV-1 Mochizuki (Gene Bank accession
#AB074760.1), DENV-2 New Guinea C (#M29095.1), DENV-3 H87
(#M93130.1) and DENV-4 H241 (#AY947539.1) were analyzed
by the epitope prediction programs Epimatrix (http://epitope.liai.org:8080/tools/matrix/iedb input?matrixClass=I,II), Margalit
(http://matgalit.huji.ac.il/), NetMHC1 (http://www.imtech.res.in/
raghava/mhc2pred), BIMAS (http://bimas.dcrt.nih.gov/molbio/hla
bind/), Peptide BindingPrediction(http://www.syfpeithi.de/Scripts
/MHCServer.dll/EpitopePrediction.htm), ProPed1 (http://www.
imtech.res.in/raghava/proped1), RANKPEP (http://bio.dfci.harvard.
edu/RANKPEP/), ANNPREP (http://www.imtech.res.in/raghava/
nhlapred/neural.html), COMPREP (http://www.imtech.res.in/
raghava/nhlapred/comp.html ), to predict mouse MHC class I
epitopes for H-2Ld, H-2Kd, H-2Dd, and MHC class II epitopes for
I-Ad. Second, we performed a consensus analysis of these predic-
tions, to rank predicted epitopes according to their probability
score (peptides with scores above 80% of the maximum binding
probability score were usually retained) and the number of times
they had been predicted by different algorithms, as this allows to
increase the reliability of the predictions (Herrera-Najera et al.,
2009). Third, we used a ClustalX sequence alignment of 400 whole
DENV polyprotein sequences from the four serotypes (about 100
sequences from each serotype, representative of a wide variety of
geographic and temporal origins) to evaluate the level of conser-
vation of the top ranking epitopes. The level of conservation was
visualized using WebLogo 3.0 (Crooks et al., 2004), in which letter
size is proportional to the level of conservation of each amino
acid in the epitope sequence. The most conserved epitopes were
selected since they most likely elicit an immune response against
all four serotypes simultaneously which have failed with other
vaccines. Fourth, we used BLAST analysis to assess the similarity of
the predicted epitopes with murine and human sequences, so that
highly similar sequences could be discarded from further evalu-
ation, to avoid the induction of potential autoimmune reactions.
From this analysis, epitopes predicted with a high probability
score, by most algorithms, conserved among DENV serotypes
and distinct from murine or human sequences were selected for
further validation in mice.
2.2. Synthetic peptides
We selected 21 of the top predicted epitopes for the evalua-tionof theirimmunogenicity(Table1), andsynthetic peptides were
purchasedfrom New England Biolabs. One additional peptide, 519-
FKNPHAKKQDVV (P1), derived from the E protein and previously
found to be immunogenic (Amexis and Young, 2007) was also syn-
thesized to be used as a positive control. The peptides were diluted
in water or 10%DMSO dependingon their amino acid composition.
2.3. Mice immunization
Three tofour Balb/cmice(6–8weeks old) pergroup were immu-
nized subcutaneously according to Herrera-Najera et al. (2009)
with a mix of 5 distinct synthetic peptides (50 g each) in Fre-
und’s complete adjuvant (v/v) (Gibco BRL). Two weeks later, mice
received a second dose of the same peptide mix in Freud’s incom-plete adjuvant (Sigma). A negative control group of mice (mock
immunized) only received two doses of the same adjuvants. Three
weeks after thelast immunization, mice were sacrificedby cervical
dislocation, blood and spleen cells were collected for the analysis
of the immune response. In subsequent experiments, mice were
immunized with subsets of 9–11 peptides mixtures as described
above, to allow for confirmation of their immunogenicity. A group
of mice was also immunized with P6 for the plaque reduction neu-
tralization test.
2.4. ELISA
Antibodies against the predicted epitopes were detected by
ELISA in sera of immunized mice. Microtiter plates were coatedovernight at 4 ◦C with each individual peptide (1g/well) in 100l
of 0.1 M carbonate buffer (Na2CO3/NaHCO3, pH 9.5). After washing
3 times with phosphate-buffered saline (PBS)-Tween 20 (0.05%),
wells were incubated for 2h at 37 ◦C with 100l of 10% bovine
serum albumin (BSA) in PBS, followed by an 1 h incubation at 37◦C
with200l of 1:100 diluted individual mouseserum in PBS-Tween.
Sera of mock-immunized mice were used as negative control, and
sera from mice immunized with peptide 519-FKNPHAKKQDVV
(P1) were used as positive control, since this peptide has been
reported as immunogenic (Amexis and Young, 2007). After wash-
ing wells with PBS-Tween, 100l of a 1:1000 dilution of alkaline
phosphatase-conjugated anti-mouse immunoglobulin IgG (Gibco
BRL) was added and incubated for 1h at 37 ◦C. The wells were
washed thrice with PBS-Tween and incubated for 30 min at 37◦
C
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Please cite this article in press as: Sánchez-Burgos,G., et al., Immunogenicity of novel Dengue virus epitopes identified by bioinformatic analysis.
Virus Res. (2010), doi:10.1016/j.virusres.2010.07.014
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Table 1
Characteristics of dengue virus peptides identified by immunoinformatics.
Peptide ID Sequence Positiona Protein MHC Allele Conservationb
P1 FKNPHAKKQDVV 519–530 E II –
P2 KPTLDIELL 318–326 E I H2-Ld
P3 CPTQGEATL 354–362 E I H2-Ld
P4 EGAMHTALT 537–545 E II IAd
P5 RGARRMAIL 687–695 E I H2-Dd
P6 DFGSVGGVL 699–707 E I H2-Kd
P7 RGPSIRTTTA 1068–1077 NS1 I H2-Dd
P8 AGPLVAGGLL 1372–1381 NS2b I H2-Dd
P9 ISYGGGWKL 1552–1560 NS3 I H2-Ld
P10 TPPGSRDPF 1792–1800 NS3 I H2-Ld
P11 AYRHAVEEL 2132–2140 NS4a I H2-Kd
P12 ASIILEFFL 2199–2207 NS4a I H2-Kd
P13 LRPASAWTL 2271–2279 NS4a I H2-Ld
P14 CYSQVNPTTL 2337–2346 NS4a I H2-Kd
P15 GSYLAGAGL 2469–2477 NS4b II IAd
P16 HAVSRGTAK 2546–2554 NS5 II IAd
P17 TYGWNLVKL 2612–2620 NS5 I H2-Kd
P18 VIPMVTQIAMTDTTP 2826–2840 NS5 II IAd
P19 YMWLGARFL 2967–2975 NS5 I H2-Ld
P20 SYSGVEGEGL 3003–3012 NS5 I H2-Kd
P21 TYQNKVVKVL 3059–3068 NS5 I H2-Kd
P22 YFHRRDLRL 3257–3265 NS5 I H2-Kd
a Position refers to the first and last amino acid covering the sequence in the polyprotein.b The level of conservation was visualized using WebLogo ( Mangada et al., 2004), in which letter height is proportional to the level of conservation of each amino acid in
the sequence and the color code indicates similar amino acid chemical properties.
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Please cite this article in press as: Sánchez-Burgos,G., et al., Immunogenicity of novel Dengue virus epitopes identified by bioinformatic analysis.
Virus Res. (2010), doi:10.1016/j.virusres.2010.07.014
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with75l of p-nitrophenylphosphate pNPP1 mg/ml (Gibco BRL)in
Tris buffer0.2 M. Thereaction was stopped byadding 25lofNaOH
1N, and the optical density (OD) at 405 nm was determined on an
ELISA plate reader ELX800 Bio-Tek. All tests were run in triplicate.
Positive antibody levels were defined as those above a cut-off OD
value set at the mean OD of negative control sera plus 3 standard
deviations.
2.5. Plaque reduction neutralization test (PRNT)
The sera of immunized mice where inactivated at 56 ◦C for
20 min and subjected to PRNT as reported previously (Sánchez-
Burgos et al., 2008). Briefly, 25l of diluted serum in 2% inactivated
Fetal Bovine Serum (FBS) in DMEM where mixed with 50 PFU of
each virus serotype stock to a final volume of 50l and incubated
at 37 ◦C for 1 hr. This mixtures where diluted to 500 l with dilu-
tion media and apply to Vero cells in 6 well plates, and incubated
at 37 ◦C, 5% CO2 for 1 hr. Infected cells where washed twice with
DMEM and overlaid with 2 ml of 5% FBS-DMEM-1% agar and incu-
bated for 6–12 days depending on the serotype at 37 ◦C, 5%CO2.
After incubation, cells where fixed with 3.7% formaldehyde in PBS
7.4 and stained with 1% crystal violet aqueous solution. PRNT50
where calculated as reported (Sánchez-Burgos et al., 2008).
2.6. Intracellular staining and flow cytometry
Pooled spleen cells were collected from each group of immu-
nized mice and epitope-specific T cell activation was measured
by staining the splenocytes following in vitro stimulation with
each individual peptide, and flow cytometry analysis as in
(Herrera-Najera et al., 2009). Cells (1×106) cultured in 100l
RPMI-1640 medium containing 10 mM l-glutamine, 100g/ml
penicillin, 100g/ml streptomycin, 20 mM sodium piruvate, 5M
-mercaptoethanol, and 10% FBS were stimulated with 10g/ml
peptide, 20g/ml phytohemagglutinine (PHA, positive control)
or not stimulated (Medium, negative control), incubated at 37 ◦C
and 5% CO2 for a total of 18h with 2M monensin (Sigma)
added during the last 5h. Cells were washed with FACS buffer(1% horse serum, 0.01% sodium azide in PBS) and then incubated
with 0.2g/l of monoclonal anti-CD3 (PerCP),0.4g/l anti-CD4
(FITC) or 0.4g/l anti-CD8 (FITC) at 4 ◦C for 30 min. All antibod-
ies were obtained from BD Pharmingen. After washing with FACS
buffer, cells were fixed in 2% formaldehyde and permeabilized
with 0.1% saponin (Sigma) for 30 min at 4 ◦C. Next, intracellular
staining was performed with0.2g/l monoclonal anti-IFN- (PE)
at 4 ◦C for 30 min. For each staining reaction, 100,000 cells were
analyzed on a FACS Calibur flow cytometer (Becton Dickinson).
Each experiment included isotype controls for each conjugated
antibody. The proportion of peptide-specific IFN--producing T
cells was calculated using WIN MDI 2.9 software and normalized
to unstimulated control cells to allow for comparisons between
groups.
2.7. Statistical analyses
Differences in antibodylevelswere assessed by ANOVA followed
by Tukey’s test when significant, using JMP 4.0 software.
3. Results
3.1. Epitope prediction
In order to identify antigens as candidate vaccines against
DENV, we analyzed the polyprotein of virus prototypes DENV-1
Mochizuki, DENV-2NGC,DENV-3 H87 and DENV-4 H241 by 9 com-
putational programs for prediction of sequences with high binding
Fig. 1. Antibody levels induced by immunogenic peptides. Mice were immunized
with a mixture of the indicated peptides, and serum samples were tested individu-
allyfor IgGagainsteach peptide byELISA. Serumof non-immunizednaïve micewere
tested against each peptide as negative controls, and the OD of these controls was
substractedto that ofimmunized mice.Peptide6 isfromproteinE andwastestedfor
neutralization. *A significant antibody level; and **themost immunogenic peptides
(Tukey post hoc tests).
affinity to H-2d alleles. We obtained a total of 83 epitopes for 2
DENV-1, 78 for DENV-2, 77 for DENV-3 and 81 for DENV-4, that 2
were predicted by more than two algorithms with a high prob- 2
ability score. Most predicted epitopes were for H-2Ld and H-2Kd2
alleles (59–63 epitopes for each serotype). We then assessed the 2
level of conservation of the predicted epitopes among the virus 2
serotypes to further select the most conserved epitopes for in 2
vivo analysis. Twenty-one predicted epitopes presenting a high 2
level of conservation among virus types were selected (Table 1). 2
Of these, 2 epitopes presented 100% conservation and 14 pre- 2
sented some variation in only 1 or 2 amino acids (Table 1). On 2
the other hand, none of the predicted epitopes showed similar- 2
ity with human or murine sequences. Overall, we thus selected 2
8 H-2Kd, 3 H-2Dd, 6 H-2Ld and 4 I-Ad epitopes. Most of the epi- 2
topes were identified in the NS5 (7/21) protein followed by E 2
(5/21), NS4a (4/21), NS3 (2/21) and 1 of each NS1, NS2b and NS4b 2
(Table 1). 2
3.2. Evaluation of peptide-specific IgG and neutralizing titers in 2
serum 2
The immunogenicity of the predicted epitopes was first tested 2
by measuring peptide-specific antibody levels in the serum from 2
mice previously immunized with different mixes consisting of 5 2
peptides. Specific IgG against each peptide were measured using 2
an ELISA assay, and considered positive if above the cut-off value 2
established usingserum from mock-immunized mice. As expected, 2
pooled serum from mice that had been immunized with the con- 2
trol peptide P1 presented elevated IgG levels against this known 2
epitope (Fig. 1). Of the 21 peptides tested, four (P10, P16, P20 y 2
P22) did not induce significant IgG levels and 17 showed posi- 2
tive immunoreactivity (not shown). To confirm this observation 2
and compare antibody levels, groups of 4 mice were immunized 2
with the immunogenic peptides and IgG levels against each epi- 2
topewas measured individually.As shown in Fig.1, 7 epitopes were 2
immunogenic, and 4 induced negligible antibody levels (ANOVA, 2
F =6.57, p < 0.0001). Peptides P12, P13, P14 and P19induced partic- 2
ularly high IgG levels (Tukey, p < 0.05). 2
Peptides P12, P13, P14 and P19 are derived from non-structural 2
proteins sequences; therefore these sera are not suitable for PRNT. 2
On the other hand, peptides P5 and P6 are located on domain III 2
from protein E, which has been implicated as receptor binding 2
domain, and several neutralizing monoclonal antibodies have their 2
epitopes in thisdomain(Wahala et al.,2009;Trirawatanaponget al.,2
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Please cite this article in press as: Sánchez-Burgos,G., et al., Immunogenicity of novel Dengue virus epitopes identified by bioinformatic analysis.
Virus Res. (2010), doi:10.1016/j.virusres.2010.07.014
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Fig. 2. Flow cytometry analysis of IFN production by T cells from immunized mice. Mice were immunized by pools of 5 peptides and their spleen cells restimulated in vitro
with the individual peptides used for the immunization. Cells were stained with anti-CD3, anti-CD4, anti-CD8 and anti-IFN antibodies and 105 cells were analyzed by flow
cytometry. For the analysis, cells were gated on CD3+ and CD4+ (top) or CD3+ and CD8+(bottom). Representative examples of unstimulated (medium) and stimulation with
an immunogenic control peptide (P1) and a test epitope (P18) are shown. The numbers in the upper right corners refer to the percentage of CD4 + or CD8+ T cells producing
IFN.
Table 2
PRNT50 titers of sera from mice immunized with peptide P6.
Mice DENV-1 DENV-2 DENV-3 DENV-4
#1 1:10 1:40 1:10 1:20
#2 1:10 1:10 NEG 1:10
#3 NEG NEG NEG NEG
Dilutions at which each serum reduced 50% of infection on Vero cells are indicated.
NEG: no neutralization at 1:10 serum dilution.
1992). Therefore, we determined the presence of neutralizing anti-3
bodies against all serotypes on the sera of three mice immunized4
with P6. Although neutralizing titers were just above the detection5
limit (1:10) in 2 out of 3 mice ( Table 2), this observation indicated6
that a neutralizing response was induced against all four serotypes.7
We did not look at neutralizing antibodies against P5 as it induced8
lower antibody levels than P6. Even P5 peptide induced also more9
T cell response than P6 (Figs. 3 and 4), whichseems to confer it less0
capacity for B cell induction (Fig. 1; Table 3).
Table 3
Immune responses induced by confirmed epitopes.
Peptide Sequence Protein Immune response elicited
P10 TPPGSRDPF NS3 Mostly CD4+
P16 HAVSRGTAK NS5
P6 DFGSVGGVL E
P13 LRPASAWTL NS4a Mostly antibodies (neutralizing for P6)
P19 YMWLGARFL NS5
P5 RGARRMAIL E Antibodies and CD8+
P22 YFHRRDLRL NS5
P20 SYSGVEGEGL NS5 No Anti bodies, but CD4+ and CD8+
P8 AGPLVAGGLL NS2b
P15 GSYLAGAGL NS4b
P18 VIPMVTQIAMTDTTP NS5
P14 CYSQVNPTTL NS4a CD4+ and CD8+ and antibodies
P12 ASIILEFFL NS4a
3.3. Identification of epitope-specific T cell populations
The immunogenicity of the selected epitopes was further char-
acterized by measuring T cell IFN- recall response from groups
of mice immunized with the synthetic peptides. We performed
immunostaining of CD4+ and CD8+ T cells followed by intracel-
lular staining of IFN- in spleen cells from immunized mice. As
Fig. 3. Quantification of IFN producing T cellsfollowingimmunization and in vitro
stimulation withthe predictedepitopes.All datawere normalizedto theirrespective
unstimulated control cells (medium) indicated by the horizontal dotted line. Each
bar represents the mean proportion of IFN-producing CD4+ (A) or CD8+ (B) T cells
induced by each peptide (P1–P22), assayed in duplicate, and from groups of three
mice immunized with different combinations of five peptides each.
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Please cite this article in press as: Sánchez-Burgos,G., et al., Immunogenicity of novel Dengue virus epitopes identified by bioinformatic analysis.
Virus Res. (2010), doi:10.1016/j.virusres.2010.07.014
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Fig. 4. Quantification of IFN producing T cellsfollowingimmunization and in vitro
stimulation with immunogenic peptides. All data were normalized to their respec-
tive unstimulated control cells(medium).Each bar represents the meanproportion
of IFN-producing CD4+ (A) or CD8+ (B) T cells induced by each peptide (P5–P22),
assayed in duplicate, and from a group of 4 mice immunized with the nine-peptide
mixture.
expected, a negligible proportion of unstimulated T cells produced
IFN-, while the immunogenic control peptide P1 induced IFN-production from a large proportion of both CD4+ and CD8+ T cells
from immunized mice (Fig. 2). Several other peptides, such as P18
also induced IFN-production froma largeproportion of bothCD4+
and CD8+ T cells (Fig. 2). Analysis of IFN- production induced by
each of the21 predictedepitopesindicated that 4 peptides (P2, P10,
P13 and P17) preferentially induced IFN- production from CD4+
T cells only, 6 peptides (P5, P6, P8, P14, P19 and P20) stimulated
IFN- production from CD8+ T cells only, and 7 peptides (P4, P12,
P15, P16, P18, P21 and P22) elicited IFN- production from both
CD4+ and CD8+ lymphocytes and were considered more promiscu-
ous epitopes (Fig. 3). To confirm the potential of some of the mostimmunogenic epitopes, we further immunized a group of 4 mice
with a mixture containing P5, P8, P10, P14, P15, P16, P18, P20 and
P22 peptides, and analyzed again the cellular immune response
by flow cytometry. These nine peptides resulted being very good
activators of IFN- production by T cells, with P10, P18 and P16
beingmore specific forCD4+, P5wasmorespecificfor CD8+ and the
other five (P8, P15, P14, P20 and P22) were confirmed to be highly
immunogenicfor both cell types (Fig. 4). The change of lymphocyte
activation profile in the case of P8, P14, P16, P18 and P20 may be
due to immunologic interference between peptides in the different
mixtures. Taken together, our results indicated that at least 13 out
of 21 (62%) predicted epitopes were highly immunogenic in mice,
andthatthey were able to stimulatea variety of immuneresponses
(Table 3).
4. Discussion 3
DF and DHF are considered major public health problems 3
due to the increased incidence, prevalence and transmission in 3
tropical and subtropical areas around the world (DengueNet1 3
– http://www.who.int/csr/disease/dengue/impact/en/index.html) 3
andthere are major efforts to develop an effective and safe vaccine 3
against these diseases (Whitehead et al., 2007). Ideally, DENV vac- 3
cines should generate immunity against all serotypes to avoid the 3
risk of development of DHF by the antibody-dependent enhance- 3
ment mechanism (Halstead, 2007; Green and Rothman, 2006), 3
and novel strategies are necessary to achieve this goal. The most 3
important step for vaccine development is the identification of an 3
immunodominant antigen(s) or epitope(s). Therefore,the complete 3
sequence of DENVgenomesand the development of computational 3
programs to predict MHC binding sequences have lead to the iden- 3
tification of an increased number of DENV epitopes (Khan et al., 3
2006; Vázquez et al., 2002; Leclerc et al., 1993; Jiang et al., 2010; 3
Wen et al., 2007; Bashyam et al., 2006; Yauch et al., 2009; Khan et 3
al., 2008). Powerful bioinformatic tools for T cell epitope predic- 3
tion from protein sequences have greatly facilitated the analysis of 3
large numbers of antigens whichcan be used for the immunization 3
of mice for the evaluation and validation of their immunogenicity 3
and antigenicity as vaccine candidates (De Groot, 2006). The use 3
of epitope-based vaccines has eliminated the need to obtain large 3
amounts of recombinant proteins for immunizations as well as the 3
riskof infectionby revertants of attenuated microorganism. As DNA 3
vaccines, few epitopes predicted by computational methods have 3
been found to induce good immune responses and protection in 3
mice model of dengue infection (Yauch et al., 2009; Khan et al., 3
2008). 3
In thisstudy,we usedseveral bioinformatictools to identifycon- 3
served epitopes from DENV polyprotein, and evaluated in vivo and 3
in vitro the immunogenicity and antigenicity of the correspond- 3
ing synthetic vaccine candidates. We identified 21 potential DENV 3
epitopes by computational analysis, and found that 13 (62%) were 3
immunogenic in mice. Of these epitopes, most were located in 3
the proteins NS5 (30.7%), E (30.7%) and to a lesser extent in NS4a 3
(15.4%), NS3, NS4b and NS2b (7.7% each), and they were able to 3
induce specific antibodies and/or T cell activation. 3
This is in agreement with previous studies which had identi- 3
fied immunogenic epitopes in almost all proteins of DENV. Some 3
DENV-specific CD4+ T cell epitopes had been identified mainly in 3
NS3 or C protein (Wen et al., 2007; Kurane et al., 1995; Gagnon et 3
al., 1996; Mangada et al., 2004), while CD8+ T cell epitopes had 3
been identified in DENV proteins C, M, E, NS2a, NS4b and NS5 3
(Roehrig, 2003; Wen et al., 2007; Yauch et al., 2009 ). Some of the 3
HLA restricted epitopes identified previously induced IFN-+ CD4+3
or CD8+ T cell responses of PBMC obtained from DF patients (Wen 3
et al., 2007) or from splenocytes harvested from mice after infec- 3
tion with DENV (Yauch et al., 2009). Antibodies to protein C, prM, 3
E, NS1, NS3 and NS4a have also been detected in sera of DENV- 3
infected patients (Churdboonchart et al., 1991; Anandarao et al., 3
2005). Thus, our results confirm the immunogenicity of epitopes 3
from non-structural proteins of DENV and strengthen the use of 3
these proteins/epitopes in vaccine design. 3
Comparison of the efficacy of the different approaches for epi- 3
tope identification is difficult, since most previous studies focused 3
on specific viral proteins or a single serotype, while we attempted 3
to cover the entire DENV polyproteins from all four serotypes. 3
For example, using a single T cell epitope prediction program 3
(RankPep),Wenet al.(2007) validatedonly fourpredicted epitopes. 3
In another study, of 106 predicted epitopes from a single DENV-2 3
strain, only 12 (11%) were found to be able to induce DENV-specific 3
CD8+ IFN-+ T cells in mice (Yauch et al., 2009). On the other hand, 3
a rather integrative study across all DENV serotypes, based in a3
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first step on evolutionary analysis aimed at identifying highly con-0
served sequences, followed by an analysis of epitope prediction as
well as epitope database searches, resulted in the identification of 2
44 sequences present in more than 80% of all sequences of each3
DENV serotypes (Khan et al., 2008). These epitopes were local-4
ized in NS5, followed by NS3, NS1, NS4b, NS4a and E proteins, and5
26 of these (59%) resulted immunogenic in humanized transgenic6
mice (Khan et al., 2008). This is quantitatively very similar to our7
results, although we focused first on epitope prediction, followed8
by an analysis of conservation across serotypes. Thus, a combina-9
tion of consensus epitope prediction with an analysis of sequence0
evolution and conservation appears as a strategy of choice for the
identification of conserved epitopes, as suggested before (Larsenet2
al., 2005; Donnes and Kohlbacher, 2005).3
It is known thatbothneutralizingantibodies andT cellresponses4
confer protection against DENVinfection (Rothman,2004; Yauch et5
al.,2009), thereforeit isimportant toinducea wide arrayof immune6
mechanisms in vaccination strategies. In that respect, the epitopes7
we identified elicit such a variety of immune responses, as sum-8
marized in Table 3. Some induce a very narrow immune response,9
based on antibodies, or T cells, respectively, while other induce a0
much broader response including the simultaneous production of
antibodies and activation of CD4+ and CD8+ T cells. Furthermore,2
the observation that peptide P6 was able to elicit a modest but3
detectable neutralizing response against all four DENV serotypes4
is very promising and suggest that optimization of the immuniza-5
tion protocol may generate a stronger neutralizing response. We6
are carried out further studies in mice sensible to DENV infec-7
tion STAT1-/- for assessing protection by different combinations8
of peptides and immune responses induced in virus infections. In9
addition we are evaluating the recognition of the predicted epi-0
topes by antibodies present in sera of confirmed dengue patients
to demonstrate their potential use as vaccine candidates against2
dengue virus. In that respect, it is interesting to note that epitope3
P14 (CYSQVNPITL) had been identified previously and found to be4
protective against DENV infectionin an animal model (Yauch et al.,5
2009).6
4.1. Conclusion7
In conclusion, a systematic bioinformatic analysis of DENV8
polyproteins withT cellprediction tools followedby sequence com-9
parisons allowed to identify 21 virus epitopes, of which 13 (62%)0
resulted immunogenic in mice. Three synthetic peptides induced
mostly IgG antibodies, and one of these from the E gene induced2
a neutralizing response. Ten peptides induced a combination of 3
humoral and cellular responses by CD4+ and CD8+ T cells, opening4
the wayto evaluate the contributionof thedifferentimmunemech-5
anisms for DENV control in animal models. These results indicate6
that ourbioinformatics strategyis a very powerful tool forthe iden-7
tification of novel antigens and vaccine candidates against DENV8
and its application to human HLA may lead to the characterization9
of a potent epitope-based vaccine.0
Acknowledgements
This work was supported by grants #SALUD-2007-01-689092
and #SEP-2004-C01-47122 from the Consejo Nacional de Cien-3
cia y Tecnología (CONACyT), Mexico to Gilma Sánchez-Burgos and4
Eric Dumonteil. The authors thank Ariana Carballo-Dzay and Ana5
Sánchez-Argáez for their technical assistance.6
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