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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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

ARTICLE IN PRESSGModel

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

http://dx.doi.org/10.1016/j.virusres.2010.07.014

http://epitope.liai.org:8080/tools/matrix/iedb_input?matrixClass=I,II

http://matgalit.huji.ac.il/

http://www.imtech.res.in/raghava/mhc2pred

http://bimas.dcrt.nih.gov/molbio/hla_bind/

http://www.syfpeithi.de/Scripts/MHCServer.dll/EpitopePrediction.htm

http://www.imtech.res.in/raghava/proped1

http://bio.dfci.harvard.edu/RANKPEP/

http://www.imtech.res.in/raghava/nhlapred/neural.html

http://www.imtech.res.in/raghava/nhlapred/comp.html














http://bimas.dcrt.nih.gov/molbio/hla_bind/


http://matgalit.huji.ac.il/








VIRUS952101–8

G. Sánchez-Burgos et al. / Virus Research xxx (2010) xxx–xxx 3

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.








VIRUS952101–8


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








VIRUS952101–8


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.








VIRUS952101–8


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


http://www.who.int/csr/disease/dengue/impact/en/index.html








VIRUS952101–8


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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Immunogenicity of Novel Dengue Virus Epitopes Identified by tic Analysis

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