Arch Virol DOI 10.1007/s00705-013-1956-4

ORIGINAL ARTICLE

Generation and characterization of potential dengue vaccine candidates based on domain III of the envelope protein and the capsid protein of the four serotypes of dengue virus Edith Suzarte • Ernesto Marcos • La´zaro Gil • Iris Valde´s • Laura Lazo • Yassel Ramos • Yusleidi Pe´rez • Viviana Falco´n • Yaremis Romero • Marı´a G. Guzma´n • Sirenia Gonza´lez Juan Kourı´ • Gerardo Guille´n • Lisset Hermida

•

Received: 29 August 2013 / Accepted: 13 December 2013 Ó Springer-Verlag Wien 2014

Abstract Dengue is currently one of the most important arthropod-borne diseases, causing up to 25,000 deaths annually. There is currently no vaccine to prevent dengue virus infection, which needs a tetravalent vaccine approach. In this work, we describe the cloning and expression in Escherichia coli of envelope domain IIIcapsid chimeric proteins (DIIIC) of the four dengue serotypes as a tetravalent dengue vaccine candidate that is potentially able to generate humoral and cellular immunity. The recombinant proteins were purified to more than 85 % purity and were recognized by anti-dengue mouse and human sera. Mass spectrometry analysis verified the identity of the proteins and the correct formation of the intracatenary disulfide bond in the domain III region. The chimeric DIIIC proteins were also serotype-specific, and in the presence of oligonucleotides, they formed aggregates that were visible by electron microscopy. These results support the future use of DIIIC recombinant chimeric

Electronic supplementary material The online version of this article (doi:10.1007/s00705-013-1956-4) contains supplementary material, which is available to authorized users. E. Suzarte  E. Marcos  L. Gil  I. Valde´s  L. Lazo  Y. Ramos  Y. Pe´rez  V. Falco´n  Y. Romero  G. Guille´n  L. Hermida (&) Center for Genetic Engineering and Biotechnology (CIGB), Avenue 31, P.O. Box 6162, Havana 6 10600, Cuba e-mail: [email protected] M. G. Guzma´n Department of Virology, PAHO/WHO Collaborating Center for the Study of Dengue and its Vector, Pedro Kourı´ Tropical Medicine Institute (IPK), P.O. Box 601, Havana, Cuba S. Gonza´lez  J. Kourı´ CINVESTAV-IPN, Mexico, D.F, Mexico

proteins in preclinical studies in mice for assessing their immunogenicity and efficacy.

Introduction Dengue is currently one of the most important arthropodborne diseases, causing up to 25,000 deaths annually. Approximately 40 % of the world’s population, living in tropical and sub-tropical countries, is at risk of dengue virus (DENV) infection [1]. The clinical spectrum of the disease ranges from an asymptomatic infection to a self-limiting febrile illness, dengue fever, to severe dengue, a clinical syndrome that typically presents with capillary permeability and can lead to dengue shock syndrome and dengue hemorrhagic fever. There is no licensed dengue vaccine, and prevention is exclusively through vector control. DENV exists as four immunologically and antigenically distinct serotypes, which belong to the genus Flavivirus of the family Flaviviridae. Infection with any one DENV serotype provides lifelong immunity to that serotype, with only transient cross-protection against the remaining three [2, 3]. Epidemiological studies suggest that pre-existing crossreactive non-neutralizing antibodies can predispose an individual to the severe form of the disease during a secondary infection through antibody-dependent enhancement [4–6]. For this reason, it is necessary for a DEN vaccine to elicit a balanced and durable immune response specific for each of the four DENV serotypes simultaneously. Several approaches for the development of tetravalent dengue vaccines are in different stages of development [7]. Among them, the recombinant subunit vaccine strategy is an economical approach that avoids the reactogenicity and unbalanced immunogenicity of live dengue vaccine

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candidates. However, many of these non-infectious subunit vaccines are poorly immunogenic and need potent immunostimulatory adjuvants. Several subunit vaccine candidates in development focus on the major structural protein on the surface of the mature dengue virions, the envelope protein (E). The domain III region (DIII) of this protein is accessible on the virion surface [8], and several studies have shown that it is targeted by neutralizing antibodies [9, 10]. In addition, DIII is involved in the recognition of the DENV receptor on target cells [10, 11]. The immunogenicity [12, 13], protective capacity [14–16], and serotype specificity of domain III [17] of the DENV envelope protein make this antigen an ideal candidate for inducing a functional humoral immune response. Cell-mediated immunity (CMI) has recently been recognized as an important factor in the protection against DENV in mice [18–20]. Our group found that the capsid protein of DENV-2 induces partial protection in the mouse model of dengue encephalitis without eliciting a functional humoral response [21]. Based on these findings, a domain III-capsid chimeric (DIIIC) protein of serotype 2 was previously expressed in a highly aggregated form and evaluated in mice, and it was shown to induce a functional immune response and provide protection against challenge with the homologous virus [22]. In this study, we produced four chimeric proteins based on domain III of the envelope protein and the capsid region corresponding to the four DENV serotypes. We describe the cloning and expression in E. coli, purification, and antigenic characterization of each construct. The chimeric proteins were successfully expressed in bacteria, properly folded and purified. They showed a serotype-specific recognition pattern and were able to aggregate in the presence of the 39M oligonucleotide (ODN 39M).

Materials and methods Bacterial strains, plasmids, antibodies, and viral antigens Escherichia coli BL21(DE3): F– ompT gal dcm lon hsdSB(rB mB ) k(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5]) was used for expressing the recombinant DIIIC 1, 3 and 4 genes [23] and the recombinant capsid of the four serotypes, while the E. coli strain Rosetta (DE3) pLysS (FompT hsdSB(RB mB ) gal dcm k(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5]) pLysSRARE (CamR) [24] was used for expressing DIIIC-2 protein. For propagating the plasmids, we used Escherichia coli strain XL1-blue [F’::Tn10 proAþBþ lacIq D(lacZ)M15/ recA1 endA1gyrA96(NaIr) thi hsdR17(r_k mþk) supE44

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relA1 lac) [25]. All strains were grown in LB medium and stored at -70 °C in the same medium supplemented with 20 % glycerol. Antibiotics were added, when necessary, at the following concentrations: ampicillin (Amp) (100 lg/ ml), kanamycin (Km) (50 lg/ml) and chloramphenicol (Cl) (34 lg/ml). Polyclonal hyperimmune murine ascitic fluids (HMAF) from the four serotypes as well as human sera were kindly provided by the Department of Virology, ‘‘Pedro Kourı´’’ Tropical Medicine Institute (IPK), Cuba. Viral preparations from suckling mouse brain infected with DENV 1-4, used as antigens for the studies of antigenicity, were also provided by IPK. The viral strains used were DENV-1 (Hawai, 27 passages in mice), DENV-2 (NGC, 27 passages in mice), DENV-3 (H87, 27 passages in mice), and DENV-4 (H241, 27 passages in mice) [26]. A similar preparation obtained from brains of non-inoculated mice was used as a negative control. The recombinant plasmids pD10, pD18, pD24 (P64kdomain III of DENV-1, 3, and 4, respectively) and pDomIII-Capsid2 (DIII-capsid of DENV-2), used as the sources of the domain III fragments, were constructed previously by our group [21, 22, 27]. The pET28a plasmid (Novagen) with minor modifications was used as the expression vector. Construction of recombinant plasmids ACC and ACDC, expressing capsid and chimeric protein DIIIC, respectively, of the four DEN serotypes The capsid gene of DENV-1, 3 and 4 from the viral strain Jamaica (isolated in 1977, AF425621, generously donated by the late Dr. Robert Shope), a primary isolate from the Nicaragua 1994 epidemic (FJ882576), and the isolate Dominica 814669 (AF326573), respectively, was amplified by reverse transcriptase polymerase chain reaction (RTPCR) using the primers listed in Table 1. Briefly, the RT-PCR amplification was performed under the following conditions: 30 min at 42 °C using 200 pg viral RNA, 50 pmol of downstream primer, 200 lM each deoxynucleotide triphosphate (dNTP), 20 U RNAse inhibitor (RNAsin, Promega, USA), and 20 U AMV RT (reverse transcriptase from Boehringer, Germany) in a final volume of 25 ll. The synthesized cDNA fragments were used as a template for the nested PCR described below. The reaction components were as follows: 5 ll of the product of RT-PCR, 50 pmol primers, 200 lM each dNTP, PCR buffer (50 mM KCl, 1.5 mM MgCl2, and 10 mM TrisHCl, pH 8.5), and 2.5 U of Thermus aquaticus DNA polymerase (Boehringer, Germany). The reaction was performed at a final volume of 50 lL using the following program: 30 cycles of 95 °C for 30 s, 50 °C for 30 s, and 72 °C for 20 s in an Eppendorf thermocycler.

Chimeric dengue virus vaccine Table 1 Primers employed for capsid and domain III cloning

The underlined sequence corresponds to enzyme restriction sites

Capsid 1 BamHI upstream

50 -GGATCCAACCAACGAAAAAAGAC-30

Capsid 1 HindIII downstream

50 -AAGCTTATCTTTTCCTTCTATTCATTATATTC-30

Capsid 3 BamHI upstream

50 -GGATCCAACCAACGGAAGAAG- 30

Capsid 3 HindIII downstream Capsid 4 BamHI upstream

50 -AAGCTTACTTTTTCCGTTTGTTG-30 50 -GGATCCAACCAACGAAAAAAGG-30

Capsid 4 HindIII downstream

50 -AAGCTTACCTTTTTCTCCCGTTCAAG-30

DomIII-1 upstream

50 -GGATCCCGACTAAAAATGGATAAACTGAC-30

DomIII-1 downstream

50 -GGATCCGCCGCCTATAGAGCCGAAGTC-30

DomIII-3 upstream

50 -GGATCCAGACTTAAGATGGACAAATTGG-30

DomIII-3 downstream

50 -GGATCCGCCACCAACCGAACCAAAATC-30

DomIII-4 upstream

50 -GGATCCAAAGTGCGTATGGAGAAATTG-30

DomIII-4 downstream

50 -GGATCCGCCACCAACCGAACCAAAATC-30

The amplified bands were purified and cloned into pGEM-T Easy Vector (Promega, USA). Positive clones were tested by restriction analysis, and the capsid genes were fully sequenced. The fragments encoding the capsid protein were extracted using a BamHI-HindIII double digestion and cloned into the modified pET28a plasmid. Positive clones identified by restriction analysis were finally conserved under the names ACC-1, 3, and 4, corresponding to capsid gene of DENV-1, DENV-3 and DENV-4, respectively. The modified pET28a plasmid was obtained previously by deleting six bases preceding the N-terminal His tag sequence and the fragment of 66 bases between the N-terminal His tag sequence and the BamHI site of the polylinker. The domain III (DIII) regions of the envelope protein genes of DENV 1, 3 and 4, cloned in plasmids pD10, pD18 and pD24, respectively, were obtained by PCR amplification using the primers described in Table 1. The PCR conditions were similar to those described previously but using 50 lg of plasmid pD as template. The bands that were obtained were cloned into pGEM-T Easy Vector (Promega, USA). Positive clones were tested by restriction analysis, and BamHI fragments containing the region encoding DIII were cloned in the corresponding ACC plasmids, which had been digested with BamHI and treated with the enzyme calf-intestinal alkaline phosphatase (CIP; Promega). The positive clones were tested by restriction analysis and sequenced. The selected clones were named ACDC-1, 3, and 4, corresponding to the DIIIC chimeric genetic constructs of DENV serotype 1, 3 and 4, respectively. In the case of serotype 2, the recombinant plasmids ACC2 and ACDC-2 were constructed by cloning BamHI-HindIII fragments of the plasmid pDomIII-Capsid2 [22] containing DIII-2 fused to capsid 2 into a modified pET28a vector. Gene expression in E. coli The E. coli strain BL21 (DE3) was transformed with ACC1- 4 and ACDC1, 3, and 4 plasmids by electroporation, while

plasmid ACDC2 was introduced into E. coli Rosetta (DE3) pLysS. An overnight LB culture supplemented with kanamycin for the variants 1, 3, and 4 of ACDC and all of the ACC plasmids were used to inoculate 5 mL or 500 ml of LB medium supplemented with kanamycin and grown in a shaker at 37 °C until the middle of the exponential phase was reached. In the case of the variant ACDC 2, the overnight culture contained kanamycin and chloramphenicol. Isopropyl b-D-1-thiogalactopyranoside (IPTG) at 0.2 mM was used for inducing the expression of recombinant genes for 4 hours. Western blot Cellular extracts expressing the recombinant genes were electrotransferred from an acrylamide gel to a Hybond-C membrane as described [28]. One clone transformed with the modified pET28a vector was used as a negative control. The membrane was blocked with 5 % skimmed milk in PBS-T (PBS and 0.05 % Tween) for 1 h at room temperature (RT), washed three times in PBS-T, and reacted with the homologous anti-DENV HMAF for 1 h at RT. After washing, the membrane was treated with peroxidase-conjugated goat antimouse IgG (Sigma, USA) at a 1/1000 dilution in PBS-T for 1 h at RT. Afterwards, the membrane was washed again, and the bands were visualized by incubation with substrate solution (0.1 % diaminobenzidine and 0.03 % H2O2 in PBS) at RT until color development was completed. Purification of DIIIC chimeric proteins Bacterial cultures (3 L), after 4 h of expression, were harvested by centrifugation at 5,000 9 g for 5 min at 4 °C, and the cellular biomass was resuspended in 30 ml of 10 mM Tris-HCl, 6 mM EDTA, pH 8 (TE buffer). The cells were disrupted in a French press (Othake, Japan) at 1500 kg/cm2, with three passes at 4 °C. After disruption of the cells, the culture was centrifuged at 10,000 9 g for 30 min at 4 °C, and the disruption supernatant was collected for subsequent saline precipitation with 30 % ammonium

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sulfate for 1 h on ice. The pellet that was obtained was solubilized with 10 mM Tris, 7 M urea, 0.5 % Tween, and 200 mM NaCl, pH 8 (buffer 1) for 1 h with magnetic stirring at RT. The urea extract was subjected to cation exchange chromatography using SP Sepharose Fast Flow (Amersham Bioscience, UK) matrix (XK 50/20 column, 10 % loading volume, 3 cm/h flow rate) previously equilibrated with buffer 1. The recombinant protein was eluted using the buffer 1 plus 750 mM NaCl (buffer 2). The eluted fraction was subjected to immobilized metal ion affinity chromatography (IMAC). The IMAC resin used was Chelating Sepharose Fast Flow (Amersham Bioscience, UK) loaded with 100 mM ZnSO4 (XK26/20 column, 15 % loading volume and 20 cm/h flow rate) previously equilibrated with buffer 2 plus 15 mM imidazole. The protein of interest was eluted with 10 mM Tris, 7 M urea, 0.5 % Tween, 200 mM imidazole, pH 8. The refolding process was performed on a Sephadex G-25 column (XK 26/40, 16 % loading volume and 50 cm/h flow rate) previously equilibrated with TE buffer. The desalted and refolded fraction was collected, and the protein concentration was determined by the bicinchoninic acid assay (BCA). The purification process of DIIIC-4 was essentially the same, but all buffers contained 10 mM b-mercaptoethanol to prevent protein aggregation. The negative purification used BL21 cells transformed with the pET28a plasmid as a primary source and followed the same purification process that was used for DIIIC1-3 proteins.

2 lg/mL of the mock-purification product, corresponding to cells transformed with the modified pET28a plasmid without insert. Plates were blocked with 3 % skimmed milk and 2 % goat serum in coating buffer for 1 h at 37 °C. Three washes with PBS-Tween 0.2 % were completed after each step of the assay. Anti-DENV HMAF polyclonal sera and human sera were serially diluted (PBS-Tween 0.1 %, 1 % skimmed milk, and 1 % goat sera) and incubated for 1 h at 37 °C with each protein preparation. Antimouse or anti-human IgG-peroxidase conjugate (Sigma, USA) was added, and the plates were incubated for 1 h at 37 °C. After washing, 0.04 % substrate solution (o-phenylenediamine in a buffer containing 2 % Na2HPO4, 1 % citric acid, and 30 % H2O2, pH 5.0) was added. Plates were kept at 25 °C for 15 min, and the reaction was stopped with 12.5 % H2SO4. Absorbance at 492 nm was read in a microplate reader (SensIdent Scan; Merck, Germany). The positive cutoff value was taken as twice the absorbance value of the negative control. Mass spectrometry

Antigenic characterization by ELISA and mass spectrometry

Protein bands were excised and destained by washing with 250 mM ammonium bicarbonate and 30 % acetonitrile. After dehydration with 90 % acetonitrile, gel pieces were caused to swell by treating with a solution containing 12.5 ng of trypsin per lL and 50 mM ammonium bicarbonate and incubated at 37 °C for 16 h. Tryptic peptides were recovered by a tandem extraction procedure, first at basic and finally at acid pH, by using ZipTip C18 microcolumns (Millipore, MA, USA). Peptides adsorbed onto the ZipTip column were washed with 5 % formic acid, eluted in 60 % acetonitrile and 1 % formic acid and loaded into a gold-covered borosilicate capillary (Micromass, UK) for mass spectrometry analysis. Mass spectra were acquired on a QTOF2TM (Micromass, UK) hybrid instrument equipped with a nanoESI ion source. Capillary and cone voltages were 900 V and 35 V, respectively. The instrument was calibrated with sodium and cesium iodide from 50 to 2000 Da. The software for data processing was MassLynx 4.2 (Micromass, UK). Protein identification based on MS/MS spectra was made using the search engine MASCOT (http://www.matrixscience.com) or by manual inspection for disulphide-bond-containing peptides.

Anti DIIIC-ELISA

Transmission electron microscopy (TEM)

Polystyrene plates (96 wells, Costar, USA) were coated with 2 lg of DIII-C chimeric protein per ml for 2 h at 37 °C in coating buffer (0.16 % Na2CO3, 0.29 % NaHCO3, pH 9.5). As a negative control, the plates were coated with

For microscopy analysis, samples freshly glow-discharged, 400-mesh with Formvar and carbon. After absorption and extensive washing

Protein analysis Protein samples were subjected to 15 % sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) as described previously [29]. Samples that were further analyzed by mass spectrometry were incubated under nonreducing conditions with 2.5 % acrylamide for 1 h at 37 °C prior to electrophoresis. Protein bands were visualized by staining with Coomassie brilliant blue R-250 (Sigma). Gels were scanned, and the purity was estimated by densitometry using ImageJ (1.41 version) software.

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were placed on a copper grid coated 2 min of sample with water, uranyl

Chimeric dengue virus vaccine

acetate stain was applied. After 4 min of staining, grids were wick dried with Whatman no. 1 filter paper and allowed to air dry for 20 min. Samples were then viewed on a JEOL JEM-1400 electron microscope (JEOL, Japan). Three different pictures were analyzed using ImageJ software (version 1.33) to determine the number of particles and their sizes. Reduction-alkylation of DIIIC recombinant proteins The reduction reaction was carried mM in TE buffer for 2 h at 37 °C, teins were immediately subjected addition of 250 mM acrylamide for

out with DTT at 100 and the chimeric proto alkylation by the 30 min at RT.

Sandwich ELISA for determining anti-DEN virus antibodies Polystyrene 96-well plates (Costar) were coated with 100 ll/well of a mixture of human anti-DENV IgG (5 lg/ml) in coating buffer (0.16 % Na2CO3 and 0.29 % NaHCO3, pH 9.5) for 2 h at 37 °C, then blocked with coating buffer containing 5 % of skimmed milk for 1 h at 37 °C and washed three times with PBS-T. The viral antigen from suckling mouse brain infected with DENV1-4 and the negative preparation were incubated overnight at 4 °C. After three washes with PBS-T, a sample of HMAF of each serotype was tested by serial dilution in PBS-T, starting at 1:1000. Plates were incubated for 1 h at 37 °C and washed as described above. Later, 1:35,000-diluted anti-mouse IgG–peroxidase conjugate (Amersham-Pharmacia) was added, and the plates were incubated for 1 h at 37 °C. After washing, 0.04 % substrate (o-phenylenediamine in 2 % [w/v] Na2HPO4, 1 % citric acid and 30 % (v/v) H2O2, pH 5.0) was added. The plates were kept for 15 min at RT, and the reaction was stopped with 12.5 % (v/v) H2SO4 (50 ll/well). An automated ELISA reader recorded the OD at 492 nm. An absorbance value twice that of the control preparation was considered a positive value. In vitro aggregation reaction The purified DIIIC proteins were subjected to the in vitro assembly procedure. Briefly, 40 lg of each recombinant protein was incubated with a single-stranded DNA oligonucleotide of 39b (39M ODN) with the sequence 5’-ATC GAC TCT CGA GCG TTC TCG GGG GAC GAT CGT CGG GGG-3’ at a protein: nucleic acid molar ratio of 4.5:1 in TE buffer. The reaction mixture was incubated for 30 min at 30 °C and then for 4 h at 4 °C.

Results Cloning and expression in E. coli of the capsid and DIII regions from the four serotypes of DENV The sequences coding for the capsid protein (C) from each DENV serotype were amplified by PCR and cloned into the modified expression vector pET28a to yield the plasmids ACC-1, ACC-2, ACC-3 and ACC-4, respectively. The ACC plasmids contained the recombinant capsid genes fused to a 6-His-tag at the N-terminal site under the control of the T7lac promoter. On the other hand, the sequences coding for the envelope domain III fragments of the four DENV serotypes (amino acids 286–426) were fused to the N-terminus of the homologous capsid gene in the corresponding ACC plasmids, generating ACDC construction. These final recombinant constructs were sequenced to confirm the presence of the DIIIC chimeric genes with a 6-His-tag at their N-terminal sites. Cells of E. coli strain BL21 (DE3) were transformed with the recombinant plasmids ACC1-4 (containing capsid genes) and ACDC 1, 3, and 4 (containing DIII-C). Plasmid ACDC2, in turn, was introduced into the Rosetta (DE3) plysS strain. In all cases, protein expression was achieved by IPTG induction and analyzed by SDS-PAGE, and a band of approximately 15-17 kDa, representing 15-18 % of the total cellular protein, was detected for the ACC family of plasmids and immunoidentified as dengue capsid with anti-DENV polyclonal HMAF (Fig. 1). For the ACDC recombinant plasmids, analysis by SDS-PAGE after IPTG induction revealed the presence of a band of approximately 28 kDa that was also immunoindentified with anti-DENV HMAF (Fig. 1) and accounted for 9-13 % of the total bacterial protein. The size of these prominent bands corresponded to the size predicted for the fusion of the recombinant domain III and the capsid region of DENV. Purification of DIIIC chimeric proteins from the four DENV serotypes E. coli cells transformed with each ACDC plasmid variant were disrupted in a French press. The recombinant DIIIC proteins were mainly found in the soluble fraction and were purified in the presence of 7 M urea. The purification process used was similar to the one developed for the DIIIC-2 variant by Marcos et al. in 2013 [30], with the addition of an initial ammonium sulfate precipitation step (Fig. 2A). This step resulted in 1.6- to 2.2-fold purification without any appreciable yield loss. Considering the high frequency of basic amino acids in the sequence of the DIIIC proteins (theoretical isoelectric

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Fig. 1 Expression in E. coli and immunodetection of capsid protein and domain III-capsid (DIIIC) chimeric protein, of the four viral serotypes: I (serotype 1), II (serotype 2), III (serotype 3) and IV (serotype 4). Panel A: analysis of capsid and DIIIC expression, using IPTG as inducer, by SDS-PAGE in a 15 % gel stained with Coomassie blue. Panel B: western blot of gel (panel A) using mouse polyclonal hyperimmune ascitic fluid (HMAF) against the

homologous DENV serotype. Lanes: (1) cell extract of E. coli BL21(DE3) transformed with pET28a, (2) cell extract of E. coli BL21(DE3) transformed with ACC plasmid, (3) cell extract of E. coli BL21(DE3) transformed with ACDC 1,3,4 and E. coli Rosetta plysS transformed with ACDC2 plasmid, (X) broad-range protein molecular weight marker (Promega)

points ranging from 10.2 to 10.6), cation exchange chromatography was carried out as a second purification step. After this procedure, the recombinant DIIIC proteins were obtained with a purity ranging from 52.8 to 80.1 % (Table 2). Given the presence of a His-tag at the N-terminal end of the chimeric proteins, immobilized metal ion affinity chromatography (IMAC) was selected as the subsequent chromatographic step. With this procedure, the purity of the DIIICs proteins reached values higher than 85 % (Table 2). Western blot analysis of this preparation using anti-DENV HMAF against the homologous serotype revealed that the contaminant bands were either aggregates or degradation fragments of DIIIC (Fig. 2B).

sequence coverage of the domain III-capsid region of the four serotypes. The correct formation of the disulphide bond in domain III between cysteine 27 and cysteine 58 in each DIIIC molecule was also verified (Online Resource 1). Additionally, in order to demonstrate the absence of free-cysteine-containing peptides, samples were incubated with acrylamide prior to electrophoresis. No signals corresponding to the addition of acrylamide (? 71 Da) were detected, confirming that all DIIIC monomers contained the disulphide bridge.

Characterization of the DIIIC proteins Mass spectrometry Each purified preparation was analyzed by mass spectrometry. After electrophoresis, bands migrating at a molecular weight of approximately 28 kDa were in-gel digested with trypsin, and the most intense signals in the MS spectra were fragmented. The sequences obtained from the MS/MS spectra confirmed the identity of each DIIIC molecule. The detected peptides accounted for 60 %-74 %

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Reactivity against murine and human polyclonal antibodies Antigenic characterization was performed by anti-recombinant protein ELISA using anti-DENV polyclonal mouse sera, HMAF, and human sera from secondary DENV infections with high viral neutralization titers. This assay showed high levels of antigenic recognition of all DIIIC variants, although the recognition of DIIIC-4 was lower (Fig. 3). All of the sera tested showed poor recognition of the negative purification, suggesting specific recognition of DIIIC proteins by human and mouse sera. The contribution of the internal disulfide linkage of DIII to the recognition of DIIIC chimeric proteins by antiDENV HMAFs was also tested. We used unmodified and

Chimeric dengue virus vaccine

Fig. 2 Purification of DIIIC-1-4. Panel A: Analysis by 15 % SDSPAGE of the purification fractions stained with Coomassie blue. Lanes: (1) French press supernatant disruption; (2) ammonium sulfate precipitation supernatant; (3) ammonium sulfate pellet in 7 M urea; (4) fraction not adsorbed to the SP Sepharose matrix in cation exchange chromatography, (5) fraction eluted in cation exchange chromatography; (6) fraction not adsorbed to the chelating Sepharose matrix, IMAC chromatography; (7) fraction eluted from IMAC chromatography; (8) broad-range protein molecular weight marker (Promega). Panel B: immunoidentification by western blot of purified DIIIC proteins with mouse polyclonal hyperimmune ascitic fluid (HMAF) against the homologous DENV serotype (I, HMAF antiDENV-1; II, HMAF anti-DENV-2; HMAF anti-DENV-3; HMAF anti-DENV-4. Lanes: (X) negative purification; (1) DIIIC-1; (2) DIIIC-2; (3) DIIIC-3; (4) DIIIC-4 Table 2 Percent purity of DIIIC1-4 at different purification steps Recombinant protein

presence of the disulfide bond in domain III. This is shown in Fig. 4A by the considerable loss of reactivity (more than 50 %) found in proteins of these serotypes after reduction and alkylation of the cysteine residues. Interestingly, the unmodified and reduced-carbamidomethylated chimeric protein DIIIC-3 showed a very similar recognition pattern by HMAF. The efficiency of the reduction process was confirmed by SDS-PAGE, which showed complete reduction of all the protein variants marked by the loss of the highest-size contaminant bands corresponding to disulphide-bridge-dependent aggregates after the reduction treatment (Fig. 4B). The antigenic specificity of recombinant proteins was evaluated by ELISA using anti-DENV HMAF against the four serotypes. The reactivity of each protein and virus was compared against the homologous and heterologous HMAF. The recognition of viruses 1, 2 and 4 by anti-DENV polyclonal mouse sera was highly cross-reactive (Fig. 5A), since all but DENV-2 showed similar titers against three different serotypes. In the specific case of DENV-2, the virus exhibited the highest response against the homologous HMAF. In the case of DENV-3, a pattern of serotype specificity was found. The recognition of DIIIC proteins was serotype-specific, with titers against the homologous HMAF over 30,000 for DIIIC-1, 2 and 3 (Fig. 5B). In general, the rate of response against the homologous vs. heterologous serotype was 4:1 or more, even for the DIIIC-4 protein, which was less recognized by the homologous HMAF. In vitro aggregation process To verify the ability of DIIIC proteins to form aggregates after incubation with ODN, each protein was incubated with ODN 39M. Examination of the mixture by electron microscopy revealed the presence of aggregates only if ODN was added (Fig. 6). The diameter of the particles ranged from 50 to 55 nm. Centrifugation of the reaction mixture showed the precipitation of 50 % of each recombinant protein.

Purity (%) Cation exchange chromatography

IMAC

Discussion

DIIIC-1

52.8

88.3

DIIIC-2

80.1

94.1

DIIIC-3

66.3

88.3

DIIIC-4

69.7

91.5

The capsid genes from serotypes 1, 2, 3 and 4 were cloned under the control of the T7lac promoter, both alone and fused to the DIII region of the gene encoding the envelope protein of the homologous virus. This system allowed acceptable levels of protein expression in the DE3 lysogenic E. coli strain and further purification at scalable levels of the future DENV vaccine candidates. In addition, the ACC and ACDC pET28a-derived vectors carry a

reduced-carbamidomethylated DIIIC proteins. The results showed that the recognition of DIIIC proteins from serotypes 1, 2 and 4 by anti-DENV mouse sera depended on the

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E. Suzarte et al. Fig. 3 Reactivity of DIIIC recombinant proteins against anti-DENV mouse and human sera, measured in ELISA. Mouse sera: polyclonal hyperimmune ascitic fluid (HMAF) produced against the homologous DENV serotype. Human sera: SH 69, SH 19, SH 101 (sera collected in the Havana 2000 DENV epidemic). Negative control: negative purification. Results are the means and standard error of the mean of three independent experiments

Fig. 4 Reactivity of recombinant chimeric proteins DIIIC against anti-DENV polyclonal mouse sera (HMAF) under reducing and nonreducing conditions. A: ELISA results of the means and standard error of the mean of three independent experiments B: Analysis by SDS-PAGE

kanamycin resistance gene, which is the preferable antibiotic resistance for protein expression under the GMP standards required for vaccine candidates based on recombinant proteins from E. coli. Analysis of expression levels revealed proper amounts of each recombinant protein, although the capsid proteins showed higher expression in comparison with the DIIIC proteins. Probably, the insertion of an additional region affected the synthesis capacity of the host cell. Nevertheless, the yields of each DIIIC were acceptable for the

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subsequent purification processes. It is worth noting that the levels obtained under the T7lac promoter were comparable to those reported by our group for the DIIIC-2 protein, but using the T5 promoter [22]. Similarly, the capsid proteins reached levels equivalent to those obtained by our group as well as by Jones et al. in 2003 [31]. To our knowledge, this is the first report describing the expression in E. coli of recombinant capsid proteins from DENV-1, 3 and 4, alone or fused to the DIII region of each homologous virus.

Chimeric dengue virus vaccine

Fig. 5 Reactivity of recombinant chimeric proteins DIIIC and dengue viruses against anti-DENV polyclonal mouse sera (HMAF) measured by ELISA. The results represent one experiment performed in triplicate

bonds. Nevertheless, the pure variant of DIIIC-4, in SDS PAGE under reducing conditions, exhibited the presence of high-molecular-weight aggregates that were resistant to SDS (Fig. 4B). The nature of such aggregation is probably based on hydrophobic interactions. Volk et al in 2007 studied the solution structure of the envelope domain III of DENV-4 and found dramatic surface electrostatic differences among DENV-2, DENV-3 and DENV-4 domain III. The last of these had more uncharged surface area relative to the other two viruses [33]. The first antigenic characterization was performed using HMAF against each DENV serotype. It has been reported that the majority of the antibodies generated upon natural DENV infection recognize conformational epitopes on the virion [34, 35] and that this response is mainly directed to the envelope protein. In our approach, the DIII region that was cloned included the 14 amino acids preceding and the 32 amino acids following domain III. The addition of these fragments provides flexibility to the DIII region [36], decreasing the probable conformational restrictions in the context of a fusion protein. Furthermore, the last 32 amino acids belong to the ‘‘stem’’, the region that connects the end of DIII with the viral transmembrane anchor. This proximal part of the stem forms an a-helical segment in the trimeric structure of the mature virion and is vital in the process of membrane fusion [37]. Peptides derived from the stem specifically block viral fusion [38], suggesting that this region could be important in the induction of neutralizing antibodies. The high reactivity of the chimeric proteins 1, 2 and 3 against the homologous HMAFs indicates a proper folding of the DIII region in the context of the homologous capsid protein. Similar results were obtained by Simmons et al in 1998 and Hermida et al. in 2004 with fusion proteins containing DIII of DENV-2 fused to the maltose binding protein of E. coli and DIII fused to the P64k protein from Neisseria meningitidis [16, 39]. In both cases, the recombinant proteins were administered to mice, eliciting a DENV-2 virus neutralizing antibody response and partial

Fig. 6 Characterization of particles of the recombinant proteins DIIIC. Morphology and size of the particles of DIIIC proteins incubated with 39M ODN were analyzed by electron microscopy.

Samples were centrifuged at 10,000 rpm for 20 minutes and negatively stained with uranyl acetate and analyzed by transmission electron microscopy. The bar represents 200 nm

Since the DIIIC-2 protein induced both humoral and CMI in mice and monkeys [22, 32], we selected the chimeric variants for further analysis. First, the purification process was established for the four chimeric proteins, basically following the process described by Marcos et al. [30]. Only few modifications were introduced for the DIIIC-4 variant, since this protein tends to aggregate even in the presence of 7 M urea and 0.5 % Tween. This behavior made it necessary to include 10 mM b-mercaptoethanol in all buffers to avoid protein aggregation related to the formation of some level of intercatenary disulphide

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protection against challenge infection with a lethal dose of DENV-2 administered by intracranial inoculation. This result suggested the proper folding of DIII, even where different proteins were used as a carrier. In the specific case of DIIIC-4, the reactivity was lower than that measured for the rest of the proteins, probably because of the aggregation pattern observed. This pattern was previously described for two p64k-DIII fusion proteins of the same serotype [40]; nevertheless, they conferred protection in mice upon challenge with an infectious virus of serotype 4. Another factor that probably contributed to the low reactivity of HMAF and human sera for DIIIC-4 could be the poor immunogenicity that DENV-4 showed as an infecting serotype. It has been suggested that the dengue 4 serotype is a naturally attenuated virus [41], and moreover, several studies of tetravalent recombinant candidates against DENV showed that serotype 4 was less immunogenic [12, 42, 43]. The presence of a disulphide bond within the structure of DIII has been correlated with its proper folding and reactivity by Abs recognizing conformational epitopes [44]. We characterized the unmodified and reduced-carbamidomethylated DIIIC proteins by ELISA against the homologous viral serotypes. The recognition of DIIIC-1, 2 and 4 depended on the presence of a disulfide bond within domain III, in accordance with the results obtained for the fusion proteins P64k-DIII of serotype 2 [45] and for the E proteins of DENV-2 [46] and DENV-1 [47], where this bond was also critical for the correct antigenic structure of the protein. Another clue about the importance of this bridge for the stability of domain III was obtained by Zidane et al., who showed that a consensus DIII deprived of its disulfide bond by mutations was predominantly unfolded at 20 °C [48]. In the case of serotype 4, to our knowledge, no evidence has been published about the influence of the disulphide bond on the reactivity of DIII of this serotype. Interestingly, the chimeric protein DIIIC-3, when unmodified, reduced and carbamidomethylated, showed very similar recognition by the homologous HMAF with respect to the native counterpart. This behavior was not related to an inefficient reduction process of DIIIC-3, since all reduced-alkylated protein variants migrated in SDS-PAGE slightly more slowly than untreated proteins and lost the largest contaminant bands after reduction. Based on this evidence, we can assert that the disulphide bond in DIII of DENV-3 is not required for the proper recognition of anti-DENV-3 antibodies generated in mice. All of these results contrast with the findings of Zidane et al., who reported that the envelope DIII of DENV-3 was the least stable and least resistant to denaturation among all of the serotypes. This less stable DIII-DENV-3 belongs to the Asian genotype II, different from our DIII of a DENV-3 variant belonging to LatinAmerican genotype III. It is possible that different

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genotypic strains of the same viral serotype may have different biophysical properties [48], or perhaps the capsid fusion improve the stability of the molecule. The serotype specificity pattern was another parameter measured in the present work. Naturally, upon primary infection, functional serotype-specific antibodies are elicited as well as nonfunctional cross-reactive ones. Such crossreactive antibodies are recalled upon secondary heterologous infection, leading to antibody-dependent enhancement of virus infection, causing a high viral load and, consequently, an increase in disease severity [49]. Therefore, a vaccine candidate against dengue should ideally induce a high serotype-specific immune response against the four serotypes to guarantee that it does not enhance subsequent infections from exposure to DENV. In the present study, we demonstrated that DIIIC proteins are preferentially recognized by the homologous HMAFs, whereas the viruses are more broadly recognized. This finding was based on the pattern obtained with the four fusion proteins P64k-DIII as well as for the MBP-DIII of serotype 2 [12, 39]. This could suggest a possible use of the DIIIC constructs in a vaccine formulation with minimal risk of inducing the immunopathological mechanisms upon vaccination. The last antigenic characterization was performed using human sera with known neutralizing capacity. The four recombinant proteins were properly recognized by the human sera, indicating the relevance of this region upon natural infection. The high density of positive charge on the capsid protein and its possible role in the encapsidation process of the viral genome led us to carry out a controlled reaction of capsid assembly in vitro, employing ODN 39M. In previous work, we reported that aggregated forms of the protein DIIIC-2 were able to induce not only a high CMI in mice but also significant protection against challenge with infectious virus [22]. As expected, in the assembly reaction, the DIIIC-1, DIIIC-3 and DIIIC-4 proteins were able to form aggregates in a similar way to the DIIIC-2 protein, and such particles were visualized by TEM. Contrary to the reported size for the DIIIC-2 particles in our previous study, the particles detected in this work corresponded to 50 nm. The possible reason may be associated to the ODN used for the aggregation reaction. We used a defined ODN of 39 bases, whereas in the previous studies a heterogeneous mixture of 50-base-long ODNs was employed. Because the encapsidation process depends on the neutralization of the positives charges on the capsid region upon ODN addition, the nature of the ODN may influence in the size of the resultant particle. Although both aggregates could have different immunogenic properties, the fact that DIIIC-2 aggregated with ODN 39M induces a functional immune response in mice (unpublished data) is a promising result.

Chimeric dengue virus vaccine

Taken together, we can conclude that DIIIC proteins for serotypes 1, and 3 are antigenically similar to the DIIIC-2 protein, so is predicted to be immunogenic in mice. In the specific case of DIIIC-4, mouse experiments will determine its functionality. Based on the results recently obtained with C2 [50] and DIIIC-2 [17] in mice, demonstrating the serotype specificity of the immune response induced by them, we can propose that present constructs would have the same potential. The generation of mainly serotypespecific antibodies is highly desirable, since the crossreactive response, directed to the prM and E proteins, is responsible of the antibody-dependent enhancement of secondary heterologous infections [51]. Nevertheless, questions regarding the generation of appropriate immune responses and protection efficacies with the tetravalent formulation containing the present proteins remain to be evaluated.

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