Immune response to synthetic peptides of dengue prM protein

Vaccine 20 (2002) 1823–1830

Immune response to synthetic peptides of dengue prM protein Susana Vázquez a,∗ , Mar´ıa Guadalupe Guzmán a , Gerardo Guillen b , Glay Chinea b , Ana Beatriz Pérez a , Maritza Pupo a , Rosmary Rodriguez a , Osvaldo Reyes b , Hilda Elisa Garay b , Iselys Delgado a , Gissel Garc´ıa a , Mayling Alvarez a a

Virology Department, “Pedro Kouri” Tropical Medicine Institute, Autopista Novia del Mediodia, km 6, P.O. Box Marianao 13, Havana, Cuba b Research Direction, Biotechnology Center, Havana, Cuba Received 20 July 2001; received in revised form 12 December 2001; accepted 12 December 2001

Abstract The immunological activities of five synthetic peptides of the prM protein of dengue-2 (DEN-2) virus containing B cell epitopes were evaluated in BALB/c mice. Two peptides elicited neutralizing antibodies against all four DEN serotypes. Virus-specific proliferative responses were demonstrated in mice immunized with four of the five peptides, demonstrating the presence of T cell epitopes. Mice immunized with three of the five peptides conjugated with bovine albumin showed statistically significant levels (P < 0.05) of protection when challenged with DEN-2 virus. These results could constitute the basis for the establishment of the role of DEN virus pre and M antigens in the development of anti-flaviviral vaccines. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Dengue virus; Synthetic peptide; prM protein

1. Introduction Dengue (DEN) viruses are single-stranded RNA viruses. The nucleic acid is contained within a nucleocapsid composed of capsid (C) protein surrounded by a lipid envelope of about 50 nm diameter in which the envelope (E) glycoprotein and the matrix (M) protein are embedded. Both the structural and non-structural proteins (NS) are encoded by a single, open reading frame of about 10.5 kb. Two forms of the M protein have been characterized: the prM in the immature virions and M protein in extracellular mature virions. The prM (18.1–19.1 kDa) is a precursor to the M structural protein (7–9 kDa) and has a glycosylation site located in the pr portion. This precursor undergoes proteolytic cleavage by a host enzyme associated with the trans-Golgi membrane. Co-transport of prM and E heterodimer through an exocytic pathway is essential and the synthesis of a proper E protein requires co-synthesis of the prM. This cleavage is inhibited by agents that destabilize the low pH of the Golgi vesicles [1]. The fragment pr has been identified only in the extracellular medium in vitro, and its function in vivo remains unknown [2]. Application of synthetic peptides as anti-DEN vaccine subunits [3,4] could allow the inclusion in the final formu∗ Corresponding author. Tel.: +53-7-246051; fax: +53-7-220633. E-mail address: [email protected] (S. V´azquez).

lation of only protective epitopes that are not involved in the immune amplification phenomenon [5,6]. Alternatively, protective epitopes of each of the four serotypes could be included. However, there are no published studies relating to the antigenic characterization of DEN pr and M proteins, using synthetic peptides. We have designed and synthesized five peptides derived from DEN-2 prM protein. Immunization of BALB/c mice with free and conjugated peptides showed the presence of B cell epitopes in all peptides and, with one exception, the presence of T cell epitopes in the peptide sequences was demonstrated by lymphoproliferation assay (LPA). Three of these peptides were capable of conferring protection in mice. This constitutes the first report of the use of synthetic peptides for the antigenic characterization of DEN pr and M proteins.

2. Materials and methods 2.1. Prediction of antigenic regions of prM protein from DEN virus Methods used to predict antigenic sites in prM protein were based only on protein sequence information, since neither the three-dimensional structure of the prM protein from any flavivirus has been determined experimentally nor

0264-410X/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 - 4 1 0 X ( 0 1 ) 0 0 5 1 5 - 1

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is there a significant sequence similarity with any protein of known three-dimensional structure. Potential antigenic regions were predicted according to the following criteria: (a) regions of high antigenic propensity according to different prediction methods based on hydrophilicity [7], flexibility [8] and accessibility profiles [9]; (b) regions predicted to form surface loops according to predictions of secondary structure and accessibility using the PHD program [10–12]; (c) regions of high sequence variability and/or containing insertions/deletion sites comparing sequences of DEN and other flaviviruses; (d) regions containing glycosylation sites in DEN or other flaviviruses. Antigenicity-related property profiles were derived using the sequence from the A15 strain of DEN-2 isolated in Cuba in 1981 [13]. Variability per amino acid position was calculated separately for three sets of sequences: first set, DEN (DEN-1–4); second set, MBV (sequences of flaviviruses transmitted by mosquitoes, including DEN, Japanese encephalitis (JEV), Kunjin (KUNV), West Nile (WNV), Murray Valley encephalitis (MVEV), Saint Louis encephalitis (SLEV) and yellow fever (YFV)); and a third set of flaviviruses (MBV + Langat (LANV), louping ill (LIV), tick-borne encephalitis (TBEV), Kyasanur Forest disease (KFDV) and Powassan (POWV)). Multiple sequence alignments were carried out with CLUSTAL-W [14]. Converting the multiple sequence alignments to HSSP format using the Predict Protein server [15] accessed the variability values per amino acid position (the VAR column in HSSP files contains the sequence variability per position on a scale of 0–100). The values were averaged over a five amino acid window and plotted. Protein sequences were extracted from the SWISSPROT database (accession numbers: P27912, P07564, P27915, P09866, P27395, P14335, P29837, P22338, P05769, Q04538, P09732, P14336, P06935, P03314) and SPTREMBL (accession number: Q82951).

were extracted with 30% acetic acid and were purified by reverse-phase high performance liquid chromatography (HPLC, Vydac C18, 10 mm × 250 mm). A cysteine at the N-terminus for coupling with bovine serum albumin (BSA) was included in each peptide. 2.4. Coupling of peptides to a carrier protein This procedure was derived from a method proposed by Harlow and Lane [20]. Briefly, 80 ␮l of the bifunctional reagent m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), 5 ␮g/␮l in dimethylformamide, was added to a solution of 2.8 mg BSA in 250 ␮l phosphate-buffered saline (PBS). The mixture was agitated at room temperature (RT) for 30 min, and then passed through a PD10 column (Pharmacia Sweden). One milligram peptide dissolved in 300 ␮l PBS, pH 7.2–7.4 was slowly added to the activated BSA solution and incubated at RT for 3 h. The peptide–BSA conjugate concentration was determined by the method of Lowry et al. [21]. 2.5. Mouse immunization Two immunization schedules were developed using groups male BALB/c mice 4–6 weeks old. The first groups were immunized intraperitoneally with 50 ␮g of the peptide–BSA conjugate (four inoculations, each 15 days apart, 20 mice for each peptide). Freund’s complete adjuvant (FCA, 100 ␮l) was used in the first dose, and Freund’s incomplete adjuvant (FIA) in the others. The second groups were immunized with free peptides (non-conjugated) in order to evaluate the T cell response (five mice for each peptide). Three immunizations were performed at days 0, 15 and 45 with FIA. Lymphocyte proliferative response (LPA) to DEN antigens was tested 8 weeks after the last dose. In all cases blood samples were obtained from mice 7 days after the last dose and the anti-peptide antibody titer of each mouse serum was determined by ELISA.

2.2. Prediction of T cell epitopes The prediction was made using two independent methods: the pattern method of Rothbard and Taylor [16], and the determination of fragments with a propensity to form amphipathic ␣-helix structures (AMPHI 7 and 11) [17]. 2.3. Peptide synthesis Peptides were prepared at the Center for Genetic Engineering and Biotechnology (CIGB, Havana city). They were synthesized according to the solid-phase method [18] on 4-methylbenzhydrylamine (MBHA) resin (1 mmol/g, Fluka) using tert-butyloxycarbonyl (t-Boc)/benzyl strategy. Peptide resin was cleaved with hydrogen fluoride using the “Low–High” procedure [19] in the presence of appropriate scavengers, and washed three times with ether. Peptides

2.6. Indirect ELISA to detect anti-peptide and anti-virus antibodies Each peptide was coated onto a 96-well microtitration plate (Maxisorb) at 10 ␮g/ml concentration in 0.1 M carbonate buffer, pH 9.6 (100 ␮l). After 18 h incubation at 4 ◦ C, sera from mice immunized with conjugated and free peptides were added at 10-fold (1/100 to 1/100,000) dilutions in PBS + 0.05% Tween 20. Binding of antibodies to the peptides was detected using peroxidase-conjugated goat anti-mouse IgG (Amersham) and o-phenylendiamine. The reaction was stopped by addition of 12.5% H2 SO4 and the absorbance at 492 nm read in an ELISA reader. Serum antibody titers were defined as the inverse of the highest dilution at which the optical density (OD) was at least twice that of the negative control. Pre-immune and anti-BSA sera

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were used as negative controls for the free and conjugated anti-peptide antibodies, respectively. Hyperimmune ascites fluids (HAF) against DEN-1 (Hawaii), DEN-2 (New Guinea C), DEN-3 (H-87), DEN-4 (H-241), SLE viruses (kindly supplied by the Microbiology and Epidemiology Institute of Praha) and normal ascites fluid (NAF, negative control) at 1/50 dilution were also tested using this ELISA. The positive criterion was an OD twice that of the negative control at this dilution. 2.7. In vitro plaque-reduction neutralization test (PRNT) PRNT was carried out according to Morens et al. [22]. Neutralizing antibodies to DEN-2 (A15 strain) were determined in pools of anti-peptide, anti-BSA and pre-immune mice sera. Heterotypic neutralizing antibodies to DEN-1, DEN-3 and DEN-4 virus were determined in those sera previously determined to be positive for DEN-2 virus. 2.8. Assays for proliferative responses of antigen-specific T cells Spleens were removed from mice 8 weeks after immunization with synthetic peptides. Spleens from control mice were also included. Single cell suspensions were prepared by perfusion of the spleens. Erythrocytes were lysed by brief exposure to lysing medium (155 mM NH4 Cl, 10 mM KHCO3 , 190 mM EDTA). Cells were then washed and viable cells were counted and adjusted to 2 × 106 cells per ml in RPMI 1640 medium supplemented with glutamine 2 mM, penicillin G (1000 IU/ml), streptomycin sulfate (100 ␮g/ml), 2-mercaptoethanol (5×10−5 M) and 10% fetal bovine serum (Gibco BRL) [23]. Proliferative responses to DEN virus antigen were tested [24]. Spleen cells (2 × 105 ) were cultured for 4 days at 37 ◦ C in 100 ␮l RPMI 1640 in the presence of 40 ␮g DEN-2 antigen purified by sucrose gradient ultracentrifugation [25], and pulsed with 1 ␮Ci [3 H]-thymidine (Amersham) per well for 18 h. Incorporation of radiolabel was measured in a liquid scintillation counter. Stimulation index was calculated using the formula: mean counts per minute (CPM) of triplicate wells incubated with the antigen/the mean CPM of triplicate controls (no antigen). A stimulation index >2 was considered significant. 2.9. Protection assay For virus protection experiments, immunized mice with conjugate peptides were injected intracranially (i.c.) with 20 ␮l of a suspension of DEN-2 (A15 strain) virus-infected suckling mouse brain containing 100 50% lethal doses (LD50 ) 30 days after the last dose. Animals were observed daily for 21 days for morbidity and mortality. Data were tested for statistical significance using Fisher’s exact test.

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3. Results 3.1. Prediction of B and T cell epitopes of prM protein from DEN virus Results of antigenicity predictions are summarized in Figs. 1 and 2 and Table 1. It is well known that antigenicity profiles and secondary structure predictions have limited success in predicting peptide sequences capable of generating anti-peptide antibodies cross-reactive with the folded protein. In this case generation of anti-peptide antibodies with any functionality such as neutralizing activity, HI, etc. presupposes that such antibodies should recognize the protein prM as it is present in the virions. During virion assembly prM–E heterodimers further oligomerize to form a complex icosahedral envelope at the surface of virions. Hence, prediction of virion cross-reactive epitopes is a still more difficult task because a significant portion of the prM surface should be involved in protein–protein interactions or facing toward the lipid bilayer. In an attempt to overcome these difficulties we made use of the evolutionary information contained in multiple sequence alignments of protein prM from various flaviviruses (Figs. 1C and 2). Sites of high sequence variability and/or insertion/deletions are likely to be located at the outer surface of virions, and more conserved residues should be functionally or structurally relevant, perhaps involved in prM–E interactions. Additionally, glycosylation sites need to be accessible at the external face of the virion envelope and hence potentially antigenic. 3.2. Design of antigenic peptides Five peptides from prM protein of DEN-2 virus, which cover 58% of the amino acid sequence (97/166 aa) were selected and chemically synthesized (Table 2). Peptide B19-6 consists of the N-terminal segment of protein prM, which lacks cysteine. It contains some antigenicity-property peaks, potential glycosylation sites in various flaviviruses and is highly variable among the viruses, with insertions/deletions. Peptides B20-2, B19-5 and B20-1 are overlapping peptides covering the region C45 -V93 . These peptides contain various cysteine residues involved in disulfide bridges in the native protein. Although this region presents some potential antigenic sites, it is highly probable that the majority of epitopes are disulfide bridge-dependent and therefore unlikely to be mimicked by linear peptides. Moreover, the high conservation of several residues in this domain is indicative of functional epitopes, perhaps involved in prM–E interactions. However, peptides B19-5 and B20-1 include the cationic cluster H85 -R90 , a region susceptible to proteolytic cleavage by furin proteases during the virion maturation process and therefore accessible at the surface. Peptide B20-3 covers the most hydrophilic segment of the shortened ectodomain of protein M.

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Fig. 1. Hydrophilicity, surface accessibility, flexibility and residue variability profiles of protein prM: (A–C) property profiles corresponding to protein prM from DEN-2; (D–F) sequence variability of protein prM. Variability was calculated for three groups of sequences: DEN, dengue-1–4; MBV, mosquito-borne virus (dengue-1–4, Japanese encephalitis virus, Kunjin virus, Murray Valley encephalitis virus, West Nile virus, yellow fever virus, St. Louis encephalitis virus); flavivirus, all sequences (MBV, Langat virus, tick-borne encephalitis virus, louping ill virus, Kyasanur Forest disease virus, Powassan virus).

Table 1 Predictions of T cell epitopes of protein prM from DEN-2 Method

Predicted T cell epitope a b

AMPHI 7a

AMPHI 11a

RT4b

RT5b

G42 -D46 , V136 -I139

D29 -L36 , D40 -E46 , Y77 -T79 , W117 -R122 , I139 -Y142

H2 -T5 , D47 -T50 , G115 -K118 , G145 -H148

H2 -R6 , E108 -S112

AMPHI (7 and 11): epitopes predicted by the method of Margalit et al. [17]. RT4 and RT5: predictions based on patterns of four and five residues of Rothbard and Taylor [16].

Table 2 Designed antigenic peptides from prM protein of DEN virus Code

Sequence

Region

B19-6 B20-2 B19-5 B20-1 B20-3

LTTRNGEPHMIVMRQEKGKSLLFKTGDGV CEDTITYKCPLLRQNEPEDIDCW RQNEPEDIDCWCNSTSTWVTYGTCTTTGEHRREKRS NSTSTWVTYGTCTTTGEHRREKRSV LETRTETWMSSEGAWKHAQRIE

3–31 45–67 57–92 69–93 103–124

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Fig. 2. Multiple sequence alignment corresponding to flaviviral protein prM; PHD sec and PHD acc: secondary structure and accessibility predictions for DEN-2 using the PHD program; E, H, and h correspond to ␤-strand, helix and trans-membrane helix, respectively; e and b are exposed and buried predictions. ID: ∗ are conserved residues, single and double dots are conservative positions; N: potential glycosylation sites.

3.3. Anti-peptide antibody responses of immunized mice by ELISA

3.4. Anti-virus antibodies against the peptides measured by ELISA

Sera of mice immunized with the conjugated peptides and free were assayed by ELISA. Results are shown in Table 3. High titers of antibodies (up to 10,000) were found in mice immunized with conjugated peptides. The titer in mice immunized with free peptides were substantially lower (1000 or lower), but readily detectable.

HAF to the four DEN serotypes, SLE and negative sera as control were assayed by indirect ELISA using the different peptides as antigens (Fig. 3). All DEN hyperimmune sera recognized the peptides; however, the highest response was observed with DEN-2 HAF. No reaction was observed with SLE HAF. 3.5. In vitro plaque-reduction neutralization test

Table 3 Serum antibody titers in mice immunized with the synthetic peptides Peptide

Antibody titers Anti-conjugated peptides (% of micea )

B19-6 B20-2 B19-5 B20-1 B20-3 a b

Anti-free peptides (% of miceb )

100

1000

10000

100000

100

1000

10 0 5 5 10

5 10 40 10 15

70 65 55 60 70

15 25 0 25 5

60 40 80 40 40

40 60 20 60 60

Number = 20. Number = 5.

Pools of anti-peptide, anti-BSA and pre-immune sera were prepared and tested by PRNT against DEN-2 virus and those positives were re-tested against the rest of the serotypes. Table 4 shows the neutralizing antibody titer of each pool of serum. Cross-reactive neutralizing antibodies were observed in sera of animals immunized with peptides B19-6 and B20-3. 3.6. Assays for proliferative responses of antigen-specific T cells A significant lymphoproliferative response to DEN-2 virus was demonstrated with all but one (20-2) of the spleen

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Fig. 3. ODs of the HAF of each dengue serotype, SLE and normal ascites fluid.

Table 4 Dengue neutralizing antibody titers of pools of anti-peptide sera Anti-serum

B19-6 B20-2 B19-5 B20-1 B20-3 BSA Pre-immune a

Neutralizing antibody titer DEN-1

DEN-2

DEN-3

DEN-4

100 NDa ND ND 110 <10 <10

180 <10 <10 <10 80 <10 <10

160 ND ND ND 80 <10 <10

160 ND ND ND 80 <10 <10

ND: not done.

T lymphocytes of mice immunized with free peptides (Fig. 4).

Fig. 4. Proliferative response to DEN-2 virus antigen (40 ␮g/ml) of: (䊐) spleen T cells from peptide immunized mice and the cellular control (䊏).

3.7. Protection against challenge The percentage survivals in peptide immunized and control animals are shown in Fig. 5. The level of protection induced by the peptides B19-6, B19-5 and B20-1 was statistically significant (P < 0.05).

Fig. 5. Percent survival in peptide immunized and control animals.

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4. Discussion Synthetic peptides representing specific regions of proteins can induce both humoral and cellular immune responses specific to those proteins [26,27] and such observations are important for the development of strategies for the rational design of future vaccines [28]. For synthetic peptides to act as effective immunogens, they must comprise two distinct sites, one to promote B cell interaction and a second to induce cognate helper T cell activity [29,30]. In our study we selected five peptides from three distinct regions of DEN-2 prM protein: one peptide (B19-6) from the N-terminal segment (residues 1–33), three overlapping peptides (B19-5, B20-1 and B20-2) from the disulfide-bridged domain (residues 34-91) and one peptide (B20-3) from the ectodomain of protein M. The selected peptides contained potential antigenic sites based on combined criteria using antigenicity profiles, prediction of accessibility and secondary structure, sequence variability and location of potential glycosylation sites. The use of the information concerning sequence variability and potential glycosylation sites is further supported by the recent determination of the X-ray crystal structure of a soluble fragment of protein E from tick-borne encephalitis virus [31]. The defined structure indicates that protein E forms head-to-tail homodimers apparently lying parallel to the viral membrane. Highly variable regions, insertions/deletions and glycosylation sites are mostly located on the outer surface of the virions. All five peptides were capable of inducing a humoral immune response in mice and specifically with two of them, peptides B19-6 and B20-3, an in vitro neutralizing antibody response was observed. Peptide B19-6 was the lone peptide inducing both neutralizing and protective response. Consistent with predicted results is that this peptide displays high values of antigenicity-related properties, constitutes the most variable region among flavivirus sequences including insertions/deletions, and contains glycosylation sites in other flaviviruses. It is likely to be the most exposed region of prM. The E protein is associated with the induction of the major neutralizing antibody in the protective response, but also prM and M proteins are able to induce a significant immune response [32–34]. The mechanism of virus neutralization in the presence of anti-prM antibodies is not completely defined, but the proximity of both prM and E proteins may facilitate the blockade by anti-prM antibodies of the E epitopes involved in the receptor-binding entrance of the virion to the cell, and this action could result in the prevention of virus penetration. Anti-pr antibodies may be involved in this steric impediment not only in the immature virions with certain infectivity but also in mature ones, in which pr protein fragments could be present. We cannot exclude the possibility that other mechanisms could be involved in viral neutralization. Most of the epitopes that induce the production of neutralizing antibodies are conformational; however, our results

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demonstrate that linear epitopes (B19-6 and B20-3 peptides) on prM protein are also able to induce a neutralizing antibody response. The prM epitopes are not immunodominants in the antibody response to the virus, but our peptides, inoculated alone, probably were more accessible to the immune system, allowing the development of neutralizing antibodies to the four serotypes. Polyclonal anti-DEN antibodies to the four serotypes were able to recognize the five peptides when tested by ELISA. The fact that a DEN-2 strain was used into the synthesis of the peptides could explain the higher titer observed with the anti-DEN-2 serum. Of all peptides, number B19-6 showed the maximum recognition by anti-DEN-2 serum. As was expected, SLE antibodies did not recognize any peptide, on account of the high percent of divergence between the sequences studied. T cell epitopes have been identified on different structural and non-structural DEN proteins such as E [4,35], C [36] and NS1, NS2a [37], NS3 [37,38]; however, prM protein has been poorly studied. In our study, we have identified T cell presence of epitopes on synthetic peptides to pr and M proteins. Virus-specific proliferative responses using spleen T lymphocytes were demonstrated with four of the five peptides when they were used to immunize BALB/c mice. T cells from B19-6, B19-5, B20-1 and B20-3 immunized mice proliferated in an in vitro blastogenesis assay when they were cultured with DEN-2 virus. However, when spleen T cells from BALB/c mice immunized with the virus were cultured with the different peptides no proliferative response to any peptide was observed. One possibility that could explain these results is that these peptides have non-immunodominant epitopes (facultative cryptic epitopes) because the T CD4 lymphocytes are able to re-stimulate themselves in front of native protein [39]. Only one peptide (B20-2) was considered as an absolute cryptic epitope. Different protection studies using synthetic peptides have been performed. In some of these the protective response was associated with neutralizing antibody activity. Studies using synthetic peptides have indicated that the neutralizing activity in vitro may not be an absolute requirement for protection [40–42]. In the present study we found that B19-6, B20-1 and B19-5 peptides induced a statistically significant protection level and only one of them (B19-6) had neutralizing activity. This work evaluates, for the first time, the humoral and cellular responses induced by synthetic peptides of pr and M proteins of DEN virus. The presence of sequential B and T cell epitopes and the importance and relevance of the study of the prM protein of flaviviruses has been demonstrated.

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