Vaccine 24 (2006) 3165–3171

A recombinant fusion protein containing the domain III of the dengue-2envelope protein is immunogenic and protective in nonhuman primates

Lisset Hermida a,∗, Lıdice Bernardo b, Jorge Martın a, Mayling Alvarez b, Irina Prado b,Carlos Lopez a, Beatriz de la C. Sierra b, Rafael Martınez a, Rosmary Rodrıguez b, Aıda Zulueta a,

Ana B. Perez b, Laura Lazo a, Delfina Rosario b, Gerardo Guillen a, Marıa G. Guzman b

a Center for Genetic Engineering and Biotechnology, Apdo 6162, Havana 10600, Cubab “Pedro Kourı” Tropical Medicine Institute, Autopista Novia del Mediodıa, km 6 P.O. Box Marianao 13, Havana, Cuba

Received 28 December 2005; received in revised form 17 January 2006; accepted 18 January 2006Available online 3 February 2006

Abstract

We have previously reported the construction and evaluation in mice of recombinant fusion proteins formed by a fragment (aa 286–426)ofdnwcr©

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f the dengue envelope protein and the P64k protein from Neisseria meningitidis. In this work we describe the immunization of Macacaascicularis monkeys with two variants of these proteins [PD3 (insertion variant) and PD5 (fusion variant)] corresponding to serotype 2. Fouroses of the proteins adjuvated in Freund’s adjuvant were administered and the kinetics of antibody induction was monitored by ELISA andeutralization tests. Monkeys receiving PD3 or PD5 developed functional antibodies (Abs) in a dose-dependent manner. Following challengeith 5 log PFU of wild type dengue-2 virus (DEN2), animals immunized with PD5 were protected from developing viremia. These results

onstitute a proof-of-concept demonstrating that a fragment of the dengue envelope protein, containing the domain III and produced as aecombinant fusion protein in Escherichia coli, induces functional and protective immunity in a nonhuman primate model.

2006 Elsevier Ltd. All rights reserved.

eywords: Macaca fascicularis; Domain III; Protection

. Introduction

Dengue epidemics caused by the four dengue viruserotypes (DEN1–DEN4) continue to pose a major publicealth problem in most tropical and subtropical regions.ccording to estimates, as many as 100 million DEN infec-

ions occur every year worldwide [16,32,44]. The incidencef dengue fever (DF) and dengue hemorrhagic fever/denguehock syndrome (DHF/DSS) is rising and there is currentlyo vaccine available to prevent the disease. The lack of an ani-al model for DF or DHF/DSS and the fact that simultaneous

mmunization is required to induce long-lasting protection

∗ Correspondence to: Division de Vacunas, Centro de Ingenierıa GeneticaBiotecnologıa, Apdo 6162, Habana 10600, Cuba. Tel.: +53 7 271 6022;

ax: +53 7 271 4764.E-mail address: lisset.hermida@cigb.edu.cu (L. Hermida).

against all four DEN serotypes have been the principal bar-rier to the development of safe and effective DEN vaccines[10,21,23,32,38]. Live attenuated, live recombinant virus orinfectious cDNA clones are being considered as vaccine can-didates [2,24,12,13]. Such vaccines may elicit long-lastingprotective immunity to DEN but interference among theserotypes, difficulties in replication and immunity induction,the potential reversion to virulence, and the elevated cost ofproduction continue to hinder their widespread use [49]. Non-replicating inactivated and recombinant subunit vaccineshave had little success to date due to the limited immuno-genicity, assurance of conformational integrity as well as theproduction cost [52]. However, manufactured recombinantsubunit vaccines are still economically attractive. The useof Escherichia coli for expressing DEN structural geneshas been limited since the majority of DEN proteins requirecomplex co- and post-translational processing [7,29,30].

264-410X/$ – see front matter © 2006 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2006.01.036

3166 L. Hermida et al. / Vaccine 24 (2006) 3165–3171

Nevertheless, the expression of flavivirus structural subunitsas recombinant fusion proteins using this expression systemhas yielded functional recombinant variants of DEN antigens.Some of these variants have proved to be immunogenic inmice, inducing neutralizing antibodies (Abs) and protectingthem against experimental lethal DEN infection. Successfulapproaches have employed protein A from Staphylococcusaureus, glutatione-s transferase and the maltose bindingprotein as carriers [43,45,46,47,48]. Unfortunately, thesafety of these carrier proteins has not yet been evaluated inhumans.

We have reported the expression of some variants of a DENenvelope (E) fragment (aa 286–426) fused to the meningo-coccal P64k protein [54]. The P64k protein, from Neisseriameningitidis, was cloned under the Trp promoter in E. coli andproduced at high levels in economic culture media. Accord-ingly, a feasible scalable purification process was set up atdevelopmental scale [11]. On the other hand, the analysis ofimmunodominant regions of P64k confirmed that there are,within the lipoil-binding domain as well as at the C-terminus,some regions that are highly exposed in the protein structure(data pending publication). It has also been demonstratedthe carrier capacity of this bacterial protein after vaccinationof terminal patients with lung cancer with human epidermalgrowth factor [8]. On the other hand, a phase I trial in humanvolunteers was conducted using the P64k alone, and its safetya

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2.2. Recombinant proteins

The design, cloning, expression and initial evaluation ofthe recombinant proteins involved in this study have been pre-viously described [54]. Briefly, the DEN2 E gene fragment,from Jamaica strain, coding for aa 286–426 of the dengue Eglycoprotein was cloned into a vector containing the sequenceof P64k with a histidine tail. Two fusion protein variants wereconstructed. The first variant (PD3) resulted from the inser-tion of the fragment into the lipoil-binding domain of P64k[18]. To generate the second variant (PD5), the fragment wasdirectly fused to the C-terminus of the same protein [54]. TheP64k protein alone was used as a control. Ion metallic affin-ity chromatography (IMAC) was used to purify the proteins[27].

2.3. Viruses

The standard strain NGC of DEN2 was used assucrose–acetone antigen [4] for immunoassay tests. The A15strain of DEN2 (a Cuban isolate from the 1981 epidemic) hasbeen passaged three times in Vero cells and was employedin the plaque reduction neutralization test (PRNT). A viralstock for use in challenge studies was prepared in Vero cellsby removing the supplemented culture medium from infectedcells (medium 199, 5% heat inactivated fetal calf serum,2owwa

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nd immunogenicity were demonstrated [35].Previously, we reported that the whole P64k protein is

equired to obtain better folding of the DEN2 envelope frag-ent and therefore a satisfactory immune response induction

n mice [18]. We also explored the influence of two alterna-ive fusion positions for the DEN1 envelope fragment withinhe P64k protein by inserting the DEN polypeptide, eitherollowing the first 45 aa of P64k (insertion variant) or at the-terminus of P64k (fusion variant) [19]. As a result, the high-st protection corresponded to the group immunized with the-terminus variant.

Having built this body of work using the mouse model,e planned to study the immunogenicity of two variants of

he P64k fusion proteins (the insertion and fusion variantsontaining the envelope fragment from DEN2) for their pro-ective capacity in a nonhuman primate model.

. Materials and methods

.1. Animals

Healthy adult monkeys (Macaca fascicularis) werebtained from CENPALAB (Havana, Cuba). All animalsere screened for previous exposure to dengue and P64krotein by detection of serum anti-DEN and anti-P64k Abs.nimals were considered naive with respect to both antigenshen antigen specific Abs were undetectable. Monkeys wereaintained in accordance with Cuban guidelines for the care

nd use of laboratory animals.

mM l-glutamine and 100 U of streptomycin and neomycin)nce the viral cytopathic effect was observed. Fresh mediumithout fetal calf serum was then added. This supernatantas harvested 48 h later, then aliquoted, stored at −70 ◦C

nd titrated by plaque formation on BHK-21 cells.

.4. Immunization

Nine monkeys that passed the screening process wereanked by weight, age and sex and then randomly assignedo three groups with three animals each one. In order to han-le the monkeys, they were anesthetized by intramuscularnjection of ketamine hydrochloride. Animals were subcuta-eously injected with 50 �g of the recombinant proteins PD3first group), PD5 (second group) and P64k (control group).oses were administered to monkeys in each group at days:, 30, 94 and 210 using complete Freund adjuvant for therst dose and incomplete Freund adjuvant for all subsequent

noculations. The adjuvants were mixed 1:1 (v/v) with thentigens. Each immunization was given in a final volume of.5 ml, injected into four different sites on the back. Bloodamples were collected at the time of, and 15 days after eachnoculation. Serum from clotted blood was stored at −20 ◦C.

.5. Challenge and virus detection

Forty-five days after receiving the last dose monkeysere subcutaneously inoculated in the upper arms with 5 logFU of the DEN2 strain A15. Blood was collected dailyor 10 days to detect viremia. For serological studies, three

L. Hermida et al. / Vaccine 24 (2006) 3165–3171 3167

additional blood samples were taken at days 15, 30 and60 post-challenge. Sera from clotted blood were stored at−70 ◦C until viremia was analyzed. The presence of virus inthe sera was determined by inoculating 100 �l of undilutedserum on to Vero cells grown in 24-well plates, shaking theplates for 1 h at 37 ◦C, and then centrifuging the plates aspreviously described [39]. Fresh supplemented medium wasadded to the wells and after 7 days of incubation, cells weretested by indirect immunofluorescence using anti-DEN2hyperimmune mouse ascitic fluid. Samples were subjectedto three blind passages in the same cell cultures before beingconsidered as negatives.

In addition, the presence of viral RNA was assessedby qualitative RNA reverse transcriptase followed by poly-merase chain reaction (RT-PCR). RNA was extracted from250 �l of serum samples using the Trizol procedure accord-ing to the manufacturer’s instructions (Gibco-BRL, USA).RT and cDNA amplification was performed as previouslydescribed [32,53].

2.6. Analysis of the antibody response

The anti-DEN2 IgG antibodies stimulated by immuniza-tion were monitored by enzyme-linked immunosorbent assay(ELISA). Briefly MAXISORP 96-well plates were coatedwEToa

rating concentration of DEN2 antigen and the mock antigenin separate wells. Serially diluted samples from sera wereincubated 1 h at 37 ◦C with either the DEN2 or the mock anti-gens. Anti-monkey IgG-peroxidase conjugate (Sigma, USA)diluted 1/10000 was incubated for 1 h at 37 ◦C. H2O2/OPDwas then added as substrate solution for 30 min. Optical den-sities (OD) were measured at 492 nm. A dilution of serum wasconsidered positive when the ratio [OD (DEN antigen)]/[OD(mock)] was two or higher.

The functionality of the antibodies was measured by neu-tralization of DEN2 infectivity by PRNT on BHK-21 cellsculture as described [33]. The Ab response to the carrier pro-tein (P64k) was measured by an indirect ELISA as previouslydescribed [35].

3. Results

3.1. Antibody response after immunization

Sera from immunized and control monkeys were ana-lyzed for anti-P64k and anti-DEN2 antibodies by ELISA.Antibodies directed to the carrier P64k protein wereestimated by end point dilution titration on plates coatedwith P64k (Fig. 1). All the animals elicited antibodies afteriaifp

F 2 virusm

ith the monoclonal Ab 4G2 which recognizes the flavivirusprotein [1,40]. Three washes with PBS containing 0.05%

ween 20 (Merck, Germany) were completed after each stepf the ELISA. Plates were blocked with 2% bovine serumlbumin, and then incubated overnight at 4 ◦C with a satu-

ig. 1. Kinetics of IgG antibodies against P64k (close circle curves) and DEN

onkeys with PD3 (A), PD5 (B) and P64k (C). Block arrows indicate days of imm

mmunization with the recombinant proteins. High titers ofnti-P64k Abs were induced as early as 2 weeks after prim-ng with PD3, PD5 or P64k. A booster effect was detectedollowing a second dose, reaching plateau levels at late timeoints.

(open square curves) after immunization and before challenge in vaccinated

unization and challenge (CH).

3168 L. Hermida et al. / Vaccine 24 (2006) 3165–3171

Table 1Neutralizing antibody titers in immunized monkeys

Monkeys Prechallenge neutralizinga titers on Day

94b (thirddosec)

109 210 (fourthdose)

226 254(challenge)

PD5 I <5 <5 <5 20 <5II <5 24 <5 80 16III <5 60 <5 320 22

PD3 I <5 <5 <5 10 <5II <5 200 10 600 40III <5 30 <5 60 22

P64k I NT NT NT <5 <5II NT NT NT <5 <5III NT NT NT <5 <5

PD5d <5 11.3 <5 80 7.1PD3 <5 18.2 <5 71.1 9.6P64k NT NT NT NT NT

NT: not tested.a Neutralizing Ab titers are the highest serum dilution which resulted in a

50% reduction in number of plaques produced by DEN2 (A15).b No neutralizing Abs were detected prior to this day.c Days in which animals were immunized.d Geometric mean titers of neutralizing Abs per group.

The majority of monkeys receiving PD3 or PD5 respondedwith DEN2-specific IgG Abs following the second dose andthis effect was boosted after the third dose. DEN2-specificIgG Abs peaked 15 days after each inoculation and declinedover time. In general, there was a trend toward an increase inthe Ab levels following the last dose.

3.2. Neutralizing antibodies after vaccination

The neutralizing antibodies generated after the last twodoses were determined by PRNT. The majority of animal serashowed neutralizing peaks 15 days after the third and fourthdoses (Table 1) coinciding with the reactivity peaks detectedby the ELISA assays. In contrast to DEN2-specific IgG Abs,neutralizing titers were notably increased after the last dose.Monkey PD3 I developed the lowest neutralizing Ab titer andonly after the fourth dose. Neutralizing Abs declined afterthe last dose but were detectable in four of the six dengueimmunized animals at the day of challenge. Monkeys PD5 IIIand PD3 II developed the highest DEN2-specific Ab titers asestimated by the different assays. No differences were foundin the Ab response induced by PD3 or PD5 (p > 0.05, byKruskal Wallis test).

3.3. Development of viremia after challenge

misap

Table 2Viremia qualitatively estimated by RT-PCR or viral isolation in Vero cellsafter challenge with 5 log10 PFU of DEN2

Groupsmonkey

Days showing positive RT-PCRa orpositive DEN2 virus amplificationb

from sera

0c 1 2 3 4 5 6 7 8 9 10

PD51d IIIIII

PD31de I +

II

III

P64k2e I + + +II + +

III + + +

Different numbers (1, 2) mean statistical differences (p < 0.05) in the num-ber of positive days determined by virus isolation (Kruskal Wallis tests).Different letters (d, e) mean statistical differences (p < 0.05) in the numberof positive days determined by RT-PCR (Kruskal Wallis tests).

a On gray, positive sera by RT-PCR.b (+) DEN2 isolation from serum.c Days upon challenge. Monkeys were challenged at Day 0.

the virus from their sera after challenge. Viremia could bedetected only 1 day after challenge for the PD3 I monkeywhile in the control group viremia lasted for a mean of 2.6days. There was no difference in terms of protection, as esti-mated by viral isolation, between PD5- and PD3-immunizedanimals. The immunized groups were protected from chal-lenge when compared to the control group. The onset ofviremia for the unprotected animals took place 3 days afterchallenge.

Sera from the three control monkeys were positive by RT-PCR following the viral challenge with a mean duration ofviremia of 5.6 days. Groups receiving PD5 or PD3 exhibiteda mean duration of viremia of 1 and 3 days, respectively,exhibiting a reduction of viremia duration with respect to thecontrol group. Monkeys PD3 II, PD5 I and PD5 III showed areduction in the number of days with positive RT-PCR afterchallenge, whereas PD5 II and PD3 III were fully protected.Interestingly, these fully protected animals at day 226 hadlower levels of neutralizing Abs than others that were onlypartially protected. The animal that responded the least (PD3I) was similarly positive if compared with the controls.

Results from viral isolation and RT-PCR analysis suggestthat the group receiving PD5 was more protected from DEN2virus challenge than the group immunized with PD3.

3c

asa

The development of viremia upon viral challenge wasonitored daily using qualitative RT-PCR and virus culture

n Vero cell monolayers (Table 2). The virus culture resultshowed that with the exception of monkey PD3 I, all thenimals that were immunized with the recombinant fusionroteins were protected, since it was not possible to recover

.4. Antibody response of the control group afterhallenge

Sera from control monkeys at Day 69 after challenge werenalyzed for anti-DEN2 antibodies by ELISA and PRNT. Ashown in Table 3, the titers reached were of the same orders those attained after immunization with the recombinant

L. Hermida et al. / Vaccine 24 (2006) 3165–3171 3169

Table 3IgG and neutralizing antibody titers in control monkeys after challenge

Monkey Day 0a Day 69

IgGb

titersNeutralizingc

titersIgG titers Neutralizingc

titers

P64k I <100 <5 25600 100II <100 <5 25600 50III <100 <5 12800 60

GMTd <100 <5 20318 67a Days after challenge.b Titers were determined by ELISA anti-DEN2.c Neutralizing Ab titers are the highest serum dilution which resulted in a

50% reduction in number of plaques produced by DEN2 (A15).d Geometric mean titers.

proteins (p > 0.05, by Kruskal Wallis test). The day analyzedcorresponded to the maximum antibody titer detected by bothtechniques (data not shown).

4. Discussion

The use of nonhuman primates for evaluating DEN vac-cine candidates is important because their Ab responses arequalitatively similar to those of human patients and theybecome viremic after subcutaneous inoculation with liveDEN virus, although in many instances the Ab titers and/orduration of viremia in patients are greater [20].

In our approach, M. fascicularis monkeys were subjectedto four spaced doses of PD3, PD5 and P64k, to follow thekinetics of the DEN2-specific antibody response and its func-tionality in terms of neutralization capacity. Freund’s adju-vant was used to induce the optimal immune response to therecombinant fusion proteins.

The analysis of the Ab response by different assaysshowed that the two variants of the DEN2 E fragment (aa286–426) fused to the P64k protein were similarly immuno-genic for monkeys, although animals from both groups exhib-ited a high variability in the quantity and quality of theanti-DEN2 Ab response. The PD5 and PD3 proteins wereawiaow

elsao1iow

DEN strain from serotype 2. Previous studies in mice [18]have shown that animals immunized with PD3 and PD5were partially protected against lethal encephalitis followingintracranial inoculation of DEN2 (strain A15). In the presentstudy, the data from viral isolation indicated that viremiawas reduced in monkeys immunized with PD5 or PD3 withrespect to control animals immunized with the P64k protein.The RT-PCR analysis suggested that monkeys immunizedwith PD5 showed, in addition to full protection in terms ofviral isolation, a remarkable reduction of days in which pos-itive RT-PCR reactions were found with respect to PD3 andcontrol groups. The incongruity of the results from RT-PCRand viral isolation could be attributed to the different sensitiv-ities reported for these techniques [25,41]. Anti-DEN2 Absinduced by PD3 and PD5 could additionally interfere within vitro viral isolation considering the demonstrated capacityof such Abs to neutralize the DEN2 infectivity. It has beenreported that high levels of Abs in DHF may inhibit virus iso-lation by the usual techniques and make the onset of viremiaundetectable [9,34]. RT-PCR could also be detecting plas-matic viral genomes from non-infective immune complexesor viral fragments. Vaccine candidates should provide forfull clearance of infective or “non-infective” plasmatic viralload upon challenge, since higher and prolonged “viremia”until the defervescence period associated to DHF/DSS humancases has been clearly shown using sensitive methods forv

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scf

ble to induce high levels of DEN2-specific IgG Abs, whichere additionally capable of neutralizing the virus. Neutral-

zing titers elicited by PD3 and PD5 were of the same orders those attained in control monkeys infected with 5 log PFUf DEN2 (challenge virus) as well as in monkeys immunizedith other vaccine candidates [3,31,37,51].Monkeys were challenged with the DEN2 strain A15 to

xplore how immunity conferred by PD3 and PD5 modu-ates infection with the homologous serotype of DEN. Theelection of the challenge virus corresponded to the fiveminoacid variation of the strain A15 with respect to thatf the E fragment (aa 286–426) from DEN2 strain Jamaica329TVP965 (M.G. Guzman, unpublished data) includedn the PD3 and PD5 constructs. Considering the small sizef the DEN polypeptide determinant in the fusion proteinse desired an immunogen capable of protecting against any

irus isolation [50] and by quantitative PCR [53].Historically, it has been assumed that protection against

EN infection is mainly conferred by neutralizing Abs.hile existing evidence strongly suggests that neutralizingbs alone can be protective [17,22], our studies in mice have

uggested that neutralizing Abs estimated by PRNT on BHK-1 cells fail to predict the outcome of DEN lethal infection19]. In the present approach, PD5 has conferred qualita-ively improved anti-DEN2 protective immunity with respecto that conferred by PD3, as deduced from protection assayesults. We did not find any difference in terms of Ab induc-ion between the recombinant fusion proteins. Interestingly,he two animals that mounted the highest Ab response (PD5II and PD3 II) were not precisely the fully protected onesn terms of viral RNA amplification after challenge. In manytudies performed in monkeys, the degree of neutralizing Abnduction has correlated with protection [3,12,13,28,31,37],hile in others the authors have not found such a relationship

5,6,14,15,36,42,51]. In a recent cohort study it has also beenemonstrated that PRNT estimated neutralizing Abs to DEN2id not prevent the outcome of DEN2 secondary infection26]. Similarly, Kliks et al. demonstrated that conventionalests lacked the ability to discriminate between low-level Abseing protective or enhancing [23]. They recommended thatssays for protective Abs might be more meaningful if neu-ralization was quantified in human monocyte culture.

Finally, our results constitute a proof-of-concept about theuitability of the fragment of the dengue envelope protein,ontaining the domain III and produced as a recombinantusion protein in E. coli, to induce a functional Abs and pro-

3170 L. Hermida et al. / Vaccine 24 (2006) 3165–3171

tective immune response in nonhuman primates. The resultsvalidate the potential value of PD5 and similar proteins asfunctional subunits. However, independently of the availabil-ity of GMP recombinant fusion proteins from the four DENserotypes some important questions remain to be answeredbefore human clinical trials can be initiated. At present ourwork is focused on determining the anamnestic response,the durability of the immunity and on defining adjuvants forhuman use.

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