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

Vaccine 24 (2006) 3165–3171

A recombinant fusion protein containing the domain III of the dengue-2 envelope protein is immunogenic and protective in nonhuman primates Lisset Hermida a,∗ , L´ıdice Bernardo b , Jorge Mart´ın a , Mayling Alvarez b , Irina Prado b , Carlos L´opez a , Beatriz de la C. Sierra b , Rafael Mart´ınez a , Rosmary Rodr´ıguez b , A´ıda Zulueta a , Ana B. P´erez b , Laura Lazo a , Delfina Rosario b , Gerardo Guill´en a , Mar´ıa G. Guzm´an b a

b

Center for Genetic Engineering and Biotechnology, Apdo 6162, Havana 10600, Cuba “Pedro Kour´ı” Tropical Medicine Institute, Autopista Novia del Mediod´ıa, km 6 P.O. Box Marianao 13, Havana, Cuba Received 28 December 2005; received in revised form 17 January 2006; accepted 18 January 2006 Available 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) of the dengue envelope protein and the P64k protein from Neisseria meningitidis. In this work we describe the immunization of Macaca fascicularis monkeys with two variants of these proteins [PD3 (insertion variant) and PD5 (fusion variant)] corresponding to serotype 2. Four doses of the proteins adjuvated in Freund’s adjuvant were administered and the kinetics of antibody induction was monitored by ELISA and neutralization tests. Monkeys receiving PD3 or PD5 developed functional antibodies (Abs) in a dose-dependent manner. Following challenge with 5 log PFU of wild type dengue-2 virus (DEN2), animals immunized with PD5 were protected from developing viremia. These results constitute a proof-of-concept demonstrating that a fragment of the dengue envelope protein, containing the domain III and produced as a recombinant fusion protein in Escherichia coli, induces functional and protective immunity in a nonhuman primate model. © 2006 Elsevier Ltd. All rights reserved. Keywords: Macaca fascicularis; Domain III; Protection

1. Introduction Dengue epidemics caused by the four dengue virus serotypes (DEN1–DEN4) continue to pose a major public health problem in most tropical and subtropical regions. According to estimates, as many as 100 million DEN infections occur every year worldwide [16,32,44]. The incidence of dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) is rising and there is currently no vaccine available to prevent the disease. The lack of an animal model for DF or DHF/DSS and the fact that simultaneous immunization is required to induce long-lasting protection ∗ Correspondence to: Divisi´ on de Vacunas, Centro de Ingenier´ıa Gen´etica y Biotecnolog´ıa, Apdo 6162, Habana 10600, Cuba. Tel.: +53 7 271 6022; fax: +53 7 271 4764. E-mail address: [email protected] (L. Hermida).

0264-410X/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2006.01.036

against all four DEN serotypes have been the principal barrier to the development of safe and effective DEN vaccines [10,21,23,32,38]. Live attenuated, live recombinant virus or infectious cDNA clones are being considered as vaccine candidates [2,24,12,13]. Such vaccines may elicit long-lasting protective immunity to DEN but interference among the serotypes, difficulties in replication and immunity induction, the potential reversion to virulence, and the elevated cost of production continue to hinder their widespread use [49]. Nonreplicating inactivated and recombinant subunit vaccines have had little success to date due to the limited immunogenicity, assurance of conformational integrity as well as the production cost [52]. However, manufactured recombinant subunit vaccines are still economically attractive. The use of Escherichia coli for expressing DEN structural genes has been limited since the majority of DEN proteins require complex co- and post-translational processing [7,29,30].

3166

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

Nevertheless, the expression of flavivirus structural subunits as recombinant fusion proteins using this expression system has yielded functional recombinant variants of DEN antigens. Some of these variants have proved to be immunogenic in mice, inducing neutralizing antibodies (Abs) and protecting them against experimental lethal DEN infection. Successful approaches have employed protein A from Staphylococcus aureus, glutatione-s transferase and the maltose binding protein as carriers [43,45,46,47,48]. Unfortunately, the safety of these carrier proteins has not yet been evaluated in humans. We have reported the expression of some variants of a DEN envelope (E) fragment (aa 286–426) fused to the meningococcal P64k protein [54]. The P64k protein, from Neisseria meningitidis, was cloned under the Trp promoter in E. coli and produced at high levels in economic culture media. Accordingly, a feasible scalable purification process was set up at developmental scale [11]. On the other hand, the analysis of immunodominant 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 demonstrated the carrier capacity of this bacterial protein after vaccination of terminal patients with lung cancer with human epidermal growth factor [8]. On the other hand, a phase I trial in human volunteers was conducted using the P64k alone, and its safety and immunogenicity were demonstrated [35]. Previously, we reported that the whole P64k protein is required to obtain better folding of the DEN2 envelope fragment and therefore a satisfactory immune response induction in mice [18]. We also explored the influence of two alternative fusion positions for the DEN1 envelope fragment within the P64k protein by inserting the DEN polypeptide, either following the first 45 aa of P64k (insertion variant) or at the C-terminus of P64k (fusion variant) [19]. As a result, the highest protection corresponded to the group immunized with the C-terminus variant. Having built this body of work using the mouse model, we planned to study the immunogenicity of two variants of the P64k fusion proteins (the insertion and fusion variants containing the envelope fragment from DEN2) for their protective capacity in a nonhuman primate model.

2. Materials and methods 2.1. Animals Healthy adult monkeys (Macaca fascicularis) were obtained from CENPALAB (Havana, Cuba). All animals were screened for previous exposure to dengue and P64k protein by detection of serum anti-DEN and anti-P64k Abs. Animals were considered naive with respect to both antigens when antigen specific Abs were undetectable. Monkeys were maintained in accordance with Cuban guidelines for the care and use of laboratory animals.

2.2. Recombinant proteins The design, cloning, expression and initial evaluation of the recombinant proteins involved in this study have been previously described [54]. Briefly, the DEN2 E gene fragment, from Jamaica strain, coding for aa 286–426 of the dengue E glycoprotein was cloned into a vector containing the sequence of P64k with a histidine tail. Two fusion protein variants were constructed. The first variant (PD3) resulted from the insertion of the fragment into the lipoil-binding domain of P64k [18]. To generate the second variant (PD5), the fragment was directly fused to the C-terminus of the same protein [54]. The P64k protein alone was used as a control. Ion metallic affinity chromatography (IMAC) was used to purify the proteins [27]. 2.3. Viruses The standard strain NGC of DEN2 was used as sucrose–acetone antigen [4] for immunoassay tests. The A15 strain of DEN2 (a Cuban isolate from the 1981 epidemic) has been passaged three times in Vero cells and was employed in the plaque reduction neutralization test (PRNT). A viral stock for use in challenge studies was prepared in Vero cells by removing the supplemented culture medium from infected cells (medium 199, 5% heat inactivated fetal calf serum, 2 mM l-glutamine and 100 U of streptomycin and neomycin) once the viral cytopathic effect was observed. Fresh medium without fetal calf serum was then added. This supernatant was harvested 48 h later, then aliquoted, stored at −70 ◦ C and titrated by plaque formation on BHK-21 cells. 2.4. Immunization Nine monkeys that passed the screening process were ranked by weight, age and sex and then randomly assigned to three groups with three animals each one. In order to handle the monkeys, they were anesthetized by intramuscular injection of ketamine hydrochloride. Animals were subcutaneously injected with 50 ␮g of the recombinant proteins PD3 (first group), PD5 (second group) and P64k (control group). Doses were administered to monkeys in each group at days: 0, 30, 94 and 210 using complete Freund adjuvant for the first dose and incomplete Freund adjuvant for all subsequent inoculations. The adjuvants were mixed 1:1 (v/v) with the antigens. Each immunization was given in a final volume of 0.5 ml, injected into four different sites on the back. Blood samples were collected at the time of, and 15 days after each inoculation. Serum from clotted blood was stored at −20 ◦ C. 2.5. Challenge and virus detection Forty-five days after receiving the last dose monkeys were subcutaneously inoculated in the upper arms with 5 log PFU of the DEN2 strain A15. Blood was collected daily for 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 and 60 post-challenge. Sera from clotted blood were stored at −70 ◦ C until viremia was analyzed. The presence of virus in the sera was determined by inoculating 100 ␮l of undiluted serum on to Vero cells grown in 24-well plates, shaking the plates for 1 h at 37 ◦ C, and then centrifuging the plates as previously described [39]. Fresh supplemented medium was added to the wells and after 7 days of incubation, cells were tested by indirect immunofluorescence using anti-DEN2 hyperimmune mouse ascitic fluid. Samples were subjected to three blind passages in the same cell cultures before being considered as negatives. In addition, the presence of viral RNA was assessed by qualitative RNA reverse transcriptase followed by polymerase chain reaction (RT-PCR). RNA was extracted from 250 ␮l of serum samples using the Trizol procedure according to the manufacturer’s instructions (Gibco-BRL, USA). RT and cDNA amplification was performed as previously described [32,53].

rating concentration of DEN2 antigen and the mock antigen in separate wells. Serially diluted samples from sera were incubated 1 h at 37 ◦ C with either the DEN2 or the mock antigens. Anti-monkey IgG-peroxidase conjugate (Sigma, USA) diluted 1/10000 was incubated for 1 h at 37 ◦ C. H2 O2 /OPD was then added as substrate solution for 30 min. Optical densities (OD) were measured at 492 nm. A dilution of serum was considered positive when the ratio [OD (DEN antigen)]/[OD (mock)] was two or higher. The functionality of the antibodies was measured by neutralization of DEN2 infectivity by PRNT on BHK-21 cells culture as described [33]. The Ab response to the carrier protein (P64k) was measured by an indirect ELISA as previously described [35].

2.6. Analysis of the antibody response

Sera from immunized and control monkeys were analyzed for anti-P64k and anti-DEN2 antibodies by ELISA. Antibodies directed to the carrier P64k protein were estimated by end point dilution titration on plates coated with P64k (Fig. 1). All the animals elicited antibodies after immunization with the recombinant proteins. High titers of anti-P64k Abs were induced as early as 2 weeks after priming with PD3, PD5 or P64k. A booster effect was detected following a second dose, reaching plateau levels at late time points.

The anti-DEN2 IgG antibodies stimulated by immunization were monitored by enzyme-linked immunosorbent assay (ELISA). Briefly MAXISORP 96-well plates were coated with the monoclonal Ab 4G2 which recognizes the flavivirus E protein [1,40]. Three washes with PBS containing 0.05% Tween 20 (Merck, Germany) were completed after each step of the ELISA. Plates were blocked with 2% bovine serum albumin, and then incubated overnight at 4 ◦ C with a satu-

3. Results 3.1. Antibody response after immunization

Fig. 1. Kinetics of IgG antibodies against P64k (close circle curves) and DEN2 virus (open square curves) after immunization and before challenge in vaccinated monkeys with PD3 (A), PD5 (B) and P64k (C). Block arrows indicate days of immunization and challenge (CH).

3168

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

Table 1 Neutralizing antibody titers in immunized monkeys Monkeys

PD5

PD3

P64k

PD5d PD3 P64k

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

Prechallenge neutralizinga titers on Day

Days showing positive RT-PCRa or positive DEN2 virus amplificationb from sera

Groups monkey

94b (third dosec )

109

210 (fourth dose)

226

254 (challenge)

I II III

<5 <5 <5

<5 24 60

<5 <5 <5

20 80 320

<5 16 22

PD51d

I II III

<5 <5 <5

<5 200 30

<5 10 <5

10 600 60

<5 40 22

I II III

PD31de

I

I II III

NT NT NT

NT NT NT

NT NT NT

<5 <5 <5

<5 <5 <5

<5 <5 NT

11.3 18.2 NT

<5 <5 NT

80 71.1 NT

7.1 9.6 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 responded with DEN2-specific IgG Abs following the second dose and this effect was boosted after the third dose. DEN2-specific IgG Abs peaked 15 days after each inoculation and declined over time. In general, there was a trend toward an increase in the Ab levels following the last dose. 3.2. Neutralizing antibodies after vaccination The neutralizing antibodies generated after the last two doses were determined by PRNT. The majority of animal sera showed neutralizing peaks 15 days after the third and fourth doses (Table 1) coinciding with the reactivity peaks detected by 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 and only after the fourth dose. Neutralizing Abs declined after the last dose but were detectable in four of the six dengue immunized animals at the day of challenge. Monkeys PD5 III and PD3 II developed the highest DEN2-specific Ab titers as estimated by the different assays. No differences were found in the Ab response induced by PD3 or PD5 (p > 0.05, by Kruskal Wallis test).

0c

1

2

3

4

5

+ + +

+ + +

6

7

8

9

10

+

II III P64k2e

I II

+

III

+

Different numbers (1, 2) mean statistical differences (p < 0.05) in the number of positive days determined by virus isolation (Kruskal Wallis tests). Different letters (d, e) mean statistical differences (p < 0.05) in the number of 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 be detected only 1 day after challenge for the PD3 I monkey while in the control group viremia lasted for a mean of 2.6 days. There was no difference in terms of protection, as estimated by viral isolation, between PD5- and PD3-immunized animals. The immunized groups were protected from challenge when compared to the control group. The onset of viremia for the unprotected animals took place 3 days after challenge. Sera from the three control monkeys were positive by RTPCR following the viral challenge with a mean duration of viremia of 5.6 days. Groups receiving PD5 or PD3 exhibited a mean duration of viremia of 1 and 3 days, respectively, exhibiting a reduction of viremia duration with respect to the control group. Monkeys PD3 II, PD5 I and PD5 III showed a reduction in the number of days with positive RT-PCR after challenge, whereas PD5 II and PD3 III were fully protected. Interestingly, these fully protected animals at day 226 had lower levels of neutralizing Abs than others that were only partially protected. The animal that responded the least (PD3 I) was similarly positive if compared with the controls. Results from viral isolation and RT-PCR analysis suggest that the group receiving PD5 was more protected from DEN2 virus challenge than the group immunized with PD3.

3.3. Development of viremia after challenge The development of viremia upon viral challenge was monitored daily using qualitative RT-PCR and virus culture in Vero cell monolayers (Table 2). The virus culture results showed that with the exception of monkey PD3 I, all the animals that were immunized with the recombinant fusion proteins were protected, since it was not possible to recover

3.4. Antibody response of the control group after challenge Sera from control monkeys at Day 69 after challenge were analyzed for anti-DEN2 antibodies by ELISA and PRNT. As shown in Table 3, the titers reached were of the same order as those attained after immunization with the recombinant

L. Hermida et al. / Vaccine 24 (2006) 3165–3171 Table 3 IgG and neutralizing antibody titers in control monkeys after challenge Monkey

Day 0a IgGb

P64k

Day 69 Neutralizingc

IgG titers

Neutralizingc titers

titers

titers

I II III

<100 <100 <100

<5 <5 <5

25600 25600 12800

100 50 60

GMTd

<100

<5

20318

67

a

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 analyzed corresponded to the maximum antibody titer detected by both techniques (data not shown).

4. Discussion The use of nonhuman primates for evaluating DEN vaccine candidates is important because their Ab responses are qualitatively similar to those of human patients and they become viremic after subcutaneous inoculation with live DEN virus, although in many instances the Ab titers and/or duration of viremia in patients are greater [20]. In our approach, M. fascicularis monkeys were subjected to four spaced doses of PD3, PD5 and P64k, to follow the kinetics of the DEN2-specific antibody response and its functionality in terms of neutralization capacity. Freund’s adjuvant was used to induce the optimal immune response to the recombinant fusion proteins. The analysis of the Ab response by different assays showed that the two variants of the DEN2 E fragment (aa 286–426) fused to the P64k protein were similarly immunogenic for monkeys, although animals from both groups exhibited a high variability in the quantity and quality of the anti-DEN2 Ab response. The PD5 and PD3 proteins were able to induce high levels of DEN2-specific IgG Abs, which were additionally capable of neutralizing the virus. Neutralizing titers elicited by PD3 and PD5 were of the same order as those attained in control monkeys infected with 5 log PFU of DEN2 (challenge virus) as well as in monkeys immunized with other vaccine candidates [3,31,37,51]. Monkeys were challenged with the DEN2 strain A15 to explore how immunity conferred by PD3 and PD5 modulates infection with the homologous serotype of DEN. The selection of the challenge virus corresponded to the five aminoacid variation of the strain A15 with respect to that of the E fragment (aa 286–426) from DEN2 strain Jamaica 1329TVP965 (M.G. Guzman, unpublished data) included in the PD3 and PD5 constructs. Considering the small size of the DEN polypeptide determinant in the fusion proteins we desired an immunogen capable of protecting against any

3169

DEN strain from serotype 2. Previous studies in mice [18] have shown that animals immunized with PD3 and PD5 were partially protected against lethal encephalitis following intracranial inoculation of DEN2 (strain A15). In the present study, the data from viral isolation indicated that viremia was reduced in monkeys immunized with PD5 or PD3 with respect to control animals immunized with the P64k protein. The RT-PCR analysis suggested that monkeys immunized with PD5 showed, in addition to full protection in terms of viral isolation, a remarkable reduction of days in which positive RT-PCR reactions were found with respect to PD3 and control groups. The incongruity of the results from RT-PCR and viral isolation could be attributed to the different sensitivities reported for these techniques [25,41]. Anti-DEN2 Abs induced by PD3 and PD5 could additionally interfere with in vitro viral isolation considering the demonstrated capacity of such Abs to neutralize the DEN2 infectivity. It has been reported that high levels of Abs in DHF may inhibit virus isolation by the usual techniques and make the onset of viremia undetectable [9,34]. RT-PCR could also be detecting plasmatic viral genomes from non-infective immune complexes or viral fragments. Vaccine candidates should provide for full clearance of infective or “non-infective” plasmatic viral load upon challenge, since higher and prolonged “viremia” until the defervescence period associated to DHF/DSS human cases has been clearly shown using sensitive methods for virus isolation [50] and by quantitative PCR [53]. Historically, it has been assumed that protection against DEN infection is mainly conferred by neutralizing Abs. While existing evidence strongly suggests that neutralizing Abs alone can be protective [17,22], our studies in mice have suggested that neutralizing Abs estimated by PRNT on BHK21 cells fail to predict the outcome of DEN lethal infection [19]. In the present approach, PD5 has conferred qualitatively improved anti-DEN2 protective immunity with respect to that conferred by PD3, as deduced from protection assay results. We did not find any difference in terms of Ab induction between the recombinant fusion proteins. Interestingly, the two animals that mounted the highest Ab response (PD5 III and PD3 II) were not precisely the fully protected ones in terms of viral RNA amplification after challenge. In many studies performed in monkeys, the degree of neutralizing Ab induction has correlated with protection [3,12,13,28,31,37], while 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 been demonstrated that PRNT estimated neutralizing Abs to DEN2 did not prevent the outcome of DEN2 secondary infection [26]. Similarly, Kliks et al. demonstrated that conventional tests lacked the ability to discriminate between low-level Abs being protective or enhancing [23]. They recommended that assays for protective Abs might be more meaningful if neutralization was quantified in human monocyte culture. Finally, our results constitute a proof-of-concept about the suitability of the fragment of the dengue envelope protein, containing the domain III and produced as a recombinant fusion 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 results validate the potential value of PD5 and similar proteins as functional subunits. However, independently of the availability of GMP recombinant fusion proteins from the four DEN serotypes some important questions remain to be answered before human clinical trials can be initiated. At present our work is focused on determining the anamnestic response, the durability of the immunity and on defining adjuvants for human use.

[15]

[16] [17]

[18]

References [1] Alison JJ, Martin DA, Karabatsos N, Roehrig JT. Detection of anti-arboviral immunoglobulin G by using a monoclonal antibodybased capture enzyme-linked immunosorbent assay. J Clin Microbiol 2000;38:1827–31. [2] Bray M, Falgout B, Zhao Lai CJ. Modern approaches to new vaccines including prevention of AIDS. In: Brown F, Chanock RM, Ginsberg H, Terner R, editors. Vaccines 89. New York: Cold Spring Harbor; 1989. p. 357–62. [3] Bray M, Men R, Lai CJ. Monkeys Immunized with intertypic chimeric dengue viruses are protected against wild-type virus challenge. J Virol 1996;70:4162–6. [4] Churdboonchart V, Bhamarapravati N, Peampramprecha S. Antibodies against dengue viral proteins in primary and secondary dengue hemorrhagic fever. Am J Trop Med Hyg 1991;44:481–93. [5] Deubel V, Kinney RM, Exp´osito JJ, Cropp CB, Vorndam AV, Monath TP, et al. Dengue 2 virus envelope protein expressed by a recombinant vaccinia virus failed to protect monkeys against dengue. J Gen Virol 1988;69:1921–9. [6] Eckels KH, Dubois DR, Summers PL, Schlesinger JJ, Shelly M, Cohen S, et al. Immunization of monkeys with baculovirus-dengue type-4 recombinants containing envelope and nonstructural proteins: evidence of priming and partial protection. Am J Trop Med Hyg 1994;50:472–8. [7] Fonseca BAL, Khoshnood K, Shope RE. Flavivirus type-specific antigens produced from fusions of a portion of the E protein gene with the E. coli trp-e gene. Am J Trop Med Hyg 1991;44:500–8. [8] Gonz´alez G, Crombet T, Catal´a M, Mirabal V, Hern´andez JC, Gonz´alez Y, et al. A novel cancer vaccine composed of humanrecombinant epidermal growth factor linked to a carrier protein: report of a pilot clinical trial. Annu Oncol 1998;9:431–5. [9] Gubler DJ, Suharyono W, Tan R, Abidin M, Sie A. Viraemia in patients with naturally acquired dengue infections. Bull World Health Organ 1981;59:623–30. [10] Gubler DJ, Meltzer M. Impact of dengue/dengue-hemorrhagic fever on the developing world. Adv Virus Res 1999;53:35–70. ´ [11] Guill´en G, Alvarez A, Silva R, Morera V, Gonz´alez S, Musacchio A, et al. Expression in Escherichia coli of the lpdA gene, protein sequence analysis and immunological characterization of the P64k protein from Neisseria meningitidis. Biotechnol Appl Biochem 1998;27:189–96. [12] Guirakhoo F, Arroyo J, Pugachev KV, Miller C, Zhang ZX, Weltzin R, et al. Construction, safety, and immunogenicity in nonhuman primates of a chimeric yellow fever-dengue virus tetravalent vaccine. J Virol 2001;75:7290–304. [13] Guirakhoo FR, Weltzin TJ, Chambers ZX, Zhang K, Soike M, Ratterree J, et al. Recombinant chimeric yellow fever-dengue type 2 virus is immunogenic and protective in nonhuman primates. J Virol 2000;74(5477–5485):16. [14] Guzm´an MG, Rodr´ıguez R, Rodr´ıguez R, Hermida L, Alvarez M, Lazo L, et al. Dengue 4 virus envelope glycoprotein expressed in Pichia pastoris induces neutralizing antibodies and partial protection

[19]

[20]

[21] [22]

[23]

[24]

[25]

[26]

[27]

[28]

[29] [30]

[31]

[32] [33]

from viral challenge in Macaca fascicularis. Am J Trop Med Hyg 2003;69(2):129–34. Halstead SB, Eckels KH, Putvatana R, Larsen LK, Marchette NJ. Selection of attenuated dengue 4 viruses by serial passage in primary kidney cells. IV. Characterization of a vaccine candidate in fetal rhesus lung cells. Am J Trop Med Hyg 1984;33:679–83. Halstead SB. Pathogenesis of dengue: challenge to molecular biology. Science 1988;239:476–81. Heinz FX, Roehrig JT. Flavivirus. In: Van Regenmortel MHV, Neurath AR, editors. Immunochemistry of viruses II. The basis for serodiagnosis and vaccines. Amsterdam: Elsevier; 1990. p. 289–305. Hermida L, Rodr´ıguez R, Lazo L, Silva R, Zulueta A, Chinea G, et al. A dengue-2 envelope fragment inserted within the structure of the P64k meningococcal protein carrier, enables a functional immune response against the virus in mice. J Virol Methods 2004;115: 41–9. Hermida L, Rodr´ıguez R, Lazo L, Bernardo L, Silva R, Zulueta A, et al. A fragment of the envelope protein from dengue-1 virus, fused in two different sites of the meningococcal P64k protein carrier, induces a functional immune response in mice. Biotechnol Appl Biochem 2003;38:1–8. Innis BL. Antibody responses to dengue virus infection. In: Gubler DJ, Kuno G, editors. Dengue and dengue hemorrhagic fever. 1st ed. New York: CAB international press; 1997. p. 221–43. Jacobs M. Dengue: emergence as a global public health problem and prospect for control. Trans R Soc Trop Med Hyg 2000;94:7–8. Kliks SC, Nimmanitya S, Nisalak A, Burke DS. Evidence that maternal dengue antibodies are important in the development of dengue hemorrhagic fever in infants. Am J Trop Med Hyg 1988;38:411–9. Kliks SC, Nisalak A, Brandt WE, Wahi L, Burke DS. Antibodydependent enhancement of dengue virus growth in human monocytes as a risk factor for dengue hemorrhagic fever. Am J Trop Med Hyg 1989;40:444–51. Lai CJ, Zhang YM, Falgout B, Bray M, Chanock R, Eckels KH. Modern approaches to new vaccines including prevention of AIDS. In: Brown F, Chanock RM, Ginsberg H, Terner R, editors. Vaccines 89. New York: Cold Spring Harbor; 1989. p. 351–6. Lanciotti RS, Calisher CH, Gubler DJ, Chang GJ, Vorndam AV. Rapid detection and typing of dengue viruses from clinical samples using reverse transcriptase-polymerase chain reaction. J Clin Microbiol 1992;30:545–51. Laoprasopwattana K, Libraty DH, Endy TP, Nisalak A, Chunsuttiwat S, Vaughn DW, et al. Dengue virus (DV) enhancing antibody activity in preillness plasma does not predict subsequent disease severity or viremia in secondary DV infection. J Infect Dis 2005;192(3):510–9. L´opez C, S´anchez J, Hermida L, Zulueta A, M´arquez G. Cysteine mediated multimerization of a recombinant dengue E fragment fused to the P64k protein by Immobilized metal ion affinity chromatography. Protein Expr Purif 2004;34:176–82. Markoff L, Pang X, Houng H, Falgout B, Olsen R, Jones E, et al. Derivation and characterization of a dengue type 1 host rangerestricted mutant that is attenuated and highly immunogenic in monkeys. J Virol 2002;76:3318–28. Marston FAO. The purification of eucaryotic polypeptides expressed in E. coli. Biochem J 1986;240:1–12. Mason PW, Zogel MV, Semproni AR. The antigenic structure of dengue type 1 virus envelope and NS1 protein expressed in E. coli. J Gen Virol 1990;71:2107–14. Men R, Wyatt L, Tokimatsu I, Arakaki S, Shameen G, Elkins R, et al. Immunization of rhesus monkeys with a recombinant of modified vaccinia virus Ankara expressing a truncated envelope glycoprotein of dengue type 2 virus induced resistance to dengue type 2 virus challenge. Vaccine 2000;18:3113–22. Monath T. Dengue: the risk to developed and developing countries. Proc Natl Acad Sci USA 1994;91:2395–400. Morens DM, Halstead SB, Repik PM. Simplified plaque reduction assay for dengue viruses by semimicro methods in BHK 21 cells:

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

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42]

[43]

comparison of the BHK suspension test with standard plaque reduction neutralization. J Clin Microbiol 1985;22:250–4. Nisalak A, Halstead SB, Singharaj P, Udomsakdi S, Nye SW, Vinijchaikul K. Observation related to pathogenesis of dengue hemorrhagic fever. III. Virologic studies of fatal disease. Yale J Biol Med 1970;42:293–310. P´erez A, Dickinson F, Cinza Z, Ruiz A, Serrano T, Sosa J, et al. Safety and preliminary immunogenicity of the recombinant outer membrane protein P64k of Neisseria meningitidis in human volunteers. Biotechnol Appl Biochem 2001;34:121–5. Putnak R, Barvir DA, Burrous JM, Dubois DR, D’Andrea VM, Hoke CH, et al. Development of a purified, inactivated, dengue-2 virus vaccine prototype in Vero cell: immunogenicity and protection in mice and rhesus monkeys. J Infect Dis 1996;174:1176–84. Raviprakash K, Porter KR, Kochel TJ, Ewing D, Simons M, Philllis I, et al. Dengue virus type 1 DNA vaccine induces protective immune responses in rhesus macaques. J Gen Virol 2000;81:1659–67. Rigau-Perez JG, Clark GG, Gubler DJ, Reiter P, Sanders EJ, Vorndam AV. Dengue and dengue haemorrhagic fever. Lancet 1998;352:971–7. Rodriguez R, Alvarez M, Guzman MG, Morier L, Kouri G. Comparison of rapid centrifugation assay with conventional tissue culture method for isolation of dengue 2 virus in C6/36-HT cells. J Clin Microbiol 2000;38:3508–10. Roehrig JT. Immunochemistry of dengue virus. In: Gubler DJ, Kuno G, editors. Dengue and dengue hemorrhagic fever. 1st ed. New York: CAB international press; 1997. p. 199–219. Rosario D, Alvarez M, D´ıaz J, Contreras R, V´azquez S, Rodr´ıguez R, et al. Rapid detection and typing of dengue viruses from clinical samples using reverse transcriptase-polymerase chain reaction. Pan Am J Public Health 1998;4:1–5. Scherer WF, Russell PK, Rosen L, Casals J, Dickerman RW. Experimental infection of chimpanzees with dengue virus. Am J Trop Med Hyg 1978;27:590–9. Seif SA, Morita K, Igarashi A. A 27 amino acid coding region of LE virus E protein in E. coli as fusion protein with glutathiones transferase elicit neutralizing antibody in mice. Virus Res 1997;43:91–106.

3171

[44] Sierra B, Garc´ıa G, P´erez AB, Morier L, Rodr´ıguez R, Alvarez M, et al. Long-term memory cellular immune response to dengue virus after a natural primary infection. Int J Infect Dis 2002;6:125–8. [45] Simmons M, Murphy GS, Hayes CG. Antibody responses of mice immunized with a tetravalent dengue recombinant protein subunit vaccine. Am J Trop Med Hyg 2001;65:159–61. [46] Simmons M, Nelson WM, Wu SJ, Hayes CG. Evaluation of the protective efficacy of a recombinant dengue envelope B domain fusion protein against dengue 2 virus infection in mice. Am J Trop Med Hyg 1998;58:655–62. [47] Srivastava AK, Morita K, Matsuo S. Japanese encephalitis virus fusion protein with protein A expressed in E. coli confers protection in mice. Microbiol Immunol 1991;35:863–70. [48] Srivastava A, Putnak JR, Warren RL, Hoke Jr CH. Mice immunized with a dengue type 2 virus E and NS1 fusion protein made in Escherichia coli are protected against lethal dengue virus infection. Vaccine 1995;13:1251–8. [49] Tolou H, Pisano MR, Deubel V. Probl`emes et perspectives en mati`ere de vaccination contre les flavivirus. Bull Inst Pasteur 1992;90:11–29. [50] Vaughn DW, Green S, Kalayanarooj S, Innis BL, Nimmannitya S, Suntayakorn S, et al. Dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity. J Infect Dis 2000;181:2–9. [51] Velzing J, Groen J, Drouet MT, Van Amerongen G, Copra C, Osterhaus ADME, et al. Induction of protective immunity against dengue virus type 2: comparison of candidate live attenuated and recombinant vaccines. Vaccine 1999;17:1312–20. [52] Venugopal K, Gould EA. Towards a new generation of flavivirus vaccines. Vaccine 1994;12:966–75. [53] Wang WK, Chao DY, Kao CL, Wu HC, Liu VC, Li CM, et al. High levels of plasma dengue viral load during defervescence in patients with dengue hemorrhagic fever: implications for pathogenesis. Virology 2003;305:330–8. [54] Zulueta A, Hermida L, Lazo L, Vald´es I, Rodr´ıguez R, L´opez C, et al. The fusion site of envelope fragments from each serotype of dengue virus in the P64k protein, influence some parameters of the resulting chimeric construct. Biochem Biophys Res Commun 2003;308:619–26.