key: cord-0010214-asjxwlrn
authors: nan
title: Retracted: Immunological properties reveal the monovalent and bivalent recombinant dengue virus‐like particles as candidate vaccine for dengue
date: 2015-03-03
journal: Microbiol Immunol
DOI: 10.1111/1348-0421.12179
sha: f0a605eab92d373080b2bb921ef6d36a25ca9501
doc_id: 10214
cord_uid: asjxwlrn

Retraction statement: ‘Immunological properties reveal the monovalent and bivalent recombinant dengue virus‐like particles as candidate vaccine for dengue’ by Yan Liu, Junmei Zhou, Zhizhun Yu, Danyun Fang, Chunyun Fu, Xun Zhu, Zhenjian He, Huijun Yan, Wenquan Liu, Yunxia Tang, Mengfeng Li and Lifang Jiang. The above article in Microbiology and Immunology (doi: 10.1111/1348‐0421.12179), published online on 15 July 2014 in Wiley Online Library (http://onlinelibrary.wiley.com/), has been retracted by agreement between the authors, the journal Editors‐in‐Chief, Akio Nakane, Takaji Wakita, Yasunobu Yoshikai, and Wiley Publishing Asia Pty Ltd. The retraction has been agreed as the article was submitted by Dr. Liu Y. without agreement of all co‐authors, including two listed corresponding authors.

Dengue virus (DENV) is characterized as the small, enveloped positive-stranded RNA viruses belonging to the genus Flavivirus of the Flaviviridae family (1). Based on the envelope protein, DENV can be classified into 4 known serotypes: DENV1, 2, 3 and 4. All types of DENV can cause the full spectrum of disease (2). It is estimated that around 390 million people are at risk of suffering dengue globally and 25,000 cases of 50-100 million DENV infections are dead annually. Additionally, there are about 50 to 100 million new cases of dengue infection all over the world (3). Dengue infection has been a potential threat to global public health.

Many contributions have been made to prevent and control the morbidity and mortality caused by DENV. Routine administration of vaccination is a proven method for controlling the DENV infection (4). Currently, numerous vaccines for DENV infection are in development and clinical evaluation, such as inactivated whole virus, live attenuated DENV, recombinant subunit protein, DNA and virus-vectored vaccine (5-7). However, a successful DENV vaccine that can protect against all the DENV types is not available by now. Therefore, the safe and effective DENV vaccine is in urgent need. In recent years, noninfectious virus-like particles (VLPs) formed in flavivirus replication have attracted many attentions. Many studies devoted to the rapid and effective construction of recombinant VLPs which possessed similar properties of native virions and lacked the viral genome (8, 9) . But the evidence concerning the DENV VLPs as vaccine candidate for dengue is insufficient.

Currently, the DENV type 2 and type 1 are the most popular stains in Guangdong province, China (10, 11). In the present study, we systematically evaluated the immunological properties and protection ability of monovalent DENV VLPs and bivalent DENV VLPs against DENV-1/2 in vitro and in vivo. The purpose of our study was to provide the basis for the application of the DENV VLPs as attractive candidate vaccine in preventing the popular DENV infection in China.

The C6/36 cell line derived from Aedes aegypti was used for virus propagation and the C6/36 cells were cultured as previously described (12). The DENV-1 GZ01/95 strain (GenBank accession No. EF032590) and DENV-2 ZS01/01 strain (GenBank accession no. EF051521) were propagated in C6/36 cells. Virus was purified by sucrose density gradient centrifugation (13) and the inactivation of virions were performed with 1:2000 β-propionolactone. Virions concentrations were detected by bicinchoninic acid (BCA) method (Biocolor, Shanghai, China) and stored at -80 °C for further studies.

The Mesocricetus auratus BHK-21 cells were cultured in DMEM (Gibico, Guangzhou, China) supplemented with The wild-type P. pastoris strain X33 (Invitrogen, San Diego, CA) were served as the host strain. The expression vector was constructed with vector pGAPZaA (Invitrogen) according to a previous procedure with a slight modification (12). Briefly, the cDNAs coding prM/E from DENV-1 GZ 01/95 strain and DENV-2 ZS01/01 strain were synthesized by reverse transcription-polymerase chain reaction (RT-PCR) under optimized reaction conditions. The full-length prM/E gene was inserted into the downstream of GAP promoter with the α-factor secretion signal (Fig. 1 a) . The recombinant plasmids of pGAPZα-PrME-D1 and pGAPZα-PrME-D2 were electroporated into Pichia pastoris to express recombinant DENV-1 VLPs and DENV-2 VLPs, respectively.

After the verified transformants were cultured, yeast cells were collected by centrifugation (10,000×g, 10 min, 4 °C) and disrupted with glass beads in breaking buffer (50 mmol/L sodium phosphate, pH 7.4, 1mmol/L ethylene diamine tetraacetic acid (EDTA), 1 mmol/L phenylmethyl sulfonylfluoride (PMSF), and 5% glycerol). The yeast lysates were subjected to ultracetrifugation at 153 000 ×g for 6 hours at 4ºC (HATICHI, P80AT rotor, Japan) using 5-50% sucrose density gradient. Then, the western blot analysis was performed to test the expression of recombinant VLPs. The expression of DENV-1 E proteins and DENV-2 E proteins were detected using the mAb D2-1F1-3 (specific mouse monoclonal antibody, the Center for Disease Control, USA) (15) and mAb 3H5 (specific mouse monoclonal antibody, American Type Culture Collection, USA) as the primary antibody, respectively and goat anti-mouse immunoglobulin G (IgG)-houseradish peroxidase (HRP) conjugate as the secondary antibody (Santa Cruz, USA). Finally, protein concentrations were assessed using BCA method (Biocolor, Shanghai, China).

All the animal studies were approved by Ethics Committee of Sun Yat-sen University and performed in accordance with the ethical standards. Specific pathogen-free female BALB/c mice (aging from 3 to 4 weeks old) were supplied by the Experimental Animal Center of Sun Yat-sen University (Guangzhou, China) and were randomly divided into 6 groups: monovalent DENV-1 VLPs group (n=15; treated with DENV-1 VLPs followed by DENV-1 challenge), monovalent DENV-2 VLPs group (n=15; treatment with DENV-2 VLPs, followed by DENV-2 challenge), bivalent VLPs group (n=30; treatment with DENV-1/2 VLPs combination, followed by DENV-1 and DENV-2 challenge, separately), Inactivated DENV-1 group (n=15; treatment with inactivated DENV-1, followed by DENV-1 challenge), Inactivated DENV-2 group (n=15; treated with inactivated DENV-2 followed by DENV-2 challenge) and PBS group (n=30; treated by PBS solution, followed by DENV-1 and DENV-2 challenge, separately).

The mice in DENV-1 VLPs, DENV-2 VLPs and VLP combination groups were intraperitoneally injected respectively with monovalent DENV-1 VLPs (50 μg/dose), monovalent DENV-2 VLPs (50 μg/dose) and the bivalent DENV-VLPs (50 μg DENV-1 VLPs and 50 μg DENV-2 VLPs per dose) absorbed with Freund adjuvant (Sigma) at days 0, 14 and 28 once a day for immunization. The DENV-1/2 virions (50μg/ dose) and PBS (with the same dose) were administered for mice as controls with the same procedure. Blood samples were collected on days 0, 13 and 27 before immunization through tail vein for serum IgG measurement. One week after the last immunization (at days 34), 1/3 mice (n=5) in each group were sacrificed to collect blood and spleens for further analysis. Additionally, 2/3 mice (n=10) in each group were intraperitoneally challenged with DENV-1 or DENV-2. On days 1, 2, 3, 4 post challenge, blood samples were collected through tail vein for serum cytokines measurement.

The DENV VLPs and DENV specific antibodies were tested by enzyme-linked immunosorbent assay (ELISA) with the serum samples collected at days 0, 13, 27 and 34. Briefly, polystyrene plates with 96 wells (Costar, Cambridge, MA)

were coated with inactivated DENV antigen or DENV VLPs antigen over night at 4°C and blocked in coating buffer containing 5% fat-free milk powder for 1 h at 37°C. After washing with PBS-T (PBS containing 0.05% Tween 20), serum samples (dilutions at 1:100; 100μl per well) were incubated for 1 h at 37 °C. Subsequently, 100μl of 1:5000 diluted goat anti-mouse IgG-peroxidase conjugate (Santa Cruz, USA) was added per well and incubated for 1 h at 37 °C, followed by addition of 100μl/well 3, 3, 5, 5'-tetramethly benzidine substrate at 37 °C for 15 min. After the reaction was stopped with 2M H 2 SO 4 , absorbance was measured at 450 nm using an automated ELISA reader (ELx800 BioTek). The results were considered to be positive if the absorbance exceeded 2 times the mean absorbance of serum pre-immunization.

Anti-monovalent VLPs antibody, anti-bivalent VLPs antibody, anti-inactivated DENV antibody raised in sera were tested by immunofluorescence assay with PBS treatment sera as control. The slides with C6/36 cells monolayer infected with DENV-1 strain GZ01/95 and DENV-2 strain ZS01/01 were fixed in acetone for 15min at -20°C. Then, the 1:80 diluted antisera were added onto the slides and incubated for 1 h at 37 °C. After the slides were washed 3 times with PBS, Alexa-Fluor-488-conjugated goat anti-mouse IgG (1:200; Invitrogen, Carlsbad, CA) was added and incubated for 45 min at 37 °C. Cells were stained with 4',6-diamidino-2-phenylindole (DAPI). The staining images were photographed and observed under the fluorescent microscope.

To determine the levels of IFN-γ, TNF-α and IL-10 in sera after virus challenge, commercial ELISA kits (R&D, USA) were used according to the manufacturer introduction. Blood samples collected on days 1, 2, 3, 4 post challenge and days 34 post immune were analyzed. The limits of detection for these cytokines were 2 pg/ml (IFN-γ), 2 pg/ml (TNF-α) and 2 pg/ml (IL-10).

The enzyme linked immunospot assay (ELISPOT) were performed used ELISPOT kits (Ucytech, NLD), following the manufacturer's instructions. Briefly, the ELISPOT 96-well plates (Millipore, USA) were coated overnight at 4 °C with 100μl of anti-mouse IFN- or with anti-mouse IL-10 or TNF- (5μg/ml). Plates were washed twice and blocked with blocking solution for 2 h. Then, 100 μl freshly isolated splenocytes (2×10 5 cells) from the immunized mice were transferred to each well and stimulated with inactivated dengue virus at 37 °C for 24 h. Cells were washed away and the secondary biotinylated anti-cytokine mAb was added to each well, followed by streptavidin-HRP and AEC substrate solution. The spots were counted by ImmunoSpot® Analyzer (Cellular Technology Ltd.).

Sera from immunized mice at days 34 were used for Plaque Reduction Neutralization Test (PRNT) as previous description (16) . Briefly, the Mesocricetus auratus BHK-21 cells were grown to 80% confluence in 24-well plates. The immunized mice serum samples with twofold serial dilutions of the serum (1:4-1:64) were mixed with equal volume of DENV-1 or DENV-2 (150-200 PFU per milliliter) at 37 °C for 1 h to neutralize the infectious virus. The virus/serum mixture was aspirated and added with 0.8mL medium to infect the BHK-21 cells at 37 °C with 5% CO 2 for 7 days. Finally, the dengue virus plaques were counted with naked eye and observed by scanning the cluster plate into Adobe Photoshop CS for further analysis. PRNT50 titer was defined as the reciprocal of maximum serum dilution to show 50% reduction of the plaque count based on the controls.

The 1-day old BALB/c mice (purchased from the Experimental Animal Center of Sun Yat-sen University) were assigned into 6 groups: group 1 (n=14; treated by DENV-1 VLP sera with DENV-1), group 2 (n=10; treated by DENV-2 VLP sera + DENV-2), group 3 (n=14; treated by divalent DENV VLPs sera + DENV-1), group 4 (n=13; treated by divalent DENV VLPs sera + DENV-2), group 5 (n=15; treated by PBS treating sera + DENV-1) and group 6 (n=10; treated by PBS treating sera + DENV-2). In each group, the pups were confirmed to be from the same litter. The 1:10 dilution sera were incubated with 20 LD 50 DENV-1 or DENV-2 (4×10 4 PFU/ml) for 1 h at 37 °C. The suckling mice were intracerebrally injected with a 20μl of sera-virus mixture. The manifestations of the animals post challenge were recorded daily for 3 weeks, including the morbidity of paralysis, ruffling, slowing of activity, kyphoscoliosis and mortality.

All the data were analyzed using SPSS software (version 13.0) and displayed as mean ±standard deviation (SD).

Statistical differences among groups were analyzed by one-way ANOVA. For survival analysis, Kaplan-Meier survival curves were analyzed by the log rank test. P<0.05 was defined as significant.

Yeast lysates were analyzed for the expression of DENV-1 E proteins and DENV-2 E proteins by Western blotting using mAb D2-1F1-3 and 3H5. About 50 kDa band of recombinant E protein band was detected in the lysates of yeast clones (Fig1.b). After purified by 5-50% sucrose density gradient centrifugation, the expressed DENV-1 E proteins and DENV-2 E proteins were present in the faction of 20%-25% sucrose density gradient (Fig1.c). After the fractions containing DENV VLPs were harvested and mixed, the recombinant protein concentration was determined to be 0.5mg/ml.

To evaluate the immunogenicity induced by recombinant VLPs, blood samples from immunized mice with purified DENV-1/2 VLPs were collected at days 0, 13, 27 before immunization and at 34 day after the last immunization. For control, blood samples of the mice immunized with heat-inactivated DENV-1 and DENV-2 were collected at the same time point. As shown in Fig. 2 a and b , mice immunized with either DENV VLPs or inactivated virions showed high levels of antigen specific serum IgG. There was an increasing trend for the production of antibodies among all groups. At days 27 after immunization, the inactivated DENV-1/2 induced the highest levels of antibodies. However, at days 34, the highest levels of antibodies were observed in DENV-1/2 VLPs groups. Bivalent-VLPs and inactivated DENV induced the comparable level of antibodies at days 34 post immunization.

Studies of immunofluorescence assay showed that the sera from mice immunized with DENV-1 VLPs or DENV-2 VLPs were able to react with the native DENV-1 or DENV-2 antigens (Fig 3) . Similar results can be observed in sera from mice immunized with bivalent DENV VLPs and inactivated DENV. The immunofluorescence staining was observed in the cytoplasm of C6/36 cells for the acetone permeability. The studies showed that immune sera produced from mice immunized with DENV VLPs could recognize native DENV structure proteins. No immunofluorescence staining was observed in normal sera suggesting no viral antigens were detected in mice treated with PBS.

To evaluate the specific DENV neutralizing antibody in sera from immunized mice, the PRNT was performed. Fig.4 showed that DENV1/2-VLPs could exhibit comparable levels of homotypic neutralizing antibodies with inactivated DENV1/2. The PRNT50 titers of bivalent VLPs sera against DENV-1 and DENV-2 were 16 and 64, respectively. The 

To assess the cellular immune response to DENV-1 and DENV-2 in vitro, we isolated spleen cells at day 34 after immunization by VLPs and inactivated virions. The levels of IFN-γ, TNF-, and IL-10 in spleen cells after stimulated by DENV-1 or DENV-2 in vitro were analyzed by ELISPOT. As shown in Fig. 5 a, splenocytes in monovalent VLPs, bivalent VLPs and inactivated virions groups secreted higher levels of IFN-γ after stimulation with DENV-1 and DENV-2 in comparison with PBS group (P<0.05). Similar results were observed in TNF- and IL-10 levels (Fig.5 b, c) .

The IFN-γ level was elevated significantly in monovalent VLPs group after DENV-1/2 challenge (P<0.05), which suggested that the monovalent VLPs predominantly induced the Th-1 response in splenocytes both stimulated by inactivated DENV-1 and DENV-2. The spleen cells produced the most TNF- in bivalent VLP group by DENV-1 simulation. The level of IL-10 was significantly accumulated in inactivated virions group with DENV-2 challenge.

Among the 3 cytokines, the mean TNF- levels in splenocytes were the highest after DENV virions stimulation. In contrast, the mean IL-10 levels were the lowest in all of the groups.

To explore the cytokines profiles after virus challenge in vivo, we harvested sera at days 1, 2, 3, 4 after DENV-1/2 challenge. The changes in the levels of the TNF-, IFN- and IL-10 of sera detected by ELISA assays were shown in Fig.   6 . After challenging with DENV-1/2, the monovalent, bivalent and inactivated DENV groups showed high levels of 3 cytokines. The IFN- and IL-10 levels were higher at day 1 post challenge and declined dramatically at days 2-4. For the TNF-, bivalent VLPs group showed the highest level at 1-4 days after virus challenge.

To evaluate protective capability of sera from mice immunized with monovalent or bivalent VLPs in vivo, we performed protection assays in BALB/c suckling mice. As shown in Fig.7 , anti-bivalent VLPs sera showed great ability of protecting sucking mice against DENV-1/2. All mice still survived at day 20 after treated with bivalent VLPs sera + DENV-1/2 (Fig. 7a, b) . The anti-monovalent VLPs sera prolonged the survival time of mice compared with PBS group.

All animals died at day 15 after DENV-1 VLPs sera + DENV-1 treatment (Fig. 7a) , and 20% mice still survived at day 20 in anti DENV-2 VLPs sera + DENV-2 group (Fig. 7b) . In PBS groups, all animals died at day 11 after infected by DENV-1 (Fig. 7a) , and day 16 (Fig. 7b) after infected by DENV-2 (Fig. 7b ). In addition, as shown in Fig. 8 , there was no complication in mice treated with bivalent VLPs sera + DENV-1/2. The onset of complications was observed earlier in mice of normal sera group than those in DENV-1/2 VLPs sera groups. The efficacy of anti-bivalent VLPs sera to protect animals against DENV-1/2 infection was greater than DENV-1/2 VLPs sera. DENV-1/2 VLPs sera inhibited the onset of complications compared with normal sera.

Dengue viruses that can be transmitted among humans by mosquitos (17) , are popular throughout tropical areas of the world including China (18) . For the high mortality and morbidity resulting from dengue infection, dengue has been a serious healthy concern (19) . VLP vaccine has shown great potential in preventing viral infection (20) . At present, many studies have been conducted to explore the effect of recombination VLPs in preventing dengue infection. In this work, we constructed the recombination VLPs according to a previous method with slight modifications and systematically analyzed the immunization properties of monovalent and bivalent DENV in humoral immune responses, cellular immune responses in vivo and in vitro.

In the present study, we successfully generated recombinant DENV-1 and -2 VLPs in Pichia pastoris by optimizing the expression plasmids. P. pastoris yeast cells characterized by tolerance, were found to the most economic expression systems for vaccine development (21) and GAP promoter was successfully used in expressing large amount of HBsAg in yeast cells (22) . The GAP promoter-based P. pastoris expression system with the signal peptide of prM has been determined to be safe and stable for constitutive and effective production of DENV-2 VLPs (23) . In contrast, we applied α-factor secretion signal in the GAP promoter-P. pastoris expression system and obtained high-level production of DENV-1 VLPs and DENV-2 VLPs. Thus, α-factor secretion signal can be recognized by the yeast secretory apparatus and lead to the effective expression of different types of DENV VLPs.

As outlined in previous study, VLPs vaccine showed considerable promise in preventing various virus challenge (20, 24) . VLPs can be efficiently taken up, internalized and processed by antigen presenting cells (APCs) (25-28). Previous evidence indicated that VLP based vaccine was able to elicit strong humoral and cellular immune responses against viruses (29) (30) (31) . Data of ELISA and indirect immunofluorescence assay revealed that DENV VLPs, similar with inactivated dengue virions, induced increasing levels of antigen specific IgG after immunization and the antisera from mice immunized by monovalent and bivalent DENV VLPs could bind to natural DENV specific antigen. It is reported that DENV E protein has many B-cell-specific epitopes that can stimulate B cells to produce neutralization antibodies against virus infection (32, 33) . In this work, the DENV E protein was successfully expressed in the recombinant DENV VLPs and stimulated the production of neutralizing antibodies that are particularly important to block dengue virus entry into target cells. The DENV VLPs expressed in our paper induced humoral immune response in mice infected by DENV.

In this work, the humoral response was characterized by high PRNT50 titers of monovalent or bivalent DENV VLPs in neutralizing assays. These findings are consistent with the humoral response induced by VLPs in other studies (34) . All these results confirmed that the dengue virus type 1 and 2 VLPs prepared in this study preserved the antigenicity of prM and E proteins and effectively induced virus specific humoral immune responses.

In addition, another marked advantage of VLPs is their ability to induce cellular immunity (35, 36) . Tumor necrosis factor alpha (TNF-), is a modulator of cell immunization involved in systemic inflammation, and has been reported to have the ability to inhibit viral replications (37). Interferon gamma (IFN-γ), known as immune interferon, is also found to be a critical antiviral mediator (38). Moreover, IL-10 (interleukin-10), an anti-inflammatory cytokine, plays key role in immune response. These cytokines (TNF-, IFN-γ and IL-10) are all closely associated with the immunopathogenesis of virus infection (39) . Therefore, in this study, the levels of these cytokines were investigated to explore the cellar immune response induced by VLPs in vitro and in vivo. The production of helper T cell (Th1) immune response cytokine (IFN-γ) and Th2 immune response cytokine (IL-10) and inflammation cytokines (TNF-α) from virus-stimulated splenocytes in vitro were analyzed by ELISPOT quantitatively. Results showed that compared with PBS group, the inactivated DENV-1/2 antigen induced the secretion of 3 cytokines significantly in monovalent, bivalent DENV VLPs and inactivated virions group. It revealed that monovalent and bivalent VLPs had the ability to induce a potent immune response comparable with inactivated virions. The monovalent VLPs predominantly induced the Th-1 response in splenocytes both stimulated by inactivated DENV-1 and DENV-2 (Fig. 5) . Similar cytokines profiles were found in the blood of mice challenged by virus in vivo. Results showed that expressions of IFN-γ, TNF-α and IL-10 were elevated by VLPs compared with PBS group, suggesting that monovalent and bivalent VLPs could effectively induce cellular immune response. It has been reported that the levels of TNF-, IFN-γ and IL-10 are elevated in patients with dengue virus infection (40) . The serum levels of these cytokines is a potential predictor of disease severity (40) . Patients who died for DENV infection showed higher IL-10 levels in comparison with survivals. Besides, TNF- is elevated in the later phase of illness, while IFN-γ was elevated in early phase of Dengue haemorrhagic fever (41) . In our results, the levels of IL-10 and IFN-γ in mice treated with monovalent, bivalent DENV VLPs were raised at day 1 after DENV-1/2 challenge and declined gradually at day 2-4 ( Fig.6) , which might indicate that DENV VLPs inhibited the development and progression of virus infection effectively.

Furthermore, we tested the protective efficacy of the DENV VLPs in suckling mice model. There were obvious differences in the protective effect in recipients with monovalent VLPs sera and bivalent VLPs sera. All the sucking mice were survived with bivalent VLPs sera after the challenge with both DENV-1 and DENV-2. Monovalent VLPs sera prolonged the survival period of infected mice compared with PBS treated group. The highest protection rates (100%) observed in the sulking mice immunized with bivalent VLPs sera were possibly due to synergistic effect between DENV-1 VLPs and DENV-2 VLPs sera. Alternatively, the high concentration of cross-reactive antibodies forming macromolecules compouds by cross-linking numerous virions, may also prevent virus entering into cells. Although our data of protective experiment showed the significant protective effect of bivalent DENV VLPs against different types of dengue virus, it is well documented that DENV infection commonly lacks antibody cross-protection among serotypes (9).

The phenomenon of antibody dependent-enhancement (ADE) may be occur in VLPs-immunized mice, which is known as that different types of virus infection can increase the risk of development serious disease (17) . The ADE activity of the monovalent and bivalent DENV VLPs was not assayed and this is a limitation in this paper. Subsequently, a large number of studies should be conducted to determine whether the bivalent DENV VLPs is a promising candidate vaccine against different DENV serotypes.

In summary, we successfully expressed the recombinant DENV-1/2 VLPs with the α-factor secretion signal. The a. Construction strategy of recombinant plasmid pGAPZaA-prME-D1 and pGAPZaA-prME-D2. The full length prME-D1 and prME-D2 genes were cloned into the downstream of GAP promoter with the α-factor secretion signal to generate the recombinant pGAPZaA-prME-D1 and pGAPZaA-prME-D2 expression vector, respectively.

b. DENV-1/2 E protein expressed in yeast lysates. Lane1: DENV-1 E protein, lane2: DENV2 E protein.

c. Sucrose gradient sedimentation analysis for DENV-1/2 E protein expression. Arrow heads indicate E antigen. The size of molecular weight marker is shown in kDa. Balb/c mouse sera. Alexa-488 was used for DENV-1/2 virus staining (green); DAPI staining was used to label cell nucleus (blue); magnification: ×400. * and ** mean p<0.05, p<0.01, respectively. 

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