Dengue virus (DENV) is a re-emerging disease transmitted by the Aedes mosquitoes and has become a major public health problem in southern China. Currently, no antiviral drug or effective vaccine exist to control this disease. The chimeric DENV structural protein vaccine cannot elicit balanced levels of protective immunity to each of the four viral serotypes; therefore, non-structural protein components may be required to construct an effective DENV vaccine. The Dengue virus non-structural 1 (DENV NS1) protein plays a critical role in viral pathogenesis and protective immunity. Therefore, immunity to Dengue 1-4 NS1 subtypes may be crucial for the prevention of severe disease. This review attempts to provide an overview about the structure and function of DENV NS1.
Animals ; Dengue ; immunology ; prevention & control ; virology ; Dengue Vaccines ; chemistry ; genetics ; immunology ; Dengue Virus ; chemistry ; genetics ; immunology ; Humans ; Viral Nonstructural Proteins ; chemistry ; genetics ; immunology

OBJECTIVETo investigate the characteristics and dynamic changes of serum neutralizing antibody response in patients with primary infection of dengue virus type 1 (DENV-1).

METHODSSerum samples were obtained from the same patients with primary infection of DENV-1 within 2 weeks after symptom onset in 2006 and in 2010. A group-specific DENV NS1 capture ELISA-based micro-neutralizing test (ELISA-MNT) capable of detecting neutralizing antibodies against all the 4 serotypes of DENV was used to test the neutralizing antibody titers against DENV in the serum samples. The neutralizing antibody titers against a standard strain and 2 clinically isolated strains of DENV-1 were detected in serum samples collected in 2010.

RESULTSCross-reactive neutralizing antibody response against all the 4 serotypes of DENV was found in both of the serum samples collected in 2006 and 2010, but the samples collected in 2006 showed stronger cross-reactive neutralizing antibody responses. The neutralizing antibody against DENV-2, rather than the anticipated DENV-1 antibody, had the highest titer in the samples collected in 2006, whereas the antibody against homologous DENV-1 had the highest titer in the samples obtained in 2010. The neutralizing antibody titers against the homologous DENV-1 was significantly higher in samples collected in 2010 (U=86.500, P=0.000), which also demonstrated significantly different neutralizing antibody titers against the 3 different strains of DENV-1 (Χ(2)=12.123, P=0.002).

CONCLUSIONThe production of cross-reactive neutralizing antibodies between the 4 serotypes of DENV is a characteristic of DENV infection, particularly during early infection, but only the homologous neutralizing antibody increases obviously over time. The titers of the neutralizing antibodies against different strains, even of the same serotype, may differ distinctly.

Antibodies, Neutralizing ; blood ; Antibodies, Viral ; blood ; Cross Reactions ; Dengue ; blood ; immunology ; Dengue Virus ; classification ; immunology ; Humans ; Neutralization Tests

To investigate the effects of DENV infection on the expression of Vascular endothelial growth factor (VEGF) in monocytes, and to explore which innate immune signaling pathway is responsible for VEGF induction. Real-time PCR was used to determine the expression levels of VEGF in DENV-infected THP-1. We found that different serotype viruses (DENV1, DENV2, DENV3) induced the VEGF expression. Moreover, VEGF expression was significantly increased in human primary monocytes infected with DENV 2. In addition, VEGF induction by DENV2 was significantly impaired by knockdown of TLR3 and interferon-beta promoter stimulator 1 (IPS-1), or by inhibition of ERK, JNK or NF-kappaB. These results demonstrated that DENV induced VEGF expression in monocytes, and the activation of TLR3, IPS-1 signal pathways were required for DENV2-triggered VEGF induction, suggesting that VEGF might be a promising therapeutic target for DHF.
Dengue ; genetics ; immunology ; virology ; Dengue Virus ; classification ; genetics ; physiology ; Humans ; MAP Kinase Signaling System ; Monocytes ; NF-kappa B ; genetics ; immunology ; Up-Regulation ; Vascular Endothelial Growth Factor A ; genetics ; immunology

Dengue virus (DENV) is the most widely transmitted arbovirus in the world. Due to the lack of diagnostic technology to quickly identify the virus serotypes in patients, severe dengue hemorrhagic fever cases caused by repeated infections remain high. To realize the rapid differential diagnosis of different serotypes of DENV infection by immunological methods, in this study, four DENV serotype NS1 proteins were expressed and purified in mammalian cells. Monoclonal antibodies (MAbs) against NS1 protein were obtained by hybridoma technology after immunizing BALB/c mice. Enzyme-linked immunosorbent assay, indirect immunofluorescence assay, dot blotting, and Western blotting were used to confirm the reactivity of MAbs to viral native NS1 and recombinant NS1 protein. These MAbs include not only the universal antibodies that recognize all DENV 1-4 serotype NS1, but also serotype-specific antibodies against DENV-1, DENV-2 and DENV-4. Double antibody sandwich ELISA was established based on these antibodies, which can be used to achieve rapid differential diagnosis of serotypes of DENV infection. Preparation of DENV serotype-specific MAbs and establishment of an ELISA technology for identifying DENV serotypes has laid the foundation for the rapid diagnosis of DENV clinical infection.
Animals ; Antibodies, Monoclonal ; Antibodies, Viral/metabolism* ; Dengue/diagnosis* ; Dengue Virus/immunology* ; Enzyme-Linked Immunosorbent Assay ; Humans ; Mice ; Mice, Inbred BALB C ; Sensitivity and Specificity ; Serogroup ; Viral Nonstructural Proteins/immunology*

OBJECTIVETo construct sub-unit vaccines of dengue virus type 1 to 4 and to analyze its immunogenicity.

METHODSEnvelope domain III s of dengue serotypes 1 and 2, as well as 3 and 4, were spliced by a linker (Gly-Gly-Ser-Gly-Ser)3 and cloned into vector pET-30a, then transformed into E. coli to express recombinant fusion proteins. The recombinant proteins were purified by high-performance liquid chromatography and mixed to immunize BALB/c mice. The neutralizing antibodies were tested by neutralizing assay, as well as in newborn mice challenged intracranially with dengue virus type 1 to 4.

RESULTSMice immunized with proteins could produce neutralizing antibodies, with titers of 1:34. 9, 1: 45.3, 1: 24.7 and 1:38.4 for DEN-1 to 4 respectively. 100% newborn mice challenged with DEN-1 or 2 in combination with sera from mice immunized with recombinant proteins were protected, whereas 83% protection was obtained when challenged with DEN-3 or 4.

CONCLUSIONThe recombinant proteins possess excellent immunogenicity to induce neutralizing antibodies and would be valuable for development of a tetravalent sub-unit vaccine.

Animals ; Antibodies, Neutralizing ; immunology ; Dengue Vaccines ; chemistry ; genetics ; immunology ; Dengue Virus ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Mice ; Mice, Inbred BALB C ; Neutralization Tests ; Recombinant Fusion Proteins ; genetics ; immunology ; Viral Envelope Proteins ; genetics ; immunology

Dengue virus (DENV) envelope [E] protein is the major surface protein of the virions that indued neutralizing antibodies. The domain III of envelope protein (EDIII) is an immunogenic region that holds potential for the development of vaccines; however, the epitopes of DENV EDIII, especially neutralizing B-cell linear epitopes, have not been comprehensively mapped. We mapped neutralizing B-cell linear epitopes on DENV-1 EDIII using 27 monoclonal antibodies against DENV-1 EDIII proteins from mice immunized with the DENV-1 EDIII. Epitope recognition analysis was performed using two set of sequential overlapping peptides (16m and 12m) that spanned the entire EDIII protein from DENV-1, respectively. This strategy identified a DENV-1 type- specific and a group-specific neutralizing epitope, which were highly conserved among isolates of DENV-1 and the four DENV serotypes and located at two regions from DENV-1 E, namely amino acid residues 309-320 and 381-392(aa 309-320 and 381-392), respectively. aa310 -319(310KEVAETQHGT319)was similar among the four DENV serotypes and contact residues on aa 309 -320 from E protein were defined and found that substitution of residues E309 , V312, A313 and V320 in DENV-2, -3, -4 isolates were antigenically silent. We also identified a DENV-1 type-specific strain-restricted neutralizing epitope, which was located at the region from DENV-1 E, namely amino acid residues 329-348 . These novel type- and group-specific B-cell epitopes of DENV EDIII may aid help us elucidate the dengue pathogenesis and accelerate vaccine design.
Amino Acid Sequence ; Animals ; Antibodies, Neutralizing ; immunology ; Dengue ; virology ; Dengue Virus ; chemistry ; genetics ; immunology ; Epitope Mapping ; Epitopes, B-Lymphocyte ; chemistry ; genetics ; immunology ; Humans ; Mice ; Molecular Sequence Data ; Viral Envelope Proteins ; chemistry ; genetics ; immunology

OBJECTIVETo study cellular and humoral immune responses to NS1 protein in mice inoculated intramuscularly with recombinant plasmid expressing dengue 2 NS1 gene.

METHODSThe eukaryotic expressing plasmid pCNX2-NS1 was injected into tibialis anterior muscle in mice. The mice were subsequently boosted with the same dose and same method twice after the initial inoculation. The mice were killed at four-week intervals and their serum and spleen cells were harvested for further test.

RESULTSDengue 2 antibodies were detectable in the sera from inoculated animals four weeks after the last boost. The changes of CD4+ T lymphocyte and CD8+ T lymphocyte were also determined by flow cytometry.

CONCLUSIONThe recombinant plasmid containing dengue 2 NS1 genes is immunogenic in intramuscularly inoculated mice. The vaccinated mice produced dengue-2 specific and long lasting immunity.

Animals ; Antibodies, Viral ; blood ; CD4-Positive T-Lymphocytes ; cytology ; immunology ; CD8-Positive T-Lymphocytes ; cytology ; immunology ; Dengue ; blood ; immunology ; virology ; Dengue Vaccines ; immunology ; Dengue Virus ; genetics ; immunology ; Female ; Flow Cytometry ; Gene Expression ; Immunization ; Mice ; Mice, Inbred BALB C ; Plasmids ; genetics ; Vaccines, Combined ; immunology ; Vaccines, DNA ; genetics ; immunology ; Viral Nonstructural Proteins ; genetics

OBJECTIVETo screen and identify dengue virus type 2 specific antigens and establish an enzyme-linked immunosorbent assay (ELISA) for detecting dengue virus type 2 antibody.

METHODSUsing the bioinformatic software DNAstar and ANTHEPROT, we analyzed the hydrophilicity, flexibility, surface probability and antigenicity of dengue virus type 1-4, Japanese encephalitis virus, and Yellow fever virus M and E protein amino acid sequences, and also evaluated the influence of secondary structure. The specific epitopes of dengue virus type 2 were predicted according to the epitope location and amino acid sequence similarity, and the epitope conservation was assessed using the sequence information of different dengue virus type 2 strains in GenBank. Based on the results of bioinformatic analysis, 5 specific epitopes were amplified and inserted into the prokaryotic expression vector pET32a, which were transferred into E. coli Rosetta (DE3) for expression of the proteins. SDS-PAGE and Western blotting were used to identify the expressed proteins and test their antigenicities. The antigen selected by Western blotting was used to establish the ELISA system for dengue virus type 2 antibody detection.

RESULTSBioinformatic analysis predicted 8 possible dengue virus type 2 specific epitopes, and 6 of them were efficiently expressed in E. coli. Western blotting confirmed 1 dengue virus type 2 specific antigen, the ELISA system for dengue virus antibody detection was successfully established using this specific antigen.

CONCLUSIONWe have obtained a dengue virus type 2 specific antigen and established an ELISA system for detection of dengue virus type 2 antibody.

Antibodies, Viral ; immunology ; Antigens, Viral ; immunology ; Computational Biology ; Dengue Virus ; classification ; immunology ; isolation & purification ; Enzyme-Linked Immunosorbent Assay ; methods ; Humans ; Immunodominant Epitopes ; Software

OBJECTIVETo establish a highly sensitive and specific assay to detect dengue virus (DENV) envelope protein domain III (EDIII) IgG antibody, and to explore its value in the diagnosis and seroepidemiological survey of dengue.

METHODSThe DENV EDIII IgG antibody capture ELISA was developed using the recombinant full-length DENV EDIII, which was prepared by Pichia yeast expression system as the capture antigen. The serum samples were collected from the same group of 35 DENV-1 patients of primary infection during disease period in 2006 and their follow-up phase in 2010; and the sensitivity of the assay was compared to that of the commercial Panbio DENV IgG ELISA.

RESULTSThe sensitivity of DENV EDIII IgG ELISA in detecting the serum samples from disease period and follow-up phase was 87% (20/23) and 94% (33/35), respectively; whereas the sensitivity of Panbio DENV IgG ELISA was 71% (25/35) and 0, respectively. The sensitivity of DENV EDIII IgG ELISA in detecting the serum samples from both periods was similar, without statistical significance (χ(2) = 0.946, P = 0.331). For serum samples from disease period, the sensitivity of DENV EDIII IgG ELISA was comparable with that of Panbio DENV IgG ELISA (χ(2) = 1.924, P = 0.165). However, DENV EDIII IgG ELISA demonstrated a significantly higher sensitivity than Panbio DENV IgG ELISA in detecting the serum samples from follow-up phase (χ(2) = 62.432, P = 0.000).

CONCLUSIONDENV EDIII IgG capture ELISA is highly sensitive in detecting IgG in the serum samples from either disease period or follow-up phase. This method might be a promising alternative for diagnosis and seroepidemiologic survey of dengue.

Antibodies, Viral ; blood ; Dengue ; diagnosis ; immunology ; virology ; Dengue Virus ; immunology ; Enzyme-Linked Immunosorbent Assay ; methods ; Humans ; Immunoglobulin G ; blood ; Protein Structure, Tertiary ; Sensitivity and Specificity ; Seroepidemiologic Studies ; Viral Envelope Proteins ; immunology

Animals ; Antibodies, Monoclonal ; immunology ; Cell Line ; Cercopithecus aethiops ; Cricetinae ; Culicidae ; Dengue Virus ; immunology ; Encephalitis Virus, Japanese ; immunology ; Encephalitis, Japanese ; immunology ; Mice ; Mice, Inbred Strains ; Vero Cells ; Viral Vaccines ; immunology