Identification of a novel infection-enhancing epitope on dengue prM using a dengue cross-reacting monoclonal antibody.

BACKGROUND: Dengue virus (DENV) infection is the most important arthropod- borne viral disease in human, but antiviral therapy and approved vaccines remain unavailable due to antibody-dependent enhancement (ADE) phenomenon. Many studies showed that pre-membrane (prM)-specific antibodies do not efficiently neutralize DENV infection but potently promote ADE infection. However, most of the binding epitopes of these antibodies remain unknown. RESULTS: In the present study, we characterized a DENV cross-reactive monoclonal antibody (mAb), 4D10, that neutralized poorly but potently enhanced infection of four standard DENV serotypes and immature DENV (imDENV) over a broad range of concentration. In addition, the epitope of 4D10 was successfully mapped to amino acid residues 14 to18 of DENV1-4 prM protein using a phage-displayed peptide library and comprehensive bioinformatics analysis. We found that the epitope was DENV serocomplex cross-reactive and showed to be highly immunogenic in Balb/c mice. Furthermore, antibody against epitope peptide PL10, like 4D10, showed broad cross-reactivity and weak neutralizing activtity with four standard DENV serotypes and imDENV but significantly promoted ADE infection. These results suggested 4D10 and anti-PL10 sera were infection-enhancing antibodies and PL10 was infection-enhancing epitope. CONCLUSIONS: We mapped the epitope of 4D10 to amino acid residues 14 to18 of DENV1-4 prM and found that this epitope was infection-enhancing. These findings may provide significant implications for future vaccine design and facilitate understanding the pathogenesis of DENV infection.

Infection and replication of avian influenza H5N1 virus in an infected human.

The highly pathogenic avian influenza H5N1 viruses usually cause severe diseases and high mortality in infected humans. However, the tissue tropism and underlying pathogenesis of H5N1 virus infection in humans have not been clearly elucidated yet. In this study, an autopsy was conducted to better understand H5N1 virus distributions in tissues of infected humans, and whether H5N1 virus can replicate in extrapulmonary tissues. We found that the lungs had the higher viral load than the spleen, whereas no detectable viruses in tissues of heart, liver, kidney, large intestine, small intestine, or brain. Specifically, the viral load was higher in the left lung (7.1 log10 copies per ml) in relation to the right lung (5.7 log10 copies per ml), resulting in more severe pathological damage in the left lung, and lung tissues contained both positive- and negative-stranded viral RNA. However, there existed a low level of H5N1 viruses in the spleen (3.8 log10 copies per ml), with the absence of positive-stranded viral RNA. Our results indicate that replication of H5N1 viruses mainly occurs in the lungs, and the degree of lung damage is highly correlated with the viral load in the lungs. The low-load viruses in the spleen might be introduced through blood circulation or other ways.

Computational prediction and identification of dengue virus-specific CD4(+) T-cell epitopes.

In this study, we tried to identify dengue virus-specific CD4(+) T-cell epitopes, which can induce PBMC (peripheral blood mononuclear cells) isolated from DF convalescent patients (dengue virus type 1 infection) to secrete IFN-gamma. PBMC of DF convalescent patients were stimulated in vitro with dengue virus-derived peptides, which were prepared based on the prediction of dengue virus-specific CD4(+) T-cell epitopes by using RANKpep online software. Subsequently, the frequency of IFN-gamma producing T cells and percentage of IFN-gamma(+) CD4(+) T cells were measured by using ELISPOT assay and ICS assay (intracellular cytokine straining), respectively. The positive response of PBMC by ELISPOT showed that the numbers of SFC (spots forming cells) ranged from 50 to 310 SFC/1x10(6) PBMC. The positive response of PBMC by ICS assay showed that the percentage of IFN-gamma(+) CD4(+) T cells ranged from 0.03 to 0.27%. As a result, C(45-57) (KLVMAFIAFLRFL), E(396-408) (SSIGKMFEATARG), NS3(23-35) (YRILQRGLLGRSQ), and NS3(141-155) (NREGKIVGLYGNGVV) were identified as dengue virus-specific CD4(+) T-cell epitopes.

Application of an oligonucleotide array assay for rapid detecting and genotyping of Chlamydia trachomatis from urogenital specimens.

An oligonucleotide array technology was established for rapidly detecting and genotyping Chlamydia trachomatis in urogenital infections. The VS1-VS2 region of the omp1 gene was used to design oligonucleotide probes. Eleven serovar-specific probes to serovars A, B, C, D, E, F, G, H, I, J, and K, and 3 group-specific probes to group B (B, Ba, D, E, L1, and L2), group C (A, C, H, I, J, K, and L3), and an intermediate group (F and G) were synthesized and spotted onto the nylon membrane. Two pairs of universal primers were designed for the nested polymerase chain reaction (PCR) amplification of the VS1-VS2 gene. Digoxigenin-labeled amplicons of the VS1-VS2 gene of C. trachomatis were hybridized to the membrane array. Hybridization signals were read by the nitroblue tetrazolium/5-bromo-4-chloro-3-indolylphosphate color development. The assay developed was tested with reference strains of C. trachomatis serovars and clinical samples. The sensitivity was evaluated for 57 samples previously found to be positive for C. trachomatis by using plasmid PCR, and 98.2% (56/57) concordance was obtained. Fourteen oligonucleotide probes were optimized by trying different reaction conditions, showing specific hybridization with the corresponding reference strains, but no cross-reactions with other urogenital microorganisms. Using this procedure, a total of 59 strains were detected from 56 chlamydial samples. Eight genotypes were found, and type D, E, F, and H were the most frequently observed types (77.9%). Three cases (5.4%) had multiple infections with serovars: 1.D/E, 2.D/F, and 3.F/K. To validate the reference strains and confirm the genotype identity as determined by the oligonucleotide array technology, we sequenced all reference strains and 10 selected specimens across variable sequence VS1 and VS2. No discrepancies were found between the array typing and the genotype identity confirmed by nucleotide sequencing of the PCR product. The findings from this study indicated that the oligonucleotide array is a simple, fast, and specific assay for directly detecting and genotyping C. trachomatis from clinical samples.

Substitution of the precursor peptide prevents anti-prM antibody-mediated antibody-dependent enhancement of dengue virus infection.

Antibody-dependent enhancement (ADE) is currently considered as the mechanism underlying the pathogenesis of severe dengue disease. Many studies have shown that precursor (pr) peptide-specific antibodies do not efficiently neutralize infection but potently promote ADE of dengue virus (DENV) infection. To explore the effect of pr peptide substitution on neutralization and ADE of DENV infection, the rabbit anti-prM polyclonal antibodies (pAbs) and anti-JEVpr/DENV-M pAbs were prepared, and the neutralization and ADE of these two pAbs were further compared. Here, we report that both anti-JEVpr/DENV-M and anti-prM pAbs exhibited broad cross-reactivity and only partial neutralization with four DENV serotypes and immature DENV. Rabbit anti-prM pAbs showed a significant enhancement in a broad range of serum dilutions. However, there was no statistically significant difference in the enhancing activity of rabbit anti-JEVpr/DENV-M pAbs at various levels of dilution. These results demonstrate that anti-prM antibody-mediated ADE can be prevented by JEV pr peptide replacement. The present study contribute further to research on the pathogenesis of DENV infection.

Secreted expression of dengue virus type 2 full-length envelope glycoprotein in Pichia pastoris.

The full-length envelope glycoprotein gene of dengue virus type 2 was cloned using an RT-PCR method from the infected C6/36 cells and inserted into pPICZaB vector. The recombinant plasmid was integrated into Pichia pastoris by electroporation and the expressed product was identified by SDS-PAGE and Western blotting. High-level secreted expression was performed by determining the Mut(+) phenotype and screening multi-copy integrants in the recombinant yeast cells. A recombinant protein with a molecular size of approximately 69 kDa was secreted into the supernatant from the yeast cells when induced with methanol. The expressed supernatant was able to bind with mouse polyclonal antibody or E-specific monoclonal antibody of dengue-2 virus. Purified E-poly (His)-tagged fusion protein was obtained from the expressed product by passing through a metal-chelating affinity chromatographic (MCAC) column. The results of Western blotting and solid-phase ELISA using dengue virus antibodies indicated that the purified recombinant E glycoprotein retained its antigenicity. High-level production of the recombinant E protein up to 100 mg/l indicates that P. pastoris is an efficient expression system for dengue virus full-length envelope glycoprotein.

Application of oligonucleotide array technology for the rapid detection of pathogenic bacteria of foodborne infections.

A rapid and accurate method for detection for common pathogenic bacteria in foodborne infections was established by using oligonucleotide array technology. Nylon membrane was used as the array support. A mutation region of the 23S rRNA gene was selected as the discrimination target from 14 species (genera) of bacteria causing foodborne infections and two unrelated bacterial species. A pair of universal primers was designed for PCR amplification of the 23S rRNA gene. Twenty-one species (genera)-specific oligonucleotide detection probes were synthesized and spotted onto the nylon membranes. The 23S rRNA gene amplification products of 14 species of pathogenic bacteria were hybridized to the oligonucleotide array. Hybridization results were analyzed with digoxigenin-linked enzyme reaction. Results indicated that nine species of pathogenic bacteria (Escherichia coli, Campylobacter jejuni, Shigella dysenteriae, Vibrio cholerae, Vibrio parahaemolyticus, Proteus vulgaris, Bacillus cereus, Listeria monocytogenes and Clostridium botulinum) showed high sensitivity and specificity for the oligonucleotide array. Two other species (Salmonella enterica and Yersinia enterocolitica) gave weak cross-reaction with E. coli, but the reaction did not affect their detection. After redesigning the probes, positive hybridization results were obtained with Staphylococcus aureus, but not with Clostridium perfringens and Streptococcus pyogenes. The oligonucleotide array can also be applied to samples collected in clinical settings of foodborne infections. The superiority of oligonucleotide array over other tests lies on its rapidity, accuracy and efficiency in the diagnosis, treatment and control of foodborne infections.

Induction of virus-neutralizing antibodies and T cell responses by dengue virus type 1 virus-like particles prepared from Pichia pastoris.

BACKGROUND: Dengue is currently a significant global health problem but no vaccines are available against the four dengue serotypes virus infections. The development of safe and effective vaccines has been hampered by the requirement of conferring complete protection against all four dengue serotypes and the lack of a convenient animal model. Virus-like particles (VLPs) have emerged as a promising subunit vaccine candidate. One strategy of vaccine development is to produce a tetravalent dengue subunit vaccine by mixing recombinant VLPs, corresponding to all four dengue virus serotypes. Towards this end, this study aimed to establish a Pichia pastoris (P. pastoris) expression system for production of dengue virus type 1 (DENV-1) VLPs and evaluate the humoral and cellular immune response of this particle in mice. METHODS: A recombinant yeast P. pastoris clone containing prM and E genes of DENV-1 was constructed and DENV-1 VLPs expressed by this clone were analyzed by sucrose density gradient centrifugation, Western blotting, and transmission electron microscope. Groups of mice were immunized by these particles plus adjuvant formulations, then mice were tested by ELISA and neutralization assay for humoral immune response, and by lymphocyte proliferation and cytokine production assays for a cellular immune response. RESULTS: Our data demonstrated that recombinant DENV-1 VLPs consisting of prM and E protein were successfully expressed in the yeast P. pastoris. Sera of VLPs immunized mice were shown to contain a high-titer of antibodies and the neutralization assay suggested that those antibodies neutralized virus infection in vitro. Data from the T lymphocyte proliferation assay showed proliferation of T cell, and ELISA found elevated secretion levels of interferon IFN-γ and IL-4. CONCLUSIONS: P. pastoris-expressed DENV-1 VLPs can induce virus neutralizing antibodies and T cell responses in immunized mice. Using P. pastoris to produce VLPs offers a promising and economic strategy for dengue virus vaccine development.

Selection and identification of B-cell epitope on NS1 protein of dengue virus type 2.

NS1 of dengue virus (DENV) is an important non-structural protein, which plays an important role in DENV replication and dengue infection. In this study, using the phage-displayed peptide library screening method and purified anti-DENV2-NS1 polyclonal antibody immunoglobulin G (IgG) as target, which was generated from the purified recombinant expressed DENV2-NS1 protein immunization on rabbit, seven B-cell epitopes of DENV2-NS1 protein were screened. Considering the results of comprehensive bioinformatic analysis on NS1 B-cell epitopes, possible dominant B-cell epitopes are located in amino acids residues 36-45, 80-89, 103-112, 121-130, 187-196, 295-304, and 315-324 of the NS1, and two epitope-based NS1 protein dodecapeptides corresponding to the predominant epitopes (PA10: (36)PESPSKLASA(45) and AA10: (187)AIKDNRAVHA(196)) were chosen for synthesis. Results of binding assay and competitive-inhibition assays indicated the two peptides were the specific epitopes of DENV2-NS1 protein. These epitopes could be useful in understanding the pathogenesis of DENV and as dengue vaccine constituents in further study.

Selection of SARS-coronavirus-specific B cell epitopes by phage peptide library screening and evaluation of the immunological effect of epitope-based peptides on mice.

Antibodies to SARS-Coronavirus (SARS-CoV)-specific B cell epitopes might recognize the pathogen and interrupt its adherence to and penetration of host cells. Hence, these epitopes could be useful for diagnosis and as vaccine constituents. Using the phage-displayed peptide library screening method and purified Fab fragments of immunoglobulin G (IgG Fab) from normal human sera and convalescent sera from SARS-CoV-infected patients as targets, 11 B cell epitopes of SARS-CoV spike glycoprotein (S protein) and membrane protein (M protein) were screened. After a bioinformatics tool was used to analyze these epitopes, four epitope-based S protein dodecapeptides corresponding to the predominant epitopes were chosen for synthesis. Their antigenic specificities and immunogenicities were studied in vitro and in vivo. Flow cytometry and ELISPOT analysis of lymphocytes as well as a serologic analysis of antibody showed that these peptides could trigger a rapid, highly effective, and relatively safe immune response in BALB/c mice. These findings might aid development of SARS diagnostics and vaccines. Moreover, the role of S and M proteins as important surface antigens is confirmed.

Secreted expression and purification of dengue 2 virus full-length nonstructural glycoprotein NS1 in Pichia pastoris.

The dengue 2 virus (DEN-2) RNA (NGC strain) was used as a substrate to produce DNA clones of the full-length NS1 genes via reverse transcriptase synthesis of cDNA followed by polymerase chain reaction amplification of the NS1 region. Products were cloned into pPICZalphaB vector for sequencing and into Pichia pastoris for expression. A recombinant protein with a molecular size of approximately 80 KDa was secreted into the supernatant from the yeast cells when induced with methanol. The expressed protein was able to bind with mouse polyclonal antibody or NS1-specific monoclonal antibody of dengue 2 virus. Purified NS1-poly(His)-tagged fusion protein was obtained from the expressed product by passing through a metal-chelating affinity chromatographic (MCAC) column. The study also verified that our purified rNS1 protein retained its antigenicity. High-level production of the rNS1 protein up to 70 mg/l indicates that P. pastoris is an efficient expression system for dengue virus full-length NS1 glycoprotein.

[Inoculation of plasmid expressing the dengue 2 NS1 gene elicits immunity in mice].

OBJECTIVE: To study cellular and humoral immune responses to NS1 protein in mice inoculated intramuscularly with recombinant plasmid expressing dengue 2 NS1 gene. METHODS: The 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. RESULTS: Dengue 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. CONCLUSION: The recombinant plasmid containing dengue 2 NS1 genes is immunogenic in intramuscularly inoculated mice. The vaccinated mice produced dengue-2 specific and long lasting immunity.

Dengue virus type 2 infects human endothelial cells through binding of the viral envelope glycoprotein to cell surface polypeptides.

The endothelial cell line ECV304, derived from human umbilical cord and identified to be susceptible to dengue virus type 2 (DEN-2) infection, was used to study the molecular mechanism of DEN-2 binding to endothelial cells. DEN-2 was found by virus overlay protein-binding assays (VOPBAs) to bind to three ECV304 cell membrane proteins with molecular masses of 29, 34 and 43 kDa. Only a single protein of 29 kDa was observed when VOPBAs were carried out using preparations of trypsin-treated ECV304 cells. Pre-incubation of live ECV304 cells in culture or cell membrane proteins in modified VOPBAs with the recombinant DEN-2 envelope glycoprotein (rEgp) inhibited DEN-2 infection and blocked virus binding to the three proteins identified. These results indicate that DEN-2 rEgp could bind to three proteins on the surface of ECV304 cells. This virus-cell interaction may be associated with the receptor complex specific for DEN-2 infection of endothelial cells.