ERG-Associated lncRNA (ERGAL) Promotes the Stability and Integrity of Vascular Endothelial Barrier During Dengue Viral Infection via Interaction With miR-183-5p.

Dengue virus (DENV) continues to be a major public health problem. DENV infection will cause mild dengue and severe dengue. Severe dengue is clinically manifested as serious complications, including dengue hemorrhagic fever and/or dengue shock syndrome (DHF/DSS), which is mainly characterized by vascular leakage. Currently, the pathogenesis of severe dengue is not elucidated thoroughly, and there are no known therapeutic targets for controlling the disease effectively. This study aimed to further reveal the potential molecular mechanism of severe dengue. In this study, the long non-coding RNA, ERG-associated lncRNA (lncRNA-ERGAL), was activated and significantly up-regulated in DENV-infected vascular endothelial cells. After knockdown of lncRNA-ERGAL, the expression of ERG, VE-cadherin, and claudin-5 was repressed; besides, cell apoptosis was enhanced, and cytoskeletal remodeling was disordered, leading to instability and increased permeability of vascular endothelial barrier during DENV infection. Fluorescence in situ hybridization (FISH) assay showed lncRNA-ERGAL to be mainly expressed in the cytoplasm. Moreover, the expression of miR-183-5p was found to increase during DENV infection and revealed to regulate ERG, junction-associated proteins, and the cytoskeletal structure after overexpression and knockdown. Then, ERGAL was confirmed to interact with miR-183-5p by luciferase reporter assay. Collectively, ERGAL acted as a miRNA sponge that can promote stability and integrity of vascular endothelial barrier during DENV infection via binding to miR-183-5p, thus revealing the potential molecular mechanism of severe dengue and providing a foundation for a promising clinical target in the future.

Zika Virus Infection Induces Acute Kidney Injury Through Activating NLRP3 Inflammasome Via Suppressing Bcl-2.

Zika virus (ZIKV) is a newly emerging flavivirus that broadly exhibits in various bodily tissues and fluids, especially in the brain, and ZIKV infection often causes microcephaly. Previous studies have been reported that ZIKV can infect renal cells and can be detected in the urine samples of infected individuals. However, whether ZIKV infection causes renal diseases and its pathogenic mechanisms remains unknown. Here, we identified that ZIKV infection resulted in acute kidney injury (AKI) in both newborn and adult mouse models by increasing the levels of AKI-related biomarkers [e.g., serum creatinine (Scr), kidney injury molecular-1 (Kim-1), and neutrophil gelatinase-associated lipocalin (NGAL)]. ZIKV infection triggered the inflammatory response and renal cell injury by activating Nod-like receptor 3 (NLRP3) inflammasome and secreting interleukin-1ß (IL-1ß). IL-1ß inhibited aquaporins expression and led to water re-absorption disorder. Furthermore, ZIKV infection induced a decreased expression of B-cell lymphoma-2 (Bcl-2) in the kidney. Overexpression of Bcl-2 attenuated ZIKV-induced NLRP3 inflammasome activation in renal cells and down-regulated PARP/caspase-3-mediated renal apoptosis. Overall, our findings demonstrated that ZIKV infection induced AKI by activating NLRP3 inflammasome and apoptosis through suppressing Bcl-2 expression, which provided potential therapeutic targets for ZIKV-associated renal diseases.

Avian Influenza A (H7N9) viruses isolated from patients with mild and fatal infection differ in pathogenicity and induction of cytokines.

Since 2013, a novel Influenza A (H7N9) virus strain has continued to circulate within poultry and causing human disease. Influenza A (H7N9) virus results in two types of infection: mild and severe. The different results of clinical findings may be related with host susceptibility and characteristics of the virus itself. In order to investigate potential pathogenesis of Influenza A (H7N9) virus, we performed pathogenecity and cytokines analysis of two isolates, A/Guangdong/6/2013 H7N9 virus (GD-6) from a patient with a mild infection, and A/Guangdong/7/2013 H7N9 virus (GD-7) from a patient with a fatal infection. We found that GD-7 replicated to higher levels than GD-6 in human peripheral blood mononuclear cells (PBMCs), lung tissues, and mice. Furthermore, GD-7 infection resulted in more severe lung damage in mice lung tissues than GD-6 infection. GD-7 elicited higher levels of interleukin-6 (IL-6) and tumor necrosis factor-α(TNF-α) than GD-6 did. In conclusion, GD-7 was more pathogenic and induced higher levels of proinflammatory cytokines than GD-6 did.

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.

Suppressed expression of miR-378 targeting gzmb in NK cells is required to control dengue virus infection.

Dengue virus (DENV) remains a major public health threat because no vaccine or drugs are available for the prevention and treatment of DENV infection, and the immunopathogenesis mechanisms of DENV infection are not fully understood. Cytotoxic molecules, such as granzyme B (GrzB), may be necessary to control viral infections. However, the exact role of GrzB during DENV infection and the mechanisms regulating GrzB expression during DENV infection are not clear. This study found that miR-27a*, miR-30e, and miR-378 were down-regulated in DENV-infected patients, and DENV infection in humans induced a significant up-regulation of GrzB in natural killer (NK) cells and CD8(+) T cells. Further investigation indicated that NK cells, but not CD8(+) T cells, were the major sources of GrzB, and miR-378, but not miR-27a* or miR-30e, suppressed GrzB expression in NK cells. Notably, we found that overexpression of miR-378 using a miR-378 agomir in DENV-infected mice inhibited GrzB expression and promoted DENV replication. These results suggest the critical importance of miR-378 in the regulation of GrzB expression and a protective role for GrzB in controlling DENV replication in vivo. Therefore, this study provides a new insight into the immunopathogenesis mechanism of DENV infection and a biological basis for the development of new therapeutic strategies to control DENV infection.

Replacement of pr gene with Japanese encephalitis virus pr using reverse genetics reduces antibody-dependent enhancement of dengue virus 2 infection.

Severe dengue is more likely found during secondary heterologous dengue virus (DENV) infection or primary infection of infants born to dengue-immune mothers and led to the hypothesis of antibody-dependent enhancement (ADE). It has been reported that pre-membrane (prM)-reactive antibodies do not efficiently neutralize DENV infection but instead potently promote ADE infection. Meanwhile, these enhancing anti-prM antibodies mainly react with the precursor (pr) peptide. To evaluate the effect of pr gene substitution on neutralization and ADE of DENV infection, a novel chimeric dengue virus (JEVpr/DENV2) was rationally constructed by replacing the DENV pr gene with Japanese encephalitis virus (JEV) pr gene, based on the full-length infectious complementary DNA (cDNA) clone of DENV2 ZS01/01. We found that chimeric JEVpr/DENV2 showed reduced virulence and good immunogenicity. In addition, anti-JEVpr/DENV2 sera showed broad cross-reactivity and efficient neutralizing activity with all four DENV serotypes and immature DENV2 (ImDENV2). Most importantly, compared with anti-DENV2 sera, anti-JEVpr/DENV2 sera showed significantly reduced enhancing activity of DENV infection in K562 cells. These results suggest that the ADE activities could be reduced by replacing the DENV pr gene with JEV pr gene. These findings may help us better understand the pathogenesis of DENV infection and provide a reference for the development of a vaccine against DENV.

Comprehensive mapping infection-enhancing epitopes of dengue pr protein using polyclonal antibody against prM.

Dengue vaccine development is considered a global public health priority, but the antibody-dependent enhancement (ADE) issues have critically restricted vaccine development. Recent findings have demonstrated that pre-membrane (prM) protein was involved in dengue virus (DENV) infection enhancement. Although the importance of prM antibodies have been well characterized, only a few epitopes in DENV prM protein have ever been identified. In this study, we screened five potential linear epitopes located at positions pr1 (1-16aa), pr3 (13-28aa), pr4 (19-34aa), pr9 (49-64aa), and pr10 (55-70aa) in pr protein using peptide scanning and comprehensive bioinformatics analysis. Then, we found that only pr4 (19-34aa) could elicit high-titer antibodies in Balb/c mice, and this epitope could react with sera from DENV2-infected patients, suggesting that specific antibodies against epitope peptide pr4 were elicited in both DENV-infected mice and human. In addition, our data demonstrated that anti-pr4 sera showed limited neutralizing activity but significant ADE activity toward standard DENV serotypes and imDENV. Hence, it seems responsible to hypothesize that anti-pr4 serum was infection-enhancing antibody and pr4 was infection-enhancing epitope. In conclusion, we characterized a novel infection-enhancing epitope on dengue pr protein, a finding that may provide new insight into the pathogenesis of DENV infection and contribute to dengue vaccine design.

Tetravalent recombinant dengue virus-like particles as potential vaccine candidates: immunological properties.

BACKGROUND: Currently, a licensed vaccine for Dengue Virus (DENV) is not yet available. Virus-like particles (VLP) have shown considerable promise for use as vaccines and have many advantages compared to many other types of viral vaccines. VLPs have been found to have high immunogenic potencies, providing protection against various pathogens. RESULTS: In the current study, four DENV-VLP serotypes were successfully expressed in Pichia pastoris, based on co-expression of the prM and E proteins. The effects of a tetravalent VLP vaccine were also examined. Immunization with purified, recombinant, tetravalent DENV1-4 VLPs induced specific antibodies against all DENV1-4 antigens in mice. The antibody titers were higher after immunization with the tetravalent VLP vaccine compared to titers after immunization with any of the dengue serotype VLPs alone. Indirect immunofluorescence assay (IFA) results indicated that sera from VLP immunized mice recognized the native viral antigens. TNF-α and IL-10 were significantly higher in mice immunized with tetravalent DENV-VLP compared to those mice received PBS. The tetravalent VLP appeared to stimulate neutralizing antibodies against each viral serotype, as shown by PRNT50 analysis (1:32 against DENV1 and 2, and 1:16 against DENV3 and 4). The highest titers with the tetravalent VLP vaccine were still a little lower than the monovalent VLP against the corresponding serotype. The protection rates of tetravalent DENV-VLP immune sera against challenges with DENV1 to 4 serotypes in suckling mice were 77, 92, 100, and 100%, respectively, indicating greater protective efficacy compared with monovalent immune sera. CONCLUSIONS: Our results provide an important basis for the development of the dengue VLP as a promising non-infectious candidate vaccine for dengue infection.

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.

Melaleuca alternifolia concentrate inhibits in vitro entry of influenza virus into host cells.

Influenza virus causes high morbidity among the infected population annually and occasionally the spread of pandemics. Melaleuca alternifolia Concentrate (MAC) is an essential oil derived from a native Australian tea tree. Our aim was to investigate whether MAC has any in vitro inhibitory effect on influenza virus infection and what mechanism does the MAC use to fight the virus infection. In this study, the antiviral activity of MAC was examined by its inhibition of cytopathic effects. In silico prediction was performed to evaluate the interaction between MAC and the viral haemagglutinin. We found that when the influenza virus was incubated with 0.010% MAC for one hour, no cytopathic effect on MDCK cells was found after the virus infection and no immunofluorescence signal was detected in the host cells. Electron microscopy showed that the virus treated with MAC retained its structural integrity. By computational simulations, we found that terpinen-4-ol, which is the major bioactive component of MAC, could combine with the membrane fusion site of haemagglutinin. Thus, we proved that MAC could prevent influenza virus from entering the host cells by disturbing the normal viral membrane fusion procedure.

Differentially expressed genes of human microvascular endothelial cells in response to anti-dengue virus NS1 antibodies by suppression subtractive hybridization.

It has been previously shown that anti-dengue virus (DENV) nonstructural protein NS1 antibodies could act as autoantibodies that direct against one or more of the host's own proteins, which has potential implications for dengue hemorrhagic fever pathogenesis. In the present study, we have employed suppression subtractive hybridization (SSH) to identify the differentially expressed genes from human microvascular endothelial cells (HMEC-1) in response to anti-dengue virus type 2 NS1 antibodies (anti-DENV2 NS1 Abs). A total of 35 clones from the SSH cDNA library were randomly selected for further analysis using bioinformatics tools after vector screening. After searching for sequence homology in NCBI GenBank database with BLASTN and BLASTX programs, 23 obtained sequences with significant matches (E-values <1×10(-4)) in the SSH library. The predicted genes in the subtracted library include immune response molecules (CD59 antigen preproprotein preproprotein, MURR1), signal transduction molecules (Nuclear casein kinase and cyclin-dependent kinase substrate 1), calcium-binding proteins (S100A6, Annexin A2 isoform 1/2), and cell-membrane component (Yip1 domain family). From these clones, 5 upregulated genes were selected for differential expression profiling by real-time RT-PCR to confirm their upregulated status. The results confirmed their differential upregulation, and thus verified the success of SSHs and the likely involvement of these genes in dengue pathogenesis.

Tim-3-expressing CD4+ and CD8+ T cells in human tuberculosis (TB) exhibit polarized effector memory phenotypes and stronger anti-TB effector functions.

T-cell immune responses modulated by T-cell immunoglobulin and mucin domain-containing molecule 3 (Tim-3) during Mycobacterium tuberculosis (Mtb) infection in humans remain poorly understood. Here, we found that active TB patients exhibited increases in numbers of Tim-3-expressing CD4(+) and CD8(+) T cells, which preferentially displayed polarized effector memory phenotypes. Consistent with effector phenotypes, Tim-3(+)CD4(+) and Tim-3(+)CD8(+) T-cell subsets showed greater effector functions for producing Th1/Th22 cytokines and CTL effector molecules than Tim-3(-) counterparts, and Tim-3-expressing T cells more apparently limited intracellular Mtb replication in macrophages. The increased effector functions for Tim-3-expressing T cells consisted with cellular activation signaling as Tim-3(+)CD4(+) and Tim-3(+)CD8(+) T-cell subsets expressed much higher levels of phosphorylated signaling molecules p38, stat3, stat5, and Erk1/2 than Tim-3- controls. Mechanistic experiments showed that siRNA silencing of Tim-3 or soluble Tim-3 treatment interfering with membrane Tim-3-ligand interaction reduced de novo production of IFN-γ and TNF-α by Tim-3-expressing T cells. Furthermore, stimulation of Tim-3 signaling pathways by antibody cross-linking of membrane Tim-3 augmented effector function of IFN-γ production by CD4(+) and CD8(+) T cells, suggesting that Tim-3 signaling helped to drive stronger effector functions in active TB patients. This study therefore uncovered a previously unknown mechanism for T-cell immune responses regulated by Tim-3, and findings may have implications for potential immune intervention in TB.

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.

Identification and immunogenicity of two new HLA-A*0201-restricted CD8+ T-cell epitopes on dengue NS1 protein.

Immunopathogenesis of dengue virus (DEN) infection remains poorly studied. Identification and characterization of human CD8(+) T-cell epitopes on DEN are necessary for a better understanding of the immunopathogenesis of dengue infection and would facilitate the development of immunotherapy and vaccines to protect from dengue infection. Here, we identified two new HLA-A*0201-restricted CD8(+) T-cell epitopes, DEN-4 NS1(990)(-998) and DEN-4 NS1(997)(-1005) that are conserved in three or four major DEN serotypes, respectively. Unexpectedly, we found that immunization of HLA-A*0201 transgenic mice with DEN-4 NS1(990)(-998) or DEN-4 NS1(997)(-1005) epitope peptide induced de novo synthesis of tumor necrosis factor (TNF)-α and IFN-γ, two important pro-inflammatory molecules that are hard to be detected directly without in vitro antigenic re-stimulation. Importantly, we demonstrated that CD8(+) T cells specifically activated by DEN-4 NS1(990)(-998) or DEN-4 NS1(997)(-1005) epitope peptide induced de novo synthesis of perforin. Furthermore, we observed that DEN-4 NS1(990)(-998) or DEN-4 NS1(997)(-1005)-specific CD8(+) T cells capable of producing large amounts of perforin, TNF-α and IFN-γ preferentially displayed CD27(+)CD45RA(-), but not CD27(-)CD45RA(+), phenotypes. This study, therefore, suggested the importance of synergistic effects of pro-inflammatory cytokines and cytotoxic molecules which were produced by dengue-specific CD8(+) T cells in immunopathogenesis or anti-dengue immunity during dengue infection.

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.

Recombinant dengue virus-like particles from Pichia pastoris: efficient production and immunological properties.

The envelope glycoprotein (E) of flavivirus is the major structural protein on the surface of the mature virions. The complexes of premembrane (prM) and E play important roles in virus assembly and fusion modulation and in potential immunity-inducing vaccines. In the present study, the cDNA encoding prM and E proteins of dengue virus type 2 (DENV-2) was subcloned into the pGAPZalphaA vector and further integrated into the genome of Pichia pastoris under the control of the glyceraldehyde-3-phosphate dehydrogenase (GAP) constitutive promoter. The high-level constitutive expression of recombinant E antigen was achieved in P. pastoris. Both the cell lysate and the culture supernatant, examined by electron microscopy, were found to contain DENV-2 virus-like particles (VLPs) with diameters of about 30 nm. After immunization of BALB/c mice, the VLPs exhibited similar efficacies as inactivated virus in terms of antibody induction and neutralization titer. These results suggest that recombinant DENV VLPs can be efficiently produced in the GAP promoter-based P. pastoris expression system. This system may be useful for the development of effective and economic dengue subunit vaccine.

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.

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.