Induction of multiple cytotoxic T lymphocyte responses in mice by a multiepitope DNA vaccine against dengue virus serotype 1.

Dengue virus (DENV) is still a major threat to human health in most tropical and subtropical countries and regions. In the present study, a multi-epitope DNA vaccine that encodes 15 immunogenic and conserved HLA-A*0201-, HLA-A*1101-, HLA-A*2402-restricted CTL epitopes from DENV serotype 1 (DENV-1) was constructed based on the eukaryotic expressing plasmid pcDNATM 3.1/myc-His(-) A. Immunization of HLA-A*0201, HLA-A*1101 and HLA-A*2402 transgenic mice with the recombinant plasmid pcDNATM 3.1/myc-His(-) A-DENV-1-Meg resulted in significantly greater IFN-γ-secreting T-cell responses against most (14/15) CTL epitopes than occurred in mice immunized with the empty plasmid pcDNATM 3.1/myc-His(-) A. Additionally, the epitope-specific T cells directed to some epitopes secreted not only IFN-γ but also IL-6 and/or TNF-α. Finally, the induced epitope-specific T cells also efficiently lysed epitope-pulsed splenocytes and DENV-1-infected splenic monocytes. The present study confirms the immunogenicity of multi-epitope DENV vaccine, suggesting that it may contribute to the development of a universal DENV vaccine.

Comparative Genomic Analysis of Enterovirus 71 Revealed Six New Potential Neurovirulence-associated Sites.

In the present study, the complete genomes of four common (4/EV71/Wenzhou/CHN/2014, 15/ EV71/Wenzhou/CHN/2014, 116/EV71/Wenzhou/ CHN/2014, and 120/EV71/Wenzhou/CHN/2014) and two virulent (11/EV71/Wenzhou/CHN/2014 and 109/EV71/Wenzhou/CHN/2014) enterovirus 71 (EV71) isolates were sequenced and described. They are 7405 bp in length and belong to EV71 sub-genotype C4 (C4a cluster). Nucleotide sequence alignment revealed six nucleotide variations (GP151→TP151, GP199→AP199, GP261→TP261, AP328→CP328, GP422→AP422, and GP437→TP437) in the two virulent isolates within the 5'UTR of the IRES element. RNA secondary structure predictions of IRES and FCE indicated that the common isolates shared similar structures, which were different from those of the virulent isolates. Moreover, the GP114→CP114 and GP151→TP151 mutations in the virulent isolates contributed to the formation of the unique RNA secondary structures in SL II. Furthermore, nucleotide/amino acid sequence alignments of 82 EV71 isolates indicated that six sites (TP488 and CP577 in the 5'UTR; AsnP57 in 2A; IleP56 in 3C; CP10 and AP47 in the 3'UTR) are potentially associated with the neurovirulence of EV71. Finally, the 3D structures of 2A were analogous, whereas the structures of VP1 and 3C were variable.

Development of reverse-transcription loop-mediated isothermal amplification assay for rapid detection and differentiation of dengue virus serotypes 1-4.

BACKGROUND: Dengue virus (DENV), the most widely prevalent arbovirus, continues to be a threat to human health in the tropics and subtropics. Early and rapid detection of DENV infection during the acute phase of illness is crucial for proper clinical patient management and preventing the spread of infection. The aim of the current study was to develop a specific, sensitive, and robust reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay for detection and differentiation of DENV1-4 serotypes. RESULTS: The method detection primers, which were designed to target the different DENV serotypes, were identified by inspection of multiple sequence alignments of the non-structural protein (NS) 2A of DENV1, NS4B of DENV2, NS4A of DENV3 and the 3' untranslated region of the NS protein of DENV4. No cross-reactions of the four serotypes were observed during the tests. The detection limits of the DENV1-4-specific RT-LAMP assays were approximately 10-copy templates per reaction. The RT-LAMP assays were ten-fold more sensitive than RT-PCR or real-time PCR. The diagnostic rate was 100% for clinical strains of DENV, and 98.9% of the DENV-infected patients whose samples were tested were detected by RT-LAMP. Importantly, no false-positives were detected with the new equipment and methodology that was used to avoid aerosol contamination of the samples. CONCLUSION: The RT-LAMP method used in our study is specific, sensitive, and suitable for further investigation as a useful alternative to the current methods used for clinical diagnosis of DENV1-4, especially in hospitals and laboratories that lack sophisticated diagnostic systems.

Identification of Mycobacterium tuberculosis PPE68-specific HLA-A*0201-restricted epitopes for tuberculosis diagnosis.

PPE68 is a Mycobacterium tuberculosis-specific protein which is absent from the vaccine strains of BCG. A panel of 14 PPE68-derived peptides predicted to bind to HLA-A*0201 was synthesized. The HLA-A*0201 restriction of these peptides was determined in T2 cell line and HLA-A*0201 transgenic mice. The specificity of peptides was assessed in pulmonary tuberculosis (TB) patients using IFN-γ enzyme-linked immunospot (ELISPOT) assay, and immunodominant peptides were further used to evaluate their diagnostic potential in HLA-A*0201-positive pulmonary TB patients. 13 out of 14 peptides were identified as high-affinity binders. Of these peptides, 12 peptides induced significant IFN-γ-secreting T cell response in transgenic mice and 9 peptides were efficiently recognized by peripheral blood mononuclear cells of 10 HLA-A*0201-positive TB patients. Four immunodominant HLA-A*0201-restricted epitopes (PPE68126-134, PPE68133-141, PPE68140-148, and PPE68148-156) were recognized by the most of 80 HLA-A*0201-positive TB patients (81, 86, 74, and 84 %, respectively). These epitopes may be used for a potential diagnosis of M. tuberculosis infection.

Japanese encephalitis virus E protein domain III immunization mediates cross-protection against Zika virus in mice via antibodies and CD8+T cells.

Zika virus (ZIKV) and Japanese encephalitis virus (JEV) are antigenically related flaviviruses that co-circulate in many countries/territories. The interaction between the two viruses needs to be determined. Recent findings by ourselves and other labs showed that JEV-elicited antibodies (Abs) and CD8+T cells exacerbate and protect against subsequent ZIKV infection, respectively. However, the impact of JEV envelope (E) protein domain III (EDIII)-induced immune responses on ZIKV infection is unclear. We show here that sera from JEV-EDIII-vaccinated mice cross-react with ZIKV-EDIII in vitro, and transfer of the same sera to mice significantly decreases death upon lethal ZIKV infection at a dose-dependent manner. Maternally acquired anti-JEV-EDIII Abs also significantly reduce the mortality of neonatal mice born to JEV-EDIII-immune mothers post ZIKV challenge. Similarly, transfer of ZIKV-EDIII-reactive IgG purified from JEV-vaccinated humans increases the survival of ZIKV-infected mice. Notably, transfer of an extremely low volume of JEV-EDIII-immune sera or ZIKV-EDIII-reactive IgG does not mediate the Ab-mediated enhancement (ADE) of ZIKV infection. Similarly, transfer of JEV-EDIII-elicited CD8+T cells protects recipient mice against ZIKV challenge. These results demonstrate that JEV-EDIII-induced immune components including Abs and T cells have protective roles in ZIKV infection, suggesting EDIII is a promising immunogen for developing effective and safety JEV vaccine.

Efficacy and safety of recombinant Mycobacterium tuberculosis ESAT-6 protein for diagnosis of pulmonary tuberculosis: a phase II trial.

BACKGROUND: This study aimed to determine the efficacy and safety of recombinant Mycobacterium tuberculosis ESAT-6 protein for diagnosis of pulmonary tuberculosis (TB). MATERIAL AND METHODS: A phase II trial was performed in 158 patients with pulmonary TB (145 initially-treated and 13 re-treated) and 133 healthy subjects. Skin testing was carried out by injecting purified protein derivative (PPD) (on left forearm) or recombinant ESAT-6 protein at a dosage of 2, 5, or 10 µg/mL (on the right forearm) in each subject. Reaction activity and adverse events were monitored at 24, 48, and 72 h following the injection. Receiver operating characteristic curves were plotted to determine the areas under the curves (AUCs) and the cut-off induration diameters for the optimal diagnostic performance. RESULTS: The reaction activity was significantly increased upon recombinant ESAT-6 injection in pulmonary TB patients compared with healthy subjects. In pulmonary TB patients, the reaction was dose-dependent, and at 48 h, 10 µg/mL recombinant ESAT-6 produced a reaction similar to that produced by PPD. The AUCs for a 10 µg/mL dosage were 0.9823, 0.9552, and 0.9266 for 24 h, 48 h, and 72 h, respectively, and the induration diameters of 4.5-5.5 mm were the optimal trade-off values between true positive rates and false positive rates. No serious adverse events occurred in any subjects. CONCLUSIONS: Recombinant ESAT-6 protein is efficacious and safe for diagnosing pulmonary TB. Based on the reaction, performance, safety, and practicability, we recommend that 10 µg/mL at 48 h with an induration cut-off value of 5.0 mm be used.

HLA-A*0201-restricted CD8+ T-cell epitopes identified in dengue viruses.

BACKGROUND: All four dengue virus (DV) serotypes (D1V, D2V, D3V and D4V) can cause a series of disorders, ranging from mild dengue fever (DF) to severe dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS). Previous studies have revealed that DV serotype-specific CD8(+) T cells are involved in controlling DV infection. Serotype cross-reactive CD8(+) T-cells may contribute to the immunopathogenesis of DHF/DSS. The aim of the study was to identify HLA-A*0201-binding peptides from four DV serotypes. We then examined their immunogenicity in vivo and cross-reactivity within heterologous peptides. METHODS: D1V-derived candidate CD8(+) T-cell epitopes were synthesized and evaluated for their affinity to the HLA-A*0201 molecule. Variant peptides representing heterologous D2V, D3V, D4V serotypes were synthesized. The immunogenicity of the high-affinity peptides were evaluated in HLA-A*0201 transgenic mice. RESULTS: Of the seven D1V-derived candidate epitopes [D1V-NS4a(56-64)(MLLALIAVL), D1V-C(46-54)(LVMAFMAFL), D1V-NS4b(562-570)(LLATSIFKL), D1V-NS2a(169-177)(AMVLSIVSL), D1V-NS4a(140-148)(GLLFMILTV), D1V-NS2a(144-152)(QLWAALLSL) and D1V-NS4b(183-191)(LLMRTTWAL)], three peptides [D1V-NS4a(140-148), D1V-NS2a(144-152) and D1V-NS4b(183-191)] had a high affinity for HLA-A*0201 molecules. Moreover, their variant peptides for D2V, D3V and D4V [D2V-NS4a(140-148)(AILTVVAAT), D3V-NS4a(140-148)(GILTLAAIV), D4V-NS4a(140-148)(TILTIIGLI), D2V-NS2a(144-152)(QLAVTIMAI), D3V-NS2a(144-152)(QLWTALVSL), D4V-NS2a(143-151)(QVGTLALSL), D2V-NS4b(182-190)(LMMRTTWAL), D3V-NS4b(182-190) (LLMRTSWAL) and D4V-NS4b(179-187)(LLMRTTWAF)] also had a high affinity for HLA-A*0201 molecules. Furthermore, CD8+ T cells directed to these twelve peptides were induced in HLA-A*0201 transgenic mice following immunization with these peptides. Additionally, cross-reactivity within four peptides (D1V-NS4b(183-191), D2V-NS4b(182-190), D3V-NS4b(182-190) and D4V-NS4b(179-187)) was observed. CONCLUSIONS: Two novel serotype-specific HLA-A*0201-restricted CD8(+) T-cell epitopes (NS4a(140-148) and NS2a(144-152)) and one cross-reactive HLA-A*0201-restricted CD8(+) T-cell epitopes which is similar to a previously identified epitope were identified in D1V-D4V. Combining prediction algorithms and HLA transgenic mice is an effective strategy to identify HLA-restricted epitopes. Serotype-specific epitopes would be used to determine the protective role of serotype-specific CD8(+) T cells, while cross-reactive epitopes may provide assistance in exploring the role of serotype cross-reactive CD8(+) T cells in the immunopathogenesis of DHF/DSS.

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.

Identification of conserved and HLA-A*2402-restricted epitopes in Dengue virus serotype 2.

In this study, we set out to identify dengue virus serotype 2 (DENV-2)-specific HLA-A*2402-restricted epitopes and determine the characteristics of T cells generated to these epitopes. We screened the full-length amino-acid sequence of DENV-2 to find potential epitopes using the SYFPEITHI algorithm. Twelve putative HLA-A*2402-binding peptides conserved in hundreds of DENV-2 strains were synthesized, and the HLA restriction of peptides was tested in HLA-A*2402 transgenic mice. Nine peptides (NS4b(228-237), NS2a(73-81), E(298-306), M(141-149), NS4a(96-105), NS4b(159-168), NS5(475-484), NS1(162-171), and NS5(611-620)) induced high levels of peptide-specific IFN-γ-secreting cells in HLA-A*2402 transgenic mice. Apart from IFN-γ, NS4b(228-237-), NS2a(73-81-) and E(298-306)-specific CD8(+) cells produced TNF-α and IL-6 simultaneously, whereas M(141-149-) and NS5(475-484-) CD8(+) cells produced only IL-6. Moreover, splenic mononuclear cells (SMCs) efficiently recognized and killed peptide-pulsed splenocytes. Furthermore, each of nine peptides could be recognized by splenocytes from DENV-2-infected HLA-A*2402 transgenic mice. The SMCs from HLA-A*2402 transgenic mice immunized with nine immunogenic peptides efficiently killed DENV-2-infected splenic monocytes. The present identified epitopes have the potential to be new diagnostic tools for characterization of T-cell immunity in DENV infection and may serve as part of a universal epitope-based vaccine.

Two novel squamous cell carcinoma antigen-derived HLA-A*0201-binding peptides induce in vitro and in vivo CD8+ cytotoxic T lymphocyte responses.

Squamous cell carcinoma antigen (SCCA) is overexpressed in many squamous cell cancers and SCCA­derived peptide-specific CD8(+) cytotoxic T lymphocytes can display cytotoxicity against tumor cells. In the present study, we screened the SCCA amino acid sequence for potential HLA-A*0201-binding CD8(+) T­cell epitopes using two predictive computational algorithms. Seven epitope candidates were selected of which SCCA(246-254)(llpneidgl), SCCA(223-231)(sledvqakv), SCCA(328­336)(vlhkafvev) and SCCA(324­332)(vlsgvlhka) significantly stabilized HLA-A*0201 molecules on T2 cells. Both SCCA(328­336) and SCCA(324-332) induced CD8(+) IFN-γ(+) T­cell responses in HLA-A*0201-positive peripheral blood mononuclear cells as assessed by intracellular cytokine staining. Consistent with this, immunization with either SCCA(328-336) or SCCA(324­332) effectively elicited CD8(+) IFN-γ(+) T cells in HLA-A*0201 transgenic mice as visualized by IFN-γ ELISPOT assay and intracellular cytokine staining. Furthermore, CD8(+) T cells induced in vitro or in vivo by SCCA(328-336) or SCCA(324-332) demonstrated in vitro cytotoxicity against peptide-pulsed T2 cells and splenocytes, respectively. These novel SCCA­derived CD8(+) T­cell epitopes described, herein, may be potentially important components for diagnostic reagents and immunotherapeutic vaccines for the treatment of squamous cell carcinomas.

CD4+ CD25+ regulatory T cells: a therapeutic target for liver diseases.

BACKGROUND: Regulatory T cells (Tregs) have been shown to play an important role in maintaining peripheral immune homeostasis by suppressing autoreactive and allergen-specific T cells and turning off the immune response after the pathogen has been cleared. However, in certain situations Tregs can impair effective immunity to some pathogens and tumour cells. OBJECTIVE: To review the role of Tregs in liver pathology and to assess the potential to enhance or inhibit their function as applied to the treatment of liver disease. METHODS: The literature was reviewed using standard indexing terms and incorporating publications up to and including those published in 2007. RESULTS/CONCLUSIONS: Tregs are therapeutic targets for modulation in autoimmune disease and may provide new opportunities for application to human liver conditions.

Genomic analysis of a Chinese isolate of Getah-like virus and its phylogenetic relationship with other Alphaviruses.

An alphavirus, M-1 strain, was isolated from a pool of culicine mosquitoes collected in Hainan island of China during an arbovirus survey in 1964. In the present study, we determined the complete nucleotide sequence of the M-1 strain using RT-PCR and RACE techniques. The M-1 genome is 11,690 nucleotides (nt) in length and contains two open reading frames (ORFs) encoding four nonstructural proteins and five structural proteins, respectively. Searches using Blast and comparison analyses suggested that M-1 is closely linked to Sagiyama virus (SAGV, AB032553) with 98% identity and Getah viruse (GETV, AY702913) with 97.8% identity in the full-length nucleotide sequence. However, compared with SAGV, there is 1 deletion (3 nucleotides in length) in the Capsid region, a deletion in the 3' untranslated region (10 nucleotides in length) and 2 insertions in the 3' untranslated region involving a total of 5 nucleotides. Interestingly, from the 5' UTR to the end of coding region, M-1 share the highest identity with GETV, even though the identity of 3' UTR drops dramatically to 76.2%. Furthermore, phylogenetic analysis based on the complete genomic sequences and sequences for structural or non-structural proteins of M-1 and 15 alphaviruses showed that M-1 grouped with GETV first and then grouped together with SAGV. Based on the comparison analysis and phylogenetic analysis, we conclude that M-1 strain can be considered as a strain that is a Chinese isolate of Getah-like virus.