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.

An optimal thoracoscopic segmentectomy approach: Combined ultra-high-definition 4K endovision systems with "no-waiting" technique in S8-9 complex segmentectomy.

Thoracoscopic segmentectomy might be an alternative to lobectomy for small size lung cancer. Precise identification of the pulmonary intersegmental plane was needed for an optimal segmentectomy. Recently, (1) the ultra-high-definition 4K systems had claimed to overcome the lack of depth perception by secondary visual cues; (2) the no-waiting procedure was induced as an alternative and optimized method for identifying the plane. It was unclear whether combined ultra-high-definition 4K endovision systems with "no-waiting" technique in thoracoscopic segmentectomy could achieve an excellent result. A 68-year-old female patient was admitted into our hospital for occasional pulmonary nodule during her routine physical examination. The nodule is located between S8 and S9 segment, and was suspected to be an early-stage lung cancer. She underwent a thoracoscopic S89 complex segmentectomy using ultra-high-definition 4K endovision systems and "no-waiting" surgical technique. The intersegmental plane was clearly detected and easily treated by the endoscopic linear cutting staplers. The patient recovered well and was discharged without complications. Combining ultra-high-definition 4K endovision systems with "no-waiting" technique seems to be an optimal thoracoscopic segmentectomy approach for the management of lung cancers.

Integrating multifunctional catalytic sites in COF@ZIF-67 derived carbon for the HER and ORR.

Covalent organic framework (COF)-derived carbons have various applications in electrochemical energy storage and conversion systems. In this work, we have constructed a highly active catalyst (Ir-COF@ZIF800) for the HER and ORR by integrating multifunctional sites (Ir nanoparticles, Co-N sites and Co nanoparticles) into core-shell COF@ZIF-67-derived carbon.

Cellulose based hyper-crosslinked polymer for efficiently recovering valuable materials from PO/SM wastewater.

Herein, a TEMPO-oxidized cellulose-grafted-polystyrene hypercrosslinked polymer (TOC-PS-HCP) was synthesized facilely by TEMPO oxidation, grafting copolymerization and post crosslinking route. Based on the structural characterization, it was confirmed that TOC-PS-HCP mainly consisted of polystyrene chain on cellulose and rigid crosslinked bridge. Additionally, the as-prepared TOC-PS-HCP displayed appropriate hydrophobicity (water contact angle = 102.44°) and high specific surface area (SBET = 601.20 m2·g--1), which could efficiently recover ethylbenzene and styrene from PO/SM wastewater. The adsorption experiment was conducted to study the recovery performance for ethylbenzene and styrene in the aqueous phase. The results showed that TOC-PS-HCP could recover ethylbenzene and styrene quickly by adsorption process, and maintain a stable recovery rate both in different aqueous conditions and recycle experiments. The adsorption experiment in the simulated wastewater solution showed that TOC-PS-HCP exhibited the greater affinity for ethylbenzene and styrene than other substrates. Furthermore, a possible mechanism for the efficient recovery of ethylbenzene and styrene was suggested on the basis of experimental and theoretical results, which may be associated with van der Waals force and π-π stacking.

Japanese encephalitis virus-primed CD8+ T cells prevent antibody-dependent enhancement of Zika virus pathogenesis.

Cross-reactive anti-flaviviral immunity can influence the outcome of infections with heterologous flaviviruses. However, it is unclear how the interplay between cross-reactive antibodies and T cells tilts the balance toward pathogenesis versus protection during secondary Zika virus (ZIKV) and Japanese encephalitis virus (JEV) infections. We show that sera and IgG from JEV-vaccinated humans and JEV-inoculated mice cross-reacted with ZIKV, exacerbated lethal ZIKV infection upon transfer to mice, and promoted viral replication and mortality upon ZIKV infection of the neonates born to immune mothers. In contrast, transfer of CD8+ T cells from JEV-exposed mice was protective, reducing the viral burden and mortality of ZIKV-infected mice and abrogating the lethal effects of antibody-mediated enhancement of ZIKV infection in mice. Conversely, cross-reactive anti-ZIKV antibodies or CD8+ T cells displayed the same pathogenic or protective effects upon JEV infection, with the exception that maternally acquired anti-ZIKV antibodies had no effect on JEV infection of the neonates. These results provide clues for developing safe anti-JEV/ZIKV vaccines.

Dengue virus envelope protein domain III-elicited antibodies mediate cross-protection against Zika virus in a mouse model.

Dengue virus (DENV) and Zika virus (ZIKV) are antigenically related mosquito-transmitted viruses which represent a big public health problem. Although the antigenic cross-reactivity between two viruses were intensively investigated at the antibody and T cell levels, how DENV envelope protein domain III (EDIII)-elicited antibodies (Abs) impact the outcome of ZIKV infection is uncertain. Here, our results show that the sera isolated from DENV-EDIII-immunized wild-type mice recognized ZIKV-EDIII and cross-neutralized ZIKV in vitro. Passive transfer of DENV-EDIII-immune sera protected 1-day-old mice against lethal ZIKV challenge. Finally, maternally acquired anti-DENV-EDIII Abs significantly increased the survival of 1-day-old mice born to DENV-EDIII-immunized mothers post ZIKV challenge. These results reveal that DENV-EDIII-induced Abs provide cross-protection against ZIKV and may not mediate the Ab-dependent enhancement of ZIKV infection at the concentration used here. The present study would contribute to the development and application of DENV-EDIII-based vaccines.

[Preparation of mouse monoclonal antibodies against EV71 capsid protein VP1].

Objective To prepare enterovirus 71 (EV71) capsid protein VP1-specific murine monoclonal antibodies (mAbs) and determine their characteristics. Methods We synthesized EV71 VP1-encoding gene and cloned it into prokaryotic plasmid pET21a. Recombinant VP1 was used to immunize BALB/c mice; the splenocytes were fused with myeloma SP2/0 cell line and the hybridoma cells which could produce VP1-specific mAbs were screened using ELISA; the hybridoma cells were inoculated intraperitoneally into mice. VP1-specific mAbs were purified from ascites using protein G affinity chromatography, and mAb purifiy and specificity were detected by SDS-PAGE and Western blotting, respectively. Based on EV71-infected Vero cells, Western blotting and indirect immunofluorescence assay (IFA) were used to assess the ability of VP1-specific mAbs to recognize EV71. Results EV71-VP1 was successfully expressed and four VP1-specific mAb-producing hybridoma cells were generated (6E, 8D, 9F and 10C). Finally, mouse mAbs secreted by the hybridoma cells recognized EV71. Conclusion Four EV71-VP1-specific murine mAbs have been developed.

[Identification and application of Mycobacterium tuberculosis esxN-specific cell epitopes in the diagnosis of pulmonary tuberculosis].

Objective To identify Mycobacterium tuberculosis ESAT-6 protein esxN-specific HLA-A*0201-restricted CTL epitopes and assess the diagnostic potential of the identified epitopes in pulmonary tuberculosis. Methods The esxN-specific HLA-A*0201-restricted CTL epitopes were predicted by the T epitope prediction software SYFPEITHI and further synthesized. The binding affinity of the candidate epitopes for HLA-A*0201 was detected using MHC-peptide complex stabilization assay. The immunogenicity of candidate epitopes were assessed using ELISPOT in HLA-A*0201 transgenic mice. Based on identified CTL epitopes, ESAT-6 and culture filtrate protein-10 (CFP-10), the ELISPOT was performed to detect the frequency of epitope/protein-specific CTL. Results In six CTL epitope candidates we tested, two epitopes, esxN15-24 (AMIRAQAASL) and esxN48-57 (VACQEFITQL), were found to have a high affinity for HLA-A*0201. In the HLA-A*0201 transgenic mice immunized with the epitope candidates, esxN48-57 induced T-cell response with a significantly high IFN-γ secretion. The IFN-γ-producing T cells directed to esxN15-24 and esxN48-57 were found to be correlated with the presence of ESAT-6 and CFP-10 in positive pulmonary tuberculosis patients. The sensitivity of these tests for the esxN15-24 and esxN48-57 epitopes was similar to that of ESAT-6 and CFP-10. Conclusion Two novel Mycobacterium tuberculosis protein esxN-derived HLA-A*0201-restricted CTL epitopes have potential for the diagnosis of tuberculosis.

The diagnostic potential of MPT63-derived HLA-A*0201-restricted CD8+ T-cell epitopes for active pulmonary tuberculosis.

MPT63 protein is found only in Mycobacterium tuberculosis complex, including M. tuberculosis and M. bovis. Detection of MPT63-specific IFN-γ-secreting T cells could be useful for the diagnosis of tuberculosis (TB) diseases. In the present study, the HLA-A*0201 restriction of ten predicted MPT63-derived CD8(+) T-cell epitopes was assessed on the basis of T2 cell line and HLA-A*0201 transgenic mice. The diagnostic potential of immunogenic peptides in active pulmonary TB patients was evaluated using an IFN-γ enzyme-linked immunospot assay. It was found that five peptides bound to HLA-A*0201 with high affinity, whereas the remaining peptides exhibited low affinity for HLA-A*0201. Five immunogenic peptides (MPT6318-26 , MPT6329-37 , MPT6320-28 , MPT635-14 and MPT6310-19 ) elicited large numbers of cytotoxic IFN-γ-secreting T cells in HLA-A*0201 transgenic mice. Each of the five immunogenic peptides was recognized by peripheral blood mononuclear cells from 45% to 73% of 40 HLA-A*0201 positive TB patients. The total diagnostic sensitivity of the five immunogenic peptides was higher than that of a T-SPOT.TB assay (based on ESAT-6 and CFP-10) (93% versus 90%). It is noticeable that the diagnostic sensitivity of the combination of five immunogenic peptides and T-SPOT.TB assay reached 100%. These MPT63-derived HLA-A*0201-restricted CD8(+) T-cell epitopes would likely contribute to the immunological diagnosis of M. tuberculosis infection and may provide the components for designing an effective TB vaccine.

Identification of cytotoxic T lymphocyte epitopes in dengue virus serotype 1.

Dengue virus (DENV) has a serious and growing impact on global health and the exact role of DENV-specific CD8(+) T-cells in DENV infection is still uncertain. In the present study, SYFPEITHI algorithm was used to screen the amino acid sequence of Dengue virus serotype 1 (DENV-1) for potential epitopes, and seven putative HLA-A*1101-restricted and five putative HLA-A*2402-restricted epitopes conserved in hundreds of DENV-1 strains were synthesized. The binding affinity of these epitope candidates to corresponding HLA molecules was evaluated using competitive peptide-binding assay. The immunogenicity and specificity of peptides were further tested in HLA-A*1101 transgenic mice, HLA-A*2402 transgenic mice and peripheral blood mononuclear cells (PBMCs) of patients infected with DENV-1. Percentage inhibition (PI) values calculated in competitive peptide-binding assay showed that six peptides (E39-47 PTLDIELLK, NS5(505-513) GVEGEGLHK, NS2b(15-23) SILLSSLLK, NS5(561-569) ALLATSIFK, NS3(99-107) AVEPGKNPK, and NS4b(159-167) VVYDAKFEK) could bind to HLA-A*1101 molecule with high affinity and five peptides (NS3472-480 QYIYMGQPL, NS4a40-48 AYRHAMEEL, NS5(880-888) DYMTSMKRF, NS3(548-556) SYKVASEGF, and NS3(22-30) IYRILQRGL) have a high affinity for HLA-A*2402 molecule. Enzyme-linked immunospot (ELISPOT) results indicated that these high-affinity peptides were recognized by splenocytes of DENV-1-infected transgenic mice and high-affinity peptide-immunized transgenic mice displayed high levels of peptide-specific IFN-γ-secreting cells. In addition, both peptide-pulsed splenocytes and DENV-1-infected splenic monocytes were efficiently killed by these peptide-specific cytotoxic T lymphocytes. Finally, except NS2b(15-23), 10 high-affinity peptides were recognized by PBMCs of patients infected with DENV-1. These identified epitopes would contribute to the understanding of the function of DENV-specific CD8(+) T-cells.

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.

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.

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.

Identification of a dengue virus-specific HLA-A*0201-restricted CD8+ T cell epitope.

In this study, a combination of epitope-prediction programs and in vitro assays was used to identify dengue virus (DENV)-specific CD8(+) T cell epitopes. Peripheral blood mononuclear cells (PBMCs) isolated from patients who recovered from dengue fever were stimulated with candidate epitope peptides derived from DENV, which were predicted by using SYFPEITHI and RANKpep epitope-prediction programs. The IFN-gamma ELISpot results and the results of intracellular staining of IFN-gamma showed that peptides NS4b_40 (TLYAVATTI), E_256 (QEGAMHTAL), NS3_205 (LPAIVREAI), NS5_210 (SRNSTHEMY), and NS3_207 (AIVREAIKR) could induce the recall response of CD8(+) T cells. Furthermore, the results of the MHC-peptide complex stabilization assay revealed that peptide NS4b_40 (TLYAVATTI) has a high affinity for HLA-A*0201 molecules. The IFN-gamma ELISpot results and staining of intracellular IFN-gamma confirmed that this peptide could induce high-level CD8(+) T cell response in HLA-A*0201 positive PBMCs. Peptide NS4b_40 (TLYAVATTI) was identified as a novel DENV-specific HLA-A*0201-restricted CD8(+) T cell epitope.