Shuguang Tan

Nanozyme-strip for rapid local diagnosis of Ebola

Ebola continues to rage in West Africa. In the absence of an approved vaccine or treatment, the priority in controlling this epidemic is to promptly identify and isolate infected individuals. To this end, a rapid, highly sensitive, and easy-to-use test for Ebola diagnosis is urgently needed. Here, by using Fe3O4 magnetic nanoparticle (MNP) as a nanozyme probe, we developed a MNP-based immunochromatographic strip (Nanozyme-strip), which detects the glycoprotein of Ebola virus (EBOV) as low as 1 ng/mL, which is 100-fold more sensitive than the standard strip method. The sensitivity of the Nanozyme-strip for EBOV detection and diagnostic accuracy for New Bunyavirus clinical samples is comparable with ELISA, but is much faster (within 30 min) and simpler (without need of specialist facilities). The results demonstrate that the Nanozyme-strip test can rapidly and sensitively detect EBOV, providing a valuable simple screening tool for diagnosis of infection in Ebola-stricken areas.

Revival of the identification of cytotoxic T-lymphocyte epitopes for immunological diagnosis, therapy and vaccine development

Immunogenic T-cell epitopes have a central role in the cellular immunity against pathogens and tumors. However, in the early stage of cellular immunity studies, it was complicated and time-consuming to identify and characterize T-cell epitopes. Currently, the epitope screening is experiencing renewed enthusiasm due to advances in novel techniques and theories. Moreover, the application of T-cell epitope-based diagnoses for tuberculosis and new data on epitope-based vaccine development have also revived the field. There is a growing knowledge on the emphasis of epitope-stimulated T-cell immune responses in the elimination of pathogens and tumors. In this review, we outline the significance of the identification and characterization of T-cell epitopes. We also summarize the methods and strategies for epitope definition and, more importantly, address the relevance of cytotoxic T-lymphocyte epitopes to clinical diagnoses, therapy and vaccine development.

Angiotensin II plasma levels are linked to disease severity and predict fatal outcomes in H7N9-infected patients

A novel influenza A (H7N9) virus of avian origin emerged in eastern China in the spring of 2013. This virus causes severe disease in humans, including acute and often lethal respiratory failure. As of January 2014, 275 cases of H7N9-infected patients had been reported, highlighting the urgency of identifying biomarkers for predicting disease severity and fatal outcomes. Here, we show that plasma levels of angiotensin II, a major regulatory peptide of the renin–angiotensin system, are markedly elevated in H7N9 patients and are associated with disease progression. Moreover, the sustained high levels of angiotensin II in these patients are strongly correlated with mortality. The predictive value of angiotensin II is higher than that of C-reactive protein and some clinical parameters such as the PaO2/FiO2 ratio (partial pressure of arterial oxygen to the fraction of inspired oxygen). Our findings indicate that angiotensin II is a biomarker for lethality in flu infections. Supplementary information The online version of this article (doi:10.1038/ncomms4595) contains supplementary material, which is available to authorized users.

CD8+ T Cell Immune Response in Immunocompetent Mice during Zika Virus Infection

ABSTRACT Zika virus (ZIKV) infection causees neurologic complications, including Guillain-Barré syndrome in adults and central nervous system (CNS) abnormalities in fetuses. We investigated the immune response, especially the CD8+ T cell response in C57BL/6 (B6) wild-type (WT) mice, during ZIKV infection. We found that a robust CD8+ T cell response was elicited, major histocompatibility complex class I-restricted CD8+ T cell epitopes were identified, a tetramer that recognizes ZIKV-specific CD8+ T cells was developed, and virus-specific memory CD8+ T cells were generated in these mice. The CD8+ T cells from these infected mice were functional, as evidenced by the fact that the adoptive transfer of ZIKV-specific CD8+ T cells could prevent ZIKV infection in the CNS and was cross protective against dengue virus infection. Our findings provide comprehensive insight into immune responses against ZIKV and further demonstrate that WT mice could be a natural and easy-access model for evaluating immune responses to ZIKV infection. IMPORTANCE ZIKV infection has severe clinical consequences, including Guillain-Barré syndrome in adults, microcephaly, and congenital malformations in fetuses and newborn infants. Therefore, study of the immune response, especially the adaptive immune response to ZIKV infection, is important for understanding diseases caused by ZIKV infection. Here, we characterized the CD8+ T cell immune response to ZIKV in a comprehensive manner and identified ZIKV epitopes. Using the identified immunodominant epitopes, we developed a tetramer that recognizes ZIKV-specific CD8+ T cells in vivo, which simplified the detection and evaluation of ZIKV-specific immune responses. In addition, the finding that tetramer-positive memory CD8+ T cell responses were generated and that CD8+ T cells can traffic to a ZIKV-infected brain greatly enhances our understanding of ZIKV infection and provides important insights for ZIKV vaccine design.

The Serum Profile of Hypercytokinemia Factors Identified in H7N9-Infected Patients can Predict Fatal Outcomes

The novel avian origin influenza A (H7N9) virus has caused severe diseases in humans in eastern China since the spring of 2013. Fatal outcomes of H7N9 infections are often attributed to the severe pneumonia and acute respiratory distress syndrome (ARDS). There is urgent need to discover biomarkers predicting the progression of disease and fatal outcome of potentially lethal flu infections, based on sound statistical analysis. We discovered that 34 of the 48 cytokines and chemokines examined in this study were significantly elevated in the plasma samples from patients infected with H7N9. We report for the first time that the levels of MIF, SCF, MCP-1, HGF, and SCGF-β are highly positively linked to disease severity and the profile of mediators MIF, SCF, MCP-1, HGF, SCGF-β, IP-10, IL-18, and IFN-γ is an independent outcome predictor.

Cross-Allele Cytotoxic T Lymphocyte Responses against 2009 Pandemic H1N1 Influenza A Virus among HLA-A24 and HLA-A3 Supertype-Positive Individuals

ABSTRACT Lack of a universal vaccine against all serotypes of influenza A viruses and recent progress on T cell-related vaccines against influenza A virus illuminate the important role of human leukocyte antigen (HLA)-restricted cytotoxic T lymphocytes (CTLs) in anti-influenza virus immunity. However, the diverse HLA alleles among humans complicate virus-specific cellular immunity research, and elucidation of cross-HLA allele T cell responses to influenza virus specificity requires further detailed work. An ideal CTL epitope-based vaccine would cover a broad spectrum of epitope antigens presented by most, if not all, of the HLAs. Here, we evaluated the 2009 pandemic influenza A (H1N1) virus-specific T cell responses among the HLA-A24+ population using a rationally designed peptide pool during the 2009 pandemic. Unexpectedly, cross-HLA allele T cell responses against the influenza A virus peptides were detected among both HLA-A11+ and HLA-A24+ donors. Furthermore, we found cross-responses in the entire HLA-A3 supertype population (including HLA-A11, -A31, -A33, and -A30). The cross-allele antigenic peptides within the peptide pool were identified and characterized, and the crystal structures of the major histocompatibility complex (MHC)-peptide complexes were determined. The subsequent HLA-A24-defined cross-allele peptides recognized by the HLA-A11+ population were shown to mildly bind to the HLA-A*1101 molecule. Together with the structural models, these results partially explain the cross-allele responses. Our findings elucidate the promiscuity of the cross-allele T cell responses against influenza A viruses and are beneficial for the development of a T cell epitope-based vaccine applied in a broader population.

An unexpected similarity between antibiotic-resistant NDM-1 and beta-lactamase II from Erythrobacter litoralis

NDM-1 (New Delhi metallo-beta-lactamase) gene encodes a metallo-beta-lactamase (MBL) with high carbapenemase activity, which makes the host bacterial strain easily dispatch the last-resort antibiotics known as carbapenems and cause global concern. Here we present the bioinformatics data showing an unexpected similarity between NDM-1 and beta-lactamase II from Erythrobacter litoralis, a marine microbial isolate. We have further expressed these two mature proteins in E. coli cells, both of which present as a monomer with a molecular mass of 25 kDa. Antimicrobial susceptibility assay reveals that they share similar substrate specificities and are sensitive to aztreonam and tigecycline. The conformational change accompanied with the zinc binding visualized by nuclear magnetic resonance, Zn2+-bound NDM-1, adopts at least some stable tertiary structure in contrast to the metal-free protein. Our work implies a close evolutionary relationship between antibiotic resistance genes in environmental reservoir and in the clinic, challenging the antimicrobial resistance monitoring.

Conserved epitopes dominate cross‐CD8+ T‐cell responses against influenza A H1N1 virus among Asian populations

Novel strains of influenza A viruses (IAVs) have emerged with high infectivity and/or pathogenicity in recent years, causing worldwide concern. T cells are correlated with protection in humans through cross‐reactive immunity against heterosubtypes of IAV. However, the different hierarchical roles of IAV‐derived epitopes with distinct levels of polymorphism in the cross‐reactive T‐cell responses against IAV remain elusive. In this study, immunodominant epitopes scattered throughout the entire proteome of 2009 pandemic influenza A H1N1 virus and seasonal IAVs were synthesized and divided into different pools depending on their conservation. The overall profile of the IAV‐specific CD8+ T‐cell immunity was detected by utilizing these peptide pools and also individual peptides. A dominant role of the conserved peptide‐specific T‐cell immunity was illuminated within the anti‐IAV responses, while the CD8+ T‐cell responses against the variable epitopes were lower than the conserved peptides. As previously demonstrated within a Caucasian population, we determined that GL9‐specific T cells, which also utilize Vβ 17 TCR (BV19), play a pivotal role in IAV‐specific T‐cell immunity within an HLA‐A2+ Asian population. Our study objectively reveals the different predominant roles of T‐cell epitopes among IAV‐specific cross‐reactive cellular immunity. This may guide the development of vaccines with cross‐T‐cell immunogenicity against heterosubtypes of IAV.

Distinct PD-L1 binding characteristics of therapeutic monoclonal antibody durvalumab

Blockade of PD-1/PD-L1 signaling pathway by monoclonal antibodies (MAbs) to release the anti-tumor activity of preexisting tumor specific T cell immunity has initiated a new era for tumor immunotherapy. Administration of anti-PD-1 MAbs (nivolumab and pembrolizumab) in either monotherapy or in combination with anti-CTLA-4 MAbs or traditional chemotherapy has achieved a tumor regression rate of 30%–50% in dealing with melanoma, non-small cell lung cancer, etc. (Larkin et al., 2015). The approval of anti-PD-L1 atezolizumab and avelumab by US Food and Drug Administration (FDA) since 2016 has provided additional choices in dealing with multiple tumors aside from anti-PD-1 and anti-CTLA-4 MAbs as immunotherapeutic medication. The structures of the two therapeutic anti-PD-1 MAbs, nivolumab and pembrolizumab, complexed with PD-1 have been reported which elucidated the molecular basis of MAb-based anti-PD-1 immunotherapy (Tan et al., 2016a, b; Na et al., 2017; Tan et al., 2017). Complex structures of avelumab and BMS-936559 with PD-L1 were also reported which contributes a better understanding of the molecular basis of MAb-based anti-PD-L1 checkpoint blockade therapy (Lee et al., 2016; Liu et al., 2017). In addition, two additional anti-PD-L1 MAbs are in clinics or phase III trials, atezolizumab and durvalumab. Durvalumab (MEDI4736) is a fully human IgG1 MAb targeting PD-L1 that was developed by AstraZeneca, and has been approved by US FDA very recently. Multiple Phase III clinical trials are still ongoing in non-small cell lung cancer, head and neck cancer, urothelial cancer, etc. (NCT02542293, NCT02369874, NCT02516241, etc.). A Phase Ib report demonstrated that durvalumab is well tolerated and showed promising anti-tumor efficacy in nonsmall cell lung cancer patients (Antonia et al., 2016). However, the molecular basis of durvalumab-based anti-PD-L1 reactivity and binding characteristics compared to the other three MAbs used in clinics has not yet been elucidated. In the present study, weexpressed the two-Ig-domain PD-L1 and single chain Fv fragment (scFv) of durvalumab as inclusion bodies in Escherichia coli cells. Soluble proteins were obtained by in vitro refolding, and the two refolded proteins survived well in gel filtration (Fig. S1). Subsequently, crystal screen was performed with the durvalumab-scFv/PD-L1 complex proteins, and well-diffractable crystals grew in 3.5 mol/L sodium formate, pH 7.0 (See more details in supplementary information). The complex structure of durvalumab-scFv/PD-L1 was determined by molecular replacement at a resolution of 2.3 Å (Table S1). The binding of durvalumab to PD-L1 involves both of its heavy chain (VH) and light chain (VL) (Fig. 1A). All of the three complementarity-determining regions (CDRs) of VH and CDR1 and CDR3 of VL contribute to interactions with PD-L1, leaving LCDR2without any contacts. Previous reports on the anti-PD-1MAbs revealed that the binding of theseMAb ismainly located on the loops of PD-1, i.e., the N-terminal loop of PD-1 for nivolumab interaction and the C’D loop for pembrolizumab. However, the binding of avelumab and BMS936559 is mainly located on the strands of the front-β-sheet face of PD-L1. Here, the binding of durvalumab on PD-L1 was also mainly located on the front β-sheet face which is constituted by A, G, F, C, and C’ strands of the IgV domain of PD-L1. A detailed analysis of the interactions between durvalumab and PD-L1 shows an unbiased contribution from VH and VL of durvalumab in binding to PD-L1. The A, G, and F strands of PD-L1 provide major hydrogen bond interactions with durvalumab (Fig. 1B). D26 of the A strand andR113 of the F strand of PD-L1were occupied by S30 of LCDR1 andE58 of HCDR2, respectively (Table S2). Especially, residues of the G strand (Y123, K124, and R125) provide multiple hydrogen bonds to both VH (F104 of HCDR3 and N51 nearby HCDR2) and VL (Y92 and S94 of LCDR3), which contribute major hydrogen bond interactions to durvalumab, 7 out of 10 hydrogen bonds in all (Table S2). Structural superimposition of the PD-1/PD-L1 complex (PDB: 4ZQK) and the durvalumab-scFv/PD-L1 complex was conducted to elucidate the molecular basis of durvalumabbased PD-1/PD-L1 intervention. The binding of durvalumab to PD-L1 shows a stereo clash with that of PD-1 (Fig. 1C). The binding surface of durvalumab and PD-1 on PD-L1 is highly overlapped that residues of PD-L1, which contributed major hydrogen bond interactions with PD-1 (D26, Y123, K124, and R125), were also occupied by durvalumab (Fig. 1D) (Zak et al., 2015). The competitive binding involves both VH and VL of durvalumab. Thus, the molecular basis of

Structural basis of cross-allele presentation by HLA-A*0301 and HLA-A*1101 revealed by two HIV-derived peptide complexes

Human leukocyte antigens (HLA) are initially classified by serotyping but recently can be re-grouped by their peptide-presentation characteristics into supertypes. Both HLA-A*0301 and HLA-A*1101 are grouped into A3 supertype. Although a number of cross-presented T cell epitopes of HLA-A*0301 and HLA-A*1101 have been identified, the molecular mechanisms of cross-presentation remain elusive. Herein, the structures of HLA-A*0301 with two HIV-derived immunodominant T cell epitopes were solved and their characteristics in comparison with HLA-A*1101 presenting the same peptides were analyzed. The comparable structures of HLA-A*0301 and HLA-A*1101 with subtle differences illustrate the common modes of cross-presented peptides and the strict HLA-restriction of T cell receptor (TCR)-recognition.