Binding and molecular basis of the bat coronavirus RaTG13 virus to ACE2 in humans and other species.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been spreading worldwide, causing a global pandemic. Bat-origin RaTG13 is currently the most phylogenetically related virus. Here we obtained the complex structure of the RaTG13 receptor binding domain (RBD) with human ACE2 (hACE2) and evaluated binding of RaTG13 RBD to 24 additional ACE2 orthologs. By substituting residues in the RaTG13 RBD with their counterparts in the SARS-CoV-2 RBD, we found that residue 501, the major position found in variants of concern (VOCs) 501Y.V1/V2/V3, plays a key role in determining the potential host range of RaTG13. We also found that SARS-CoV-2 could induce strong cross-reactive antibodies to RaTG13 and identified a SARS-CoV-2 monoclonal antibody (mAb), CB6, that could cross-neutralize RaTG13 pseudovirus. These results elucidate the receptor binding and host adaption mechanisms of RaTG13 and emphasize the importance of continuous surveillance of coronaviruses (CoVs) carried by animal reservoirs to prevent another spillover of CoVs.

An engineered bispecific human monoclonal antibody against SARS-CoV-2.

The global severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic requires effective therapies against coronavirus disease 2019 (COVID-19), and neutralizing antibodies are a promising therapy. A noncompeting pair of human neutralizing antibodies (B38 and H4) blocking SARS-CoV-2 binding to its receptor, ACE2, have been described previously. Here, we develop bsAb15, a bispecific monoclonal antibody (bsAb) based on B38 and H4. bsAb15 has greater neutralizing efficiency than these parental antibodies, results in less selective pressure and retains neutralizing ability to most SARS-CoV-2 variants of concern (with more potent neutralizing activity against the Delta variant). We also selected for escape mutants of the two parental mAbs, a mAb cocktail and bsAb15, demonstrating that bsAb15 can efficiently neutralize all single-mAb escape mutants. Furthermore, prophylactic and therapeutic application of bsAb15 reduced the viral titer in infected nonhuman primates and human ACE2 transgenic mice. Therefore, this bsAb is a feasible and effective strategy to treat and prevent severe COVID-19.

Molecular Basis of Arthritogenic Alphavirus Receptor MXRA8 Binding to Chikungunya Virus Envelope Protein.

Arthritogenic alphaviruses, such as Chikungunya virus (CHIKV), cause severe and debilitating rheumatic diseases worldwide, resulting in severe morbidity and economic costs. Recently, MXRA8 was reported as an entry receptor. Here, we present the crystal structures of the mouse MXRA8, human MXRA8 in complex with the CHIKV E protein, and the cryo-electron microscopy structure of human MXRA8 and CHIKV virus-like particle. MXRA8 has two Ig-like domains with unique structural topologies. This receptor binds in the "canyon" between two protomers of the E spike on the surface of the virion. The atomic details at the interface between the two binding entities reveal that both the two domains and the hinge region of MXRA8 are involved in interaction with CHIKV E1-E2 residues from two protomers. Notably, the stalk region of MXRA8 is critical for CHIKV virus entry. This finding provides important information regarding the development of therapeutic countermeasures against those arthritogenic alphaviruses.

De novo design of protein interactions with learned surface fingerprints.

Physical interactions between proteins are essential for most biological processes governing life1. However, the molecular determinants of such interactions have been challenging to understand, even as genomic, proteomic and structural data increase. This knowledge gap has been a major obstacle for the comprehensive understanding of cellular protein-protein interaction networks and for the de novo design of protein binders that are crucial for synthetic biology and translational applications2-9. Here we use a geometric deep-learning framework operating on protein surfaces that generates fingerprints to describe geometric and chemical features that are critical to drive protein-protein interactions10. We hypothesized that these fingerprints capture the key aspects of molecular recognition that represent a new paradigm in the computational design of novel protein interactions. As a proof of principle, we computationally designed several de novo protein binders to engage four protein targets: SARS-CoV-2 spike, PD-1, PD-L1 and CTLA-4. Several designs were experimentally optimized, whereas others were generated purely in silico, reaching nanomolar affinity with structural and mutational characterization showing highly accurate predictions. Overall, our surface-centric approach captures the physical and chemical determinants of molecular recognition, enabling an approach for the de novo design of protein interactions and, more broadly, of artificial proteins with function.

Characterization of CD8+ T cells in immune-privileged organs of ZIKV-infected Ifnar1-/- mice.

Zika virus (ZIKV) infection caused neurological complications and male infertility, leading to the accumulation of antigen-specific immune cells in immune-privileged organs (IPOs). Thus, it is important to understand the immunological responses to ZIKV in IPOs. We extensively investigated the ZIKV-specific T cell immunity in IPOs in Ifnar1-/- mice, based on an immunodominant epitope E294-302 tetramer. The distinct kinetics and functions of virus-specific CD8+ T cells infiltrated into different IPOs were characterized, with late elevation in the brain and spinal cord. Single epitope E294-302-specific T cells can account for 20-60% of the total CD8+ T cells in the brain, spinal cord, and testicle and persist for at least 90 days in the brain and spinal cord. The E294-302-specific TCRαßs within the IPOs are featured with the majority of clonotypes utilizing TRAV9N-3 paired with diverse TRBV chains, but with distinct αß paired clonotypes in 7 and 30 days post-infection. Specific chemokine receptors, Ccr2 and Ccr5, were selectively expressed in the E294-302-specific CD8+ T cells within the brain and testicle, indicating an IPO-oriented migration of virus-specific CD8+ T cells after infection. Overall, this study adds to the understanding of virus-specific CD8+ T cell responses for controlling and clearing ZIKV infection in IPOs.IMPORTANCEThe immune-privileged organs (IPOs), such as the central nervous system and testicles, presented pathogenicity and inflammation after Zika virus (ZIKV) infection with infiltrated CD8+ T cells. Our data show that CD8+ T cells keep up with virus increases and decreases in immune-privileged organs. Furthermore, our study provides the first ex vivo comparative analyses of the composition and diversity related to TCRα/ß clonotypes across anatomical sites and ZIKV infection phases. We show that the vast majority of TCRα/ß clonotypes in tissues utilize TRAV9N-3 with conservation. Specific chemokine expression, including Ccr2 and Ccr5, was found to be selectively expressed in the E294-302-specific CD8+ T cells within the brain and testicle, indicating an IPO-oriented migration of the virus-specific CD8+ T cells after the infection. Our study adds insights into the anti-viral immunological characterization and chemotaxis mechanism of virus-specific CD8+ T cells after ZIKV infection in different IPOs.

Molecular Basis for the Recognition of HIV Nef138-8 Epitope by a Pair of Human Public T Cell Receptors.

Cross-recognized public TCRs against HIV epitopes have been proposed to be important for the control of AIDS disease progression and HIV variants. The overlapping Nef138-8 and Nef138-10 peptides from the HIV Nef protein are HLA-A24-restricted immunodominant T cell epitopes, and an HIV mutant strain with a Y139F substitution in Nef protein can result in immune escape and is widespread in Japan. Here, we identified a pair of public TCRs specific to the HLA-A24-restricted Nef-138-8 epitope using PBMCs from White and Japanese patients, respectively, namely TD08 and H25-11. The gene use of the variable domain for TD08 and H25-11 is TRAV8-3, TRAJ10 for the α-chain and TRBV7-9, TRBD1*01, TRBJ2-5 for the ß-chain. Both TCRs can recognize wild-type and Y2F-mutated Nef138-8 epitopes. We further determined three complex structures, including TD08/HLA-A24-Nef138-8, H25-11/HLA-A24-Nef138-8, and TD08/HLA-A24-Nef138-8 (2F). Then, we revealed the molecular basis of the public TCR binding to the peptide HLA, which mostly relies on the interaction between the TCR and HLA and can tolerate the mutation in the Nef138-8 peptide. These findings promote the molecular understanding of T cell immunity against HIV epitopes and provide an important basis for the engineering of TCRs to develop T cell-based immunotherapy against HIV infection.

Pneumonia Severity and Phase Linked to Virus-Specific T Cell Responses with Distinct Immune Checkpoints during pH1N1 Infection.

The detailed features and the longitudinal variation of influenza-specific T cell responses within naturally infected patients and the relationship with disease severity remain uncertain. In this study, we characterized the longitudinal influenza-specific CD4+ and CD8+ T cell responses, T cell activation, and migration-related cytokine/chemokine secretion in pH1N1-infected patients with or without viral pneumonia with human PBMCs. Both the influenza-specific CD4+ and CD8+ T cells presented higher responses in patients with severe infection than in mild ones, but with distinct longitudinal variations, phenotypes of memory markers, and immune checkpoints. At 7 ± 3 d after onset of illness, effector CD8+ T cells (CD45RA+CCR7-) with high expression of inhibitory immune receptor CD200R dominated the specific T cell responses. However, at 21 ± 3 d after onset of illness, effector memory CD4+ T cells (CD45RA-CCR7-) with high expression of PD1, CTLA4, and LAG3 were higher among the patients with severe disease. The specific T cell magnitude, T cell activation, and migration-related cytokines/chemokines possessed a strong connection with disease severity. Our findings illuminate the distinct characteristics of immune system activation during dynamic disease phases and its correlation with lung injury of pH1N1 patients.

Cross-species recognition of SARS-CoV-2 to bat ACE2.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has emerged as a major threat to global health. Although varied SARS-CoV-2-related coronaviruses have been isolated from bats and SARS-CoV-2 may infect bat, the structural basis for SARS-CoV-2 to utilize the human receptor counterpart bat angiotensin-converting enzyme 2 (bACE2) for virus infection remains less understood. Here, we report that the SARS-CoV-2 spike protein receptor binding domain (RBD) could bind to bACE2 from Rhinolophus macrotis (bACE2-Rm) with substantially lower affinity compared with that to the human ACE2 (hACE2), and its infectivity to host cells expressing bACE2-Rm was confirmed with pseudotyped SARS-CoV-2 virus and SARS-CoV-2 wild virus. The structure of the SARS-CoV-2 RBD with the bACE2-Rm complex was determined, revealing a binding mode similar to that of hACE2. The analysis of binding details between SARS-CoV-2 RBD and bACE2-Rm revealed that the interacting network involving Y41 and E42 of bACE2-Rm showed substantial differences with that to hACE2. Bats have extensive species diversity and the residues for RBD binding in bACE2 receptor varied substantially among different bat species. Notably, the Y41H mutant, which exists in many bats, attenuates the binding capacity of bACE2-Rm, indicating the central roles of Y41 in the interaction network. These findings would benefit our understanding of the potential infection of SARS-CoV-2 in varied species of bats.

Tumor cell-intrinsic PD-1 receptor is a tumor suppressor and mediates resistance to PD-1 blockade therapy.

The programmed cell death 1 (PD-1) receptor on the surface of immune cells is an immune checkpoint molecule that mediates the immune escape of tumor cells. Consequently, antibodies targeting PD-1 have shown efficacy in enhancing the antitumor activity of T cells in some types of cancers. However, the potential effects of PD-1 on tumor cells remain largely unknown. Here, we show that PD-1 is expressed across a broad range of tumor cells. The silencing of PD-1 or its ligand, PD-1 ligand 1 (PD-L1), promotes cell proliferation and colony formation in vitro and tumor growth in vivo. Conversely, overexpression of PD-1 or PD-L1 inhibits tumor cell proliferation and colony formation. Moreover, blocking antibodies targeting PD-1 or PD-L1 promote tumor growth in cell cultures and xenografts. Mechanistically, the coordination of PD-1 and PD-L1 activates its major downstream signaling pathways including the AKT and ERK1/2 pathways, thus enhancing tumor cell growth. This study demonstrates that PD-1/PD-L1 is a potential tumor suppressor and potentially regulates the response to anti-PD-1/PD-L1 treatments, thus representing a potential biomarker for the optimal cancer immunotherapeutic treatment.

N-glycosylation of PD-1 promotes binding of camrelizumab.

PD-1 is a highly glycosylated inhibitory receptor expressed mainly on T cells. Targeting of PD-1 with monoclonal antibodies (MAbs) to block the interaction with its ligand PD-L1 has been successful for the treatment of multiple tumors. However, polymorphisms at N-glycosylation sites of PD-1 exist in the human population that might affect antibody binding, and dysregulated glycosylation has been observed in the tumor microenvironment. Here, we demonstrate varied N-glycan composition in PD-1, and show that the binding affinity of camrelizumab, a recently approved PD-1-specific MAb, to non-glycosylated PD-1 proteins from E. coli is substantially decreased compared with glycosylated PD-1. The structure of the camrelizumab/PD-1 complex reveals that camrelizumab mainly utilizes its heavy chain to bind to PD-1, while the light chain sterically inhibits the binding of PD-L1 to PD-1. Glycosylation of asparagine 58 (N58) promotes the interaction with camrelizumab, while the efficiency of camrelizumab to inhibit the binding of PD-L1 is substantially reduced for glycosylation-deficient PD-1. These results increase our understanding of how glycosylation affects the activity of PD-1-specific MAbs during immune checkpoint therapy.

Binding mode of the side-by-side two-IgV molecule CD226/DNAM-1 to its ligand CD155/Necl-5.

Natural killer (NK) cells are important component of innate immunity and also contribute to activating and reshaping the adaptive immune responses. The functions of NK cells are modulated by multiple inhibitory and stimulatory receptors. Among these receptors, the activating receptor CD226 (DNAM-1) mediates NK cell activation via binding to its nectin-like (Necl) family ligand, CD155 (Necl-5). Here, we present a unique side-by-side arrangement pattern of two tandem immunoglobulin V-set (IgV) domains deriving from the ectodomains of both human CD226 (hCD226-ecto) and mouse CD226 (mCD226-ecto), which is substantially different from the conventional head-to-tail arrangement of other multiple Ig-like domain molecules. The hybrid complex structure of mCD226-ecto binding to the first domain of human CD155 (hCD155-D1) reveals a conserved binding interface with the first domain of CD226 (D1), whereas the second domain of CD226 (D2) both provides structural supports for the unique architecture of CD226 and forms direct interactions with CD155. In the absence of the D2 domain, CD226-D1 exhibited substantially reduced binding efficacy to CD155. Collectively, these findings would broaden our knowledge of the interaction between NK cell receptors and the nectin/Necl family ligands, as well as provide molecular basis for the development of CD226-targeted antitumor immunotherapeutics.

Clinical and Immunological Characteristics of Human Infections With H5N6 Avian Influenza Virus.

BACKGROUND: H5N6 avian influenza virus (AIV) has caused sporadic, recurring outbreaks in China and Southeast Asia since 2013, with 19 human infections and 13 deaths. Seventeen of these infections occurred since December 2015, indicating a recent rise in the frequency of H5N6 cases. METHODS: To assess the relative threat of H5N6 virus to humans, we summarized and compared clinical data from patients infected with H5N6 (n = 19) against data from 2 subtypes of major public health concern, H5N1 (n = 53) and H7N9 (n = 160). To assess immune responses indicative of prognosis, we compared concentrations of serum cytokines/chemokines in patients infected with H5N6, H5N1, H7N9, and 2009 pandemic H1N1 and characterized specific immune responses from 1 surviving and 2 nonsurviving H5N6 patients. RESULTS: H5N6 patients were found to have higher incidences of lymphopenia and elevated alanine aminotransferase and lactate dehydrogenase levels compared with H5N1 and H7N9 patients. Hypercytokinemia was detected at substantially higher frequencies from H5N6 patients compared to those infected with other AIV subtypes. Evaluation of adaptive immunity showed that both humoral and cellular responses could be detected in the H5N6-infected survivor, but cellular responses were absent in the nonsurvivors. In addition, the surviving patient had lower concentrations of both pro- and anti-inflammatory cytokines/chemokines compared to the nonsurvivors. CONCLUSIONS: Our results support that H5N6 virus could potentially be a major public health threat, and suggest it is possible that the earlier acquisition of cellular immunity and lower concentrations of cytokines/chemokines contributed to survival in our patient. Analysis of more patient samples will be needed to draw concrete conclusions.

Prolonged Evolution of Virus-Specific Memory T Cell Immunity after Severe Avian Influenza A (H7N9) Virus Infection.

Since 2013, influenza A H7N9 virus has emerged as the most common avian influenza virus subtype causing human infection, and it is associated with a high fatality risk. However, the characteristics of immune memory in patients who have recovered from H7N9 infection are not well understood. We assembled a cohort of 45 H7N9 survivors followed for up to 15 months after infection. Humoral and cellular immune responses were analyzed in sequential samples obtained at 1.5 to 4 months, 6 to 8 months, and 12 to 15 months postinfection. H7N9-specific antibody concentrations declined over time, and protective antibodies persisted longer in severely ill patients admitted to the intensive care unit (ICU) and patients presenting with acute respiratory distress syndrome (ARDS) than in patients with mild disease. Frequencies of virus-specific gamma interferon (IFN-γ)-secreting T cells were lower in critically ill patients requiring ventilation than in patients without ventilation within 4 months after infection. The percentages of H7N9-specific IFN-γ-secreting T cells tended to increase over time in patients ≥60 years or in critically ill patients requiring ventilation. Elevated levels of antigen-specific CD8+ T cells expressing the lung-homing marker CD49a were observed at 6 to 8 months after H7N9 infection compared to those in samples obtained at 1.5 to 4 months. Our findings indicate the prolonged reconstruction and evolution of virus-specific T cell immunity in older or critically ill patients and have implications for T cell-directed immunization strategies.IMPORTANCE Avian influenza A H7N9 virus remains a major threat to public health. However, no previous studies have determined the characteristics and dynamics of virus-specific T cell immune memory in patients who have recovered from H7N9 infection. Our findings showed that establishment of H7N9-specific T cell memory after H7N9 infection was prolonged in older and severely affected patients. Severely ill patients mounted lower T cell responses in the first 4 months after infection, while T cell responses tended to increase over time in older and severely ill patients. Higher levels of antigen-specific CD8+ T cells expressing the lung-homing marker CD49a were detected at 6 to 8 months after infection. Our results indicated a long-term impact of H7N9 infection on virus-specific memory T cells. These findings advance our understanding of the dynamics of virus-specific memory T cell immunity after H7N9 infection, which is relevant to the development of T cell-based universal influenza vaccines.

Two classes of protective antibodies against Pseudorabies virus variant glycoprotein B: Implications for vaccine design.

Pseudorabies virus (PRV) belongs to the Herpesviridae family, and is an important veterinary pathogen. Highly pathogenic PRV variants have caused severe epidemics in China since 2011, causing huge economic losses. To tackle the epidemics, we identified a panel of mouse monoclonal antibodies (mAbs) against PRV glycoprotein B (gB) that effectively block PRV infection. Among these 15 mAbs, fourteen of them block PRV entry in a complement-dependent manner. The remaining one, 1H1 mAb, however can directly neutralize the virus independent of complement and displays broad-spectrum neutralizing activities. We further determined the crystal structure of PRV gB and mapped the epitopes of these antibodies on the structure. Interestingly, all the complement-dependent neutralizing antibodies bind gB at the crown region (domain IV). In contrast, the epitope of 1H1 mAb is located at the bottom of domain I, which includes the fusion loops, indicating 1H1 mAb might neutralize the virus by interfering with the membrane fusion process. Our studies demonstrate that gB contains multiple B-cell epitopes in its crown and base regions and that antibodies targeting different epitopes block virus infection through different mechanisms. These findings would provide important clues for antiviral drug design and vaccine development.

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

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.

Clinical, immunological and bacteriological characteristics of H7N9 patients nosocomially co-infected by Acinetobacter Baumannii: a case control study.

BACKGROUND: Bacterial co-infection of patients suffering from influenza pneumonia is a key element that increases morbidity and mortality. The occurrence of Acinetobacter baumannii co-infection in patients with avian influenza A (H7N9) virus infection has been described as one of the most prevalent bacterial co-infections. However, the clinical and laboratory features of this entity of H7N9 and A. baumannii co-infection have not been systematically investigated. METHODS: We collected clinical and laboratory data from laboratory-confirmed H7N9 cases co-infected by A. baumannii. H7N9 patients without bacterial co-infection and patients with A. baumannii-related pneumonia in the same hospital during the same period were recruited as controls. The antibiotic resistance features and the corresponding genome determinants of A. baumannii and the immune responses of the patients were tested through the respiratory and peripheral blood specimens. RESULTS: Invasive mechanical ventilation was the most significant risk factor for the nosocomial A. baumannii co-infection in H7N9 patients. The co-infection resulted in severe clinical manifestation which was associated with the dysregulation of immune responses including deranged T-cell counts, antigen-specific T-cell responses and plasma cytokines. The emergence of genome variations of extensively drug-resistant A. baumannii associated with acquired polymyxin resistance contributed to the fatal outcome of a co-infected patient. CONCLUSIONS: The co-infection of H7N9 patients by extensively drug-resistant A. baumannii with H7N9 infection is an important issue which deserves attention. The dysfunctions of immune responses were associated with the co-infection and were correlated with the disease severity. These data provide useful reference for the diagnosis and treatment of H7N9 infection.

Cross-immunity Against Avian Influenza A(H7N9) Virus in the Healthy Population Is Affected by Antigenicity-Dependent Substitutions.

BACKGROUND: The emergence of infections by the novel avian influenza A(H7N9) virus has posed a threat to human health. Cross-immunity between A(H7N9) and other heterosubtypic influenza viruses affected by antigenicity-dependent substitutions needs to be investigated. METHODS: We investigated the cellular and humoral immune responses against A(H7N9) and 2009 pandemic influenza A(H1N1) virus (A[H1N1]pdm09), by serological and T-cell-specific assays, in a healthy population. The molecular bases of the cellular and humoral antigenic variability of A(H7N9) were illuminated by structural determination. RESULTS: We not only found that antibodies against A(H7N9) were lacking in the studied population, but also revealed that both CD4+ and CD8+ T cells that cross-reacted with A(H7N9) were at significantly lower levels than those against the A(H1N1)pdm09 peptides with substitutions. Moreover, individual peptides for A(H7N9) with low cross-reactivity were identified. Structural determination indicated that substitutions within these peptides influence the antigenic variability of A(H7N9) through both major histocompatibility complex (MHC) binding and T-cell receptor docking. CONCLUSIONS: The impact of antigenicity-dependent substitutions on cross-reactivity of T-cell immunity against the novel influenza virus A(H7N9) in the healthy population benefits the understanding of immune evasion of influenza viruses and provides a useful reference for universal vaccine development.

Nosocomial Co-Transmission of Avian Influenza A(H7N9) and A(H1N1)pdm09 Viruses between 2 Patients with Hematologic Disorders.

A nosocomial cluster induced by co-infections with avian influenza A(H7N9) and A(H1N1)pdm09 (pH1N1) viruses occurred in 2 patients at a hospital in Zhejiang Province, China, in January 2014. The index case-patient was a 57-year-old man with chronic lymphocytic leukemia who had been occupationally exposed to poultry. He had co-infection with H7N9 and pH1N1 viruses. A 71-year-old man with polycythemia vera who was in the same ward as the index case-patient for 6 days acquired infection with H7N9 and pH1N1 viruses. The incubation period for the second case-patient was estimated to be <4 days. Both case-patients died of multiple organ failure. Virus genetic sequences from the 2 case-patients were identical. Of 103 close contacts, none had acute respiratory symptoms; all were negative for H7N9 virus. Serum samples from both case-patients demonstrated strong proinflammatory cytokine secretion but incompetent protective immune responses. These findings strongly suggest limited nosocomial co-transmission of H7N9 and pH1N1 viruses from 1 immunocompromised patient to another.

VP2 dominated CD4+ T cell responses against enterovirus 71 and cross-reactivity against coxsackievirus A16 and polioviruses in a healthy population.

Enterovirus 71 (EV71)-associated hand-foot-mouth disease has become a major threat to public health in the Asia-Pacific region. Although T cell immunity is closely correlated with clinical outcomes of EV71 infection, little is known about T cell immunity baseline against EV71 and T cell immunogenecity of EV71 Ags in the population, which has restricted our understanding of immunoprotection mechanisms. In this study, we investigated the cellular immune responses against the four structural Ags of EV71 and determined the immunohierarchy of these Ags in healthy adults. A low frequency of EV71-responsive T cells was detected circulating in peripheral blood, and broad T cell immune responses could be identified in most of the subjects after in vitro expansion. We demonstrated that the VP2 Ag with broad distribution of immunogenic peptides dominates T cell responses against EV71 compared with VP1, VP3, and VP4. Furthermore, the responses were illuminated to be mainly single IFN-γ-secreting CD4(+) T cell dependent, indicating the previous natural acute viral infection of the adult population. Conservancy analysis of the immunogenic peptides revealed that moderately variant peptides were in the majority in coxsackievirus A16 (CV-A16) whereas most of the peptides were highly variant in polioviruses. Less efficient cross-reactivity against CV-A16 might broadly exist among individuals, whereas influences derived from poliovirus vaccination would be limited. Our findings suggest that the significance of VP2 Ag should be addressed in the future EV71-responsive immunological investigations. And the findings concerning the less efficient cross-reactivity against CV-A16 and limited influences from poliovirus vaccination in EV71-contacted population would contribute to a better understanding of immunoprotection mechanisms against enteroviruses.

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 Vß 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.