Distinct Immunogenicity and Efficacy of Poxvirus-Based Vaccine Candidates against Ebola Virus Expressing GP and VP40 Proteins.

Zaire and Sudan ebolavirus species cause a severe disease in humans and nonhuman primates (NHPs) characterized by a high mortality rate. There are no licensed therapies or vaccines against Ebola virus disease (EVD), and the recent 2013 to 2016 outbreak in West Africa highlighted the need for EVD-specific medical countermeasures. Here, we generated and characterized head-to-head the immunogenicity and efficacy of five vaccine candidates against Zaire ebolavirus (EBOV) and Sudan ebolavirus (SUDV) based on the highly attenuated poxvirus vector modified vaccinia virus Ankara (MVA) expressing either the virus glycoprotein (GP) or GP together with the virus protein 40 (VP40) forming virus-like particles (VLPs). In a human monocytic cell line, the different MVA vectors (termed MVA-EBOVs and MVA-SUDVs) triggered robust innate immune responses, with production of beta interferon (IFN-ß), proinflammatory cytokines, and chemokines. Additionally, several innate immune cells, such as dendritic cells, neutrophils, and natural killer cells, were differentially recruited in the peritoneal cavity of mice inoculated with MVA-EBOVs. After immunization of mice with a homologous prime/boost protocol (MVA/MVA), total IgG antibodies against GP or VP40 from Zaire and Sudan ebolavirus were differentially induced by these vectors, which were mainly of the IgG1 and IgG3 isotypes. Remarkably, an MVA-EBOV construct coexpressing GP and VP40 protected chimeric mice challenged with EBOV to a greater extent than a vector expressing GP alone. These results support the consideration of MVA-EBOVs and MVA-SUDVs expressing GP and VP40 and producing VLPs as best-in-class potential vaccine candidates against EBOV and SUDV.IMPORTANCE EBOV and SUDV cause a severe hemorrhagic fever affecting humans and NHPs. Since their discovery in 1976, they have caused several sporadic epidemics, with the recent outbreak in West Africa from 2013 to 2016 being the largest and most severe, with more than 11,000 deaths being reported. Although some vaccines are in advanced clinical phases, less expensive, safer, and more effective licensed vaccines are desirable. We generated and characterized head-to-head the immunogenicity and efficacy of five novel vaccines against EBOV and SUDV based on the poxvirus MVA expressing GP or GP and VP40. The expression of GP and VP40 leads to the formation of VLPs. These MVA-EBOV and MVA-SUDV recombinants triggered robust innate and humoral immune responses in mice. Furthermore, MVA-EBOV recombinants expressing GP and VP40 induced high protection against EBOV in a mouse challenge model. Thus, MVA expressing GP and VP40 and producing VLPs is a promising vaccine candidate against EBOV and SUDV.

Tumor Suppressor p53 Stimulates the Expression of Epstein-Barr Virus Latent Membrane Protein 1.

Epstein-Barr virus (EBV) is associated with multiple human malignancies. EBV latent membrane protein 1 (LMP1) is required for the efficient transformation of primary B lymphocytes in vitro and possibly in vivo The tumor suppressor p53 plays a seminal role in cancer development. In some EBV-associated cancers, p53 tends to be wild type and overly expressed; however, the effects of p53 on LMP1 expression is not clear. We find LMP1 expression to be associated with p53 expression in EBV-transformed cells under physiological and DNA damaging conditions. DNA damage stimulates LMP1 expression, and p53 is required for the stimulation. Ectopic p53 stimulates endogenous LMP1 expression. Moreover, endogenous LMP1 blocks DNA damage-mediated apoptosis. Regarding the mechanism of p53-mediated LMP1 expression, we find that interferon regulatory factor 5 (IRF5), a direct target of p53, is associated with both p53 and LMP1. IRF5 binds to and activates a LMP1 promoter reporter construct. Ectopic IRF5 increases the expression of LMP1, while knockdown of IRF5 leads to reduction of LMP1. Furthermore, LMP1 blocks IRF5-mediated apoptosis in EBV-infected cells. All of the data suggest that cellular p53 stimulates viral LMP1 expression, and IRF5 may be one of the factors for p53-mediated LMP1 stimulation. LMP1 may subsequently block DNA damage- and IRF5-mediated apoptosis for the benefits of EBV. The mutual regulation between p53 and LMP1 may play an important role in EBV infection and latency and its related cancers.IMPORTANCE The tumor suppressor p53 is a critical cellular protein in response to various stresses and dictates cells for various responses, including apoptosis. This work suggests that an Epstein-Bar virus (EBV) principal viral oncogene is activated by cellular p53. The viral oncogene blocks p53-mediated adverse effects during viral infection and transformation. Therefore, the induction of the viral oncogene by p53 provides a means for the virus to cope with infection and DNA damage-mediated cellular stresses. This seems to be the first report that p53 activates a viral oncogene; therefore, the discovery would be interesting to a broad readership from the fields of oncology to virology.

Expression of interferon regulatory factor 7 correlates with the expression of Epstein-Barr Virus latent membrane protein 1 and cervical lymph node metastasis in nasopharyngeal cancer.

Interferon regulatory factor 7 (IRF7) has oncogenic properties in several malignancies such as Epstein-Barr virus (EBV)-associated lymphoma. However, there is no evidence whether IRF7 is associated with the oncogenesis of nasopharyngeal cancer (NPC), the pathogenesis of which is closely associated with EBV. Herein, we report that expression of IRF7 was increased in normal nasopharyngeal cells that expressed the EBV principal oncoprotein, latent membrane protein 1 (LMP1). In addition, IRF7 was mainly expressed in the nucleus in both normal nasopharyngeal cells and nasopharyngeal cancer cells that expresses LMP1. On immunohistochemical analysis, IRF7 was predominantly localized in the nucleus in biopsy samples of NPC tissues. In total, IRF7 expression was detected with 36 of 49 specimens of these tissues. Furthermore, the expression score of IRF7 correlated with the expression score of LMP1. Moreover, the expression score of IRF7 is associated with cervical lymph-node metastasis, which reflects the highly metastatic nature of this cancer. Taken together, our results suggest that expression of IRF7 is one of the metastatic effectors of LMP1 signalling in EBV-associated NPC.

Expression and Immunogenicity of VP40 Protein of ZEBOV.

BACKGROUND: EBOV outbreaks continue to threaten the world due to the absence of effective vaccines and therapeutics. Easy-to-use and rapid diagnostic tests for EBOV are highly desired for prevention and control of the EVD epidemic. METHODS: Escherichia coli expression system was used to express VP40 protein of Zaire Ebola virus (ZEBOV) as water-soluble protein upon optimization of temperature, time, and IPTG concentration. VP40 protein was purified through Ni-NTA affinity chromatography and applied to immunize rabbits for immunogenicity analysis. Rabbit polyclonal antibodies against VP40 protein was produced and antibody response was analyzed using Western blot, enzyme-linked immunosorbent assay (ELISA), and immunoperoxidase monolayer assay (IPMA). RESULTS: Recombinant full-length VP40 protein of ZEBOV was expressed in E. coli Rosetta (DE3) cells as water-soluble protein. Analysis of antibody responses showed that rabbit polyclonal antibodies against VP40 protein could react specifically with this E. coli-expressed protein in Western blot and ELISA, and antibody titers in ELISA reached 1:25600. Besides, the produced rabbit polyclonal antibodies bound to VP40 proteins eukaryotically expressed by transfecting pcDNA-eGFP-VP40 into BHK-21 cells in IPMA. CONCLUSION: These results show that the prokaryotically expressed VP40 protein has high immunogenicity and can be used as diagnostic antigen in ELISA and other immunoassays. The strategy used in this study might be a potential way for preparing diagnostic agents for prevention and control of exotic diseases.

Expression and purification of native and functional influenza A virus matrix 2 proton selective ion channel.

Influenza A virus displays one of the highest infection rates of all human viruses and therefore represents a severe human health threat associated with an important economical challenge. Influenza matrix protein 2 (M2) is a membrane protein of the viral envelope that forms a proton selective ion channel. Here we report the expression and native isolation of full length active M2 without mutations or fusions. The ability of the influenza virus to efficiently infect MDCK cells was used to express native M2 protein. Using a Calixarene detergents/surfactants based approach; we were able to solubilize most of M2 from the plasma membrane and purify it. The tetrameric form of native M2 was maintained during the protein preparation. Mass spectrometry shows that M2 was phosphorylated in its cytoplasmic tail (serine 64) and newly identifies an acetylation of the highly conserved Lysine 60. ELISA shows that solubilized and purified M2 was specifically recognized by M2 antibody MAB65 and was able to displace the antibody from M2 MDCK membranes. Using a bilayer voltage clamp measurement assay, we demonstrate a pH dependent proton selective ion channel activity. The addition of the M2 ion channel blocker amantadine allows a total inhibition of the channel activity, illustrating therefore the specificity of purified M2 activity. Taken together, this work shows the production and isolation of a tetrameric and functional native M2 ion channel that will pave the way to structural and functional characterization of native M2, conformational antibody development, small molecules compounds screening towards vaccine treatment.

A new strategy for full-length Ebola virus glycoprotein expression in E.coli.

Ebola virus (EBOV) causes severe hemorrhagic fever in humans and non-human primates with high rates of fatality. Glycoprotein (GP) is the only envelope protein of EBOV, which may play a critical role in virus attachment and entry as well as stimulating host protective immune responses. However, the lack of expression of full-length GP in Escherichia coli hinders the further study of its function in viral pathogenesis. In this study, the vp40 gene was fused to the full-length gp gene and cloned into a prokaryotic expression vector. We showed that the VP40-GP and GP-VP40 fusion proteins could be expressed in E.coli at 16 °C. In addition, it was shown that the position of vp40 in the fusion proteins affected the yields of the fusion proteins, with a higher level of production of the fusion protein when vp40 was upstream of gp compared to when it was downstream. The results provide a strategy for the expression of a large quantity of EBOV full-length GP, which is of importance for further analyzing the relationship between the structure and function of GP and developing an antibody for the treatment of EBOV infection.

Mouse model for acute Epstein-Barr virus infection.

Epstein-Barr Virus (EBV) infects human B cells and drives them into continuous proliferation. Two key viral factors in this process are the latent membrane proteins LMP1 and LMP2A, which mimic constitutively activated CD40 receptor and B-cell receptor signaling, respectively. EBV-infected B cells elicit a powerful T-cell response that clears the infected B cells and leads to life-long immunity. Insufficient immune surveillance of EBV-infected B cells causes life-threatening lymphoproliferative disorders, including mostly germinal center (GC)-derived B-cell lymphomas. We have modeled acute EBV infection of naive and GC B cells in mice through timed expression of LMP1 and LMP2A. Although lethal when induced in all B cells, induction of LMP1 and LMP2A in just a small fraction of naive B cells initiated a phase of rapid B-cell expansion followed by a proliferative T-cell response, clearing the LMP-expressing B cells. Interfering with T-cell activity prevented clearance of LMP-expressing B cells. This was also true for perforin deficiency, which in the human causes a life-threatening EBV-related immunoproliferative syndrome. LMP expression in GC B cells impeded the GC reaction but, upon loss of T-cell surveillance, led to fatal B-cell expansion. Thus, timed expression of LMP1 together with LMP2A in subsets of mouse B cells allows one to study major clinically relevant features of human EBV infection in vivo, opening the way to new therapeutic approaches.

A clinicopathological study of primary central nervous system lymphomas & their association with Epstein-Barr virus.

BACKGROUND & OBJECTIVES: Primary central nervous system lymphomas (PCNSLs) are relatively uncommon, accounting for 2-3 per cent of primary brain tumours. Majority of these are diffuse large B cell lymphomas (DLBCL) occurring both in immunocompromised and immunocompetent patients. We undertook this study to classify PCNSL into germinal centre (GC) and non-germinal centre (NGC) type based on Hans classification and to find the role of Epstein-Barr virus (EBV) in pathogenesis both by conventional immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH). METHODS: The consecutive cases of PCNSL during a 10 years period were analysed by IHC for CD45, CD20, CD3, B-cell lymphoma 2 and 6 (Bcl-2 and Bcl-6), B-cell specific octamer binding protein-1 (BOB-1), multiple myeloma oncogene-1 (MUM-1), EBV latent-membrane protein 1 (LMP-1), cyclin-D1, CD10, CD5 and CD23, as well as by CISH for EBV. RESULTS: During a period of 10 years, 65 PCNSL were diagnosed which comprised 0.69 per cent (65/9476) of all intracranial tumours. The mean age of presentation was 49 yr with sex ratio (M:F) of 1.4:1. Most common location was supratentorial region with predominant involvement of frontal lobe. Single lesions were seen in 38 (58.4%) and multifocal lesions in 27 (41.5%) patients. None of the patients were immunocompromised. All cases were B cell immunophenotype and were DLBCL except one case of follicular lymphoma. According to Hans classification, majority of them were NGC (n=51, 79.6%) and 13 (20.3%) were GC type. Bcl-2 expression was noted in 34 (52.3%) tumours. EBV was positive in three (4.6%) cases; two were detected both by IHC and CISH and one case by CISH only. INTERPRETATION & CONCLUSIONS: In Indian population, PCNSL occurs mainly in immunocompetent patients, and a decade earlier than in western population. Immunophenotyping revealed that all cases were DLBCL with predominance of NGC type. No prognostic difference was seen between GC and NGC DLBCL. Association of EBV was rare and this virus was possibly not involved in the pathogenesis of PCNSL in immunocompetent individuals. CISH was an easy, economical and less cumbersome method for detection of EBV in PCNSL.

Differential expression of EBV proteins LMP1 and BHFR1 in EBV­associated gastric and nasopharyngeal cancer tissues.

Epstein­Barr virus (EBV) infection is associated with the development of T cell lymphoma, nasopharyngeal cancer (NPC), and EBV­associated gastric cancer (EBVaGC). This study assessed the expression of the EBV­associated proteins latent membrane protein 1 (LMP1) and BamHI­A rightward frame 1 (BARF1) in NPC and EBVaGC tissue specimens and determined their association with clinicopathological data, microvessel density (MVD) and micro­lymphatic vessel density (MLVD). This study collected 600 gastric cancer and 75 NPC tissue samples. EBV infection was assessed using in situ hybridization, and LMP1 and BARF1 expression was assessed using immunohistochemistry. The levels of MVD and MLVD were assessed using immunostaining of vascular endothelial growth factor (VEGF)­C, CD34, and lymphatic vessel endothelial receptor 1 (LYVE­1). Among the 600 gastric cancer cases, 30 were positive for EBV infection, which was shown to be associated with the age of patients (P=0.073), tumor differentiation (P<0.0001), tumor location (P<0.0001) and lymph node metastasis (P<0.0001). In these 30 EBVaGC cases, only one case was weakly positive for LMP1, but 17 cases were BARF1 positive. BARF1 expression was associated with lymph node metastasis of EBVaGC and the level of MLVD. Furthermore, 61 (81%) of 75 NPC patients were EBV positive, among which 38 cases were LMP­1 positive (62.3%) and LMP1 expression was associated with tumor­node­metastasis stage (P=0.011) and lymph node metastasis (P=0.041). MLVD was significantly higher in LMP1­positive cases than LMP1­negative cases. There were only 8 (13.3%) cases positive for BARF1 expression. In conclusion, EBV infection exhibits a role in gastric cancer and NPC development; however, expression of EBV­associated proteins LMP1 and BARF1 have differential functions during tumorigenesis of these two types of cancer.

Production of the virus-like particles of nipah virus matrix protein in Pichia pastoris as diagnostic reagents.

The matrix (M) protein of Nipah virus (NiV) is a peripheral protein that plays a vital role in the envelopment of nucleocapsid protein and acts as a bridge between the viral surface and the nucleocapsid proteins. The M protein is also proven to play an important role in production of virus-like particles (VLPs) and is essential for assembly and budding of NiV particles. The recombinant M protein produced in Escherichia coli assembled into VLPs in the absence of the viral surface proteins. However, the E. coli produced VLPs are smaller than the native virus particles. Therefore, the aims of this study were to produce NiV M protein in Pichia pastoris, to examine the structure of the VLPs formed, and to assess the potential of the VLPs as a diagnostic reagent. The M protein was successfully expressed in P. pastoris and was detected with anti-myc antibody using Western blotting. The VLPs formed by the recombinant M protein were purified with sucrose density gradient ultracentrifugation, high-performance liquid chromatography (HPLC), and Immobilized Metal Affinity Chromatography (IMAC). Immunogold staining and transmission electron microscopy confirmed that the M protein assembled into VLPs as large as 200 nm. ELISA revealed that the NiV M protein produced in P. pastoris reacted strongly with positive NiV sera demonstrating its potential as a diagnostic reagent. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1038-1045, 2016.

Expression and purification of the matrix protein of Nipah virus in baculovirus insect cell system.

Nipah virus (NiV) causes fatal respiratory illness and encephalitis in humans and animals. The matrix (M) protein of NiV plays an important role in the viral assembly and budding process. Thus, an access to the NiV M protein is vital to the design of viral antigens as diagnostic reagents. In this study, recombinant DNA technology was successfully adopted in the cloning and expression of NiV M protein. A recombinant expression cassette (baculovirus expression vector) was used to encode an N-terminally His-tagged NiV M protein in insect cells. A time-course study demonstrated that the highest yield of recombinant M protein (400-500 µg) was expressed from 107 infected cells 3 days after infection. A single-step purification method based on metal ion affinity chromatography was established to purify the NiV M protein, which successfully yielded a purity level of 95.67% and a purification factor of 3.39. The Western blotting and enzyme-linked immunosorbent assay (ELISA) showed that the purified recombinant M protein (48 kDa) was antigenic and reacted strongly with the serum of a NiV infected pig.

Expression of LMP and EBNA genes in Epstein-Barr virus-associated lymphomas in Hu-PBL/SCID mice.

Transplantation of peripheral blood lymphocytes (PBLs) from healthy humans with latent Epstein-Barr virus (EBV) infection into severe combined immunodeficiency (SCID) mice results in development of EBV-associated human B-cell lymphoma. However, the expression of EBV genes in relation to lymphoma development has not been reported. We investigated latent membrane protein (LMP) and EBV nuclear antigen (EBNA) gene expression in PBLs from EBV-positive blood donors and induced-lymphoma cells from SCID mice to elucidate the functions and effects of the EBV genome in the occurrence and development of lymphoma. PBLs were isolated from 9 healthy blood donors and transplanted into SCID mice. Gene expression levels of LMP-1, LMP-2A, and LMP-2B and EBNA-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C and EBNA-LP were monitored by real-time quantitative-polymerase chain reaction (qRT-PCR) in cells from nine EBV-induced lymphomas and in matched lymphocytes from healthy subjects. LMP-1, EBNA-1 and EBNA-2 protein levels were detected by western blotting. As a result, LMP-1, LMP-2A and LMP-2B mRNA levels were upregulated 256-, 38- and 331-fold, respectively, in the EBV-induced lymphoma cells compared with the controls, while EBNA-1 and EBNA-3A mRNA levels were upregulated 1157- and 1154-fold, respectively. EBNA-2, EBNA-3B, EBNA-3C and EBNA-LP mRNAs were detected in lymphoma cells, but not in lymphocytes from EBV-positive blood donors. LMP-1 and EBNA-2 proteins were not expressed in lymphocytes from EBV-positive blood donors, according to western blotting. Weak EBNA-1 expression was observed in lymphocytes from blood donors with latent EBV infection, while LMP-1, EBNA-1 and EBNA-2 protein levels were significantly upregulated in EBV-induced lymphoma cells, consistent with mRNA expression levels detected by qRT-PCR. In conclusion, LMP-1, LMP-2A, LMP-2B, EBNA-1 and EBNA-3A were upregulated in EBV-induced lymphoma cells, while EBNA-2, EBNA-3B, EBNA-3C and EBNA-LP were absent in lymphocytes from humans with latent EBV infection, but were positively expressed in EBV-induced lymphoma cells.

Epstein-Barr virus-encoded LMP1 increases miR-155 expression, which promotes radioresistance of nasopharyngeal carcinoma via suppressing UBQLN1.

OBJECTIVE: Epstein-Barr virus (EBV)-encoded latent membrane protein (LMP1) can drive aberrant expression of miR-155 in nasopharyngeal carcinoma (NPC). In this study, we investigated the regulation of miR-155 expression over UBQLN1 and studied their effects on radio-sensitivity of NPC. MATERIALS AND METHODS: MiR-155, LMP1 and ubiquilin-1 expression were measured in 40 cases of NPC cases. The regulative role of miR-155 over UBQLN1 was investigated using a dual luciferase assay, qRT-PCR and Western blot analysis. The effect of miR-155-UBQLN1 axis on radio-sensitivity was explored using loss-and-gain study. The activation of PI3K/Akt pathway and the expression change of some important genes regulating cell cycle, cell proliferation and epithelial-to-mesenchymal transition (EMT) were measured. RESULTS: MiR-155 was significantly increased in radio-resistant NPC tissues and was negatively correlated to ubiqulin-1 expression. LMP1 overexpression led to significantly higher miR-155 expression. MiR-155 had two binding sites with 3'UTR of UBQLN1 and could decrease it expression. MiR-155 overexpression increased survival fraction of CNE-2 cells after exposure to 6 Gy and decreased cell apoptosis. It also partly abrogated the inhibiting effect of UBQLN1. Through decreasing ubiqulin-1, miR-155 changed the cell cycle to a more radio-resistant model. The miR-155-UBQLN1 axis affected the activation of PI3K/Akt pathway in NPC cells and changed the expression of some important genes regulating the cell cycle, cell proliferation and EMT. CONCLUSIONS: This study found that aberrant miR-155 expression driven by LMP1 can modulate radio-sensitivity of the NPC cell at least partly through targeting UBQLN1.

The full-length DNA sequence of Epstein Barr virus from a human gastric carcinoma cell line, SNU-719.

The consistent presence of Epstein-Barr virus (EBV) in malignant cells of EBV-associated gastric carcinoma (EBVaGC) suggests it plays an important role during the development of EBVaGC. However, the entire genomic sequence of EBV from EBVaGC has yet to be determined. This study first determined, annotated, and analyzed the full genomic sequence of EBV from the naturally infected gastric carcinoma cell line SNU-719 using next-generation sequencing and comparative analyses. In consistent with the notion that EBV sequence isolates better reflect their geographic area than tissue origin, the SNU-719 EBV (named as GC1) was categorized as an East Asian type I EBV. Compared with the prototype B95.8 sequence, SNU-719 EBV contained 1372 variations, with 937 and 435 within coding and non-coding regions, respectively. Of the 937 variations, 465 were non-synonymous changes, while 472 synonymous changes included partial internal deletions in the coding regions of LMP1 and gp350. The RNAseq transcriptome revealed that multiple BART transcripts comprised the majority of EBV RNA reads. The SNU-719 EBV expressed high levels of BART, LF3, BHLF1, and BNLF2. Evidence of RNA editing at multiple sites in the host chromosome was found; however, no evidence of genome integration was seen. The annotated SNU-719 EBV sequence will be a useful reference in future EBVaGC studies.

LMP1 promotes nasal NK/T-cell lymphoma cell function by eIF4E via NF-κB pathway.

Nasal natural killer T-cell lymphoma (NKTL) is a highly malignant tumor that is closely associated with Epstein-Barr virus (EBV) infection. Latent membrane protein 1 (LMP1) is encoded by EBV and plays an important role in EBV-induced cell transformation. Therefore, we assessed the function of LMP1 as a stimulant of NKTL progression and the underlying mechanism. A human EBV-positive NKTL cell line (SNK-6) was transfected with pcDNA3.1-LMP1, LV-LMP1 shRNA or LV-eukaryotic translation initiation factor 4E (eIF4E)-shRNA. Then, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to assess the proliferation of SNK-6 cells, and cell migration and invasion were analyzed by transwell chamber assay. Flow cytometry was used to analyze the cell cycle and apoptosis. The results showed LMP1 was highly expressed in SNK-6 cells compared with control groups. Following pretreatment with LMP1 shRNA, the proliferation of SNK-6 cells was inhibited and resulted in a G0/G1 phase arrest. A reduction in invasion and migration was also observed. LMP1 silencing promoted cell apoptosis. Further mechanistic analysis suggested that LMP1 overexpression induced the expression of eIF4E, while eIF4E-shRNA dramatically attenuated the increase in cell proliferation, invasion, migration and the inhibition of apoptosis triggered by LMP-1 upregulation. Moreover, the effect of LMP1 on eIF4E expression was mediated by the NF-κB pathway. Therefore, this finding may provide a potential target against NKTL.

Lung CD8+ T Cell Impairment Occurs during Human Metapneumovirus Infection despite Virus-Like Particle Induction of Functional CD8+ T Cells.

UNLABELLED: Human metapneumovirus (HMPV) is a major cause of respiratory disease in infants, the elderly, and immunocompromised individuals worldwide. There is currently no licensed HMPV vaccine. Virus-like particles (VLPs) are an attractive vaccine candidate because they are noninfectious and elicit a neutralizing antibody response. However, studies show that serum neutralizing antibodies are insufficient for complete protection against reinfection and that adaptive T cell immunity is important for viral clearance. HMPV and other respiratory viruses induce lung CD8(+) T cell (TCD8) impairment, mediated by programmed death 1 (PD-1). In this study, we generated HMPV VLPs by expressing the fusion and matrix proteins in mammalian cells and tested whether VLP immunization induces functional HMPV-specific TCD8 responses in mice. C57BL/6 mice vaccinated twice with VLPs and subsequently challenged with HMPV were protected from lung viral replication for at least 20 weeks postimmunization. A single VLP dose elicited F- and M-specific lung TCD8s with higher function and lower expression of PD-1 and other inhibitory receptors than TCD8s from HMPV-infected mice. However, after HMPV challenge, lung TCD8s from VLP-vaccinated mice exhibited inhibitory receptor expression and functional impairment similar to those of mice experiencing secondary infection. HMPV challenge of VLP-immunized µMT mice also elicited a large percentage of impaired lung TCD8s, similar to mice experiencing secondary infection. Together, these results indicate that VLPs are a promising vaccine candidate but do not prevent lung TCD8 impairment upon HMPV challenge. IMPORTANCE: Human metapneumovirus (HMPV) is a leading cause of acute respiratory disease for which there is no licensed vaccine. Virus-like particles (VLPs) are an attractive vaccine candidate and induce antibodies, but T cell responses are less defined. Moreover, HMPV and other respiratory viruses induce lung CD8(+) T cell (TCD8) impairment mediated by programmed death 1 (PD-1). In this study, HMPV VLPs containing viral fusion and matrix proteins elicited epitope-specific TCD8s that were functional with low PD-1 expression. Two VLP doses conferred sterilizing immunity in C57BL/6 mice and facilitated HMPV clearance in antibody-deficient µMT mice without enhancing lung pathology. However, regardless of whether responding lung TCD8s had previously encountered HMPV antigens in the context of VLPs or virus, similar proportions were impaired and expressed comparable levels of PD-1 upon viral challenge. These results suggest that VLPs are a promising vaccine candidate but do not prevent lung TCD8 impairment upon HMPV challenge.

Structural Analysis of the Roles of Influenza A Virus Membrane-Associated Proteins in Assembly and Morphology.

UNLABELLED: The assembly of influenza A virus at the plasma membrane of infected cells leads to release of enveloped virions that are typically round in tissue culture-adapted strains but filamentous in strains isolated from patients. The viral proteins hemagglutinin (HA), neuraminidase (NA), matrix protein 1 (M1), and M2 ion channel all contribute to virus assembly. When expressed individually or in combination in cells, they can all, under certain conditions, mediate release of membrane-enveloped particles, but their relative roles in virus assembly, release, and morphology remain unclear. To investigate these roles, we produced membrane-enveloped particles by plasmid-derived expression of combinations of HA, NA, and M proteins (M1 and M2) or by infection with influenza A virus. We monitored particle release, particle morphology, and plasma membrane morphology by using biochemical methods, electron microscopy, electron tomography, and cryo-electron tomography. Our data suggest that HA, NA, or HANA (HA plus NA) expression leads to particle release through nonspecific induction of membrane curvature. In contrast, coexpression with the M proteins clusters the glycoproteins into filamentous membrane protrusions, which can be released as particles by formation of a constricted neck at the base. HA and NA are preferentially distributed to differently curved membranes within these particles. Both the budding intermediates and the released particles are morphologically similar to those produced during infection with influenza A virus. Together, our data provide new insights into influenza virus assembly and show that the M segment together with either of the glycoproteins is the minimal requirement to assemble and release membrane-enveloped particles that are truly virus-like. IMPORTANCE: Influenza A virus is a major respiratory pathogen. It assembles membrane-enveloped virus particles whose shapes vary from spherical to filamentous. Here we examine the roles of individual viral proteins in mediating virus assembly and determining virus shape. To do this, we used a range of electron microscopy techniques to obtain and compare two- and three-dimensional images of virus particles and virus-like particles during and after assembly. The virus-like particles were produced using different combinations of viral proteins. Among our results, we found that coexpression of one or both of the viral surface proteins (hemagglutinin and neuraminidase) with the viral membrane-associated proteins encoded by the M segment results in assembly and release of filamentous virus-like particles in a manner very similar to that of the budding and release of influenza virions. These data provide novel insights into the roles played by individual viral proteins in influenza A virus assembly.

The influenza m2 cytoplasmic tail changes the proton-exchange equilibria and the backbone conformation of the transmembrane histidine residue to facilitate proton conduction.

The influenza M2 protein forms an acid-activated tetrameric proton channel important for the virus lifecycle. Residue His37 in the transmembrane domain is responsible for channel activation and proton selectivity. While the structure and dynamics of His37 have been well studied in TM peptide constructs, it has not been investigated in the presence of the full cytoplasmic domain, which increases the proton conductivity by 2-fold compared to the TM peptide. We report here (13)C and (15)N chemical shifts of His37 in the cytoplasmic-containing M2(21-97) and show that cationic histidines are already present at neutral pH, in contrast to the TM peptide, indicating that the cytoplasmic domain shifts the protonation equilibria. Quantification of the imidazole (15)N intensities yielded two resolved proton dissociation constants (pKa's) of 7.1 and 5.4, which differ from the TM result but resemble the M2(18-60) result, suggesting cooperative proton binding. The average His37 pKa is higher for M2(21-97) than for the shorter constructs. We attribute this higher pKa to direct and indirect effects of the cytoplasmic domain, which is rich in acidic residues. 2D (13)C-(13)C correlation spectra reveal seven His37 Cα-Cß cross peaks at different pH, some of which are unique to the cytoplasmic-containing M2 and correspond to more ideal α-helical conformations. Based on the pH at which these chemical shifts appear and their side chain structures, we assign these conformations to His37 in differently charged tetramers. Thus, the cytoplasmic domain facilitates proton conduction through the transmembrane pore by modifying the His37-water proton exchange equilibria and the His37 backbone conformational distribution.

Histone deacetylase inhibitor romidepsin induces efficient tumor cell lysis via selective down-regulation of LMP1 and c-myc expression in EBV-positive diffuse large B-cell lymphoma.

We investigated the role of the histone deacetylase inhibitor, romidepsin, in Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (DLBCL), an aggressive non-Hodgkin lymphoma with poor clinical outcomes. We used EBV-positive and EBV-negative DLBCL cell lines and generated two EBV-transfected cell lines, LY7/EBV and U2932/EBV. Romidepsin was cytotoxic to cultured EBV-positive cells via the activation of the caspase cascade. Moreover, in vivo mice xenograft models demonstrated the cytotoxicity of romidepsin to EBV-positive DLBCL cells. Romidepsin induced cytotoxicity via the reduction of LMP1 and c-myc expression in EBV-positive cells. Inhibiting either LMP1 or c-myc using small inhibitory RNAs caused partial cytotoxicity in EBV-positive Farage and U2932/EBV lines. The dual inhibition of LMP1 and c-myc showed a synergistic cytotoxic effect in EBV-positive cells similar in magnitude to that of romidepsin alone. In addition, either double blockade of LMP1 and c-myc activity or romidepsin single treatment activated EBV lytic cycle in EBV-positive cells. In conclusion, romidepsin exerts strong anti-tumor activity in EBV-positive DLBCL via the inhibition of both LMP1 and c-myc. Our findings indicate that romidepsin might be a promising treatment for EBV-positive DLBCL.

High-Yield Expression of M2e Peptide of Avian Influenza Virus H5N1 in Transgenic Duckweed Plants.

Avian influenza is a major viral disease in poultry. Antigenic variation of this virus hinders vaccine development. However, the extracellular domain of the virus-encoded M2 protein (peptide M2e) is nearly invariant in all influenza A strains, enabling the development of a broad-range vaccine against them. Antigen expression in transgenic plants is becoming a popular alternative to classical expression methods. Here we expressed M2e from avian influenza virus A/chicken/Kurgan/5/2005(H5N1) in nuclear-transformed duckweed plants for further development of avian influenza vaccine. The N-terminal fragment of M2, including M2e, was selected for expression. The M2e DNA sequence fused in-frame to the 5' end of ß-glucuronidase was cloned into pBI121 under the control of CaMV 35S promoter. The resulting plasmid was successfully used for duckweed transformation, and western analysis with anti-ß-glucuronidase and anti-M2e antibodies confirmed accumulation of the target protein (M130) in 17 independent transgenic lines. Quantitative ELISA of crude protein extracts from these lines showed M130-ß-glucuronidase accumulation ranging from 0.09-0.97 mg/g FW (0.12-1.96 % of total soluble protein), equivalent to yields of up to 40 µg M2e/g plant FW. This relatively high yield holds promise for the development of a duckweed-based expression system to produce an edible vaccine against avian influenza.