Cross-Protective Peptide Vaccine against Influenza A Viruses Developed in HLA-A*2402 Human Immunity Model

Background: The virus-specific cytotoxic T lymphocyte (CTL) induction is an important target for the development of a broadly protective human influenza vaccine, since most CTL epitopes are found on internal viral proteins and relatively conserved. In this study, the possibility of developing a strain/subtype-independent human influenza vaccine was explored by taking a bioinformatics approach to establish an immunogenic HLA-A24 restricted CTL epitope screening system in HLAtransgenic mice. Methodology/Principal Findings: HLA-A24 restricted CTL epitope peptides derived from internal proteins of the H5N1 highly pathogenic avian influenza A virus were predicted by CTL epitope peptide prediction programs. Of 35 predicted peptides, six peptides exhibited remarkable cytotoxic activity in vivo. More than half of the mice which were subcutaneously vaccinated with the three most immunogenic and highly conserved epitopes among three different influenza A virus subtypes (H1N1, H3N2 and H5N1) survived lethal influenza virus challenge during both effector and memory CTL phases. Furthermore, mice that were intranasally vaccinated with these peptides remained free of clinical signs after lethal virus challenge during the effector phase. Conclusions/Significance: This CTL epitope peptide selection system can be used as an effective tool for the development of a cross-protective human influenza vaccine. Furthermore this vaccine strategy can be applicable to the development o

Cross-Reactive T Cells Are Involved in Rapid Clearance of 2009 Pandemic H1N1 Influenza Virus in Nonhuman Primates

In mouse models of influenza, T cells can confer broad protection against multiple viral subtypes when antibodies raised against a single subtype fail to do so. However, the role of T cells in protecting humans against influenza remains unclear. Here we employ a translational nonhuman primate model to show that cross-reactive T cell responses play an important role in early clearance of infection with 2009 pandemic H1N1 influenza virus (H1N1pdm). To “prime” cellular immunity, we first infected 5 rhesus macaques with a seasonal human H1N1 isolate. These animals made detectable cellular and antibody responses against the seasonal H1N1 isolate but had no neutralizing antibodies against H1N1pdm. Four months later, we challenged the 5 “primed” animals and 7 naive controls with H1N1pdm. In naive animals, CD8+ T cells with an activated phenotype (Ki-67+ CD38+) appeared in blood and lung 5–7 days post inoculation (p.i.) with H1N1pdm and reached peak magnitude 7–10 days p.i. In contrast, activated T cells were recruited to the lung as early as 2 days p.i. in “primed” animals, and reached peak frequencies in blood and lung 4–7 days p.i. Interferon (IFN)-γ Elispot and intracellular cytokine staining assays showed that the virus-specific response peaked earlier and reached a higher magnitude in “primed” animals than in naive animals. This response involved both CD4+ and CD8+ T cells. Strikingly, “primed” animals cleared H1N1pdm infection significantly earlier from the upper and lower respiratory tract than the naive animals did, and before the appearance of H1N1pdm-specific neutralizing antibodies. Together, our results suggest that cross-reactive T cell responses can mediate early clearance of an antigenically novel influenza virus in primates. Vaccines capable of inducing such cross-reactive T cells may help protect humans against severe disease caused by newly emerging pandemic influenza viruses

HLA restriction of cell-mediated lysis of influenza virus-infected human cells.

MURINE T lymphocytes that mediate the lysis of virus-infected cells show specificity both for the viral cell surface antigens and for the H-2K or D antigens of the major histo-compatibility complex1-8. The cytotoxic T lymphocytes and the target cell must share H-2K or D products. The experiments reported here demonstrate that there is a similar requirement for partial HLA identity between human cytotoxic lymphocytes and influenza virus-infected target cells. © 1977 Nature Publishing Group

Mutation of the alpha 2 domain disulfide bridge of the class I molecule HLA-A*0201. Effect on maturation and peptide presentation.

A combination of saturation and site-directed mutagenesis was utilized to disrupt the alpha 2 domain disulfide bridge of HLA-A*0201. Mutation of cysteine 101 to a serine (C101S) or of cysteine 164 to alanine (C164A) decreased the rate of maturation of the heavy chain, the total amount of mature heavy chain within the cell, and the level of surface expression. Cells expressing these genes and loaded with a synthetic peptide derived from the influenza A matrix protein (58-66) were recognized poorly by HLA-A*0201-restricted, peptide-specific CTLs. Cells expressing mutant HLA-A*0201 loaded with a synthetic peptide derived from the HIV-1 pol protein (476-484) were not recognized by pol IV-9-specific CTLs. Mutant C164A cells infected with influenza virus were partially recognized by influenza matrix peptide-specific CTLs, while C101S cells were not lysed. Surprisingly, endogenous peptide loading of cells expressing mutant HLA-A*0201 using a minigene coding for either the influenza A matrix peptide 58-66, or HIV-1 pol peptide 476-484, resulted in efficient CTL recognition. This suggests different structural constraints for peptide binding in the endoplasmic reticulum during biosynthesis and for binding to exported molecules on the cells surface

The spectrum of tev gamma rays from the crab nebula

The spectrum of gamma rays from the Crab Nebula has been measured in the energy range 500 GeV-8 TeV at the Whipple Observatory by the atmospheric Cerenkov technique. Two methods of analysis that were used to derive spectra, in order to reduce the chance of calibration errors, gave good agreement, as did analysis of observations made with changed equipment several years apart. It is concluded that stable and reliable energy spectra can now be made in the TeV range. The spectrum can be represented in this energy range by the power-law fit, J = (3.20 ± 0.17 ± 0.6) × (E/1 TeV)-2.49±0-06±0-04 m-2 s-1 TeV-1, or by the following form, which extends much better to the GeV domain: J = (3.25 ± 0.14 ± 0.6) × 10-7 E-2.44±0-06±0.04-0.151 log10 E m-2 s-1 TeV-1 (E in TeV) The integral flux above 1 TeV is (2.1 ± 0.2 ± 0.3) × 10-7 m-2 s-1. Using the complete spectrum of the Crab Nebula, the spectrum of relativistic electrons is deduced, and the spectrum of the inverse Compton emission that they would generate is in good agreement with the observed gamma-ray flux from 1 GeV to many TeV, if the magnetic field in the region where these scattered photons originate (essentially the X-ray-emitting region, around 0.4 pc from the pulsar) is ∼16 nT (160 μG), in reasonable agreement with the field deduced by Aharonian and Atoyan. If the same field strength were present throughout the nebula, there would be no clear need for an additional radiation source in the GeV domain such as has recently been suggested; the results give an indication that the magnetic field is well below the often-assumed equipartition strength (35-60 nT). Further accurate gamma-ray spectral measurements over the range from 1 GeV to tens of TeV have the potential to probe the growth in the magnetic field in the inner region of the nebula. © 1998. The American Astronomical Society. All rights reserved

Differential Phenotypic and Functional Profiles of TcCA-2 -Specific Cytotoxic CD8+ T Cells in the Asymptomatic versus Cardiac Phase in Chagasic Patients

It has been reported that the immune response mediated by T CD8+ lymphocytes plays a critical role in the control of Trypanosoma cruzi infection and that the clinical symptoms of Chagas disease appear to be related to the competence of the CD8+ T immune response against the parasite. Herewith, in silico prediction and binding assays on TAP-deficient T2 cells were used to identify potential HLA-A*02:01 ligands in the T. cruzi TcCA-2 protein. The TcCA-2-specific CD8+ T cells were functionality evaluated by Granzyme B and cytokine production in peripheral blood mononuclear cells (PBMC) from Chagas disease patients stimulated with the identified HLA-A*02:01 peptides. The specific cells were phenotypically characterized by flow cytometry using several surface markers and HLA-A*02:01 APC-labeled dextramer loaded with the peptides. In the T. cruzi TcCA-2 protein four T CD8+ epitopes were identified which are processed and presented during Chagas disease. Interestingly, a differential cellular phenotypic profile could be correlated with the severity of the disease. The TcCA-2-specific T CD8+ cells from patients with cardiac symptoms are mainly effector memory cells (TEM and TEMRA) while, those present in the asymptomatic phase are predominantly naive cells (TNAIVE). Moreover, in patients with cardiac symptoms the percentage of cells with senescence features is significantly higher than in patients at the asymptomatic phase of the disease. We consider that the identification of these new class I-restricted epitopes are helpful for designing biomarkers of sickness pathology as well as the development of immunotherapies against T. cruzi infection.This work was supported by grants SAF2012-35777 and SAF2013-48527-R from Programa Estatal I+D+i (MINECO); Network of Tropical Diseases Research RICET, grants RD12/0018/0021 and RD12/0018/0018 (MSSSI, Spain) and FEDER. MS and BC were also supported by grant FIS, 2009SGR385 from ISCIII (MSSSI, Spain). Coauthor Concepción Marañón is employed by Genomic Medicine Department, GENYO. Centre for Genomics and Oncological Research: Pfizer / University of Granada / Andalusian Regional Government. Centre for Genomics and Oncological Research: Pfizer / University of Granada / Andalusian Regional Government provided support in the form of salaries for author Concepción Marañón, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific role of this author is articulated in the author contributions section.Peer reviewe

The TeV spectrum of Markarian 501

The energy spectrum of the active galactic nucleus Markarian 501 has been determined from 0.3 to 10 TeV with the Whipple Observatory Cerenkov Imaging Telescope, by using both small zenith angle and large zenith angle data taken between 1997 February 14 and June 8. The TeV emission from Mrk 501 was unprecedentedly high, allowing a statistically accurate spectrum to be derived. In contrast to previously measured TeV spectra, the spectrum over this energy region is not well described by a simple power law. Instead, the spectrum exhibits significant curvature and can be well fitted by a parabolic spectrum proportional to E-2.22 ± 0.04 ± 0.05 - (0.47 ± 0.07) log10E, where the first set of errors is statistical and the second systematic and E is in units of TeV. Simple power-law fits to the TeV data are also inconsistent with upper limits from EGRET observations that temporally overlap a subset of the TeV observations. The data show a statistically significant signal above energies of 7 TeV. This energy, combined with variability timescales, yields a Doppler beaming factor, δ, of at least 1.5