Identification of Class I HLA T Cell Control Epitopes for West Nile Virus

The recent West Nile virus (WNV) outbreak in the United States underscores the importance of understanding human immune responses to this pathogen. Via the presentation of viral peptide ligands at the cell surface, class I HLA mediate the T cell recognition and killing of WNV infected cells. At this time, there are two key unknowns in regards to understanding protective T cell immunity: 1) the number of viral ligands presented by the HLA of infected cells, and 2) the distribution of T cell responses to these available HLA/viral complexes. Here, comparative mass spectroscopy was applied to determine the number of WNV peptides presented by the HLA-A*11:01 of infected cells after which T cell responses to these HLA/WNV complexes were assessed. Six viral peptides derived from capsid, NS3, NS4b, and NS5 were presented. When T cells from infected individuals were tested for reactivity to these six viral ligands, polyfunctional T cells were focused on the GTL9 WNV capsid peptide, ligands from NS3, NS4b, and NS5 were less immunogenic, and two ligands were largely inert, demonstrating that class I HLA reduce the WNV polyprotein to a handful of immune targets and that polyfunctional T cells recognize infections by zeroing in on particular HLA/WNV epitopes. Such dominant HLA/peptide epitopes are poised to drive the development of WNV vaccines that elicit protective T cells as well as providing key antigens for immunoassays that establish correlates of viral immunity. © 2013 Kaabinejadian et al

Direct Interrogation of Viral Peptides Presented by the Class I HLA of HIV-Infected T Cells

Identification of CD8+ cytotoxic T lymphocyte (CTL) epitopes has traditionally relied upon testing of overlapping peptide libraries for their reactivity with T cells in vitro. Here, we pursued deep ligand sequencing (DLS) as an alternative method of directly identifying those ligands that are epitopes presented to CTLs by the class I human leukocyte antigens (HLA) of infected cells. Soluble class I HLA-A*11:01 (sHLA) was gathered from HIV-1 NL4-3-infected human CD4+ SUP-T1 cells. HLA-A*11:01 harvested from infected cells was immunoaffinity purified and acid boiled to release heavy and light chains from peptide ligands that were then recovered by size-exclusion filtration. The ligands were first fractionated by high-pH high-pressure liquid chromatography and then subjected to separation by nano-liquid chromatography (nano-LC)–mass spectrometry (MS) at low pH. Approximately 10 million ions were selected for sequencing by tandem mass spectrometry (MS/MS). HLA-A*11:01 ligand sequences were determined with PEAKS software and confirmed by comparison to spectra generated from synthetic peptides. DLS identified 42 viral ligands presented by HLA-A*11:01, and 37 of these were previously undetected. These data demonstrate that (i) HIV-1 Gag and Nef are extensively sampled, (ii) ligand length variants are prevalent, particularly within Gag and Nef hot spots where ligand sequences overlap, (iii) noncanonical ligands are T cell reactive, and (iv) HIV-1 ligands are derived from de novo synthesis rather than endocytic sampling. Next-generation immunotherapies must factor these nascent HIV-1 ligand length variants and the finding that CTL-reactive epitopes may be absent during infection of CD4+ T cells into strategies designed to enhance T cell immunity

A shared MHC supertype motif emerges by convergent evolution in macaques and mice, but is totally absent in human MHC molecules

The SIV-infected rhesus macaque (Macaca mulatta) is the most established model of AIDS disease systems, providing insight into pathogenesis and a model system for testing novel vaccines. The understanding of cellular immune responses based on the identification and study of Major Histocompatibility Complex (MHC) molecules, including their MHC:peptide-binding motif, provides valuable information to decipher outcomes of infection and vaccine efficacy. Detailed characterization of Mamu-B*039:01, a common allele expressed in Chinese rhesus macaques, revealed a unique MHC:peptide-binding preference consisting of glycine at the second position. Peptides containing a glycine at the second position were shown to be antigenic from animals positive for Mamu-B*039:01. A similar motif was previously described for the Dd mouse MHC allele, but for none of the human HLA molecules for which a motif is known. Further investigation showed that one additional macaque allele, present in Indian rhesus macaques, Mamu-B*052:01, shares this same motif. These “G2” alleles were associated with the presence of specific residues in their B pocket. This pocket structure was found in 6% of macaque sequences but none of 950 human HLA class I alleles. Evolutionary studies using the “G2” alleles points to common ancestry for the macaque sequences, while convergent evolution is suggested when murine and macaque sequences are considered. This is the first detailed characterization of the pocket residues yielding this specific motif in nonhuman primates and mice, revealing a new supertype motif not present in humans

Mineral nutrition of peas (Pisum sativum L.3, varieties asgrow 40 and okaw

Deficiencies of the macronutrients and boron. The presente work was carried out in order to study: a - The effect of the omission and presence of the macronutrients and boron on the growth of the plants - variety Asgrow 40; b - deficiency symptoms of macronutrients and boron in the same variety; c - the effects of the deficiencies of each macronutrient and boron on the dry matter production and on the chemical composition of the plant-variety Asgrow 40. I - Deficiencies of the macronutrients Young peas plants (Pisum sativum, L.), variety Asgrow 40 were grown in pots containing pure quartz. Several times a day they were irrigated by percolation with nutrient solution. The treatments were: complete solution and deficient solution, in which each one of the macronutrient was omitted as well boron. Soon as the malnutrition symptoms apperared, the plants were harvested and divided into: roots, stalks, inferior and superior leaves, flowers, husk containing the seeds. The dry matter was analysed chemically. Conclusions: 1 - Symptoms of malnutrition were observed for N, P, K, Ca and B; 2 - The omission of the macronutrients and boron affected the dry matter production, excepted for magnesium and or sulphur; 3 - The nutrient content in the dry matter, expressed in porcentages (%) and or parts per million (ppm) in deficient leaves and healthy leaves were:O presente trabalho teve o objetivo de estudar alguns aspectos da nutrição da ervilha (Pisum sativum, L.), variedade Okaw ("Torta-de-Flor-Roxa") e Asgrow 40 no que concerne: 1 - Efeitos das omissões dos macronutrientes e do boro, na obtenção de um quadro sintomatológico das carências, na variedade Asgrow 40. 2 - Efeitos das carências dos macronutrientes e do boro na produção de matéria seca e composição química na variedade Asgrow 40. Sementes de ervilha (Pisum sativum, L.) da variedade Asgrow 40, foram postas a germinar em silica, mantendo-se um teor de umidade adequado. Passou-se a regar as plântulas com soluções nutritivas com omissões dos nutrientes, entre 20 e 25 dias após a germinação. Tão logo se delineou o quadro sintomatológico das carências, as plantas foram coletadas, mensuradas em altura (cm) e peso da matéria seca (g). Divididas em raízes, caules, folhas inferiores e superiores, flores, vagens e analisadas. Os dados mostram que: 1 - As omissões dos macronutrientes com excessão do magnésio e do enxofre, apresentaram sintomas visuais de deficiência características. 2 - A produção da matéria seca foi afetada em todos os tratamentos com omissão de nutrientes, com excessão daqueles nos quais foram omitidos o magnésio e/ou enxofre. 3 - Os teores dos nutrientes expressos em porcentagem e/ou partes por milhão em folhas apresentando ou não sintomas de carências foram

A Recombinant Influenza A Virus Expressing Domain III of West Nile Virus Induces Protective Immune Responses against Influenza and West Nile Virus

West Nile virus (WNV) continues to circulate in the USA and forms a threat to the rest of the Western hemisphere. Since methods for the treatment of WNV infections are not available, there is a need for the development of safe and effective vaccines. Here, we describe the construction of a recombinant influenza virus expressing domain III of the WNV glycoprotein E (Flu-NA-DIII) and its evaluation as a WNV vaccine candidate in a mouse model. FLU-NA-DIII-vaccinated mice were protected from severe body weight loss and mortality caused by WNV infection, whereas control mice succumbed to the infection. In addition, it was shown that one subcutaneous immunization with 105 TCID50 Flu-NA-DIII provided 100% protection against challenge. Adoptive transfer experiments demonstrated that protection was mediated by antibodies and CD4+T cells. Furthermore, mice vaccinated with FLU-NA-DIII developed protective influenza virus-specific antibody titers. It was concluded that this vector system might be an attractive platform for the development of bivalent WNV-influenza vaccines

MHC-I peptides get out of the groove and enable a novel mechanism of HIV-1 escape

Major histocompatibility complex class I (MHC-I) molecules play a crucial role in immunity by capturing peptides for presentation to T cells and natural killer (NK) cells. The peptide termini are tethered within the MHC-I antigen-binding groove, but it is unknown whether other presentation modes occur. Here we show that 20% of the HLA-B*57:01 peptide repertoire comprises N-terminally extended sets characterized by a common motif at position 1 (P1) to P2. Structures of HLA-B*57:01 presenting N-terminally extended peptides, including the immunodominant HIV-1 Gag epitope TW10 (TSTLQEQIGW), showed that the N terminus protrudes from the peptide-binding groove. The common escape mutant TSNLQEQIGW bound HLA-B*57:01 canonically, adopting a dramatically different conformation than the TW10 peptide. This affected recognition by killer cell immunoglobulin-like receptor (KIR) 3DL1 expressed on NK cells. We thus define a previously uncharacterized feature of the human leukocyte antigen class I (HLA-I) immunopeptidome that has implications for viral immune escape. We further suggest that recognition of the HLA-B*57:01-TW10 epitope is governed by a 'molecular tension' between the adaptive and innate immune systems