Browsing by MeSH term "Antigens, Viral"
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Publication A monomeric uncleaved respiratory syncytial virus F antigen retains prefusion-specific neutralizing epitopes(American Society for Microbiology (ASM), 2014-10) Swanson, Kurt A; Balabanis, Kara; Xie, Yuhong; Aggarwal, Yukti; Palomo-Sanz, Concepcion; Mas-Lloret, Vicente; Metrick, Claire; Yang, Hui; Shaw, Christine A; Melero, Jose Antonio; Dormitzer, Philip R; Carfi, Andrea; Plan Nacional de I+D+i (España); Ministerio de Economía y Competitividad (España)Respiratory syncytial virus (RSV) is the leading infectious cause of severe respiratory disease in infants and a major cause of respiratory illness in the elderly. There remains an unmet vaccine need despite decades of research. Insufficient potency, homogeneity, and stability of previous RSV fusion protein (F) subunit vaccine candidates have hampered vaccine development. RSV F and related parainfluenza virus (PIV) F proteins are cleaved by furin during intracellular maturation, producing disulfide-linked F1 and F2 fragments. During cell entry, the cleaved Fs rearrange from prefusion trimers to postfusion trimers. Using RSV F constructs with mutated furin cleavage sites, we isolated an uncleaved RSV F ectodomain that is predominantly monomeric and requires specific cleavage between F1 and F2 for self-association and rearrangement into stable postfusion trimers. The uncleaved RSV F monomer is folded and homogenous and displays at least two key RSV-neutralizing epitopes shared between the prefusion and postfusion conformations. Unlike the cleaved trimer, the uncleaved monomer binds the prefusion-specific monoclonal antibody D25 and human neutralizing immunoglobulins that do not bind to postfusion F. These observations suggest that the uncleaved RSV F monomer has a prefusion-like conformation and is a potential prefusion subunit vaccine candidate. Importance: RSV is the leading infectious cause of severe respiratory disease in infants and a major cause of respiratory illness in the elderly. Development of an RSV vaccine was stymied when a clinical trial using a formalin-inactivated RSV virus made disease, following RSV infection, more severe. Recent studies have defined the structures that the RSV F envelope glycoprotein adopts before and after virus entry (prefusion and postfusion conformations, respectively). Key neutralization epitopes of prefusion and postfusion RSV F have been identified, and a number of current vaccine development efforts are focused on generating easily produced subunit antigens that retain these epitopes. Here we show that a simple modification in the F ectodomain results in a homogeneous protein that retains critical prefusion neutralizing epitopes. These results improve our understanding of RSV F protein folding and structure and can guide further vaccine design efforts.Publication A viral, transporter associated with antigen processing (TAP)-independent, high affinity ligand with alternative interactions endogenously presented by the nonclassical human leukocyte antigen E class I molecule(American Society for Biochemistry and Molecular Biology (ASBMB), 2012-10-12) Lorente, Elena; Infantes, Susana; Abia, David; Barnea, Eilon; Beer, Ilan; Garcia, Ruth; Lasala, Fatima; Jimenez, Mercedes; Mir-Gerrero, Carmen; Morreale, Antonio; Admon, Arie; Lopez, Daniel; Ministerio de Ciencia e Innovación (España); Fundación para la Investigación y la Prevención del Sida en España; Israel Science FoundationThe transporter associated with antigen processing (TAP) enables the flow of viral peptides generated in the cytosol by the proteasome and other proteases to the endoplasmic reticulum, where they complex with nascent human leukocyte antigen (HLA) class I. Later, these peptide-HLA class I complexes can be recognized by CD8(+) lymphocytes. Cancerous cells and infected cells in which TAP is blocked, as well as individuals with unusable TAP complexes, are able to present peptides on HLA class I by generating them through TAP-independent processing pathways. Here, we identify a physiologically processed HLA-E ligand derived from the D8L protein in TAP-deficient vaccinia virus-infected cells. This natural high affinity HLA-E class I ligand uses alternative interactions to the anchor motifs previously described to be presented on nonclassical HLA class I molecules. This octameric peptide was also presented on HLA-Cw1 with similar binding affinity on both classical and nonclassical class I molecules. In addition, this viral peptide inhibits HLA-E-mediated cytolysis by natural killer cells. Comparison between the amino acid sequences of the presenting HLA-E and HLA-Cw1 alleles revealed a shared structural motif in both HLA class molecules, which could be related to their observed similar cross-reactivity affinities. This motif consists of several residues located on the floor of the peptide-binding site. These data expand the role of HLA-E as an antigen-presenting molecule.Publication Antigen processing of a short viral antigen by proteasomes(American Society for Biochemistry and Molecular Biology (ASBMB), 2006-10-13) Lopez, Daniel; Calero, Olga; Jimenez, Mercedes; García-Calvo, Margarita; Del Val, Margarita; Instituto de Salud Carlos III; Comunidad de Madrid (España); Ministerio de Educación y Ciencia (España)Mass spectrometry (MS)-based methods coupled to reverse phase chromatography separation are a useful technology to analyze complex peptide pools that are comprised of different peptides with unrelated sequences. In antigen presentation, proteasomes generate a set of short peptides that are closely related and overlapping and in some instances may even have identical retention times and identical masses. In these situations, micro-liquid chromatography-MS/MS focused on each theoretical parent ion followed by manual interpretation optimizes the identification of generated peptides. The results suggest that the degradation of short antigens by the proteasome occurs by sequential cleavage.Publication Characterization of a Prefusion-Specific Antibody That Recognizes a Quaternary, Cleavage-Dependent Epitope on the RSV Fusion Glycoprotein(Public Library of Science (PLOS), 2015-07) Gilman, Morgan S A; Moin, Syed M; Mas-Lloret, Vicente; Chen, Man; Patel, Nita K; Kramer, Kari; Zhu, Qing; Kabeche, Stephanie C; Kumar, Azad; Palomo-Sanz, Concepcion; Beaumont, Tim; Baxa, Ulrich; Ulbrandt, Nancy D; Melero, Jose Antonio; Graham, Barney S; McLellan, Jason S; Cancer Resarch Institute (Estados Unidos); Ministerio de Economía y Competitividad (España); NIH - National Institute of Allergy and Infectious Diseases (NIAID) (Estados Unidos)Prevention efforts for respiratory syncytial virus (RSV) have been advanced due to the recent isolation and characterization of antibodies that specifically recognize the prefusion conformation of the RSV fusion (F) glycoprotein. These potently neutralizing antibodies are in clinical development for passive prophylaxis and have also aided the design of vaccine antigens that display prefusion-specific epitopes. To date, prefusion-specific antibodies have been shown to target two antigenic sites on RSV F, but both of these sites are also present on monomeric forms of F. Here we present a structural and functional characterization of human antibody AM14, which potently neutralized laboratory strains and clinical isolates of RSV from both A and B subtypes. The crystal structure and location of escape mutations revealed that AM14 recognizes a quaternary epitope that spans two protomers and includes a region that undergoes extensive conformational changes in the pre- to postfusion F transition. Binding assays demonstrated that AM14 is unique in its specific recognition of trimeric furin-cleaved prefusion F, which is the mature form of F on infectious virions. These results demonstrate that the prefusion F trimer contains potent neutralizing epitopes not present on monomers and that AM14 should be particularly useful for characterizing the conformational state of RSV F-based vaccine antigens.Publication Characterization of an enhanced antigenic change in the pandemic 2009 H1N1 influenza virus haemagglutinin(Cambridge University Press, 2014-05) Garcia-Barreno, Blanca; Delgado-Romero, Teresa; Benito, Sonia; Casas Flecha, Inmaculada; Pozo Sanchez, Francisco; Cuevas, Maria Teresa; Mas-Lloret, Vicente; Trento, Alfonsina; Rodriguez-Frandsen, Ariel; Falcón, Ana; Ortín, Juan; Nieto, Amelia; Melero, Jose Antonio; Instituto de Salud Carlos III; Plan Nacional de I+D+i (España)Murine hybridomas producing neutralizing mAbs specific to the pandemic influenza virus A/California/07/2009 haemagglutinin (HA) were isolated. These antibodies recognized at least two different but overlapping new epitopes that were conserved in the HA of most Spanish pandemic isolates. However, one of these isolates (A/Extremadura/RR6530/2010) lacked reactivity with the mAbs and carried two unique mutations in the HA head (S88Y and K136N) that were required simultaneously to eliminate reactivity with the murine antibodies. This unusual requirement directly illustrates the phenomenon of enhanced antigenic change proposed previously for the accumulation of simultaneous amino acid substitutions at antigenic sites of the influenza A virus HA during virus evolution (Shih et al., Proc Natl Acad Sci USA, 104 , 6283-6288, 2007). The changes found in the A/Extremadura/RR6530/2010 HA were not found in escape mutants selected in vitro with one of the mAbs, which contained instead nearby single amino acid changes in the HA head. Thus, either single or double point mutations may similarly alter epitopes of the new antigenic site identified in this work in the 2009 H1N1 pandemic virus HA. Moreover, this site is relevant for the human antibody response, as shown by competition of mAbs and human post-infection sera for virus binding. The results are discussed in the context of the HA antigenic structure and challenges posed for identification of sequence changes with possible antigenic impact during virus surveillance.Publication Engineering, Structure and Immunogenicity of the Human Metapneumovirus F Protein in the Postfusion Conformation(Public Library of Science (PLOS), 2016-09-09) Mas-Lloret, Vicente; Rodriguez, Laura; Olmedillas Cela, Eduardo; Cano, Olga; Palomo-Sanz, Concepcion; Terrón-Orellana, Maria Carmen; Luque, Daniel; Melero, Jose Antonio; McLellan, Jason S; Ministerio de Economía y Competitividad (España); National Institutes of Health (Estados Unidos)Human metapneumovirus (hMPV) is a paramyxovirus that is a common cause of bronchiolitis and pneumonia in children less than five years of age. The hMPV fusion (F) glycoprotein is the primary target of neutralizing antibodies and is thus a critical vaccine antigen. To facilitate structure-based vaccine design, we stabilized the ectodomain of the hMPV F protein in the postfusion conformation and determined its structure to a resolution of 3.3 Å by X-ray crystallography. The structure resembles an elongated cone and is very similar to the postfusion F protein from the related human respiratory syncytial virus (hRSV). In contrast, significant differences were apparent with the postfusion F proteins from other paramyxoviruses, such as human parainfluenza type 3 (hPIV3) and Newcastle disease virus (NDV). The high similarity of hMPV and hRSV postfusion F in two antigenic sites targeted by neutralizing antibodies prompted us to test for antibody cross-reactivity. The widely used monoclonal antibody 101F, which binds to antigenic site IV of hRSV F, was found to cross-react with hMPV postfusion F and neutralize both hRSV and hMPV. Despite the cross-reactivity of 101F and the reported cross-reactivity of two other antibodies, 54G10 and MPE8, we found no detectable cross-reactivity in the polyclonal antibody responses raised in mice against the postfusion forms of either hMPV or hRSV F. The postfusion-stabilized hMPV F protein did, however, elicit high titers of hMPV-neutralizing activity, suggesting that it could serve as an effective subunit vaccine. Structural insights from these studies should be useful for designing novel immunogens able to induce wider cross-reactive antibody responses.Publication Estrategias de diagnóstico en España(Ministerio de Sanidad y Consumo (España), 1999-09) Echevarria, Juan EmilioEl virus del sarampión (VS) pertenece al género Morbillivirus de la familia Paramy- xoviridae, al igual que otros virus patógenos para el hombre, como el virus de la parotidi- tis y los virus de la parainfluenza 2 y 4 (gé- nero Rubulavirus), los virus de la parain- fluenza 1 y 3 (género Param~xovirus) y el virus respiratorio sincitial (género Pneumo- virus). Como todos los miembros de esta fa- milia, tiene como genoma un RNA no seg- mentado y de polaridad negativa de 15.893 pares de bases que se recubre de proteínas para formar una nucleocápside de simetría helicoidal. Entre estas proteínas, la más abundante es la nucleoproteína (N), estando también presentes la fosfoproteína (P) y la RNA polimerasa RNA dependiente (L), que ha de estar presente en el virión ya que, al tratarse de un RNA de polaridad negativa, éste debe transcribirse en un mRNA como paso previo a la síntesis de cualquier proteí- na vírica y ninguna enzima celular es capaz de transcribir RNA. Esta nucleoproteína se encuentra dentro de una envuelta lipoprotei- ca en la que la parte lipídica procede de la célula huésped y las proteínas son aportadas por el virus, siendo éstas la hemaglutinina (H) y la proteína de fusión (F). Ambas diri- gen la entrada del virus en las células, que se realiza por la interacción de H con un recep- tor celular, de naturaleza no del todo escla- recida, y la posterior fusión de la envuelta li- pídica con la membrana celular a través de H y F, permitiendo el acceso de la nucleo- cápside al citoplasma celular. Trabando la nucleocápside y la envuelta se encuentra la proteína matriz (M), que interacciona con ambas, dando consistencia al virión, el cual se presenta al microscopio electrónico como muy pleomórfico, abundando en las prepa- raciones las nucleocápsides libres proceden- tes de la ruptura de los viriones a causa de su gran fragilidad.Publication Exogenous, TAP-independent lysosomal presentation of a respiratory syncytial virus CTL epitope.(Wiley, 2012-11) Ramos, Manuel; Garcia-Barreno, Blanca; Lopez, Daniel; Melero, Jose Antonio; Val, Margarita del; Johnstone, Carolina; Ministerio de Ciencia e Innovación (España); Instituto de Salud Carlos IIIRespiratory syncytial virus causes lower respiratory tract infections in infancy and old age, affecting also immunocompromised patients. The viral fusion protein is an important vaccine candidate eliciting antibody and cell-mediated immune responses. CD8(+) cytotoxic T lymphocytes (CTLs) are known to have a role in both lung pathology and viral clearance. In BALB/c mice, the fusion protein epitope F249-258 is presented to CTLs by the murine major histocompatibility complex (MHC) class I molecule K(d). In cells infected with recombinant vaccinia viruses encoding the fusion protein, F249-258 is presented by MHC class I molecules through pathways that are independent of the transporters associated with antigen processing (TAP). We have now found that F249-258 can be generated from non-infectious virus from an exogenous source. Antigen processing follows a lysosomal pathway that appears to require autophagy. As a practical consequence, inactivated virus suffices for in vivo priming of virus-specific CTLs.Publication Influence of respiratory syncytial virus strain differences on pathogenesis and immunity(Springer, 2013) Melero, Jose Antonio; Moore, Martin L; Instituto de Salud Carlos IIIMolecular epidemiology studies have provided convincing evidence of antigenic and sequence variability among respiratory syncytial virus (RSV) isolates. Circulating viruses have been classified into two antigenic groups (A and B) that correlate with well-delineated genetic groups. Most sequence and antigenic differences (both inter- and intra-groups) accumulate in two hypervariable segments of the G-protein gene. Sequences of the G gene have been used for phylogenetic analyses. These studies have shown a worldwide distribution of RSV strains with both local and global replacement of dominant viruses with time. Although data are still limited, there is evidence that strain variation may contribute to differences in pathogenicity. In addition, there is some but limited evidence that RSV variation may be, at least partially, immune (antibody) driven. However, there is the paradox in RSV that, in contrast to other viruses (e.g., influenza viruses) the epitopes recognized by the most effective RSV-neutralizing antibodies are highly conserved. In contrast, antibodies that recognize strain-specific epitopes are poorly neutralizing. It is likely that this apparent contradiction is due to the lack of a comprehensive knowledge of the duration and specificities of the human antibody response against RSV antigens. Since there are some data supporting a group- (or clade-) specific antibody response after a primary infection in humans, it may be wise to consider the incorporation of strains representative of groups A and B (or their antigens) in future RSV vaccine development.Publication Mpox, herpes, and enteroviruses: Differential diagnosis(Wiley, 2024-01) Guillén-Calvo, Laura; Negredo, Anabel; Sánchez-Mora, Patricia; Molero, Paqui; Vazquez, Ana; Cabrerizo, Maria; Orviz, Eva; Ledesma, Juan; Estrada, Vicente; Tarrago Asensio, David; Sánchez-Seco, María Paz; Instituto de Salud Carlos III; Ministerio de Ciencia e Innovación (España); Unión Europea. Comisión Europea. NextGenerationEU; Centro de Investigación Biomédica en Red - CIBERINFEC (Enfermedades Infecciosas); Centro de Investigación Biomédica en Red - CIBERESP (Epidemiología y Salud Pública)Publication Multiple proteases process viral antigens for presentation by MHC class I molecules to CD8(+) T lymphocytes.(Elsevier, 2002-10) Val, Margarita del; Lopez, Daniel; Unión Europea; Comunidad de Madrid (España); Instituto de Salud Carlos IIIRecognition by CD8(+) cytotoxic T lymphocytes of any intracellular viral protein requires its initial cytosolic proteolytic processing, the translocation of processed peptides to the endoplasmic reticulum via the transporters associated with antigen processing, and their binding to nascent major histocompatibility complex (MHC) class I molecules that then present the antigenic peptides at the infected cell surface. From initial assumptions that the multicatalytic and ubiquitous proteasome is the only protease capable of fully generating peptide ligands for MHC class I molecules, the last few years have seen the identification of a number of alternative proteases that contribute to endogenous antigen processing. Trimming by non-proteasomal proteases of precursor peptides produced by proteasomes is now a well-established fact. In addition, proteases that can process antigens in a fully proteasome-independent fashion have also been identified. The final level of presentation of many viral epitopes is probably the result of interplay between different proteolytic activities. This expands the number of tissues and physiological and pathological situations compatible with antigen presentation, as well as the universe of pathogen-derived sequences available for recognition by CD8(+) T lymphocytes.Publication Need for tripeptidyl-peptidase II in major histocompatibility complex class I viral antigen processing when proteasomes are detrimental(American Society for Biochemistry and Molecular Biology (ASBMB), 2006-12-29) Guil, Sara; Rodríguez-Castro, Marta; Aguilar, Francisco; Villasevil, Eugenia M; Antón, Luis C; Val, Margarita del; Ministerio de Educación y Ciencia (España); Instituto de Salud Carlos III; Fundación Ramón Areces; Comunidad de Madrid (España)CD8(+) T lymphocytes recognize infected cells that display virus-derived antigenic peptides complexed with major histocompatibility complex class I molecules. Peptides are mainly byproducts of cellular protein turnover by cytosolic proteasomes. Cytosolic tripeptidyl-peptidase II (TPPII) also participates in protein degradation. Several peptidic epitopes unexpectedly do not require proteasomes, but it is unclear which proteases generate them. We studied antigen processing of influenza virus nucleoprotein epitope NP(147-155), an archetype epitope that is even destroyed by a proteasome-mediated mechanism. TPPII, with the assistance of endoplasmic reticulum trimming metallo-aminopeptidases, probably ERAAP (endoplasmic reticulum aminopeptidase associated with antigen processing), was crucial for nucleoprotein epitope generation both in the presence of functional proteasomes and when blocked by lactacystin, as shown with specific chemical inhibitors and gene silencing. Different protein contexts and subcellular targeting all allowed epitope processing by TPPII as well as trimming. The results show the plasticity of the cell's assortment of proteases for providing ligands for recognition by antiviral CD8(+) T cells. Our observations identify for the first time a set of proteases competent for antigen processing of an epitope that is susceptible to destruction by proteasomes.Publication Structural, antigenic and immunogenic features of respiratory syncytial virus glycoproteins relevant for vaccine development(Elsevier, 2017) Melero, Jose Antonio; Mas-Lloret, Vicente; McLellan, Jason SExtraordinary progress in the structure and immunobiology of the human respiratory syncytial virus glycoproteins has been accomplished during the last few years. Determination of the fusion (F) glycoprotein structure folded in either the prefusion or the postfusion conformation was an inspiring breakthrough not only to understand the structural changes associated with the membrane fusion process but additionally to appreciate the antigenic intricacies of the F protein. Furthermore, these developments have opened new avenues for structure-based designs of promising hRSV vaccine candidates. Finally, recent advances in our knowledge of the attachment (G) glycoprotein and its interaction with cell-surface receptors have revitalized interest in this molecule as a vaccine, as well as its role in hRSV immunobiology.Publication The DC receptor DNGR-1 mediates cross-priming of CTLs during vaccinia virus infection in mice(American Society for Clinical Investigation (ASCI), 2012-05) Iborra, Salvador; Izquierdo-Fernandez, Helena Maria; Martinez-Lopez, Maria; Blanco-Menendez, Noelia; Reis e Sousa, Caetano; Sancho, David; Ministerio de Ciencia e Innovación (España); Unión Europea. Comisión Europea. European Research Council (ERC)In order to prime T cells, DCs integrate signals emanating directly from pathogens and from their noxious action on the host. DNGR-1 (CLEC9A) is a DC-restricted receptor that detects dead cells. Therefore, we investigated the possibility that DNGR-1 affects immunity to cytopathic viruses. DNGR-1 was essential for cross-presentation of dying vaccinia virus-infected (VACV-infected) cells to CD8(+) T cells in vitro. Following injection of VACV or VACV-infected cells into mice, DNGR-1 detected the ligand in dying infected cells and mediated cross-priming of anti-VACV CD8(+) T cells. Loss of DNGR-1 impaired the CD8+ cytotoxic response to VACV, especially against those virus strains that are most dependent on cross-presentation. The decrease in total anti-VACV CTL activity was associated with a profound increase in viral load and delayed resolution of the primary lesion. In addition, lack of DNGR-1 markedly diminished protection from infection induced by vaccination with the modified vaccinia Ankara (MVA) strain. DNGR-1 thus contributes to anti-VACV immunity, following both primary infection and vaccination. The non-redundant ability of DNGR-1 to regulate cross-presentation of viral antigens suggests that this form of regulation of antiviral immunity could be exploited for vaccination.Publication Unusual viral ligand with alternative interactions is presented by HLA-Cw4 in human respiratory syncytial virus-infected cells.(Wiley, 2011-05) Infantes, Susana; Lorente, Elena; Cragnolini, Juan José; Ramos, Manuel; Garcia, Ruth; Jimenez, Mercedes; Iborra, Salvador; Val, Margarita del; Lopez, Daniel; Instituto de Salud Carlos IIIShort viral antigens bound to human major histocompatibility complex (HLA) class I molecules are presented on infected cells. Vaccine development frequently relies on synthetic peptides to identify optimal HLA class I ligands. However, when natural peptides are analyzed, more complex mixtures are found. By immunoproteomics analysis, we identify in this study a physiologically processed HLA ligand derived from the human respiratory syncytial virus matrix protein that is very different from what was expected from studies with synthetic peptides. This natural HLA-Cw4 class I ligand uses alternative interactions to the anchor motifs previously described for its presenting HLA-Cw4 class I molecule. Finally, this octameric peptide shares its C-terminal core with the H-2D(b) nonamer ligand previously identified in the mouse model. These data have implications for the identification of antiviral cytotoxic T lymphocyte responses and for vaccine development.Publication Virological surveillance of influenza viruses in the WHO European Region in 2019/20 - impact of the COVID-19 pandemic(European Centre for Disease Prevention and Control (ECDC), 2020-11) Melidou, Angeliki; Pereyaslov, Dmitriy; Hungnes, Olav; Prosenc, Katarina; Alm, Erik; Adlhoch, Cornelia; Fielding, James; Sneiderman, Miriam; Martinuka, Oksana; Celentano, Lucia Pastore; Pebody, Richard; WHO European Region influenza surveillance network; Pozo Sanchez, Francisco; Casas Flecha, InmaculadaThe COVID-19 pandemic negatively impacted the 2019/20 WHO European Region influenza surveillance. Compared with previous 4-year averages, antigenic and genetic characterisations decreased by 17% (3,140 vs 2,601) and 24% (4,474 vs 3,403). Of subtyped influenza A viruses, 56% (26,477/47,357) were A(H1)pdm09, 44% (20,880/47,357) A(H3). Of characterised B viruses, 98% (4,585/4,679) were B/Victoria. Considerable numbers of viruses antigenically differed from northern hemisphere vaccine components. In 2020/21, maintaining influenza virological surveillance, while supporting SARS-CoV-2 surveillance is crucial.