Fátima Lasala

C-Type Lectins DC-SIGN and L-SIGN Mediate Cellular Entry by Ebola Virus in cis and in trans

ABSTRACT Ebola virus is a highly lethal pathogen responsible for several outbreaks of hemorrhagic fever. Here we show that the primate lentiviral binding C-type lectins DC-SIGN and L-SIGN act as cofactors for cellular entry by Ebola virus. Furthermore, DC-SIGN on the surface of dendritic cells is able to function as a trans receptor, binding Ebola virus-pseudotyped lentiviral particles and transmitting infection to susceptible cells. Our data underscore a role for DC-SIGN and L-SIGN in the infective process and pathogenicity of Ebola virus infection.

Acute respiratory distress syndrome after convalescent plasma use: treatment of a patient with Ebola virus disease contracted in Madrid, Spain

BACKGROUND In the current epidemic of Ebola virus disease, health-care workers have been transferred to Europe and the USA for optimised supportive care and experimental treatments. We describe the clinical course of the first case of Ebola virus disease contracted outside of Africa, in Madrid, Spain. METHODS Herein we report clinical, laboratory, and virological findings of the treatment of a female nurse assistant aged 44 years who was infected with Ebola virus around Sept 25-26, 2014, while caring for a Spanish missionary with confirmed Ebola virus disease who had been medically evacuated from Sierra Leone to La Paz-Carlos III University Hospital, Madrid. We also describe the use of experimental treatments for Ebola virus disease in this patient. FINDINGS The patient was symptomatic for 1 week before first hospital admission on Oct 6, 2014. We used supportive treatment with intravenous fluids, broad-spectrum antibiotics, and experimental treatments with convalescent plasma from two survivors of Ebola virus disease and high-dose favipiravir. On day 10 of illness, she had acute respiratory distress syndrome, possibly caused by transfusion-related acute lung injury, which was managed without mechanical ventilation. Discharge was delayed because of the detection of viral RNA in several bodily fluids despite clearance of viraemia. The patient was discharged on day 34 of illness. At the time of discharge, the patient had possible subacute post-viral thyroiditis. None of the people who had contact with the patient before and after admission became infected with Ebola virus. INTERPRETATION This report emphasises the uncertainties about the efficacy of experimental treatments for Ebola virus disease. Clinicians should be aware of the possibility of transfusion-related acute lung injury when using convalescent plasma for the treatment of Ebola virus disease. FUNDING La Paz-Carlos III University Hospital.

In Vitro Evaluation of Cyanovirin-N Antiviral Activity, by Use of Lentiviral Vectors Pseudotyped with Filovirus Envelope Glycoproteins

Cyanovirin-N (CV-N) has been shown to inhibit Ebola Zaire virus (EboZV) infection, both in vitro and in vivo, through its ability to bind to oligomannoses-8/9 on the EboZV surface glycoprotein (GP). Here, we report the in vitro potency of CV-N to inhibit EboZV GP- and Marburg virus GP-pseudotyped viruses (EC50 approximately 40-60 nmol/L and approximately 6-25 nmol/L, respectively) from mediating gene transduction into HeLa cells. In addition, we provide evidence that CV-N can effectively inhibit DC-SIGN-mediated EboZV infection. Our data emphasize both the utility of GP-pseudotyped vectors in the assessment of compounds that affect cell entry by filovirus and the use of CV-N as a reagent for the probing of carbohydrate-dependent interactions at viral entry.

Role of the C-type lectins DC-SIGN and L-SIGN in Leishmania interaction with host phagocytes.

Abstract Leishmaniasis is a parasitic disease that courses with cutaneous or visceral clinical manifestations. The amastigote stage of the parasite infects phagocytes and modulates the effector function of the host cells. Our group has described that the interaction between Leishmania and immature monocyte-derived dendritic cells (DCs) takes place through dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN), a C-type lectin that specifically recognizes fungal, viral and bacterial pathogens. The DC-SIGN-mediated recognition of Leishmania amastigotes does not induce DC maturation, and the DC-SIGN ligand/s on Leishmania parasites is/are still unknown. We have also found that the DC-SIGN-related molecule L-SIGN, specifically expressed in lymph node and liver sinusoidal endothelial cells, acts as a receptor for L. infantum, the parasite responsible for visceral leishmaniasis, but does not recognize L. pifanoi, which causes the cutaneous form of the disease. Therefore, DC-SIGN and L-SIGN differ in their ability to interact with Leishmania species responsible for either visceral or cutaneous leishmaniasis. A deeper knowledge of the parasite-C-type lectin interaction may be helpful for the design of new DC-based therapeutic vaccines against Leishmania infections.

Autochthonous Crimean-Congo Hemorrhagic Fever in Spain.

Crimean-Congo hemorrhagic fever (CCHF) is a widely distributed, viral, tickborne disease. In Europe, cases have been reported only in the southeastern part of the continent. We report two autochthonous cases in Spain. The index patient acquired the disease through a tick bite in the province of Ávila - 300 km away from the province of Cáceres, where viral RNA from ticks was amplified in 2010. The second patient was a nurse who became infected while caring for the index patient. Both were infected with the African 3 lineage of this virus. (Funded by Red de Investigación Cooperativa en Enfermedades Tropicales [RICET] and Efficient Response to Highly Dangerous and Emerging Pathogens at EU [European Union] Level [EMERGE].).

Multiple, Non-conserved, Internal Viral Ligands Naturally Presented by HLA-B27 in Human Respiratory Syncytial Virus-infected Cells

Cytotoxic T lymphocyte (CTL)-mediated death of virus-infected cells requires prior recognition of short viral peptide antigens that are presented by human leukocyte antigen (HLA) class I molecules on the surface of infected cells. The CTL response is critical for the clearance of human respiratory syncytial virus (HRSV) infection. Using mass spectrometry analysis of complex HLA-bound peptide pools isolated from large amounts of HRSV-infected cells, we identified nine naturally processed HLA-B27 ligands. The isolated peptides are derived from six internal, not envelope, proteins of the infective virus. The sequences of most of these ligands are not conserved between different HRSV strains, suggesting a mechanism to explain recurrent infection with virus of different HRSV antigenic subgroups. In addition, these nine ligands represent a significant fraction of the proteome of this virus, which is monitored by the same HLA class I allele. These data have implications for vaccine development as well as for analysis of the CTL response.

Natural HLA-B*2705 Protein Ligands with Glutamine as Anchor Motif: IMPLICATIONS FOR HLA-B27 ASSOCIATION WITH SPONDYLOARTHROPATHY*

Background: HLA-B27 is strongly associated with ankylosing spondylitis (AS). Results: A change in the position (P) 2 anchor motif was detected in the 3% of HLA-B27 ligands identified, which showed significant homology to pathogenic bacterial sequences. Conclusion: Unusual HLA-B*2705 ligands bind with different conformations to both AS-associated and non-AS-associated HLA-B27 subtypes. Significance: This could be sufficient to initiate autoimmune damage in patients with AS-associated subtypes. The presentation of short viral peptide antigens by human leukocyte antigen (HLA) class I molecules on cell surfaces is a key step in the activation of cytotoxic T lymphocytes, which mediate the killing of pathogen-infected cells or initiate autoimmune tissue damage. HLA-B27 is a well known class I molecule that is used to study both facets of the cellular immune response. Using mass spectrometry analysis of complex HLA-bound peptide pools isolated from large amounts of HLA-B*2705+ cells, we identified 200 naturally processed HLA-B*2705 ligands. Our analyses revealed that a change in the position (P) 2 anchor motif was detected in the 3% of HLA-B*2705 ligands identified. B*2705 class I molecules were able to bind these six GlnP2 peptides, which showed significant homology to pathogenic bacterial sequences, with a broad range of affinities. One of these ligands was able to bind with distinct conformations to HLA-B27 subtypes differentially associated with ankylosing spondylitis. These conformational differences could be sufficient to initiate autoimmune damage in patients with ankylosing spondylitis-associated subtypes. Therefore, these kinds of peptides (short, with GlnP2, and similar low affinity to all HLA-B27 subtypes tested but with unlike conformations in differentially ankylosing spondylitis-associated subtypes) must not be excluded from future researches involving potential arthritogenic peptides.

Multiple Viral Ligands Naturally Presented by Different Class I Molecules in Transporter Antigen Processing-Deficient Vaccinia Virus-Infected Cells

ABSTRACT The transporter associated with antigen processing (TAP) delivers the viral proteolytic products generated by the proteasome in the cytosol to the endoplasmic reticulum lumen that are subsequently recognized by cytotoxic T lymphocytes (CTLs). However, several viral epitopes have been identified in TAP-deficient models. Using mass spectrometry to analyze complex human leukocyte antigen (HLA)-bound peptide pools isolated from large numbers of TAP-deficient vaccinia virus-infected cells, we identified 11 ligands naturally presented by four different HLA-A, HLA-B, and HLA-C class I molecules. Two of these ligands were presented by two different HLA class I alleles, and, as a result, 13 different HLA-peptide complexes were formed simultaneously in the same vaccinia virus-infected cells. In addition to the high-affinity ligands, one low-affinity peptide restricted by each of the HLA-A, HLA-B, and HLA-C class I molecules was identified. Both high- and low-affinity ligands generated long-term memory CTL responses to vaccinia virus in an HLA-A2-transgenic mouse model. The processing and presentation of two vaccinia virus-encoded HLA-A2-restricted antigens took place via proteasomal and nonproteasomal pathways, which were blocked in infected cells with chemical inhibitors specific for different subsets of metalloproteinases. These data have implications for the study of the effectiveness of early empirical vaccination with cowpox virus against smallpox disease.

Diversity of natural self-derived ligands presented by different HLA class I molecules in transporter antigen processing-deficient cells.

The transporter associated with antigen processing (TAP) translocates the cytosol-derived proteolytic peptides to the endoplasmic reticulum lumen where they complex with nascent human leukocyte antigen (HLA) class I molecules. Non-functional TAP complexes and viral or tumoral blocking of these transporters leads to reduced HLA class I surface expression and a drastic change in the available peptide repertoire. Using mass spectrometry to analyze complex human leukocyte antigen HLA-bound peptide pools isolated from large numbers of TAP-deficient cells, we identified 334 TAP-independent ligands naturally presented by four different HLA-A, -B, and -C class I molecules with very different TAP dependency from the same cell line. The repertoire of TAP-independent peptides examined favored increased peptide lengths and a lack of strict binding motifs for all four HLA class I molecules studied. The TAP-independent peptidome arose from 182 parental proteins, the majority of which yielded one HLA ligand. In contrast, TAP-independent antigen processing of very few cellular proteins generated multiple HLA ligands. Comparison between TAP-independent peptidome and proteome of several subcellular locations suggests that the secretory vesicle-like organelles could be a relevant source of parental proteins for TAP-independent HLA ligands. Finally, a predominant endoproteolytic peptidase specificity for Arg/Lys or Leu/Phe residues in the P1 position of the scissile bond was found for the TAP-independent ligands. These data draw a new and intricate picture of TAP-independent pathways.

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

Background: Individuals with nonfunctional transporter associated with antigen processing (TAP) present ligands generated by TAP-independent processing pathways associated with classical HLA class I molecules. Results: A vaccinia virus ligand is efficiently presented by nonclassical HLA-E using alternative interactions. Conclusion: Nonclassical HLA-E presents viral ligands. Significance: This expands the role of HLA-E as an antigen-presenting molecule. The 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.