Protein sequence database for pathogenic arenaviruses

BACKGROUND: Arenaviruses are a family of rodent-borne viruses that cause several hemorrhagic fevers. These diseases can be devastating and are often lethal. Herein, to aid in the design and development of diagnostics, treatments and vaccines for arenavirus infections, we have developed a database containing protein sequences from the seven pathogenic arenaviruses (Junin, Guanarito, Sabia, Machupo, Whitewater Arroyo, Lassa and LCMV). RESULTS: The database currently contains a non-redundant set of 333 protein sequences which were manually annotated. All entries were linked to NCBI and cited PubMed references. The database has a convenient query interface including BLAST search. Sequence variability analyses were also performed and the results are hosted in the database. CONCLUSION: The database is available at and can be used to aid in studies that require proteomic information from pathogenic arenaviruses

Polyfunctional CD4+ T cell responses to a set of pathogenic arenaviruses provide broad population coverage

Background. Several arenaviruses cause severe hemorrhagic fever and aseptic meningitis in humans for which no licensed vaccines are available. A major obstacle for vaccine development is pathogen heterogeneity within the Arenaviridae family. Evidence in animal models and humans indicate that T cell and antibody-mediated immunity play important roles in controlling arenavirus infection and replication. Because CD4+T cells are needed for optimal CD8+T cell responses and to provide cognate help for B cells, knowledge of epitopes recognized by CD4+T cells is critical to the development of an effective vaccine strategy against arenaviruses. Thus, the goal of the present study was to define and characterize CD4+T cell responses from a broad repertoire of pathogenic arenaviruses (including lymphocytic choriomeningitis, Lassa, Guanarito, Junin, Machupo, Sabia, and Whitewater Arroyo viruses) and to provide determinants with the potential to be incorporated into a multivalent vaccine strategy. Results. By inoculating HLA-DRB1*0101 transgenic mice with a panel of recombinant vaccinia viruses, each expressing a single arenavirus antigen, we identified 37 human HLA-DRB1*0101-restricted CD4+T cell epitopes from the 7 antigenically distinct arenaviruses. We showed that the arenavirus-specific CD4+T cell epitopes are capable of eliciting T cells with a propensity to provide help and protection through CD40L and polyfunctional cytokine expression. Importantly, we demonstrated that the set of identified CD4+T cell epitopes provides broad, non-ethnically biased population coverage of all 7 arenavirus species targeted by our studies. Conclusions. The identification of CD4+T cell epitopes, with promiscuous binding properties, derived from 7 different arenavirus species will aid in the development of a T cell-based vaccine strategy with the potential to target a broad range of ethnicities within the general population and to protect against both Old and New World arenavirus infection. © 2010 Kotturi et al; licensee BioMed Central Ltd

Structural Basis for a Neutralizing Antibody Response Elicited by a Recombinant Hantaan Virus Gn Immunogen

Hantaviruses are a group of emerging pathogens capable of causing severe disease upon zoonotic transmission to humans. The mature hantavirus surface presents higher-order tetrameric assemblies of two glycoproteins, Gn and Gc, which are responsible for negotiating host cell entry and constitute key therapeutic targets. Here, we demonstrate that recombinantly derived Gn from Hantaan virus (HTNV) elicits a neutralizing antibody response (serum dilution that inhibits 50% infection [ID50], 1:200 to 1:850) in an animal model. Using antigen-specific B cell sorting, we isolated monoclonal antibodies (mAbs) exhibiting neutralizing and non-neutralizing activity, termed mAb HTN-Gn1 and mAb nn-ITN-Gn2, respectively. Crystallographic analysis reveals that these mAbs target spatially distinct epitopes at disparate sites of the N-terminal region of the HTNV Gn ectodomain. Epitope mapping onto a model of the higher order (Gn-Gc)(4) spike supports the immune accessibility of the mAb HTN-Gn1 epitope, a hypothesis confirmed by electron cryo-tomography of the antibody with virus-like particles. These data define natively exposed regions of the hantaviral Gn that can be targeted in immunogen design. IMPORTANCE The spillover of pathogenic hantaviruses from rodent reservoirs into the human population poses a continued threat to human health. Here, we show that a recombinant form of the Hantaan virus (HTNV) surface-displayed glycoprotein, Gn, elicits a neutralizing antibody response in rabbits. We isolated a neutralizing (HTN-Gn1) and a non-neutralizing (nn-ITN-Gn2) monoclonal antibody and provide the first molecular-level insights into how the Gn glycoprotein may be targeted by the antibody-mediated immune response. These findings may guide rational vaccine design approaches focused on targeting the hantavirus glycoprotein envelope.Peer reviewe

Strand-Specific Quantitative Reverse Transcription-Polymerase Chain Reaction Assay for Measurement of Arenavirus Genomic and Antigenomic RNAs.

Arenaviruses are bi-segmented, single-stranded RNA viruses that cause significant human disease. The manner in which they regulate the replication of their genome is not well-understood. This is partly due to the absence of a highly sensitive assay to measure individual species of arenavirus replicative RNAs. To overcome this obstacle, we designed a quantitative reverse transcription (RT)-PCR assay for selective quantitation of each of the lymphocytic choriomeningitis virus (LCMV) genomic or antigenomic RNAs. During the course of assay design, we identified a nonspecific priming phenomenon whereby, in the absence of an RT primer, cDNAs complementary to each of the LCMV replicative RNA species are generated during RT. We successfully circumvented this nonspecific priming event through the use of biotinylated primers in the RT reaction, which permitted affinity purification of primer-specific cDNAs using streptavidin-coated magnetic beads. As proof of principle, we used the assay to map the dynamics of LCMV replication at acute and persistent time points and to determine the quantities of genomic and antigenomic RNAs that are incorporated into LCMV particles. This assay can be adapted to measure total S or L segment-derived viral RNAs and therefore represents a highly sensitive diagnostic platform to screen for LCMV infection in rodent and human tissue samples and can also be used to quantify virus-cell attachment

Nonspecific conversion of arenavirus RNA species into cDNA during RT.

<p>(A) RNA was extracted from i) MC57 cells 7 d after infection with LCMV, ii) cell-free supernatant collected from MC57 cells 18 hr pi with LCMV, or iii) sucrose-banded LCMV particles collected from Vero E6 cells at 48 hr pi. Each sample was subjected to standard RT-PCR targeting the vRNAs or vcRNAs of the L or S segment, as indicated, using the primers described in Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120043#pone.0120043.t001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120043#pone.0120043.t002" target="_blank">2</a>. In each case, the RT was performed with: i) the RT enzyme and an RT primer, ii) the RT enzyme but no RT primer, or iii) no RT enzyme and no RT primer. It should be noted that the data shown in panel (A) were obtained during the linear and/or plateau phase of the PCR reaction. Therefore, these data are not suitable for exact quantitation, but rather reflect the presence or absence of a particular cDNA. (B). RNA extracted from the samples listed in panel (A) of this figure were subjected to QRT-PCR targeting the vRNAs or vcRNAs of the L or S segment, as indicated, using the primers and probes described in Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120043#pone.0120043.t001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120043#pone.0120043.t003" target="_blank">3</a>. Similar to panel (A), in each case, the RT was performed with: i) the RT enzyme and an RT primer, ii) the RT enzyme but no RT primer, or iii) no RT enzyme and no RT primer. For each particular viral RNA species, data are presented as mean ± SD relative to the value obtained using the RT primer.</p

RT primers.

<p>RT primers.</p

Validation of TaqMan primer-probe sets.

<p>Each L or S segment-specific TaqMan primer-probe set was tested for its ability to accurately measure known quantities (50, 5x10³, 5x10⁵, or 5x10⁷ copies) of a DNA standard control plasmid encoding the target sequence. Depicted are the amplification plots and standard curve fit lines for the primer-probe sets specific for S segment vcRNA (A) or L segment vcRNA (B).</p