Generation and in vitro characterization of cytokine-encoding murine coronavirus vectors.

<p>(A) Schematic representation of MHV A59 genome and construction of cytokine-encoding vectors. (B, C) Growth kinetics of the indicated MHV vectors in 17ECl20 packaging cells (B) and macrophages (Mph) (C). Cells were infected at an MOI of 1 and titres in supernatants were determined at the indicated time points. (D) Transduction in bone marrow-derived DCs with murine coronavirus vectors. DCs from B6 mice were transduced with the indicated vectors at a multiplicity of infection (MOI) of 1. Cells were harvested 24 h later and EGFP expression on CD11c⁺ cells was assessed. Pooled data from three independent experiments with values indicating mean percentage ±SEM of EGFP⁺CD11c⁺ cells. (E) Cytokine production induced by Flt3L and IL-2 encoding vectors. DCs or macrophages were transduced at a MOI of 1 and concentration of cytokines in the supernatants was determined by ELISA. Representative data from one out of three independent experiments.</p

In vivo maturation of DCs following vaccination with coronavirus vectors.

<p>B6 mice were i.v. immunized with 10⁶ pfu of the indicated viral vectors or left untreated (mock). (A, B) Transduction of DCs as assessed by EGFP expression. Spleens were collected after 24 h, digested with collagenase and low-density cells were analyzed by flow cytometry. Values in dot plots (A) and bar graph (B) show the mean percentage ± SEM of EGFP⁺IA^bhigh cells gated on CD11c⁺ cells. Data from two independent experiments with three mice per group (n=6). (C, D) Cytokine concentration in serum and spleen homogenates at 24 h post immunization with Flt3L (C) and IL-2 (D) vectors. Pooled data from three independent experiments with three mice per group (mean ±SEM, n=9). (E) Representative histograms showing expression of the DC maturation markers CD40 and CD86 on EGFP⁺CD11c⁺ cells transduced with the indicated vectors. (F) Mean fluorescence values ±SEM (n=9) of CD40 and CD86 expression in transduced EGFP⁺CD11c⁺ cells (+) and non-transduced EGFP⁺CD11c^- cells (-) cells (*, p< 0.05; **, p< 0.01).</p

Prophylactic and therapeutic tumor immunity against a peripheral solid tumor.

<p>(A) B6 mice were i.v. immunized with 10⁵ pfu of the different vectors or received PBS as control. Seven days later, 5×10⁵ gp-recombinant Lewis lung carcinoma cells were injected s.c. on the left flank. (B) Assessment of therapeutic tumor immunity induced by 10⁵ pfu of the different vectors applied on day 4 post s.c. inoculation with 5×10⁵ gp-recombinant Lewis lung carcinoma cells. Tumor growth was monitored on the indicated days. Values indicate mean tumor volume ±SEM (n=9 mice). Values in parentheses indicate number of growing tumors of inoculated tumors. Statistical analysis in (B) was performed using one way ANOVA with Bonferroni multiple comparison test (**, p< 0.01; ***, p< 0.001; ns, non-significant).</p

Evaluation of CD8⁺ T cell response induced by cytokine-encoding coronavirus vectors.

<p>(A) Induction of gp34-specific CD8⁺ T cells. B6 mice were immunized i.v. with 10⁵ pfu of the indicated vectors. At day 7 post immunization, splenocytes were analyzed for expression of CD8 and reactivity with H2-K^b/gp34-tetramers, and were tested for gp34-specific IFN-γ production. Values indicate mean percentages of tet⁺ cells ± SEM (upper row) or mean percentages of IFN-γ⁺ cells ± SEM (lower row) in the CD8⁺ T-cell compartment (pooled data of 3 independent experiments, n=12 mice). (B and C) Duration of vector-induced CD8⁺ T cell responses. B6 mice were immunized i.v. with 10⁵ pfu of the indicated vectors or infected with 200 pfu of LCMV WE. Frequencies (B) and total numbers (C) of splenic CD8⁺ tet-gp34-binding T cells were determined at the indicated time points (mean percentages or total numbers of tet-gp34⁺ cells ±SEM, n=6-12 mice per time point); nd, not detectable. (D) Induction of gp33-specific CD8⁺ T cells as determined by tetramer analysis on day 7 post i.v. immunization with the indicated vectors (mean percentages of tet-gp33⁺ cells ± SEM, n=8 mice). (E) Expansion of gp33-specific P14 TCR transgenic CD8⁺ T cells. One day before immunization with 10⁵ pfu of the indicated vectors, CD45.2⁺ B6 mice had received 10⁵ CD45.1⁺ P14 splenocytes. Expansion of CD45.1⁺CD8⁺ T cells in spleens was assessed on day 7 post immunization (mean percentages of CD45.1⁺CD8⁺ T cells ±SEM, n=8 mice) (*, p< 0.05; **, p< 0.01; ***, p< 0.001; comparison with gp control vector).</p

Coronavirus vector-induced immunity against a metastasizing tumor.

<p>(A, B) B6 mice were i.v. immunized with the indicated doses of the different vectors or infected with 200 pfu LCMV WE. PBS was administered as negative control. Seven days later mice were challenged with 5×10⁵ B16F10-gp melanoma cells or the parental B16F10 cells. (A) Representative microphotographs of lungs on day 12 post tumor inoculation. (B) Number of metastatic foci per lung was determined 12 days after challenge (pooled data from three independent experiments, mean ±SEM, n=6-9 mice per time point). (C, D) Assessment of therapeutic tumor immunity. B6 mice received 5×10⁵ B16F10-gp melanoma cells i.v. at day 0 and were vaccinated 10 days later with 10⁵ pfu of the indicated vectors or left untreated. (C) Representative microphotographs of affected lungs on day 20 after tumor inoculation. (D) Disease severity was quantified by black pixel counting on lung surfaces on day 20. Values represent mean percentage ±SEM (n=6-8 mice) of affected lung surface. Statistical analysis in (D) was performed using one way ANOVA with Tukey’s post analysis (**, p< 0.01; ns, non-significant).</p

Analysis of SARS-CoV gene expression in hDCs using a recSARS-CoV expressing <i>Renilla</i> luciferase.

<p><b>A.</b> The genome structure of recombinant SARS-CoV and HCoV-229E viruses expressing <i>Renilla</i> luciferase (HCoV-229E-luc and SARS-CoV-luc) is shown. White boxes represent ORFs encoding virus replicase and accessory proteins, grey boxes represent ORFs encoding virus structural proteins. The regions of HCoV-229E ORF4a/b and SARS-CoV ORF7a are enlarged to illustrate the ORF encoding <i>Renilla</i> luciferase and surrounding nucleotides. Nucleotide numbers depict CoV nucleotides at the border to non-CoV sequences. The dashed line in the upper panel depicts nucleotides derived from restriction sites <i>BamHI</i> and <i>EcoRI</i> that have been introduced to facilitate cloning of the recombination plasmid containing the <i>Renilla</i> luciferase gene. <b>B.</b> Pairwise comparison of the replication of recSARS-CoV and HCoV-229E with the corresponding luciferase encoding viruses. RecSARS-CoV/SARS-CoV-luc and HCoV-229E/HCoV-229E-luc were used to infect Vero-E6 and Huh-7 cells, respectively, at an MOI of 0.01. The culture supernatants were harvested at 24, 48 and 72 hrs p.i and the titers of virus in the supernatants determined by plaque assay. The peak viral titres for recSARS-CoV/SARS-CoV-luc (at 72 hours p.i) and HCoV-229E/HCoV-229E-luc (at 48 hours p.i) are shown. The average titers from 3 independent experiments are shown together with error bars. <b>C.</b> Analysis of SARS-CoV-luc- and HCoV-229E-luc-mediated <i>Renilla</i> luciferase expression in infected (MOI = 1) Vero-E6, Huh-7 and hDCs. <i>Renilla</i> luciferase expression was assessed at 12 hours (black bars) and 24 hours p.i. (white bars), The fold increase in <i>Renilla</i> luciferase expression levels in virus-infected cells represents the ratio of luciferase activity in virus-infected cells compared to that in mock-infected cells.</p

SARS-CoV genome nucleotide sequence comparison.

a<p>GenBank accession number SARS-CoV Frankfurt-1: AY291315; SARS-CoV HKU-39849: AY278491; SARS-CoV HKU-39849 UOB: JQ316196 and recSARS-CoV HKU-39849: JN854286.</p>b<p>Introduced nucleotide change to create <i>Sfi</i>I restriction site for cloning purposes.</p

Characterization of a candidate tetravalent vaccine based on 2'-O-methyltransferase mutants

<div><p>Dengue virus (DENV) is one of the most widespread arboviruses. The four DENV serotypes infect about 400 million people every year, causing 96 million clinical dengue cases, of which approximately 500’000 are severe and potentially life-threatening. The only licensed vaccine has a limited efficacy and is only recommended in regions with high endemicity. We previously reported that 2’-<i>O</i>-methyltransferase mutations in DENV-1 and DENV-2 block their capacity to inhibit type I IFNs and render the viruses attenuated <i>in vivo</i>, making them amenable as vaccine strains; here we apply this strategy to all four DENV serotypes to generate a tetravalent, non-chimeric live-attenuated dengue vaccine. 2’-<i>O</i>-methyltransferase mutants of all four serotypes are highly sensitive to type I IFN inhibition in human cells. The tetravalent formulation is attenuated and immunogenic in mice and cynomolgus macaques and elicits a response that protects from virus challenge. These results show the potential of 2’<i>-O</i>-methyltransferase mutant viruses as a safe, tetravalent, non-chimeric dengue vaccine.</p></div

Construction of a vaccinia virus based SARS-CoV reverse genetic system.

<p>The genome structure of SARS-CoV is shown at the top of the figure. Nine cDNA clones produced from the genomic RNA of SARS-CoV isolate HKU-39849 are shown below. The region of the SARS-CoV genome encompassed by each clone is indicated by the nucleotide number (using the recSARS-CoV sequence; GenBank: JN854286) at the beginning and end of each clone. Restriction enzyme sites used to join the clones are shown, with restriction enzymes sites added to the clones shown in bold. The cDNA fragments isolated from the clones and gpt PCR products covering regions of the genome unstable as cDNA clones were ligated with each other and vaccinia virus DNA to produce two vaccinia virus recombinant clones spanning nts 1–20288 and 20272–29727 of the SARS-CoV genome respectively. The first 2012 nts of the former vaccinia virus recombinant was derived from the SARS-CoV isolate Frankfurt-1 (shaded in dark grey). Vaccinia virus mediated homologous recombination was then used to reconstitute the SARS-CoV subgenomic fragments, introducing regions of cDNA that were unstable in <i>E. coli</i> and repairing errors (*) introduced during the cloning process. This resulted in the vaccinia virus clones vSARS-CoV-5prime and vSARS-CoV-3prime. The SARS-CoV cDNA fragments were isolated from the two vaccinia virus recombinants by restriction enzyme digestion and then joined using unique <i>SfiI</i> and <i>BglI</i> sites that had been introduced into the cDNA. The ligated cDNA fragments were used as a template for <i>in vitro</i> transcription using a T7 polymerase promoter introduced at the 5′ end of the SARS-CoV 5′ cDNA clone to produce a RNA transcript representing the SARS-CoV genome.</p

Recovery and analysis of recSARS-CoV by RT-PCR and plaque assay.

<p><b>A.</b> Immunofluorescence microscopy analysis of Vero-E6 cells infected with P0 virus harvests obtained from cells electroporated with full-length recSARS-CoV RNA. Cells were fixed at 8 hours post infection (p.i.) and stained for nonstructural protein 3 (green) as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032857#pone.0032857-Snijder1" target="_blank">[40]</a>. Nuclei were stained using Hoechst 33258 (blue). <b>B.</b> RT-PCR analysis and restriction enzyme digestion confirm the recovery of recSARS-CoV. Vero-E6 cells were infected with P0 culture medium from cells transfected with recSARS-CoV RNA, (harvested 48 hours post transfection, lanes 2–5), SARS-CoV Frankfurt-1 (SARS-CoV, lanes 6 and 7) or mock infected (mock, lanes 8 and 9). At 48 hours p.i., RNA was isolated from culture supernatants (lanes 4 and 5) or infected Vero-E6 cells (lanes 2, 3, 6, 7, 8 and 9). The RNA samples were used for RT-PCR analysis. Lanes 2, 4, 6 and 8 show the products obtained from RT-PCR reactions designed to amplify a genomic region containing a <i>BglI</i> restriction site that had been engineered into recSARS-CoV genome at the cDNA level, whereas lanes 3, 5, 7, and 9 show corresponding control reactions without reverse transcriptase. The RT-PCR products shown in lanes 2, 4 and 6 were then further analyzed by <i>Bgl</i>I digestion to verify the presence of this marker mutation in the recSARS-CoV progeny. The 2.5 kb PCR products derived from recSARS-CoV (lanes 11 and 12) were digested into two expected fragments of similar size (1266 bp and 1285 bp), whereas the wildtype PCR product remains undigested. The sizes of the PCR products were determined by comparison to a 1 Kb Plus DNA ladder (Invitrogen) (lanes 1 and 10). Bacteriophage λ DNA was cleaved with <i>Bgl</i>I (lane 14) as a digestion control. <b>C.</b> Comparative plaque assays of different SARS-CoV variants on Vero-E6 cells. Upon complete CPE, progeny virus was harvested from Vero-E6 cells infected with recSARS-CoV, SARS-CoV-Frankfurt-1, the original SARS-CoV HKU-39849 (HKU), and the SARS-CoV HKU-39849 used to produce the recSARS-CoV cDNAs used in this study (HKU-B). Tenfold serial dilutions were plated on Vero-E6 cells under a semisolid overlay and cell layers were fixed and stained with crystal violet after two days.</p