Virus-specific antisera used in this study have been characterized before62 (link) and were pools of sera derived from mice vaccinated with either YF17D61 (link) (Stamaril, Sanofi-Pasteur), JE SA 14-14-261 (link), or DENV-2 NGC23 (link). All sera were incubated at 56 °C for 30 min to inactivate complement and split into aliquots before storage.
Stamaril
Manufactured by Sanofi
Sourced in France
Stamaril is a sterile, freeze-dried vaccine used for the prevention of yellow fever. It contains the live, attenuated yellow fever virus.
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Lab products found in correlation
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem, by Merck Group (1 mentions)
Fsme immun, by Pfizer (1 mentions)
Encepur, by GlaxoSmithKline (1 mentions)
C6 36, by Thermo Fisher Scientific (1 mentions)
Hek293t, by Thermo Fisher Scientific (1 mentions)
Bhk 21, by Thermo Fisher Scientific (1 mentions)
C6 36, by American Type Culture Collection (1 mentions)
Mrc 5, by American Type Culture Collection (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Hek293t, by American Type Culture Collection (1 mentions)
Bhk 21, by American Type Culture Collection (1 mentions)
Dmem media, by Thermo Fisher Scientific (1 mentions)
32 protocols using stamaril
1
Virus-Specific Antisera Characterization
2
YFV Vaccine Antibody Response Monitoring
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Human sera before and after vaccination with the YFV Vaccine Stamaril® (Sanofi) were derived from a YF-17D vaccination study, approved by the responsible institutional review board of the Medical Faculty, LMU Munich; (IRB #86–16). In this study, blood was taken from healthy adults directly before the vaccination (d0) as an individual reference and on day 7, 14 and 28 post vaccination (dpv) to determine the titre of neutralising antibodies. Serum was collected in S-Monovettes (Z-Gel; Sarstedt, Nuembrecht, Germany) and separated from whole blood by centrifugation at 2500 × g for 10 min. Samples were frozen and kept at -80°C until use. To probe the reliability of the FluoRNT at later time points after the vaccination blood samples were taken from 15 healthy volunteers with a history of being vaccinated with Stamaril® between 4 month and up to 19 years ago (approved by the IRB of the Medical Faculty, LMU Munich, 24062006GP).
3
Propagation and Titration of Yellow Fever Virus
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YF-17D virus (Stamaril, Sanofi Pasteur; reconstituted as recommended by the manufacturer) was propagated in Vero (ATCC CCL-81) cells grown in DMEM containing 10% FCS, glutamine and antibiotics (penicillin and streptomycin). Cell monolayers were seeded 24 hours earlier and infected with at a multiplicity of infection (MOI) of 0.001/cell in DMEM 2% FCS; infectious supernatants were harvested when cytopathic effect was more than 60% (day 6 p.i), freeze-thawed and clarified of cell debris at 2000 rpm for 15 min at 4C°. Viral stocks were stored as single use aliquots at -80C°. For determining virus infectious titers, we used an Immuno Focus assay (IFA) recently developed in our lab [22 (link)]. In brief, virus stocks were serially diluted (ten-fold) in DMEM and adsorbed for 1h at 37 C° onto Vero (ATCC CCL-81) cell monolayers in twenty four-well plates; cells were then overlaid with media containing 0.9% methyl cellulose and incubated at 37 C° for 3 days. Following fixation and permeabilization, a monoclonal antibody directed against NS3 from YF-17D virus was used for detection of virus antigens within infected cells; the foci of infection (analogous to plaques) were visualized using HRP-substrate reaction and counted. When virus titers in the brains were determined, the IFA was performed on serial 10-fold dilutions of homogenized and clarified 10% organ suspensions.
4
Antiviral Screening in Dengue and Yellow Fever
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Antiviral assay : Trityl compounds were prepared as stock solution of 100 mm in dimethyl sulfoxide (DMSO) and then were diluted to a final assay concentration of 20, 10, 5 μm . Dilutions were made by adding the appropriate amount of DMSO to lyophilized compound to achieve stock concentrations of 10 to 100 mm . Dissolved compounds were stored at −20 °C and brought to rt prior to assay. All compounds were well dissolved in DMSO, and precipitates were not observed upon thawing.
Cell culture : Vero cells were grown in Dulbecco′s modified Eagle′s medium (DMEM, Sigma‐Aldrich) with 10 % FBS (Sigma‐Aldrich). Cells were plated into 96‐well plates at a density of 103 cells per well.
Virus culture : Cells were then infected with dengue virus (serotype D2 (strain New Guinea C), ATCC Number: VR‐1584), and yellow fever vaccine (17 D strain Asibi; grown from a dose of Sanofi Pasteur STAMARIL (1000IU from embryonated chicken eggs) and adapted to Vero cells (Farleigh and Bugert, 2014, unpublished results) at a moi of 1, or left uninfected to determine compound toxicity.
Cell viability was determined after seven days using CellTiterGlo PROMEGA, following the manufacturer's instructions. All experiments were performed in triplicate.
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5
Propagation and Titration of YF-17D Virus
6
Immune response to yellow fever vaccine
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The study cohort consisted of 76 subjects (42 f, 34 m; age range, 18–82 years) from whom PBMC samples were obtained 14–54 days (median, 25 days) after vaccination with the YF virus strain 17D-204 (STAMARIL®; Sanofi Pasteur). Blood samples of ten healthy donors obtained at the Austrian Red Cross and the Department of Virology, Medical University of Vienna, were used for cell sorting experiments. In these samples, previous YF vaccination was confirmed by YF virus neutralization assays. As a control, samples of ten YF-seronegative individuals (6 f, 4 m; age range, 22–47 years) who had no history of YF vaccination or infection were analysed.
7
In vitro stimulation of splenocytes
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For in vitro stimulation, a ZIKV E peptide pool (JPT Peptide Technologies GmbH, Berlin, Germany) and an MHC I Haplotype class-restricted YFV-17D NS3 peptide47 (link) (sequence ATLTYRML, Eurogentec, Seraing, Belgium) were used. For the production of cell lysates, Vero E6 cells were infected with YFV-17D (Stamaril®, SanofiPasteur) or Zika virus (BeH819015). Two days after infection, cells were subjected to serial freeze-thaw cycles, inactivated by 254 nm UV irradiation overnight and the diluted cell lysate (50 µg/ml) was used as antigen for the stimulation of splenocytes. Non-infected Vero E6 cell lysate was used as a negative control.
8
Comparison of Live and Inactivated Vaccines
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The following vaccines were used in the trial: Stamaril (Sanofi), live, attenuated YFV 17D strain produced in pathogen-free chick embryo cells, 0.5 ml, not less than 1,000 IU. The vaccine was provided in freeze dried powder form and reconstituted with provided saline solution and was administered subcutaneously. IXIARO (Valneva), inactivated, alum-adjuvanted, Vero cell-derived vaccine based on JEV strain SA14-14-2, 0.5 ml. The vaccine was provided in the form of pre-filled syringe attached without needle containing an 0.5 mL dose and was administered intramuscularly according to the manufacturer’s label. FSME IMMUN (Pfizer), inactivated, alum-adjuvanted, chick embryo cell derived vaccine based on the Neudörfl strain, 0.5 ml. The vaccine was provided as a pre-filled syringe attached without needle containing a 0.5 mL dose and was administered intramuscularly according to the manufacturer’s label. All vaccines were obtained from Apoteket AB, Karolinska University Hospital, Solna, Sweden and kept at the Phase-1 unit, KTA, Karolinska University Hospital, at its Huddinge site.
9
Generation of YF17D Reporter Viruses
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The live-attenuated yellow fever vaccine strain 17D (YF17D; Stamaril®, Sanofi-Pasteur) was purchased via the Pharmacy of the University Hospital Leuven and propagated on Vero E6 cells to generate a virus stock. The generation of the YF17D-derived reporter viruses YF17D/mCherry and YF17D/Nluc expressing, respectively, the red fluorescent protein mCherry and the bioluminescent (furimazine-converting) Nanoluciferase (NLuc, see Supplementary Fig. S1 A) has been described elsewhere.27 (link),28 (link) Virus stocks were titrated by plaque assay on BHK21 J cells.18 (link),27 (link),28 (link) The synthetic DNA construct PLLAV-YF17D/mCherry, encoding a full-length infectious cDNA clone of the YF17D/mCherry virus (Supplementary Fig. S1 B) expressed under the control of the SV40 promoter,29 (link) was grown in E. coli EPI300-T cells (Epicenter) and purified as endotoxin-free supercoiled plasmid DNA using standard alkaline lysis and affinity chromatography techniques as described previously.28 (link) PLLAV-YF17D/mCherry_CMV-eGFP is a derivative of PLLAV-YF17D/mCherry in which a CMV promoter-driven GFP reporter cassette was inserted in the plasmid backbone as a second cistron that is expressed upon transfection independent of YF17D replication (Supplementary Fig. S1 B and C).
10
Zika Virus Strain Isolation and Propagation
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ZIKV strains MR766,21 (link) SL160224 (link), and BeH81901545 (link) were obtained from the European Virus Archive (EVA) (EVA; http://www.european-virus-archive.com/viruses/zika-virus-strain-mr766 ), Prof. Martijn van Hemert, University of Leiden, The Netherlands and Prof. A. Merits, University of Tartu, Estonia.YFV-17D, Stamaril® (Sanofi-Pasteur) was passaged two times on BHK-21J cells before use.
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