Cells and viruses

Metavac®-RSV is a recombinant bivalent live-attenuated vaccine candidate generated via reverse genetics, as previously described33,36. Briefly, Metavac®-RSV is a recombinant HMPV A1 C-85473 attenuated by deletion of SH gene and expressing a GFP reporter gene and a RSV-F gene that was inserted into the viral genome between F and M2 genes.

LLC-MK2 cells (ATCC CCL-7) were maintained in minimal essential medium (MEM, Life Technologies) supplemented with 10% fetal bovine serum (Wisent, St. Bruno, QC, Canada) and 1% penicillin/streptomycin (10,000 U/mL). To establish initial viral seed batch, Metavac®-RSV was rescued and amplified by five serial passages on LLC-MK2 cell monolayers in infection medium (OptiMEM (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 1% penicillin/streptomycin and 0.5 µg/mL acetylated trypsin (T6763, Sigma-Aldrich, Saint Louis, MO, USA), following established protocols36. Initial viral stocks were titrated as 50% tissue culture infectious doses (TCID50), as previously described33,35,72. HEP-2 (ATCC CCL-23) cells were cultivated in MEM medium supplemented with 5% FBS, 1% Pen/Strep, and 2% L-Glu.

Vero cells (Vero WHO 88020401, Nuvonis Technologies GmbH, Vienna, Austria) were maintained in serum-free medium OptiPRO SFM (Gibco, USA) supplemented with 4 mM GlutaMAX (Gibco, USA) in a T175 flask at 37 °C and 5% CO273. Metavac®-RSV production on Vero cells was performed in infection medium (OptiPRO SFM (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 4 mM GlutaMAX (Gibco, USA) and 0.5 µg/mL acetylated trypsin (T6763, Sigma-Aldrich, Saint Louis, MO, USA).

In vitro replicative assay

Confluent LLC-MK2 or Vero cells monolayers in 24-well plates were washed with PBS 1X and infected with an initial stock of Metavac®-RSV at a multiplicity of infection (MOI) of 0.01 in infection media. After an adsorption time of 1.5 h at 37 °C, the infection media was removed and replaced by fresh infection medium supplemented with acetylated trypsin. Trypsin was added at 2 and 4 dpi at 0.5 µg/mL. Samples of supernatants or scraped infected cells and supernatants (total fraction) were collected every 24 h for a duration of 7 days in three independent wells to measure infectious titers by endpoint TCID50 titrations. Images of representative cytopathic effects were taken daily using bright-field and fluorescent microscopy (10x magnification, EVOS M5000 Cell Imaging System, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA).

Serial passages and viral genome sequencing

Six serial passages of Metavac®-RSV (P + 1 to P + 6) were completed by infection of Vero cells monolayers in 24-well plates at low MOI (10−3-10−4) in fresh infection medium. The initial viral seed batch of Metavac®-RSV was used for infection of the first passage, and supernatants harvested after 7 days of production were then used for the following successive passages. Viral RNA sequencing was made from samples of supernatant of selected passages P + 1, P + 4, and P + 6. Library preparation was performed from the RNA extracted from samples using the Illumina Stranded Total RNA Prep with Ribo-Zero Plus kit, following the manufacturer’s recommendations. The prepared libraries were sequenced on an Illumina NovaSeq platform, using a 2 × 150 bp paired-end configuration.

The reference proteome was constructed through the combination of proteins extracted from the HMPV genome (OL794355.1, minus SH) obtained from NCBI, supplemented with GFP protein (ADQ48006.1) and RSV-F protein (P03420.1). For contamination detection and removal, two distinct Kraken2 databases were employed. The bacterial and archaeal database was built from NCBI on August 2, 2024. Additionally, a database of vertebrate model organisms was constructed on September 18, 2024, incorporating genomic assemblies from multiple model organisms (GCF_015476345.1, GCF_002263795.3, GCA_018104995.1, GCF_011100685.1, GCF_001704415.2, GCF_034190915.1, GCF_015252025.1, GCF_000002035.6, GCF_018350175.1, GCF_016699485.2, GCF_000001405.40, GCF_037993035.1, GCF_017639785.1, GCF_000001635.27, GCF_011764305.1, GCF_009806435.1, GCF_016772045.2, GCF_036323735.1, GCA_004025045.1, GCF_000003025.6). The processing of raw fastq files was performed using the nexomis/viral-assembly nextflow74 pipeline (version 1.2.0). Briefly, read trimming was conducted using fastp (PMID: 30423086) with the pipeline default parameters, followed by an iterative classification process using Kraken275 to eliminate potential host and bacterial contamination. The pipeline retained only unclassified reads for further analysis. Trimmed reads were also mapped to the Metavac®-RSV sequence using Bowtie276 to identify anchored reads. These anchored reads were then merged with the unclassified reads to generate a set of cleaned reads. De novo assembly was performed on the cleaned reads using SPAdes77 in “rnaviral” mode78. The resulting contigs were evaluated through reference proteome mapping using miniprot79, with the contig showing maximum similarity, with the maximum number of proteins being selected for annotation and reorientation as necessary.

Variant analysis was conducted using the nexomis/viral-variant pipeline (version 1.1.1) with default parameters. Cleaned reads were mapped to the Metavac®-RSV sequence reference using BWA-MEM80, followed by duplicate marking using Picard tools (“Picard Toolkit.” 2019. Broad Institute, GitHub Repository. https://broadinstitute.github.io/picard/; Broad Institute). Alignment files underwent local realignment using ABRA281, and variants were subsequently called using sav_call (https://github.com/nexomis/sav_call). The final variant analysis incorporated sequence-specific annotation and filtering through the “call.py” script from the sav_call project (Commit: 822331 d). To ensure high-confidence variants, stringent filtering criteria were applied, with indels below 10% frequency and SNPs below 3% frequency being excluded from the final analysis.

Confocal microscopy

LLC-MK2 cells grown on Lab-Tek II chamber slides (Thermo Fisher Scientific) were infected with a MOI of 0.05 of P + 1 or P + 6 Metavac®-RSV produced on Vero cells. After 3 days of infection, infected cells were fixed with 4% paraformaldehyde in PBS 1X, washed and stained with anti-RSV-F Palivizumab (Synagis®, AstraZeneca) and anti-HMPV-F mAb (HMPV24, Abcam ab94800) antibodies used as primary antibodies in PBS-T at 1:5000 and 1:500 dilutions, as previously described36. Nuclei were counterstained with DNA-binding fluorochrome 4,6-diamidinon-2-phenylindole (DAPI, Invitrogen). After staining, the coverslips were mounted with Fluoromount G (Cliniscience) and images were taken using a confocal inverted microscope (Leica Confocal SP5 X), then processed by ImageJ software.

Animal studies

Viral stock of Metavac® and Metavac®-RSV were produced on static Vero cells cultured in 24-well plates. Total fraction (cell and supernatant) was harvested, clarified, and concentrated by ultracentrifugation, as previously described36. Viral stocks for animal studies were titrated in PFU/mL. For the vaccination study, 6–8 week-old Sigmodon hispidus female and male cotton rats (Sigmovir Biosystems, Inc., Rockville MD), randomly housed in groups of 5 per cage (included three females and two males) were immunized twice with a 28-day interval via intranasal (IN) route with 3.7 × 105 PFU of Metavac®-RSV, or Metavac® and infected 3 weeks after the second immunization with 105 PFU of RSV A/A2 (RSV, ATCC, Manassas, VA) or HMPV TN/94-49/A2 (HMPV42). As control groups, cotton rats were inoculated IN with PBS twice (on days 0 and 28) or infected IN with 105 PFU of RSV or HMPV once (on day 0) and challenged with 105 PFU of RSV or HMPV 7 weeks later. Additional control groups were immunized twice with a 28-day interval IM with formalin-inactivated (FI)-RSV (Lot#100) or FI-HMPV, both diluted 1:100 in PBS, and challenged 3 weeks later with RSV or HMPV. All animals were sacrificed 5 days post-challenge (day 54 of the study) to harvest nasal tissues for viral titration and lungs for viral titration, histopathology, and qPCR analysis. Cotton rats (n = 5) were eyebled for serum collection before the first and the second immunization and before the challenge (at day 0, 28, and 49). Sera were used to validate initial seronegative status and to measure neutralizing antibodies via neutralization assays and IgG titers via ELISA assays. To assess replication of Metavac®-RSV in the cotton rat model and measure inflammatory response to immunization, an additional group of five animals was inoculated IN with 3.7 × 105 PFU of Metavac®-RSV and sacrificed 5 days later for collection of nasal tissues for viral titration and lungs for viral titration, histopathology, and qPCR analysis. For manipulation of live animals (e.g., IN administration and blood collection), cotton rats were anesthetized via isoflurane inhalation. Euthanasia of animals was performed by carbon dioxide asphyxiation followed by assuring death, according to the guidelines provided in the Guide (2011) and the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals: 2020 edition.

Ethics

Animal study was performed according with the National Institutes of Health guidelines and Sigmovir Institutional Animal Care and Use Committee’s approved animal study protocol (IACUC Protocol #15).

Neutralization assays

To evaluate the production of a neutralizing antibody response, heat inactivated sera samples were diluted 1:10 and serially diluted further 1:4. Diluted samples were incubated with 25-50 PFU of RSV A (RSV A2, lot #102313 SSM), RSV B (RSV B 18537, ATCC VR-1580, lot #032417 SSM), or HMPV A (HMPV TN/94-49/A2, lot #030116 SSM) for 1 h at room temperature and inoculated in duplicates onto confluent HEp-2 for RSV, or LLC-MK2 for HMPV. After 2 h incubation at 37 °C in a 5% CO2 incubator, the wells were overlaid with 0.75% methylcellulose medium and incubated for 5 days for RSV A, and 7 days for RSV B or for HMPV A.

To read RSV neutralizing antibodies titers, the overlay was removed, and the cells were fixed with 0.1% crystal violet stain and then rinsed and air-dried. For HMPV, the overlay was removed, the cells were fixed with acetone/methanol for 1 h, air-dried, and immuno-stained using anti-HMPV antibody (Curia Biologics, DS7, custom-made), followed by HRP-conjugated secondary antibody (Jackson Immuno Research, 109-035-088). An AEC Chromogen detection solution (Sigma-Aldrich, AEC101) was added to each well and incubated at room temperature for 30 min. Visible RSV or HMPV plaques were counted. The corresponding neutralizing antibody titers were determined at the 60% reduction endpoint of the virus control using the software “plqrd.manual.entry” (Sigmovir Biosystems, Inc).

IgG quantification

Purified RSV A2 F protein (Sino Biological, 11049) or methanol/acetone fixed HMPV-infected LLC-MK2 cells (in-house, lot #041922) were coated onto a 96-well ELISA plate overnight. The next day, plates were incubated in blocking solution for 1 h at room temperature and subsequently washed. Diluted sera (1:500 in duplicates) along with the positive and negative controls (in-house naive or hyperimmune sera) were added to the wells and incubated at room temperature for 1 h. After washing the plates, chicken-anti-cotton rat IgG-HRP (Immunology Consultants Laboratory Inc., CCOT-25P) diluted 1:20,000 was added to all the wells and incubated for 30 min at room temperature. TMB substrate solution was added to all the wells and incubated at room temperature for 15 min before TMB-Stop solution was added to all of the wells. Optical density (OD) at 450 nm was recorded, and the geometric mean of the duplicate wells was calculated. OD means were then converted to arbitrary units and normalized on the non-vaccinated group for representation.

Viral titrations

To evaluate viral titers in the respiratory airways of challenged animals, lung and nose tissues were homogenized and samples were clarified by centrifugation and diluted in EMEM. Confluent LLC-MK2 monolayers for HMPV titration or Hep-2 monolayers for RSV titration were infected in duplicates with diluted homogenates in 24-well plates and incubated 2 h at 37 °C in a 5% CO2 incubator. The wells were overlaid with 0.75% methylcellulose medium. For RSV titration, after 5 days of incubation, the overlay was removed and the infected Hep-2 were fixed for 2 h with 0.1% crystal violet stain, then rinsed and air-dried. For HMPV titration, after 7 days of incubation, the overlays were removed, the infected LLC-MK2 were fixed for 1 h with acetone and methanol and air-dried for immunostaining as described above. Visible plaques were counted, and viral titers were expressed as plaque-forming units per gram of tissue. Viral titers were calculated as geometric mean ± standard error.

Histopathology analysis

To measure pulmonary inflammation, lungs were dissected and inflated with 10% neutral buffered formalin to their normal volume, and then immersed in the same fixative solution. Following fixation, the lungs were embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). Slides were scored blind on a 0-4 severity scale for each of the four parameters of pulmonary inflammation evaluated: peribronchiolitis, perivasculitis, interstitial pneumonia, and alveolitis. The scores were subsequently converted to a 0–100% histopathology scale and cumulated for graphical representation.

Cytokines quantification

Cytokines quantification was performed by RT-qPCR from total RNA extracted from lung homogenate (flash frozen lung lingular lobes). Total RNA was extracted using the RNeasy purification kit (Qiagen). One μg of total RNA was used to prepare cDNA using Super Script II RT (Invitrogen) and oligo dT primer (1 μl, Invitrogen). For real-time PCR reactions, the Bio-Rad iQTM SYBR Green Supermix were used in a final volume of 25 μl, with final primer concentrations of 0.5 μM. Reactions were set up in duplicates in 96-well trays. Amplifications were performed on a Bio-Rad iCycler for one cycle of 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 10 s, and 72 °C for 15 s. The baseline cycles and cycle threshold (Ct) were calculated by the iQ5 software in the PCR baseline-subtracted curve fit mode. Relative quantitation of DNA was applied to all samples. The standard curves were developed using serially diluted cDNA samples, most enriched in the transcript of interest. The Ct values were plotted against log10 cDNA dilution factor. These curves were used to convert the Ct values obtained for different samples to relative expression units. These relative expression units were then normalized to the level of β-actin mRNA (“housekeeping gene”) expressed in the corresponding sample.

Agitated cultures for viral production

Agitated cultures of Vero cells were realized in TubeSpin® 50 mL (TPP®) incubated in a Kühner incubator (ISF1-X, Kühner, Birsfelden, Switzerland) at 37 °C and 5% CO2 or in 2 L single-use stirred-tank bioreactor (Univessel® SU, Sartorius, Göttingen, Germany), with a temperature of 37 °C, a pH of 7.2, and a dissolved oxygen (pO2) of 40% monitored by Biostat B controller (Sartorius, Göttingen, Germany). Cytodex1 microcarriers (Cytiva, USA) were prepared according to the manufacturer’s instructions, and Vero cells were inoculated on 2 g/L Cytodex1 microcarriers at 1 × 105 cells/mL in a half final volume of culture medium OptiPro SFM supplemented with GlutaMAX (half volume meaning 5 mL in TubeSpin® 50 mL or 1 L in Univessel® SU bioreactor). To protect cells from shear stress, 0.1% Pluronic® F68 (Sigma-Aldrich Co., MO, USA) was added to the culture medium. The following day, fresh media was added up to a final volume of 10 mL in TubeSpin® or 2 L in bioreactor and cells were cultivated for 4 days before virus inoculation.

For trypsin optimization in TubeSpin®, 5 mL of culture medium was removed and replaced by 5 mL of Metavac®-RSV at MOI 0.01 in fresh medium with acetylated trypsin at 0.5 or 1 µg/mL. Depending on the conditions, trypsin was then added to the medium every day or every 2 days at the same final concentration. For MOI and volume at infection optimization in TubeSpin®, 5 mL of culture medium was removed the day of infection and replaced by 5 mL of Metavac®-RSV at MOI 0.005 or 0.01 in fresh medium with acetylated trypsin at 1 µg/mL. Alternatively, 5 mL of culture medium was removed the day of infection, Metavac®-RSV inoculation at MOI 0.005 or 0.01 was made in this volume and incubated for 3 h before addition of 5 mL of fresh infection medium. Trypsin at 1 µg/mL was added to the culture every 2 days during 4 days of viral production. Daily samples of cell and microcarrier suspension were collected to perform bioprocess samples analysis, such as cell count and viral titration.

For aeration strategy evaluation in a bioreactor, Vero cells were cultivated in Univessel® SU set to a temperature of 37 °C and pH of 7.2. pO2 was monitored at 40% of air saturation, and O2, CO2, N2, and air gases were added either by overlay or sparger pipe. The setpoint of stirring agitation using two marine-type impellers was set to 70 rpm when overlay gassing, or 85 rpm when gases were added by sparger pipe. Four days after cell seeding, exchange of half of the media was performed, and Metavac®-RSV was inoculated at an MOI of 0.01 in the presence of acetylated trypsin at a final concentration of 1 µg/mL. Trypsin at the same concentration was then added every 2 days during the 4 days of viral production.

Following the best production parameters identified at small-scale and in 2 L Univessel® SU bioreactor, optimized Metavac®-RSV production was conducted in 2 L stirred-tank bioreactor with setpoints of: 37 °C, pH of 7.2, pO2 of 40% controlled by sparger gassing and stirring agitation of 85 rpm using two marine-type impellers. Four days after cell seeding, half of the media was removed and Metavac®-RSV was inoculated at an MOI of 0.01 in the presence of acetylated trypsin at a final concentration of 0.5 µg/mL. After 3 h, 1 L of fresh infection media was added to reach a final volume of 2 L. During the 4 days of viral production, trypsin was added every two days. When the glucose concentration dropped below 2 g/L, a glucose supplementation (Glucose solution, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) was performed with a target glucose concentration of 2.5 g/L.

Daily samples of cell and microcarrier suspension were collected to perform bioprocess samples analysis, such as cell count, microscopic observation, and glucose measurement. Samples were also aliquoted and stored at −80 °C for further viral titration.

Bioprocess samples analysis

To perform cell counting in stirred cultures, 1 mL of cell and microcarrier suspension was collected. After sedimentation, 850 µL of supernatant were discarded. The microcarriers were washed with 1 mL of PBS 1X and 150 µL of trypsin TrypLE 10X (A1217701, Gibco, USA) was mixed to the pellet for 15 min at 37 °C to dissociate cells and microcarriers. Cell counting was then performed with trypan blue on kova® slides (Kova International, California, USA).

Glucose concentration was analysed using One Touch Verio Reflect (LifeScan, USA) from the supernatant.

Bright-field and fluorescent microscopy was performed using EVOS™ M5000 Cell Imaging System (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) on the total (cells and supernatant) fraction.

Infectious titers in supernatant or in total fraction were determined using the TCID50 method using Vero cells in 96-well plates. Samples were thawed on ice and diluted (supernatant fraction) or thawed on ice and centrifuged to discard the cell pellet before dilution (total fraction). Before infection, the medium was discarded from the cells and samples were diluted in series in the infection medium. Infected plates were incubated for 8 days at 37 °C with 5% CO2, and 1% final trypsin was added every two days. TCID50 titers were then calculated using the Reed and Muench method.

Transmission electron microscopy

After production in a 2 L Univessel® SU bioreactor or in static 24-well plates, Metavac®-RSV particles were concentrated by ultracentrifugation, resuspended in NaCl (0.9%) and filtered at 0.45 μm. Viral suspensions were adsorbed on 200-mesh nickel grids coated with formvar-C for 10 min at room temperature. Then, grids with suspensions were colored with Uranyless (Delta Microscopies, Mauressac, France) for 1 min and observed on a transmission electron microscope (Jeol 1400 JEM, Tokyo, Japan) equipped with a Gatan camera (Orius 1000) and Digital Micrograph software. Diameter of Metavac®-RSV particles was measured on transmission electron microscopy pictures (n = 58 for bioreactor production and n = 30 for static production).

Infection of reconstituted human airway epithelium

In vitro reconstituted human airway epithelium (HAE), derived from healthy donors’ primary nasal cells (MucilAir™), was purchased from Epithelix (Plan-les-Ouates, Switzerland). HAEs were infected by instillation at the apical side of Metavac®-RSV produced in a bioreactor at an MOI of 0.1. After 2 h of incubation at 37 °C and 5% CO2, the inoculum was removed carefully. Infected HAEs were monitored by fluorescent microscopic observation at 3, 5, and 7 dpi using EVOS™ M5000 Cell Imaging System (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). At 3, 5, and 7 dpi, apical washes with warm OptiMEM were performed in order to extract viral RNA (QIAamp Viral RNA kit, Qiagen, Hilden, Germany) and viral gene quantification was performed by RT-qPCR targeting the HMPV-M gene or RSV-F gene.

Statistical analysis

Statistical analyses were performed with GraphPad Prism 10 using t-test, one-way or two-way ANOVA with Dunnett posttest or Tukey posttest when comparing to a control group.