Human Blood
Blood samples from healthy donors were obtained from the French blood bank (Etablissement Français du Sang) as part of an agreement with the Institut Pasteur (C CPSL UNT, number 15/EFS/023). The study was approved by the Ethics Review Committee (Comité de protection des personnes) of Île-de-France VII. Fifty milliliter blood samples were obtained from twelve donors. All blood samples were collected before 2019 and PBMC were isolated using Ficoll gradient and immediately frrozen.
Monkeys
Cynomolgus macaques (M. fasicularis), aged 37–40 months and originating from Mauritian AAALAC-certified breeding centres, were used in this study. All macaques were housed in IDMIT infrastructure facilities (CEA, Fontenay-aux-Roses), under BSL-2 and BSL-3 containment when necessary (animal facility authorization D92-032-02, Prefecture des Hauts de Seine, France) and in compliance with European Directive 2010/63/EU, the French regulations and the Standards for Human Care and Use of Laboratory Animals of the Office for Laboratory Animal Welfare (OLAW, assurance number A5826-01, United States). The protocols were approved by the institutional ethical committee ‘Comité d’Ethique en Expérimentation Animale du Commissariat à l’Energie Atomique et aux Energies Alternatives’ (CEtEA 44) under statement number A20-011. The study was authorized by the ‘Research, Innovation and Education Ministry’ under registration number APAFIS#24434-2020030216532863v1.
The animals were healthy and seronegative for SIV, type D retrovirus, and simian T-cell lymphotropic virus type 1 at the time of infection and were housed in single cages within level 3 biosafety facilities after infection. At the inclusion in the study the average weight of the monkeys was between 3 and 6 kg. All monkeys were young adults with an average age of 3–5 years at inclusion. Both males and females were used. Sample collection was performed in random order. The investigators were not blinded while the animal handlers were blinded to group allocation.
Viruses
For the in vivo studies, SARS-CoV-2 virus (hCoV-19/France/lDF0372/2020 strain) was isolated by the National Reference Center for Respiratory Viruses (Institut Pasteur) as previously described31. Virus stocks used in vivo were produced by two passages on mycoplasma-free Vero E6 cells in Dulbecco’s modified Eagle’s medium (DMEM) without FBS, supplemented with 1% penicillin (10,000 U ml−1) and streptomycin (10,000 μg ml−1) and 1 μg ml−1 TPCK-trypsin at 37 °C in a humidified CO2 incubator and titrated on Vero E6 cells.
Tissue collections and processing
Whole venous blood was collected in ethylenediaminetetraacetic acid (EDTA) tubes. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density-gradient centrifugation. Cells were either immediately stained for flow cytometry or cryopreserved in 90% foetal bovine serum (FBS) and 10% dimethyl sulfphoxide (DMSO) and stored in liquid nitrogen.
The BAL fluids (BALF) were collected by immediate gentle aspiration after each aliquot and pooled in a sterile heparinate lithium container. BALF samples were centrifuged at 350 g for 10 min. The cells were washed with PBS and then mononuclear cells (MC) were separated by standard density-gradient centrifugation. The percentage of BAL fluid recovered was approximately 75% of the instilled fluid. The alveolar cellularity ranged from 50000 to 200000 cells per ml of fluid recovered. No statistically significant difference in cellularity was noticed between baseline and post-infection broncho-alveolar lavage (data not shown). To rule out potential side-effects, we have verified that repeated bronchoalveolar lavages in uninfected animals do not affect the percentages of cells among BALMC. During the experiment, the majority of BALMC that were recovered were macrophages (more than 80%).
Sars-CoV-2 viral RNA quantification in nasopharyngeal and tracheal samples
Upper respiratory (nasopharyngeal and tracheal) specimens were collected with swabs (Universal transport medium, Copan; or Viral Transport Medium, CDC, DSR-052-01). Tracheal swabs were performed by insertion of the swab above the tip of the epiglottis into the upper trachea at approximately 1.5 cm of the epiglottis. All specimens were stored between 2 °C and 8 °C until analysis. Viral copy numbers were determined by quantitative RT-PCR with a plasmid standard concentration range containing an rdrp gene fragment including the RdRp-IP4 RT–PCR target sequence. The limit of detection was estimated to be 460 copies as described previously30. The protocol describing the procedure is also available on the WHO website (https://www.who.int/docs/default-source/coronaviruse/real-time-rt-pcr-assays-for-the-detection-of-sars-cov-2-institut-pasteur-paris.pdf?sfvrsn=3662fcb6_2).
Sars-CoV-2 viral RNA quantification in BALF cells
Sars-CoV-2 viral RNA quantification in BALF cells we performed as follow: Cryopreserved BALF cells were quick-thawed, centrifuged, and washed in 2% BSA solution in D-PBS. Cells were blocked for 5 min in 2% BSA and then incubated at room temperature for 30 min with anti-NKG2a/c Ab (clone z199) to isolate simian NK cells by positive selection using magnetic beads (Miltenyi biotec). The negative fraction, consisting predominantly of macrophages, was then centrifuged and total RNA was extracted using RNeasy mini KIT (Qiagen). Primer and probes for the real-time quantitative reverse transcriptase and polymerase chain reaction (RT–qPCR) were the same as described above and available on the WHO website (https://www.who.int/docs/default-source/coronaviruse/real-time-rt-pcr-assays-for-the-detection-of-sars-cov-2-institut-pasteur-paris.pdf?sfvrsn=3662fcb6_2). Relative quantification of the viral genome was performed by one-step RT–qPCR using the RNA to ct 1 step kit (applied biosystem, 4392938). Thermal cycling was performed in a StepOnePlus Real-Time PCR System (Applied Biosystems) in MicroAmp Fast Optical 96-well reaction plates. 2 Δ Δ Ct was calculated based on the mean of Ct measured for viral RNA using the nCoV-IP4 primers and 18s RNA Cts obtained for each monkey.
Polychromatic flow cytometry
PBMCs and BALF cells were stained as previously described87. The antibodies are listed in Supplementary method Table 1. The anti-NKG2A antibody used recognizes both NKG2A and NKG2C on simian cells88. Flow cytometry acquisitions were done on a LSRFortessa (BD Biosciences) or Symphony (BD Biosciences). Intracellular staining was performed using BD Cytofix/Cytoperm™. The data were further analyzed using FlowJo 10.4.2 software (FlowJo, LLC, Ashland, OR, USA). Multiparametric analyses were performed using FlowJo plugins Phenograph (version 3.0), UMAP (version 3.1). Phenograph was performed using a K mean of 50. UMAP was performed using a value for nearest neighbors of 25 and a minimal value of 0.25.
Cell culture
K562 (human HLA class I–negative erythroleukemia) (ATCC® CCL-243) as well as HLA-E transduced K562 cells were maintained in RPMI 1640 (Life Technologies) supplemented with 10% heat-inactivated foetal calf serum (FCS), 2 mM, l-glutamine, 100 U/ml penicillin, and 100µg/ml streptomycin.
NK cells were maintained in RPMI 1640 with Glutamax (Life Technologies) supplemented with 10% heat-inactivated FCS, 2 mM, l-glutamine, 100 U/ml penicillin, 100µg/ml streptomycin and 100 IU/ml of IL-2 and 10 ng/mL of IL-15.
Antibody against SARS-CoV-2 Spike protein
The human monoclonal antibody Cv2.3194, which targets the SARS-CoV-2 receptor binding domain, was cloned from a blood memory B cell of a COVID19 convalescent, produced as a recombinant IgG1 by transient transfection of Freestyle 293-F cells (Thermo Fisher Scientific), and purified by affinity chromatography using Protein G Sepharose® 4 Fast Flow beads (Citivia)89.
Immunofluorescence staining
Macrophages were isolated from bronchoalveolar lavage using positive selection with or the anti-CD64 monoclonal antibody-PE conjugated (clone10.1 Bd bioscience, USA) reveals by anti-PE microbeads (Miltenyi Biotec (USA), the staining and the positive selection of the cells was done according to the supplier’s instructions. Immediately after positive selection, cells were cultured at the concentration of 5*105cells/ml in a 24-well tissue culture plate (Nunc,Roskilde, Denmark) in RPMI 1640 supplemented with FBS and GM-CSF, 10 ng/ml; IL-3,10 ng/ml; M-CSF, 100 ng/ml; IL-4 during 16 hours (h). Cells were then fixed with PFA 4% and staining was performed. Membrane permeabilization was induced by Triton X-100 and were stained with mouse anti-CD163 monoclonal antibody (1 : 200, ab-100909, Abcam, USA) and anti-SARS-CoV-2 Spike protein (see below). After 2 hours of incubation, the excess of unbound primary antibody was washed off with primary buffered saline solution of PBS. The primary antibodies were detected by secondary donkey anti-mouse polyclonal antibody with Alexa Fluor® 488 and Streptavidin coupled with Alexa 568 for 1 h at 37 °C (1 : 200, Thermo Fisher Scientific, USA.). The sections were mounted using Vectashield with DAPI (4'6-diamidino-2-phenylindole, Vector Laboratories, USA). Immunofluorescence was analysed under an CELENA® X High Content Imaging System (LOGOS BIOSYSTEMS, south Korea)
Peptides and HLA-E Stabilization assay
Synthetic peptides biotinylated or not were purchased from Proimmune (United Kingdom) and dissolved in DMSO at the concentration of 2mg/ml. The peptides used were VL9 (VMAPRTVLL), HSP60 (QMRPVSRVL), The sequence of the coronavirus peptide used in the study are described in the figure 5. K562 and K562-E*0101 cells were incubated with synthetic peptides (3–300µM) at 37°C for 15–20 hours in serum-free AIM-V medium (GIBCO BRL) at a concentration of 1–3 106 cells/ml. Control cultures were kept at 37 °C for over 16 hours without peptides. Cells were then harvested, washed in PBS and cell-surface expression of HLA-E was determined by incubation with PE-conjugated anti-HLA-E antibody, washed twice with PBS and fixed in 100 μl of Cytofix (BD Biosciences). Cells were acquired using a LSR II (BD Biosciences), and FlowJo software (version 9.6.4, Tree Star, Ashland, OR) was used for all analyses. Results were expressed either directly as mean fluorescent intensity (MFI).
NK cell isolation
Humans NKG2a+ NK-cells were purified (>90%) from frozen PBMCs by positive selection with antibody-coated magnetic beads (Myltenyi Biotec). NHP NKG2a/c+ NK-cells were purified (>90%) from freshly isolated tissues (ie BALF and blood) by positive selection with antibody-coated magnetic beads (Myltenyi Biotec). NK cells (106 cell/mL) were cultured in RPMI 1640 containing GlutaMAX, 10% FCS, penicillin (10 IU/mL) and streptomycin (10 μg/mL) in the presence of IL-2 (Miltenyi Biotec) at 100 IU/mL and IL-15 at 5ng/ml (Miltenyi Biotec) (Culture media). Cells were left unstimulated over night before putting them in contact with their target cells.
MHC-E dependent NK cell viral suppressor assays
NK cell activity was determined through expression of cell surface CD107a, as previously described. K562-E*0101 cells were incubated with 50 µM of a given peptide at 26°C for 15–20 h. NK cells were co-cultured with 2 × 104 K562-E*0101 target cells pulsed or not with peptides at a NK cell/target cell ratio of 5:1. Target cells were in parallel cultured in the absence of NK cells. Anti-CD107a antibody was added at the start of the assay, and GolgiStop and GolgiPlug (both BD Biosciences) were added 1 hour after start of the stimulation. After 8 hours of culture, cell suspensions were stained for viability and cytotoxic activity was analyzed through measurement of CD107a expression by flow cytometry.
Statistics
The GraphPad Prism 7 (GraphPad Software, San Diego, CA) was used to analyze data and to perform statistical analyses. Statistical test used are described in the legend for each figure, Values of p < 0.05 were considered significant. NK cell populations were analyzed by the uniform manifold approximation and projection method provided by the FlowJo plugin version 3.1. The heatmap was calculated as Raw z score of the indicated markers within the clusters using FLASKI90. Dendrograms present the clustering of samples (columns) and markers (row), which is based on hierarchical clustering with Euclidean distance metric and average linkage.
Data availability
The authors declare that all other data supporting the findings of this study are available within the article and its raw data files, or are available from the authors upon request.