Ethics and biosafety statement animal studies
The NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) humanized mice (hu-mice) were supplied by the Jackson Laboratories (Bar Harbor, ME, USA) under MTA #1720. Five donors whose HLA typing is recapitulated in the supplemental table S1 provided hematopoietic stem cells for human immune system reconstitution of the mice. The level of human immune cells reconstitution reached an average of 70%. The hu-mice were housed in Mondor Institute of Biomedical Research infrastructure facilities (U955 INSERM-Paris East Creteil University, Ile-de-France, France). The protocols were approved by the institutional ethical committee “Comité d’Ethique Anses/ENVA/UPEC (CEEA-016)” under statement number 20-043 #25329. The study was authorized by the “Research, Innovation and Education Ministry” under registration number 25329-2020051119073072 v4.
Cynomolgus macaques (Macaca fascicularis), aged 37-58 months (8 females and 13 males) and originating from Mauritian AAALAC certified breeding centers were used in this study. All animals were housed in IDMIT facilities (CEA, Fontenay-aux-roses), under BSL-3 containment (Animal facility authorization #D92-032-02, Préfecture 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, US). 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.
Production and quality assurance of the αCD40.RBD vaccine Vectors and sequences for humanized anti-human CD40 12E12 IgG4 and control human IgG4 antibodies have been described previously 1,2,3. GenBank sequences HQ738666.1 and KP684037 describe the human IgG4 chimeric forms of the 12E12, anti-human CD40 H and L chains. Methods for expression vectors and protein production and purification, via transient or stable CHO-S (Chinese Hamster Ovary cells) transfection and quality assurance including CD40 binding specificity were as are described. CHO-optimized codons encoding SARS-CoV-2 RBD residues 173-591 of sequence ID: QIC50514.1 with appended residues encoding a C-tag (EPEA) and a stop codon were inserted between the vector Nhe I and Not I sites positioned distal to the H chain C-terminal codon. Expression plasmids encoding the antibody H chain RBD fusion and the L chain were transiently transfected into Expi-CHO cells with TransIT-PRO Pro reagent (Mirus Bio) using the manufacturers protocol. The product was purified by protein A affinity capture of the culture medium followed by elution with a gradient of 1M L-Arginine monohydrochloride in H2O, from pH 8.0 and pH 1.8. Product was formulated in phosphate buffered saline (pH 7.4) with 125 mM cyclodextrin (average MW 1420). The LPS value was .037 ng/mg. Using a solid phase assay direct binding assay previously described 3 these was no significant difference in the CD40 binding affinity of anti-CD40 12E12 (EC50 30 pM) versus anti-CD40 12E12-RBD (EC50 35 pM).
DREP-S vaccine constructs were made by cloning the sequences encoding S of SARS-CoV-2 spike protein into the Semliki Forest Virus (SFV) DREP plasmid vector backbone 3 using BamHI and SpeI restriction sites 4. The S construct encodes the surface glycoprotein of SARS-CoV-2 (Wuhan-Hu-1) with an 18-aa deletion in the cytoplasmic tail (D18). The synthesis of the construct with the appropriate restriction sites was ordered from Twist bioscience. The spike variant was codon optimized for human expression and the construct’s sequence was confirmed by sequencing. Plasmid DNA of the DREP-S vaccine candidate was purified from bacterial cultures using the EndoFree Plasmid Maxi or Giga Kit (QIAGEN) and the concentration and purity was measured on a NanoDrop One (ThermoFisher).
Binding of αCD40.RBD vaccine to non-human primate cells and activation PBMC assays
PBMC from 3 naïve macaques were isolated and stained for 15 min with anti-CD11b-V450 (ICRF44, BD), anti-CD3-V500 (SP34-2, BD), anti-CD11c-BV605 (3.9, BioLegend), anti-CD8-BV650 (BW135/80, Miltenyi Biotec), anti-CD20-SB702 (2H7, Invitrogen), anti-CD163-APC (GHI/61, BioLegend), anti-CD14-A700 (M5E2, BioLegend), anti-HLA-DR-APC-H7 (L243, BD), anti-CD4-FITC (L200, BD), anti-CD45-PerCP (D058-1283, BD) and anti-CD40-AF594 (12E12) or αCD40.RBD-AF594. Next, cells were washed twice with PBS and acquired on the ZE5 flow cytometer (Biorad). Moreover, a part of these PMBCs were also incubated 18 hours with culture medium (RPMI 1640 media with L-Glutamax supplemented with Penicillin / Streptomycin and 10% of fetal calf serum (FBS)) and stimulated with αCD40.RBD (10 µg/mL) or LPS (100 ng/mL, Invivogen). Next, cells were washed in PBS and incubated 15min with LIVE/DEAD fixable Blue Dead Cell marker (Life Technologies), anti-CD11b-V450 (ICRF44, BD), anti-CD3-V500 (SP34-2, BD), anti-CD86-BV605 (2331, BD), anti-CD11c-APC (3.9, BioLegend), anti-CD20-SB702 (2H7, Invitrogen), anti-CD80-BV786 (L307.4, BD), anti-CD8-BV650 (BW135/80, Miltenyi Biotec), anti-CD14-A700 (M5E2, BioLegend), anti-HLA-DR-APC-H7 (L243, BD), anti-CD4-FITC (L200, BD), anti-CD45-PerCP (D058-1283, BD), anti-CD69-PE-Cy7 (FN50, BD) and anti-CD40-AF594 (42G5). Next, cells were washed twice with PBS and acquired on the ZE5 flow cytometer (Biorad). Analysis was performed on FlowJo v.10 software. For activation markers, results were expressed as fold change geometric MFI, obtained by dividing the geometric MFI measure in αCD40.RBD or LPS stimulation by the background geometric MFI measure in control stimulation (incubation with medium only).
Vaccination of humanized mice
The hu-mice received immunizations at week 0, 3, and 5. The priming injection was an intraperitoneal administration of 10 μg of αCD40-RDB adjuvanted with 50 μg of polyinosinic-polycytidylic acid (Poly-IC; Invivogen) combined or not with an intramuscular injection of DREP-S (10 μg). Then hu-mice received booster i.p injections of αCD40-RDB (10 μg) plus Poly-IC (50 μg). Blood was collected at weeks 0 (before immunization), 3, and 6. Hu-mice were euthanized at week 6.
T-cells response in hu-mice
To analyze the SARS-CoV-2 RBD protein-specific T cell using functional recall assay, we used fifteen-mer peptides (n = 70) overlapping by 11 amino acids (aa) and covering the vaccine RBD sequence (aa281-571 from Spike) synthesized by JPT Peptide Technologies (Berlin, Germany) and used at a final concentration of 1 µg/mL. We also used HLA class I PE labelled tetramers purchased from ProImmune Ltd (Oxford, UK). We used the following two specificities: SARS-CoV-2 A*0201 KIA (KIADYNYKL), SARS-CoV-2 A*0301 KCY (KCYGVSPTK).
Cryopreserved hu-mice spleen cells from 6 weeks after the priming immunization (one week after final immunization) were thawed and counted. Cells were rested overnight in RPMI 1640 media with L-Glutamax supplemented with Penicillin / Streptomycin and 10% of human serum. Subsequently, cells from HLA-A*0201 and HLA-A*0301 donors were pooled together for the mock group and group 2 plus 3 vaccinated hu-mice, then cultured at 0.6x106 cells per condition with 1µg/mL of 15-mers peptides JPT Peptide Technologies (Berlin, Germany). As a negative control no stimulant was added, and as a positive control 1 µL of DynabeadsTM CD3/CD28 (ThermoFischer Scientific) were used. IL-2 (100 IU/mL, R&D System) was added on day 2, half of the volume of each culture well was refreshed with fresh media containing IL-2 (10 U/mL) at day 5 and with fresh media without IL-2 at day 7. On day 8, cells were re-stimulated: no stimulant was added in the negative control, 100 ng/mL Staphylococcal enterotoxin B (LL-122, Cliniscience) was added in the positive control and 15-mers peptides in the condition of interest. BD GolgiPlug (Becton Dickinson France) was added in all conditions and the culture was continued for additional 18 hours. Next, spleen cells were washed using FACS buffer (PBS, supplemented with 1% FBS) and incubated with tetramer-PE (ProImmune Ltd, Oxford, UK), Live dead fixable Aqua Dead marker (Life Technologies) and the following antibodies: anti-hCD3-A700 (UCHT1, Sony), anti-h-CD4-BV605 (RPA-T4, Sony), anti-hCD8-APC-Cy7 (SK1, Sony) for 30 minutes. Following fixation and permeabilization, spleen cells were stained with intracellular antibodies: anti-hIFNγ-PerCPCy5.5 (B27, Sony), anti-hIL-2-BV421 (17H12, Sony), anti-hTNFα-PC7 (Mab11, Sony) for 30 minutes. Stained cells were acquired on the LSRII flow cytometer (BD Biosciences). FlowJo v.10.7.1 software was used for data analysis (TreeStar, Inc., Ashland, OR).
SARS-CoV-2 S protein-specific B cell analysis
Hu-mice PBMC from 3 weeks after the priming immunization and hu-mice PBMC and spleen cells from 6 weeks (one week after the last recall injection) were incubated first with the biotinylated SARS-CoV-2 S protein for 30 min at 4°C. After a washing step, cells were stained for 30 min at 4°C with streptavine-AF700 (ThermoFisher Scientific), anti-human (h) CD45-PeCy7 (HI30, Sony), anti-mouse (m) CD45-BV711 (30F11, Sony), anti-hCXCR4-Pe-Dazzle (12G5, eBiosciences), anti-hCCR10-PE (314305; R&D System), anti-CD3-FITC (SK7, Biolegend), anti-CD14-FITC (M5E2, Sony), anti-IgM-FITC (MHM-88, Biolegend) antibodies and the following B cell-specific antibodies: anti-hCD19-PacBlue (HIB19, Sony), anti-hCD20-APC (2H7, Sony), anti-hIgG-BV786 (G18-145, BD Biosciences), anti-hCD38-APC-H7 (HIT2, Sony). Staining on spleen cells also included a viability marker (LiveDead aqua or yellow stain ThermoFisher Scientific). Cells were washed twice with FACS buffer (PBS 1% FCS) and acquired on the LSRII flow cytometer (BD Biosciences). Analyses were performed on FlowJo v.10.7.1.
Non-human primate study design
Convalescent cynomolgus macaques previously exposed to SARS-CoV-2 and used to assess hydroxychloroquine (HCQ) and azithromycin (AZTH) antiviral efficacy. None of the AZTH neither HCQ nor the combination of HCQ and AZTH showed a significant effect on viral replication 5. Six months (24-26 weeks) post infection (p.i.), twelve of these animals were randomly assigned in two experimental groups. The convalescent vaccinated group (n=6) received 200 ug of αCD40.RBD vaccine by subcutaneous (SC) route diluted in PBS and without any adjuvant. The other six convalescent animals were used as controls and received the equivalent volume of PBS by SC. The two groups of convalescent animals were sampled at week 2 and 4 following vaccine or PBS injection for anti-SARS-CoV-2 immune response evaluation. Additional six age matched (43.7 months +/-6.76) cynomolgus macaques from same origin were included in the study as controls naÏve from any exposure to SARS-CoV-2.
Evaluation of anti-Spike, anti-RBD and neutralizing IgG antibodies
Anti-Spike IgG from human and NHP sera were titrated by multiplex bead assay. Briefly, Luminex beads were coupled to the Spike protein as previously described 6 and added to a Bio-Plex plate (BioRad). Beads were washed with PBS 0.05% tween using a magnetic plate washer (MAG2x program) and incubated for 1h with serial diluted individual serum. Beads were then washed and anti-NHP IgG-PE secondary antibody (Southern Biotech, clone SB108a) was added at a 1:500 dilution for 45 min at room temperature. After washing, beads were resuspended in a reading buffer 5 min under agitation (800 rpm) on the plate shaker then read directly on a Luminex Bioplex 200 plate reader (Biorad). Average MFI from the baseline samples were used as reference value for the negative control. Amount of anti-Spike IgG was reported as the MFI signal divided by the mean signal for the negative controls. Human sera from convalescent patients who were hospitalized with virologically confirmed COVID-19 were collected three months after symptoms recovery and used as controls for the titration of anti-Spike antibodies.
Anti-RBD and anti-Nucleocapside (N) IgG were titrated using a commercially available multiplexed immunoassay developed by Mesoscale Discovery (MSD, Rockville, MD) as previously described 7. Briefly, antigens were spotted at 200−400 μg/mL in a proprietary buffer, washed, dried and packaged for further use (MSD® Coronavirus Plate 2). Then, plates were blocked with MSD Blocker A following which reference standard, controls and samples diluted 1:500 and 1:5000 in diluent buffer were added. After incubation, detection antibody was added (MSD SULFO-TAGTM Anti-Human IgG Antibody) and then MSD GOLDTM Read Buffer B was added and plates read using a MESO QuickPlex SQ 120MM Reader. Results were expressed as arbitrary unit (AU)/mL.
The MSD pseudo-neutralization assay was used to measure antibodies neutralizing the binding of the spike protein to the ACE2 receptor. Plates were blocked and washed as above, assay calibrator (COVID- 19 neutralizing antibody; monoclonal antibody against S protein; 200 μg/mL), control sera and test sera samples diluted 1:10 and 1:100 in assay diluent were added to the plates. Following incubation of the plates, an 0.25 μg/mL solution of MSD SULFO-TAGTM conjugated ACE-2 was added after which plates were read as above. Electro-chemioluminescence (ECL) signal was recorded and results expressed as 1/ECL.
Antigen specific T cell assays using non-human primate cells
To analyze the SARS-CoV-2 protein-specific T cell using functional assay, 15-mer peptides (n = 70) overlapping by 11 amino acids (aa) and covering the vaccine RBD sequence (n=70, aa 281-571 from Spike) and the SARS-CoV-2 Nucleoprotein sequence (n=102, aa 1-419 from N) synthesized by JPT Peptide Technologies (Berlin, Germany) and used at a final concentration of 2 µg/mL.
IFNy ELISpot assay of PBMC was performed using the Monkey IFNy ELISpot PRO kit (Mabtech Monkey IFNy ELISPOT pro, #3421M-2APT) according to the manufacturer’s instructions. PBMC were stimulated with RBD or N sequence overlapping peptide pools at a final concentration of 2 μg/mL. Plates were incubated for 18 h at 37°C in an atmosphere containing 5% CO2, then washed 5 times with PBS and incubated for 2 h at 37°C with a biotinylated anti-IFNy antibody. After 5 washes, spots were developed by adding 0.45 μm-filtered ready-to-use BCIP/NBT-plus substrate solution and counted with an automated ELISpot reader ELRIFL04 (Autoimmun Diagnostika GmbH, Strassberg, Germany). Spot forming units (SFU) per 1.0×106 PBMC are means of duplicates for each animal.
T-cell responses were also characterized by measurement of the frequency of PBMC expressing IL-2 (PerCP5.5, MQ1-17H12, BD), IL-17a (Alexa700, N49-653, BD), IFN-γ (V450, B27, BD), TNF-α (BV605, Mab11, BioLegend), IL-13 (BV711, JES10-5A2, BD), CD137 (APC, 4B4, BD) and CD154 (FITC, TRAP1, BD) upon stimulation with the two peptide pools. CD3 (APC-Cy7, SP34-2, BD), CD4 (BV510, L200, BD) and CD8 (PE-Vio770, BW135/80, Miltenyi Biotec) antibodies was used as lineage markers. One million of PBMC were cultured in complete medium (RPMI1640 Glutamax+, Gibco; supplemented with 10 % FBS), supplemented with co-stimulatory antibodies (FastImmune CD28/CD49d, Becton Dickinson). Then cells were stimulated with S or N sequence overlapping peptide pools at a final concentration of 2 μg/mL. Brefeldin A was added to each well at a final concentration of 10µg/mL and the plate was incubated at 37°C, 5% CO2 during 18 h. Next, cells were washed, stained with a viability dye (LIVE/DEAD fixable Blue dead cell stain kit, ThermoFisher), and then fixed and permeabilized with the BD Cytofix/Cytoperm reagent. Permeabilized cell samples will be stored at -80 °C before the staining procedure. Antibody staining was performed in a single step following permeabilization. After 30 min of incubation at 4°C, in the dark, cells were washed in BD Perm/Wash buffer then acquired on the ZE5 flow cytometer (Biorad). Analysis was performed on FlowJo v.10 software.
Experimental infection of macaques with SARS-CoV-2
Four weeks after immunization, all animals were exposed to a total dose of 106 pfu of SARS-CoV-2 virus (hCoV-19/France/ lDF0372/2020 strain; GISAID EpiCoV platform under accession number EPI_ISL_406596) via the combination of intranasal and intra-tracheal routes (0.25 mL in each nostril and 4.5 mL in the trachea, i.e. a total of 5 mL; day 0), using atropine (0.04 mg/kg) for pre-medication and ketamine (5 mg/kg) with medetomidine (0.05 mg/kg) for anesthesia. Nasopharyngeal, tracheal and rectal swabs, were collected at 1, 2, 3, 4, 6, 9, 14 and 20 days post exposure (d.p.exp.) while blood was taken at 2, 4, 6, 9, 14 and 20 d.p.exp. Bronchoalveolar lavages (BAL) were performed using 50 mL sterile saline at 3 d.p.exp in order to be close to the peak of viral replication and to be able to observe a difference between the vaccinated and control groups. In our earlier study 5, we found that at later time-points, viral loads in the BAL were very low or negative. Chest CT was performed at baseline and at 2 and 6 d.p.exp. on anesthetized animals using tiletamine (4 mg/kg) and zolazepam (4 mg/kg). Lesions were scored as we previously described 5. Blood cell counts, haemoglobin and haematocrit were determined from EDTA blood using a DXH800 analyzer (Beckman Coulter).
Virus quantification in cynomolgus macaque samples
Upper respiratory (nasopharyngeal and tracheal) and rectal specimens were collected with swabs (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 by RT-qPCR 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 2.67 log10 copies of SARS-CoV-2 gRNA per mL and the limit of quantification was estimated to be 3.67 log10 copies per mL. SARS-CoV-2 E gene subgenomic mRNA (sgRNA) levels were assessed by RT-qPCR using primers and probes previously described (Corman et al., 2020; Wölfel et al., 2020): leader-specific primer sgLeadSARSCoV2-F CGATCTCTTGTAGATCTGTTCTC, E-Sarbeco-R primer ATATTGCAGCAGTACGCACACA and E-Sarbeco probe HEX-ACACTAGCCATCCTTACTGCGCTTCG-BHQ1. The protocol describing the procedure for the detection of SARS-CoV-2 is 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). The limit of detection was estimated to be 2.87 log10 copies of SARS-CoV-2 sgRNA per mL and the limit of quantification was estimated to be 3.87 log10 copies per mL.
Viral dynamics modeling
For the structure of the model, we started from previously published models 8,9 where we added a compartment for the inoculum to be able to distinguish between the injected virus (Vs) and the virus produced de novo (VI and VNI). The model included uninfected target cells (T) that can be infected (I1) and produce virus after an eclipse phase (I2). The virus generated can be infectious (VI) or non infectious (VNI). The model can be written as a set of differential equations as follows:
Using the concentration of viral load, measuring VS, VI and VNI, we estimated the viral infectivity (β) and the loss rate of infected cells (δ). The effect of each intervention group (convalescent macaques vaccinated or not and previously uninfected macaques) was tested on the viral infectivity and the loss rate of infected cells. Furthermore, individual variation of β, ρ and δ was estimated through random effects. Maximum likelihood estimation was performed using a stochastic approximation EM algorithm implemented in the software Monolix (www.lixoft.com). The duration of the eclipse phase (1/κ) and the clearance of the virus (c) were estimated by profile likelihood. The production of viral particles by infected cells (ρ) has been fixed to 19,000 in trachea and 36,000 in nasopharynx copies per productively infected cell per day according to previous estimations 5. The proportion of infectious virus (m) has been fixed to 1/1000 according to previous work 5. The initial concentration of target cells, that are the epithelial cells expressing the ACE2 receptor, was assumed to be 1.33x105 cells/mL in the nasopharynx and 2.25x104 cells/mL in trachea (Gonçalves et al., 2020). The initial concentration of the inoculum was assumed to be 2.3x109 copies/mL corresponding to 106 pfu (Table 1).
Differences between unmatched groups were compared using an unpaired t-test or the Mann-Whitney U test (Graphpad Prism 8.0), and differences between matched groups were compared using a paired t-test or the Wilcoxon signed-rank test (Graphpad Prism 8.0). Viral kinetic parameter was compared using log-rank tests (Graphpad Prism 8.0). Correlation between viral and immune parameter was determined using nonparametric Spearman correlation (Graphpad Prism 8.0).