key: cord-0290968-c74lgd5i
authors: Ku, Min-Wen; Bourgine, Maryline; Authié, Pierre; Lopez, Jodie; Nemirov, Kirill; Moncoq, Fanny; Noirat, Amandine; Vesin, Benjamin; Nevo, Fabien; Blanc, Catherine; Souque, Philippe; Tabbal, Houda; Simon, Emeline; Le Dudal, Marine; Guinet, Françoise; Fiette, Laurence; Mouquet, Hugo; Anna, François; Martin, Annette; Escriou, Nicolas; Majlessi, Laleh; Charneau, Pierre
title: Intranasal Vaccination with a Lentiviral Vector Strongly Protects against SARS-CoV-2 in Mouse and Golden Hamster Preclinical Models
date: 2020-09-03
journal: bioRxiv
DOI: 10.1101/2020.07.21.214049
sha: dc702f15fe3dec6a154a79ac2e9d659eeb362a07
doc_id: 290968
cord_uid: c74lgd5i

To develop a vaccine candidate against COVID-19, we generated a Lentiviral Vector (LV), eliciting neutralizing antibodies against the Spike glycoprotein of SARS-CoV-2. Systemic vaccination by this vector in mice, in which the expression of the SARS-CoV-2 receptor hACE2 has been induced by transduction of respiratory tract cells by an adenoviral vector, conferred only partial protection, despite an intense serum neutralizing activity. However, targeting the immune response to the respiratory tract through an intranasal boost with this LV resulted in > 3 log10 decrease in the lung viral loads and avoided local inflammation. Moreover, both integrative and non-integrative LV platforms displayed a strong vaccine efficacy and inhibited lung deleterious injury in golden hamsters, which are naturally permissive to SARS-CoV-2 replication and restitute the human COVID-19 physiopathology. Our results provide evidence of marked prophylactic effects of the LV-based vaccination against SARS-CoV-2 and designate the intranasal immunization as a powerful approach against COVID-19. Highlights A lentiviral vector encoding for Spike predicts a promising COVID-19 vaccine Targeting the immune response to the upper respiratory tract is key to protection Intranasal vaccination induces protective mucosal immunity against SARS-CoV-2 Lung anti-Spike IgA responses correlate with protection and reduced inflammation

). These epitopes elicited CD8 + -but not CD4 + -T cells, as assessed by intracellular 146 cytokine staining ( Figure 2C ). The predominant CD8 + phenotype of these T cells is in accordance with 147 the favored orientation of LV-encoded antigens to the MHC-I presentation pathway (Hu et al., 2011) . 148 We also identified S:441-455 (LDSKVGGNYNYLYRL), S:671-685 (CASYQTQTNSPRRAR) and 149 S:991-1005 (VQIDRLITGRLQSLQ) subdominant epitopes, which gave rise to < 2000 SFU / spleen 150 in ELISPOT assay ( Figure 2B) . 151

Establishment of a murine model expressing hACE2 in the respiratory tracts 152

As S CoV-2 does not interact well with murine ACE2, wild-type laboratory mice are not permissive to 153 replication of SARS-CoV-2 clinical isolates. Therefore, we sought to develop a murine model in which 154 human ACE2 (hACE2) expression was induced in the respiratory tracts and pulmonary mucosa to 155 evaluate the LV::S FL vaccine efficacy. This method has been successfully used to establish the 156 expression of human DPP4 for the study of mouse infection with MERS-CoV (Zhao et al., 2014) and 157 also for hACE2 during the preparation of this manuscript . We generated an Ad5 158 vector to deliver the gene encoding for hACE2 under the transcriptional control of the CMV promoter 159 (Ad5::hACE2) in an episomal form. We first checked in vitro the potential of the Ad5::hACE2 vector 160 to transduce HEK293T cells by Reverse Transcriptase (RT)-PCR ( Figure 3A ). To achieve in vivo 161 transduction of respiratory tract cells, we instilled intranasally (i.n.) 2.5 × 10 9 Infectious Genome Units 162 (IGU) of Ad5::hACE2 into C57BL/6 mice. Four days later, the hACE2 protein expression was 163 detectable in the lung cell homogenate by Western Blot ( Figure 3B ). To get more insights into the in 164 vivo expression profile of a transgene administered under these conditions, we instilled i.n. the same 165 dose of an Ad5::GFP reporter vector into C57BL/6 mice. As evaluated by flow cytometry, 4 days post 166 instillation, the GFP reporter was expressed not only in the lung epithelial EpCam + cells, but also in 167 lung immune cells, as tracked by the CD45 pan-hematopoietic marker ( Figure 3C ), showing that 168 through this approach the transduction was efficiently achieved in epithelial cells, although not 169 restricted to these cells. 170

To evaluate the permissibility of such hACE2-transduced mice to SARS-CoV-2 infection, 4 days 171 after i.n. pretreatment with either Ad5::hACE2 or an empty Ad5 control vector, C57BL/6 mice were 172 inoculated i.n. with 1 × 10 5 TCID 50 (Median Tissue Culture Infectious Dose) of a SARS-CoV-2 clinical 173 isolate (BetaCoV/France/IDF0372/2020), which was isolated in January 2020 from a COVID-19 174 patient by the National Reference Centre for Respiratory Viruses (Institut Pasteur, Paris, France) 175 (Lescure et al., 2020) . The lung viral loads, determined at 2 days post inoculation (dpi) by reverse 176 transcription and quantitative real-time PCR (qRT-PCR), were as high as (4.4 ± 1.8) × 10 9 copies of 7 SARS-CoV-2 RNA in Ad5::hACE2-pretreated mice, compared to only (6.2 ± 0.5) × 10 5 copies in 178 empty Ad5-pretreated mice, or (4.0 ± 2.9) × 10 5 copies in un-pretreated mice ( Figure 3D ). In the latter 179 two control groups, these copy numbers corresponded to the input viral RNA, as determined in 180

Ad5::hACE2-pretreated mice, inoculated with equivalent amounts of heat-killed viral particles ( Figure  181 3D). At 4 dpi, the lung viral loads were maintained in Ad5::hACE2-pretreated mice (2.8 ± 1.3 × 10 9 182 copies), whereas a drop to (1.7 ± 2.3) × 10 4 or (3.9 ± 5.1) × 10 3 copies was observed in empty Ad5-183 pretreated or non-pretreated mice, respectively. In Ad5::hACE2-pretreated mice, the viral loads were 184 still detectable at 7 dpi ((1.33 ± 0.9) × 10 6 copies). 185

Ad5::hACE-2 i.n. instillation induced CD45 + cell recruitment to the lungs ( Figure 3E ). However, 186 no pro-inflammatory effect was seen with a lower dose of 4 x 10 8 IGU/mouse ( Figure 3E ), which still 187 conferred full permissibility to SARS-CoV-2 replication ( Figure 3F ), and this dose was therefore 188 chosen for the subsequent experiments described below. In this model of permissive mice, SARS-CoV-189 2 infection resulted in widespread infiltration of the lung interstitium by mononuclear inflammatory 190 cells, i.e., lymphocytes and macrophages, at 3 dpi ( Figure S2 ). 191

These results show that pretreatment of mice with appropriate doses of Ad5::hACE2 can render 192 mice permissive to SARS-CoV-2 replication without inducing Ad5-mediated inflammation, thus 193 providing a valuable model for vaccine or drug studies. 194

To investigate the prophylactic potential of LV::S FL against SARS-CoV-2, C57BL/6 mice (n = 4-196 5/group) were injected i.p. with a single dose of 1 × 10 7 TU of LV::S FL or a negative control LV (sham). 197 At week 7 post immunization, mice were pretreated with Ad5::hACE2, and 4 days later, inoculated i.n. 198 with 1 × 10 5 TCID 50 of SARS-CoV-2 ( Figure S3A ). At 3 dpi, the lung viral loads in LV::S FL -vaccinated 199 mice were reduced by ~ 6.5 folds, i.e., mean ± SD of (5.5 ± 3.8) × 10 8 SARS-CoV-2 RNA copies 200 compared to (3.1 ± 1.9) × 10 9 or (4.3 ± 3.0) × 10 9 copies in the un-or sham-vaccinated mice, 201 respectively ( Figure S3B ). Therefore, a single i.p. LV::S FL injection provided partial protection in the 202 lung, despite intense serum NAb activity. 203

To further improve the prophylactic effect, we evaluated the prime-boost or prime-target 204 approaches. C57BL/6 mice (n = 4-5/group) were primed i.p. with 1 × 10 7 TU of LV::S FL or a control 205 LV at week 0, and then boosted at week 3 with: (i) 1 × 10 7 TU of the same LV via the i.p. route 206 ("LV::S FL i.p.-i.p.", prime-boost), or (ii) with 3 × 10 7 TU via the i.n. route ("LV::S FL i.p.-i.n.", prime-207 target) to attract the mediators of systemic immunity to the lung mucosa ( Figure 4A ). Systemic 208 boosting with LV::S FL via i.p. resulted in a significant increase in the anti-S CoV-2 IgG titers, which was 209 more obvious when the binding was evaluated against the foldon-trimerized full-length S ( Figure 4B , 210 left) than against the S1 or RBD fragments ( Figure S4A ). This observation may suggest that the 211 concerned B-cell epitopes are of conformational type. In contrast, mucosal targeting with LV::S FL via 212 8 i.n. did not lead to a statistically significant improvement of anti-S CoV-2 IgG titers at the systemic level 213 ( Figure 4B left, Figure S4A ). In terms of serum neutralization potential, even though a trend to increase 214 was observed after i.p. or i.n. boost, the differences did not reach statistical significance ( Figure 4B All mice were then pretreated with Ad5::hACE2 and challenged i.n. with 0.3 × 10 5 TCID 50 of 217 SARS-CoV-2 at week 4 post prime. At 3 dpi, the lung viral loads were significantly lower in LV::S FL 218 i.p.-i.p. immunized mice, i.e., mean ± SD (2.3 ± 3.2) × 10 8 , than in sham-vaccinated mice (13.7 ± 7.5) 219

× 10 8 copies of SARS-CoV-2 RNA, ( Figure 4C ). This viral load reduction was similar to that obtained 220 with a single LV::S FL administration ( Figure S3B ). Most importantly, after i.n. LV::S FL target 221 immunization, > 3 log10 decrease in viral loads was observed and 2 out of 5 mice harbored undetectable 222 lung viral loads as determined by qRT-PCR assay. Anti-S CoV-2 IgG were detected in the clarified lung 223 As evaluated by qRT-PCR in the total lung homogenates of the protected "int LV::S FL i.p.-i.n. Low", 282 "int LV::S FL i.p.-i.n. High" and "NILV::S FL i.m.-i.n." groups, substantial decreases were observed at 4 283 dpi in the expression of inflammatory IFN-g and IL-6 cytokines, anti-inflammatory IL-10 cytokine, 284 and CCL2, CCL3 and CXCL10 chemokines, compared to their unprotected sham-vaccinated 285 counterparts ( Figure 7A) . The other inflammatory mediators tested were not significantly modified 286 ( Figure S6 ). 287

In sham-vaccinated and challenged hamsters, overall marked multifocal degenerative changes of 288 the bronchial/bronchiolar epithelium, moderate effacement of the epithelium, associated with mild to 289 moderate mixed inflammation in the airway lumen, and minimal multifocal interstitial mononuclear 290 cell inflammation at both time-points ( Figure 7B ). At 4 dpi, effacement of the respiratory epithelium 291 and mixed inflammation in the alveoli were more severe; moderate fibrin deposits were also noted. In We first evaluated the efficacy of several LV each encoding for one of the variants of S, i.e., the S1 337 domain alone (LV::S1), the S1-S2 ecto-domain, devoid of the transmembrane and C-terminal short 338 internal tail (LV::S1-S2), or full-length, membrane anchored protein (LV::S FL ). Even though a single 339 administration of each of these LV was able to induce high anti-S CoV-2 Ab titers, only LV::S FL induced 340 highly functional Nabs with neutralizing activities similar to those found in a cohort of symptomatic 341 SARS-CoV-2 patients. This finding predicted a protective potential of the humoral responses induced 342 by the LV::S FL vector. In addition, strong anti-S CoV-2 CD8 + T-cell responses were also observed in the 343 spleen of mice as early as 2 weeks after a single LV::S FL injection, as detected against numerous MHC-344 I-restricted immunogenic regions that we identified in C57BL/6 (H-2 b ) mice. 345

In the transduced mouse model which allows high rates of SARS-CoV-2 replication, vaccination by Methods 508

Construction of transfer pFLAP plasmids coding for S FL , S1-S2, or S1 proteins 509 A codon-optimized full-length S (1-1273) sequence was amplified from pMK-RQ_S-2019-nCoV 510 and inserted between BamHI and XhoI sites of pFlap-ieCMV-WPREm. Sequences encoding for S1-511 S2 (1-1211) or S1 (1-681) were amplified by PCR from the pFlap-ieCMV-S FL -WPREm plasmid and 512 sub-cloned into pFlap-ieCMV-WPREm between the BamHI and XhoI restriction sites ( Figure S1 ). 513

Each of the PCR products were inserted between the native human ieCMV promoter and a mutated 514

Woodchuck Posttranscriptional Regulatory Element (mWPRE) sequence, in which the atg starting 515 codon was mutated to avoid transcription of the downstream truncated "X" protein of Woodchuck 516

Hepatitis Virus, in order to improve the vector safety. Plasmids were amplified in Escherichia coli 517 DH5a in Lysogeny Broth supplemented with 50 µg/ml of kanamycin and purified using the 518 NucleoBond Xtra Maxi EF Kit (Macherey Nagel) and resuspended in Tris-EDTA Endotoxin-Free 519 buffer overnight. Plasmid were quantified with a NanoDrop 2000c spectrophotometer (Thermo Ficher, 520

Illkirch, France), aliquoted and stored at -20°C. Plasmid DNA were verified by enzymatic digestion 521 and by sequencing the region proximal to the transgene insertion sites. 522

Non-replicative LV were produced in Human Embryonic Kidney (HEK)-293T cells, as previously 524 detailed (Zennou et al., 2000) . Briefly, lentiviral particles were produced by transient calcium 525 phosphate co-transfection of HEK293T cells with the vector plasmid pTRIP/sE, a VSV-G Indiana 526 envelope plasmid and an encapsidation plasmid (p8.74 or pD64V for the production of integration-527 proficient or integration-deficient vectors respectively). Supernatants were harvested at 48h post 528 transfection, clarified by 6-minute centrifugation at 2500 rpm at 4°C. LV were aliquoted and stored at 529 -80°C. Vector titers were determined by transducing 293T cells treated with aphidicolin. The titer, 530

proportional to the efficacy of nuclear gene transfer, is determined as Transduction Unit (TU)/ml by 531 qPCR on total lysates at day 3 post transduction, by use of forward 5'-TGG AGG AGG AGA TAT 532 GAG GG-3' and reverse 5'-CTG CTG CAC TAT ACC AGA CA-3' primers, specific to pFLAP 533 plasmid and forward 5'-TCT CCT CTG ACT TCA ACA GC-3' and reverse 5'-CCC TGC ACT TTT 534 TAA GAG CC-3' primers specific to the host housekeeping gene gadph as previously described 535 (Iglesias et al., 2006) . 536

Female C57BL/6JRj mice (Janvier, Le Genest Saint Isle, France) were used between the age of 6 538 and 10 weeks. Male Mesocricetus auratus golden hamsters (Janvier, Le Genest Saint Isle, France) were 539 purchased mature, i.e. 80-90 gr weight. At the beginning of the immunization regimen they weighed 540 100 to 120 gr. Experimentation on animals was performed in accordance with the European and French 541 Supplemental information titles and legends 717 Figure S1 . Maps of plasmids used for production of LV encoding S FL , S1-S2 or S1 antigens. 718 Figure S2 . Lung histology in mice pretreated with Ad5::hACE2 and inoculated with SARS-719

CoV-2. Histological analysis in C57BL/6 mice, pretreated with PBS or Ad5::hACE2, followed by i.n. 720 inoculation of 1 × 10 5 TCID 50 of SARS-CoV-2. Analysis was performed at 3 dpi. Lung, HE&S stain, 721

Original magnification: x10, scale bar: 100 µm. Br: Bronchi or bronchiole. Bv: Blood vessel. Arrow: 722

Mononuclear inflammatory cell infiltration. 723 Table S1 . Sequences of primers and probes for SARS-CoV-2 viral load determination. 750 Table S2 . Sequences of primers used to quantitate mouse cytokines and chemokines by qRT-751 

Samples from the lung were fixed in formalin for at least 7 days and routinely embedded in paraffin

Microscopic 712 changes were qualitatively described and when applicable scored semi-quantitatively, using: (i) 713 distribution qualifiers (i.e., focal, multifocal, locally extensive or diffuse), and (ii) a five-scale severity 714 grade

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guidelines (Directive 86/609/CEE and Decree 87-848 of 19 October 1987) subsequent to approval by 542 the Institut Pasteur Safety, Animal Care and Use Committee, protocol agreement delivered by local 543 ethical committee (CETEA #DAP20007) and Ministry of High Education and Research 544 APAFIS#24627-2020031117362508 v1. Animals were vaccinated with the indicated TU of LV and 545 sera were collected at various time points post immunization to monitor binding and neutralization 546 activities. Previous to i.m. or i.n. instillations, animals were anesthetized by i.p. injection of a mixture 547 of Ketamine (Imalgene, 50 mg/kg) and Xylazine (Rompun, 50 mg/kg). 548

Hamsters or Ad5::hACE2-pretreated mice were anesthetized by i.p. injection of mixture Ketamine 550 and Xylazine, transferred into a biosafety cabinet 3 where they were inoculated i.n. with 0.3 or 1 × 10 5 551 TCID 50 of the BetaCoV/France/IDF0372/2020 SARS-CoV-2 clinical isolate (Lescure et al., 2020) , 552 amplified in VeroE6 cells. The strain BetaCoV/France/IDF0372/2020 was supplied by the National 553Reference Centre for Respiratory Viruses hosted by Institut Pasteur (Paris, France) and headed by Pr. 554Sylvie van der Werf. The human sample from which strain BetaCoV/France/IDF0372/2020 was 555 isolated has been provided by Dr. X. Lescure and Pr. Y. Yazdanpanah from the Bichat Hospital, Paris, 556France. The viral inoculum was contained in 20 µl for mice and in 50 µl for hamsters. Animals were 557 then housed in an isolator in BioSafety Level 3 animal facilities of Institut Pasteur. The organs and 558 fluids recovered from the animals infected with live SARS-CoV-2 were manipulated following the 559 approved standard operating procedures of these facilities. 560

Codon-optimized nucleotide fragments encoding a stabilized foldon-trimerized version of the 562 SARS-CoV-2 S ectodomain (a.a. 1 to 1208), the S1 monomer (a.a. 16 to 681) and the RBD subdomain 563 (amino acid 331 to 519) both preceded by a murine IgK leader peptide and followed by an 8xHis Tag 564 were synthetized and cloned into pcDNA™3.1/Zeo (+) expression vector (Thermo Fisher). Proteins 565 were produced by transient co-transfection of exponentially growing Freestyle™ 293-F suspension 566 cells (Thermo Fisher) using polyethylenimine (PEI)-precipitation method as previously described 567 (Lorin and Mouquet, 2015) . Recombinant S CoV-2 proteins were purified by affinity chromatography 568 using the Ni Sepharose® Excel Resin according to manufacturer's instructions (Thermo Fisher). 569Protein purity was evaluated by in-gel protein silver-staining using Pierce Silver Stain kit (Thermo 570 Fisher) following SDS-PAGE in reducing and non-reducing conditions using NuPAGE™ 3-8% Tris-571Acetate gels (Life Technologies). Purified proteins were dialyzed overnight against PBS using Slide-572 A-Lyzer® dialysis cassettes (10 kDa MW cut-off, Thermo Fisher). Protein concentration was 573 determined using the NanoDrop™ One instrument (Thermo Fisher). 574 20 ELISA 575Ninety-six-well Nunc Polysorp plates (Nunc, Thermo Ficher) were coated overnight at 4 °C with 576 100 ng/well of purified S CoV-2 proteins in carbonate-bicarbonate buffer (pH 9.6). The next day, plates 577 were blocked with carbonate buffer containing 1% BSA for 2 h at 37°C. Wells were then washed with 578 PBS containing 0.05% Tween 20 (PBS-T), 1:100-diluted sera or 1:10-diluted lung homogenates in 579 PBS-T containing 1% BSA and four serial ten-to-ten dilutions were added and incubated during 2h at 580 37°C. After PBS-T washings, plates were incubated with 1,000-fold diluted peroxydase-conjugated 581 goat anti-mouse IgG (Jackson ImmunoResearch Europe Ltd, Cambridgeshire, United Kingdom) for 1 582 h. Plates were revealed by adding 100 µl of 3,3',5,5'-tetramethylbenzidine chromogenic substrate 583 (Eurobio Scientific). Following a 30 min incubation, reaction was stopped by adding 100 µl of 2N 584 H2SO4 and optical densities were measured at 450nm/620nm on a PR3100 reader. 

In order to map the immuno-dominant epitopes of S CoV-2 , peptides spanning the whole S FL 607 (Mimotopes, Australia) were pooled by 16, each containing 15 a.a. residues overlapping by 10 a.a. 608 21 Peptides were dissolved in DMSO at a concentration of 2 mg/ml and diluted before use at 1 µg/ml for 609 ELISPOT or 2-5 µg/ml for ICS (Intracellular Cytokine Staining) in RRMI-1640 medium supplemented 610 with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 1 x 10 -4 M non-essential amino-acids, 611 1% vol/vol HEPES, 1 x 10 -3 M sodium pyruvate and 5 × 10 -5 M of b-mercapto-ethanol. IFN-g 612 ELISPOT and ICS assays were performed as described previously ( using Adeno-X rapid Maxi purification kit and concentrated with the Amicon Ultra-4 10k centrifugal 632 filter unit. Vectors were resuspended and stored à -80°C in PIPES buffer pH 7.5, supplemented with 633 2.5% glucose. Ad5 were titrated using a qPCR protocol, as described (Gallaher and Berk, 2013) . 634

Expression of hACE2 in the lungs of Ad5::hACE2-transduced mice was assessed by Western 636Blotting. One million cells from lung cell suspension were resolved on 4 -12 % NuPAGE Bis-Tris 637 protein gels (Thermo Fisher), then transferred onto a nitrocellulose membrane (Biorad, France). The 638 nitrocellulose membrane was blocked in 5 % non-fat milk in PBS-T for 2 hours at room temperature 639 and probed overnight with goat anti-hACE2 primary Ab at 1 µg/mL (AF933, R&D systems). 640Following three wash intervals of 10 minutes with PBS-T, the membrane was incubated for 1 hour at 641 room temperature with HRP-conjugated anti-goat secondary Ab and HRP-conjugated anti-b-actin 642 22 (ab197277, Abcam). The membrane was washed with PBS-T thrice before visualization with enhanced 643 chemiluminescence via the super signal west femto maximum sensitivity substrate (ThermoFisher) on 644ChemiDoc XRS+ (Biorad, France). PageRuler Plus prestained protein ladder was used as size 645 reference. 646 Table S1 . In vitro transcribed RNA derived from 656 plasmid "pCI/SARS-CoV envelope" was synthesized using T7 RiboMAX Express Large Scale RNA 657 production system (Promega), then purified by phenol/chloroform extractions and two successive 658 precipitations with isopropanol and ethanol. RNA concentration was determined by optical density 659 measurement, then RNA was diluted to 10 9 genome equivalents/µL in RNAse-free water containing 660 100µg/mL tRNA carrier, and stored in single-use aliquots at -80°C. Serial dilutions of this in vitro 661 transcribed RNA were prepared in RNAse-free water containing 10µg/ml tRNA carrier and used to 662 establish a standard curve in each assay. Thermal cycling conditions were: (i) reverse transcription at 663 55°C for 10 min, (ii) enzyme inactivation at 95°C for 3 min, and (iii) 45 cycles of 664 denaturation/amplification at 95°C for 15 s, 58°C for 30 s. Products were analyzed on an ABI 7500 665 Fast real-time PCR system (Applied Biosystems). PFU assay was performed as recently described 666 (Case et al., 2020) . 667

Lungs from individual mice were treated with 400 U/ml type IV collagenase and DNase I (Roche) 669 for a 30-minute incubation at 37°C and homogenized by use of GentleMacs (Miltenyi Biotech) mAbs. (Table S2 , S3). The following thermal profile was used: a single cycle 700 of polymerase activation for 3 min at 95°C, followed by 40 amplification cycles of 15 sec at 95°C and 701 30 sec 60°C (annealing-extension step). Mice b-globin or hamster ribosomal protein L18 (RLP18) was 702 used as an endogenous reference control to normalize differences in the amount of input nucleic acid. 703The average C T values were calculated from the technical replicates for relative quantification of target 704 cytokines/chemokines. The differences in the C T cytokines/chemokines amplicons and the C T of the 705 endogenous reference control, termed DC T, were calculated to normalize for differences in the quantity 706 of nucleic acid. The DC T of the experimental condition compared relatively to the PBS-immunized 707 individuals using the comparative DDC T method. The fold change in gene expression was further 708 calculated using 2 −ΔΔC T . 709Lung Histopathology 710