). 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 