Brain and Lung Cross-Protection against Ancestral or Emerging SARS-CoV-2 by Intranasal 1 Lentiviral Vaccination in a New hACE2 Transgenic Murine Model 2

COVID-19 vaccines already in use or in clinical development may have reduced efficacy against 18 emerging SARS-CoV-2 variants. Although the neurotropism of SARS-CoV-2 is well established, the vaccine strategies currently developed have not taken into account the protection of the central nervous system. Here, we generated a transgenic mouse strain expressing the human Angiotensin Converting 21 Enzyme 2, with unprecedented brain as well as lung permissibility to SARS-CoV-2 replication. Using this 22 stringent transgenic model, we demonstrated that a non-integrative lentiviral vector, encoding for the spike 23 glycoprotein of the ancestral Wuhan SARS-CoV-2, used in intramuscular prime and intranasal boost elicits sterilizing protection of lung and brain against both the Wuhan and the most genetically distant Manaus P.1 SARS-CoV-2 variants. Beyond the induction of strong neutralizing antibodies, the mechanism underlying this broad protection spectrum involves a robust protective spike-specific CD8 + T-cell immunity, 27 unaffected by the recent mutations accumulated in the emerging SARS-CoV-2 variants. murine and human CD8 + T-cell epitopes identified on S CoV-2 sequence are preserved in the mutated S P.1 (Table S1). These observations indicate the strong potential of NILV at inducing full protection of lungs and brain against ancestral and emerging SARS-CoV-2 variants by eliciting strong B and T cell-responses. In contrast to the B-cell epitopes which are targets of NAbs 20 , the so far identified T- cell epitopes have not been impacted by mutations accumulated in the S CoV-2 of the emerging variants.

capable of gaining access to the brain through neural dissemination or hematogenous route 17 . Therefore, it 48 is critical to focus on the protective properties of COVID-19 vaccine candidates, not only in the respiratory 49 tract, but also in the brain. 50 New variants of SARS-CoV-2 resulting from mutations accumulating in the envelop spike glycoprotein 51 of SARS-CoV-2 (SCoV-2) have been identified by genome sequencing in diverse geographical locations 52 throughout the world. SCoV-2 is composed of S1 and S2 subunits. The former harbors a Receptor Binding 53 Domain (RBD) that encompasses the Receptor Binding Motif (RBM), which is the main functional motif 54 interacting with human Angiotensin Converting Enzyme 2 (hACE2) 18,19 . RBD and RBM are prone to 55 mutations that can further improve the fitness of SCoV-2 for binding to hACE2. A crucial consideration for 56 such mutations is the alteration in RBD/RBM B-cell epitopes, which can lead to the escape of SARS-CoV-57 2 variants from the action of neutralizing antibodies (NAbs) raised in individuals previously infected with 58 ancestral SARS-CoV-2 or immunized with SCoV2-based vaccines. In October-December 2020, B1.1.7, 59 B1.351 and P.1 variants have been respectively identified in the UK, South Africa and Manaus (Brazil), 60 with the latter being the most genetically distant 20 . 61 We have recently established the high performance of a non-integrative lentiviral vector (NILV) 62 encoding the full-length sequence of SCoV-2 of the ancestral Wuhan strain when used in systemic prime 63 followed by intranasal (i.n.) boost 21 . LVs allow transgene insertion up to 5 kb in length and offer 64 outstanding potential for gene transfer to the nuclei of host cells 22,23,24,25 . LVs display in vivo tropism for 65 immune cells, notably dendritic cells 26 . They are non-replicative, non-cytopathic and scarcely 66 inflammatory 27 . These vectors induce long-lasting B-and T-cell immunity 22,23,24,25 . LVs are pseudo-67 typed with the surface glycoprotein of Vesicular Stomatitis Virus, to which the human population has 68 limited exposure. This prevents these vectors from being targeted by preexisting immunity in humans, 69 7 changes in lung inflammation could be found between the LV::SCoV-2-vaccinated and sham groups ( Figure  149 S2C). 150 Therefore, an i.m.-i.n. prime-boost with ILV::SCoV-2 prevents SARS-CoV-2 replication in both lung 151 and CNS anatomical areas and inhibits virus-mediated lung infiltration, as well as neuro-inflammation. 152 Requirement of i.n. boost for full protection of brain in B6.K18-hACE2 IP-THV mice 153 To go further in the characterization of the protective properties of LV, in the following experiments in 154 B6.K18-hACE2 IP-THV mice, we used the safe and non-integrative version of LV. The observed protection 155 of the brain against SARS-CoV-2 may reflect the benefits of the i.n. route of vaccination against this 156 respiratory and neurotropic virus. To address this question, B6.K18-hACE2 IP-THV mice were vaccinated 157 with NILV::SCoV-2: (i) i.m. wk 0 and i.n. wk5, as a positive control, (ii) i.n. wk 0, or (iii) i.m. wk 5. Sham-158 vaccinated controls received i.n. an empty NILV at wks 0 and 5 ( Figure 3A). At wk 7, notable proportions 159 of IFN-g-producing CD8 + T cells, specific to several SCoV-2 epitopes, were detected in the lungs ( Figure 3B Mice were then challenged with SARS-CoV-2 at wk 7 and viral loads were determined in the brain or 165 lungs by E-or Esg-specific qRT-PCR at 3dpi ( Figure 3D). In this stringent pre-clinical model a single i.n. 166 or i.m. injection of NILV::SCoV-2, albeit effective, did not induce full protection in all animals of each group. 167 Only i.m. prime followed by i.n. boost conferred full protection of the brain and lungs in all animals, 168 showing the requirement of an i.n. boost to reach full protection. Correlated with the protection levels were 169 the titers of serum and lung anti-SCoV-2 IgG and IgA ( Figure 3E), as well as the serum SARS-CoV-2 170 neutralizing activity ( Figure 3F). 171 On immuno-histological examination, we detected higher numbers of CD3 + T cells per mm 2 of olfactory 172 bulbs of NILV::SCoV-2 i.m.-i.n. vaccinated and protected mice than in the sham individuals ( Figure 4A). As 173 expected with this LV vaccine, the T-cell response of protected animals was polarized towards the CD8 + 174 compartment, as evidenced by the higher proportion of CD8 + T cells in the olfactory bulbs ( Figure 4B) and 175 by the presence of CD8 + -but not CD4 + -specific reactive T cells in the spleen ( Figure S3A). No specifically 176 reacting CD4 + T cells was found in the lung either ( Figure S3B), and, in the olfactory bulb, CD4 + T cells 177 had no distinctive activated or migratory phenotype, as assessed by their surface expression of CD69 or 178 CCR7 ( Figure S3C). In line with the absence of CCR7 expression on these T cells, and unlike Murine 179 Hepatitis Virus (MHV) infection 38 , we saw no up-regulation of CCL19 and CCL21 (CCR7 ligands) in the 180 brain, regardless of the protected status of the mice ( Figure S3D). Compared to the NILV::SCoV-2 i.m.-i.n. 181 protected group, there were higher amounts of neutrophils (CD11b + Ly6C + Ly6G + ) in the olfactory bulbs 8 ( Figure 4C) and inflammatory monocytes (CD11b + Ly6C + Ly6G -) in the brain ( Figure 4D) of unprotected 183 mice, as a biomarker of inflammation. 184 Lung histological sections of infected mice detected, at 3 dpi, areas of various size exhibiting mild to 185 moderate interstitial inflammation accompanied by alveolar exudates, peribronchiolar infiltration and 186 minimal to moderate alterations of the bronchiolar epithelium. At this time-point, lesions from the 187 NILV::SCoV-2 i.m.-i.n. vaccinated and sham groups did not differ in severity or extension, indicating that 188 the immune arsenal that contributed to virus eradication had not yet been resorbed ( Figure S4  protection of the brain and lungs against SARS-CoV-2 Manaus P.1 ( Figure 5B). 203 The markedly decreased ability of the sera of NILV::SCoV-2-vaccinated mice to neutralize SB1.351 or 204 SManaus P.1 pseudo-viruses, compared to SWuhan, SD614G or SB1.117 pseudo-viruses, ( Figure 5C), raised the 205 possibility of T-cell involvement in this total protection. To evaluate this possibility, we vaccinated 206 following the same protocol ( Figure 5A), C57BL/6 WT or µMT KO mice. The latter are deficient in mature 207 B-cell compartment and therefore lack Ig/antibody response 40 . To make these non-transgenic mice 208 permissive to SARS-CoV-2 replication, they were pre-treated 4 days before the SARS-CoV-2 challenge 209 with 3 × 10 8 IGU of an adenoviral vector serotype 5 encoding hACE2 (Ad5::hACE2) 21 . Determination of 210 lung viral loads at 3 dpi showed complete protection of the lungs in vaccinated WT mice as well as a highly 211 significant protection in vaccinated µMT KO mice ( Figure 5E). This observation determines that B-cell 212 independent and antigen-specific cellular immunity, i.e. T-cell response, plays a remarkable role in LV-213 mediated protection. This is consistent with the strong CD8 + T-cell responses induced by LV::SCoV-2 at the 214 systemic level ( Figure 5F) and in the lungs ( Figure 3B, C), and the recruitment of CD8 + T cells in the 9 murine and human CD8 + T-cell epitopes identified on SCoV-2 Wuhan sequence are preserved in the mutated 217 SCoV-2 Manaus P.1 (Table S1). These observations indicate the strong potential of NILV at inducing full 218 protection of lungs and brain against ancestral and emerging SARS-CoV-2 variants by eliciting strong B 219 and T cell-responses. In contrast to the B-cell epitopes which are targets of NAbs 20 , the so far identified T-220 cell epitopes have not been impacted by mutations accumulated in the SCoV-2 of the emerging variants. 221 Discussion 222 LV-based platforms emerged recently as a powerful vaccination approach against COVID-19, notably 223 when used as a systemic prime followed by mucosal i.n. boost, inducing sterilizing immunity against lung 224 SARS-CoV-2 infection in preclinical animal models 21 . In the present study, to investigate the efficacy of 225 our vaccine candidates, we generated a new transgenic mouse model, using the LV-based transgenesis 226 approach 41 . The ILV used in this strategy encodes for hACE2 under the control of the cytokeratin K18 227 promoter, i.e., the same promoter as previously used by Perlman's team to generate B6.K18-ACE2 2Prlmn/JAX 228 mice 30 , with a few adaptations to the lentiviral FLAP transfer plasmid. However, the new B6.K18-229 hACE2 IP-THV mice have certain distinctive features, as they express much higher levels of hACE2 mRNA 230 in the brain and display markedly increased brain permissibility to SARS-CoV-2 replication, in parallel 231 with a substantial brain inflammation and development of a lethal disease in <4 days post infection. These 232 distinctive characteristics can arise from differences in the hACE2 expression profile due to: (i) alternative 233 insertion sites of ILV into the chromosome compared to naked DNA, and/or (ii) different effect of the 234 Woodchuck Posttranscriptional Regulatory Element (WPRE) versus the alfalfa virus translational enhancer 235 30 , in B6.K18-hACE2 IP-THV and B6.K18-ACE2 2Prlmn/JAX animals, respectively. Other reported hACE2 236 humanized mice express the transgene under: (i) murine ACE2 promoter, without reported hACE2 mRNA 237 expression in the brain 42 , (ii) "hepatocyte nuclear factor-3/forkhead homologue 4" (HFH4) promoter, i.e., 238 "HFH4-hACE2" C3B6 mice, in which lung is the principal site of infection and pathology 43, 44 , and (iii) 239 "CAG" mixed promoter, i.e. "AC70" C3H × C57BL/6 mice, in which hACE2 mRNA is expressed in 240 various organs including lungs and brain 45 . Comparison of AC70 and B6.K18-hACE2 IP-THV mice could 241 yield information to assess the similarities and distinctions of these two models. The B6.K18-hACE2 IP-THV 242 murine model not only has broad applications in COVID-19 vaccine studies, but also provides a unique 243 rodent model for exploration of COVID-19-derived neuropathology. Based on the substantial permissibility 244 of the brain to SARS-CoV-2 replication and development of a lethal disease, this pre-clinical model can be 245 considered as even more stringent than the golden hamster model. 246 The source of neurological manifestations associated with COVID-19 in patients with comorbid 247 conditions can be: (i) direct impact of SARS-CoV-2 on CNS, (ii) infection of brain vascular endothelium 248 and, (iii) uncontrolled anti-viral immune reaction inside CNS. ACE2 is expressed in human neurons, 249 astrocytes and oligodendrocytes, located in middle temporal gyrus and posterior cingulate cortex, which 250 may explain the brain permissibility to SARS-CoV-2 in patients 46 . Previous reports have demonstrated 251 that respiratory viruses can invade the brain through neural dissemination or hematogenous route 17 . Besides 252 that, the direct connection of olfactory system to the CNS via the frontal cortex also represents a plausible 253 route for brain invasion 47 . Neural transmission of viruses to the CNS can occur as a result of direct neuron 254 invasion through axonal transport in the olfactory mucosa. Subsequent to intraneuronal replication, the 255 virus spreads to synapses and disseminate to anatomical CNS zones receiving olfactory tract projections 9, 256 48, 49, 50 . However, the detection of viral RNA in CNS regions without connection with olfactory mucosa 257 suggests the existence of another viral entry into the CNS, including migration of SARS-CoV-2-infected 258 immune cells crossing the hemato-encephalic barrier or direct viral entry pathway via CNS vascular 259 endothelium 16 . Although at steady state, viruses cannot penetrate into the brain through an intact blood-260 brain barrier 48 , inflammation mediators which are massively produced during cytokine/chemokine storm, 261 notably TNF-α and CCL2, can disrupt the integrity of blood-brain barrier or increase its permeability, 262 allowing paracellular blood-to-brain transport of the virus or virus-infected leukocytes 4, 23 . The use of the 263 highly stringent B6.K18-hACE2 IP-THV mice demonstrated the importance of i.n. booster immunization for 264 inducing sterilizing protection of CNS by our LV-based vaccine candidate developed against SARS-CoV-265 2. Olfactory bulb may control viral CNS infection through the action of local innate and adaptive immunity 266 51 . In line with these observations, we detected increased frequencies of CD8 + T cells at this anatomically 267 strategic area in i.m.-i.n. vaccinated and protected mice. In addition, substantial reduction in the 268 inflammatory mediators was also found in the brain of the i.m.-i.n. vaccinated and protected mice, as well 269 as decreased proportions of neutrophils and inflammatory monocytes respectively in the olfactory bulbs 270 and brain. Regardless of the mechanism of the SARS-CoV-2 entry into the brain, we provide evidence of 271 the full protection of the CNS against SARS-CoV-2 by i.n. booster immunization with NILV::SCoV-2. leading to some contagiousness. The partial resistance of the variants to the NAbs generated by the first-292 generation vaccines may exacerbate this issue in the future, avoiding a complete containment of the 293 outbreak by mass vaccination. The sterilizing protection of the brain and lungs against the ancestral and the 294 most distant variants of SARS-CoV-2 conferred by a i.m.-i.n. prime-boost with NILV::SCov-2 provides a 295 promising COVID-19 vaccine candidate of second generation. This vaccine candidate can be used to induce 296 long-term protection or to broaden the specificity of the protection in previously vaccinated persons or in 297 COVID-19 convalescents against SARS-CoV-2 emerging variants. Protection of the brain, so far not 298 directly addressed by other vaccine strategies, has also to be taken into account, considering the multiple 299 and sometimes severe neuropathological manifestations associated with COVID-19. 300 Methods 302

Construction and production of LV 303
A codon-optimized prefusion S sequence (1-1262) (Table S2) was amplified from pMK-RQ_S-2019-304 nCoV and inserted into pFlap by restriction/ligation between BamHI and XhoI sites, between the native 305 human ieCMV promoter and a mutated Woodchuck Posttranscriptional Regulatory Element (WPRE) 306 sequence. The atg starting codon of WPRE was mutated (mWPRE) to avoid transcription of the 307 downstream truncated "X" protein of Woodchuck Hepatitis Virus for safety concerns ( Figure S5). Plasmids 308 were amplified and used to produce LV as previously described 21 . 309

Mice 310
Female C57BL/6JRj mice (Janvier, Le Genest Saint Isle, France) were used between the age of 7 and 311 12 wks. µMT KO mice were bred at Institut Pasteur animal facilities and were a kind gift of Dr P. Vieira 312 (Institut Pasteur). Transgenic B6.K18-ACE2 2Prlmn/JAX mice (JAX stock #034860) were from Jackson 313 Laboratories and were a kind gift of Dr J. Jaubert (Institut Pasteur). Transgenic B6.K18-hACE2 IP-THV mice 314 were generated and bred, as detailed below, at the CIGM of Institut Pasteur. During the immunization 315 period transgenic mice were housed in individually-ventilated cages under specific pathogen-free 316 conditions. Mice were transferred into individually filtered cages in isolator for SARS-CoV-2 inoculation 317 at the Institut Pasteur animal facilities. Prior to i.n. injections, mice were anesthetized by i.p. injection of 318 Ketamine (Imalgene, 80 mg/kg) and Xylazine (Rompun, 5 mg/kg). 319

Mouse Transgenesis 320
The human K18 promoter (GenBank: AF179904.1 nucleotide 90 to 2579) was amplified by nested PCR 321 from A549 cell lysate, as previously described 54, 55 . The "i6x7" intron (GenBank: AF179904.1 nucleotide 322 2988 to 3740) was synthesized by Genscript. The K18 JAX (originally named K18i6x7PA) promoter includes 323 the K18 promoter, the i6x7 intron at 5′ and an enhancer/polyadenylation sequence (PA) at 3' of the hACE2 324 gene. The K18 IP-ThV promoter, instead of PA, contains the stronger wild-type WPRE element at 3 'of the 325 hACE2 gene. Unlike the K18 JAX construct which harbors the 3' regulatory region containing a polyA 326 sequence, the K18 IP-ThV construct uses the polyA sequence already present within the 3' Long Terminal 327 Repeats (LTR) of the lentiviral plasmid. The i6x7 intronic part was modified to introduce a consensus 5' 328 splicing donor and a 3' donor site sequence. The AAGGGG donor site was further modified for the 329 AAGTGG consensus site. Based on a consensus sequence logo 56 , the poly-pyrimidine tract preceding 330 splicing acceptor site (TACAATCCCTC in original sequence GenBank: AF179904.1 and TTTTTTTTTTT 331 in K18 JAX ) was replaced by CTTTTTCCTTCC to limit incompatibility with the reverse transcription step 332 during transduction. Moreover, original splicing acceptor site CAGAT was modified to correspond to the 333 consensus sequence CAGGT. As a construction facilitator, a ClaI restriction site was introduced between 334 14 the promoter and the intron. The construct was inserted into a pFLAP plasmid between the MluI and BamHI 335 sites. hACE2 gene cDNA was introduced between the BamHI and XhoI sites by restriction/ligation. 336 Integrative LV::K18-hACE2 was produced as described in 21 and concentrated by two cycles of 337 ultracentrifugation at 22,000 rpm 1h 4°C. 338 ILV of high titer (4.16 × 10 9 TU/ml) carrying K18-hACE2 IP-THV was used in transgenesis by subzonal 339 micro-injection under the pellucida of fertilized eggs, and transplantation into the pseudo-pregnant 340 B6CBAF1 females. LV allows particularly efficient transfer of the transgene into the nuclei of the fertilized 341 eggs 41 . At N0 generation, ≈ 11% of the mice, i.e., 15 out of 139, had at least one copy of the transgene per 342 genome. Eight N0 hACE2 + males were crossed with female WT C57BL/6 mice. At N1 generation, ≈ 62% 343 of the mice, i.e., 91 out of 147, had at least one copy of the transgene per genome. 344

Genotyping and quantitation of hACE2 gene copy number/genome in transgenic mice 345
Genomic DNA (gDNA) from transgenic mice was prepared from the tail biopsies by phenol-chloroform 346 extraction. Sixty ng of gDNA were used as a template of qPCR with SYBR Green using specific primers 347 listed in Table S3. Using the same template and in the same reaction plate, mouse pkd1 (Polycystic Kidney 348 Disease 1) and gapdh were also quantified. All samples were run in quadruplicate in 10 µl reaction as 349 follows: 10 min at 95°C, 40 cycles of 15 s at 95°C and 30 sec at 60°C. To calculate the transgene copy 350 number, the 2 −ΔΔCt method was applied using the pkd1 as a calibrator and gapdh as an endogenous control. 351 The 2 −ΔΔCt provides the fold change in copy number of the hACE2 gene relative to pkd1 gene.

Humoral and T-cell immunity, Inflammation 359
As recently detailed elsewhere 21 , T-splenocyte responses were quantitated by IFN-g ELISPOT and anti-360 S IgG or IgA Abs were detected by ELISA by use of recombinant stabilized SCoV-2. NAb quantitation was 361 performed by use of LV particles pseudo-typed with SCoV-2 from the diverse variants, as previously 362 described 57,58 . The qRT-PCR quantification of inflammatory mediators in the lungs and brain of mice was 363 performed on total RNA extracted by TRIzol reagent, as recently detailed 21 . CCL19 and CCL21 expression 364 were verified using the following primer pairs: forward primers were 5'-CTG CCT CAG ATT ATC TGC 365 CAT-3' for CCL19 and 5'-AAG GCA GTG ATG GAG GGG-3' for CCL21; reverse primers were 5'-366 AGG TAG CGG AAG GCT TTC AC -3' for CCL19 and 5'-CGG GGT AAG AAC AGG ATT G -3' for 15 CCL21. 368

Determination of viral loads in the organs 377
Organs from mice were removed aseptically and immediately frozen at -80°C. RNA from circulating 378 SARS-CoV-2 was prepared from lungs as recently described 21 . Briefly, lung homogenates were prepared 379 by thawing and homogenizing of the organs in lysing matrix M (MP Biomedical) with 500 μl of ice-cold 380 PBS using a MP Biomedical Fastprep 24 Tissue Homogenizer. RNA was extracted from the supernatants 381 of lung homogenates centrifuged during 10 min at 2000g. Missing neutralization step with AVL 382 buffer/carrier RNA here then extraction with Qiagen RNeasy kit These RNA preparations were used to 383 determine viral loads by E-specific qRT-PCR. 384 Alternatively, total RNA was prepared from lungs or other organs using lysing matrix D (MP 385 Biomedical) containing 1 mL of TRIzol reagent and homogenization at 30 s at 6.0 m/s twice using MP 386 Biomedical Fastprep 24 Tissue Homogenizer. Total RNA was extracted using TRIzol reagent 387 (ThermoFisher). These RNA preparations were used to determine viral loads by Esg-specific qRT-PCR, 388 hACE2 expression level or inflammatory mediators. 389 SARS-CoV-2 E gene 60 or E sub-genomic mRNA (Esg RNA) 37 , was quantitated following reverse 390 transcription and real-time quantitative TaqMan® PCR, using SuperScriptTM III Platinum One-Step qRT-391 PCR System (Invitrogen) and specific primers and probe (Eurofins) ( Table S4). The standard curve of Esg 392 mRNA assay was performed using in vitro transcribed RNA derived from PCR fragment of "T7 SARS-393 CoV-2 Esg mRNA". The in vitro transcribed RNA was synthesized using T7 RiboMAX Express Large 394 Scale RNA production system (Promega) and purified by phenol/chloroform extraction and two successive 395 precipitations with isopropanol and ethanol. Concentration of RNA was determined by optical density 396 measurement, diluted to 10 9 genome equivalents/μL in RNAse-free water containing 100μg/mL tRNA 397 carrier, and stored at -80°C. Serial dilutions of this in vitro transcribed RNA were prepared in RNAse-free 398 water containing 10μg/ml tRNA carrier to build a standard curve for each assay. PCR conditions were: (i) 399 reverse transcription at 55°C for 10 min, (ii) enzyme inactivation at 95°C for 3 min, and (iii) 45 cycles of 400 denaturation/amplification at 95°C for 15 s, 58°C for 30 s. PCR products were analyzed on an ABI 7500 401 Fast real-time PCR system (Applied Biosystems). 402

Cytometric analysis of immune lung and brain cells 403
Isolation and staining of lung innate immune cells were largely detailed recently 21 . Cervical lymph 404 nodes, olfactory bulb and brain from each group of mice were pooled and treated with 400 U/ml type IV 405 collagenase and DNase I (Roche) for a 30-minute incubation at 37°C. Cervical lymph nodes and olfactory 406 bulbs were then homogenized with glass homogenizer while brains were homogenized by use of 407 GentleMacs (Miltenyi Biotech). Cell suspensions were then filtered through 100 μm-pore filters, washed 408 and centrifuged at 1200 rpm during 8 minutes. Cell suspensions from brain were enriched in immune cells 409 on Percoll gradient after 25 min centrifugation at 1360 g at RT, without brakes. The recovered cells from 410 lungs were stained as recently described elsewhere 21 . The recovered cells from brain were stained by 411 appropriate mAb mixture as follows.