key: cord-0893291-9pw95oiw
authors: Ku, Min-Wen; Authié, Pierre; Bourgine, Maryline; Anna, François; Noirat, Amandine; Moncoq, Fanny; Vesin, Benjamin; Nevo, Fabien; Lopez, Jodie; Souque, Philippe; Blanc, Catherine; Chardenoux, Sébastien; Lafosse, llta; Hardy, David; Nemirov, Kirill; Guinet, Françoise; Vives, Francina Langa; Majlessi, Laleh; Charneau, Pierre
title: Full Brain and Lung Prophylaxis against SARS-CoV-2 by Intranasal Lentiviral Vaccination in a New hACE2 Transgenic Mouse Model or Golden Hamsters
date: 2021-02-03
journal: bioRxiv
DOI: 10.1101/2021.02.03.429211
sha: 763bd3a4813ea7430bf67903fc67c5e2718a48b9
doc_id: 893291
cord_uid: 9pw95oiw

Non-integrative, non-cytopathic and non-inflammatory lentiviral vectors are particularly suitable for mucosal vaccination and recently emerge as a promising strategy to elicit sterilizing prophylaxis against SARS-CoV-2 in preclinical animal models. Here, we demonstrate that a single intranasal administration of a lentiviral vector encoding a prefusion form of SARS-CoV-2 spike glycoprotein induces full protection of respiratory tracts and totally avoids pulmonary inflammation in the susceptible hamster model. More importantly, we generated a new transgenic mouse strain, expressing the human Angiotensin Converting Enzyme 2, with unprecedent brain permissibility to SARS-CoV-2 replication and developing a lethal disease in <4 days post infection. Even though the neurotropism of SARS-CoV-2 is now well established, so far other vaccine strategies under development have not taken into the account the protection of central nervous system. Using our highly stringent transgenic model, we demonstrated that an intranasal booster immunization with the developed lentiviral vaccine candidate achieves full protection of both respiratory tracts and brain against SARS-CoV-2.

glycoprotein of Vesicular Stomatitis Virus, to which the human population has limited exposure, avoiding 51 these vectors to be targeted by preexisting immunity in humans, which is in net contrast to adenoviral 52 vectors (Rosenberg et al., 1998; Schirmbeck et al., 2008) . The safety of LV has been established in human 53 in a phase I/II Human Immunodeficiency Virus (HIV)-1 vaccine trial (2011-006260-52 EN). 54

6 Assessment of lung viral load by a sub-genomic ECoV-2 RNA (Esg) qRT-PCR, which was reported to be 134 an indicator of active viral replication (Chandrashekar et al., 2020; Tostanoski et al., 2020; Wolfel et al., 135 2020) (Tostanoski et al., 2020) , showed total absence of replicating virus in the three vaccinated groups 136 versus a mean ± SD of (1.24 ± 0.99) × 10 9 copies of Esg RNA of SARS-CoV-2/lungs in the sham-137 vaccinated group ( Figure 1E ). Many publications use PFU counting to determine viral loads. We noticed 138 that large amounts of NAbs in the lungs of vaccinated individuals, even though not necessarily spatially 139 in contact with circulating viral particles in alive animal, can come to contact with and neutralize viral 140 particles in the lung homogenates in vitro. In this case the PFU assay underestimates the amounts of 141 cultivable viral particles ( Figure S2A ). 142

At 4 dpi, as evaluated by qRT-PCR in total lung homogenates, a substantial decreased in inflammation 143 was detected in NILV::SDF2P-vaccinated hamsters compared to their sham-vaccinated counterparts, 144 regardless of the immunization regimen, i.e., i.m.-i.n. prime-boost or single i.n. injection given at wk 0 or 145 5 ( Figure 2A ). On lung histopathological examination, sham-vaccinated controls demonstrated lung 146 infiltration and interstitial syndrome ( Figure 2B -D), severe alveolo-interstitial inflammation leading to 147 dense pre-consolidation areas ( Figure 2E ), accompanied by bronchiolar lesions, with images of 148 bronchiolar epithelium desquamation into the lumen ( Figure 2F -H). In vaccinated groups the lesions were 149 minimal, although some degree of alveolar infiltration could be seen ( Figure 2I ). 150

These data collectively indicated that a single i.n. administration of NILV::SDF2P was as protective as a 151 systemic prime and i.n. boost regimen, conferred sterilizing pulmonary immunity against SARS-CoV-2 152 and readily prevented lung inflammation and pathogenic tissue injury in the susceptible hamster model. 153

These data also showed the long-term feature of the conferred protection because 7 weeks after a single 154 injection of the vaccine, the protection potential remained complete. 155

Generation of new hACE2 transgenic mice with substantial brain permissibility to SARS-CoV-2 156 replication 157

No hACE2 transgenic mice were available in Europe until September 2020. To set up a mouse model 158 permissive to SARS-CoV-2 replication allowing assessment of our vaccine candidates, based on the 159 previously produced B6.K18-ACE2 2Prlmn/JAX mice (McCray et al., 2007), we generated C57BL/6 transgenic 160 mice with an LV (Nakagawa and Hoogenraad, 2011) carrying the hACE2 gene under the human cytokeratin 161 18 promoter, namely "B6.K18-hACE2 IP-THV ". The permissibility of these mice to SARS-CoV-2 replication 162 was evaluated after one generation backcross to WT C57BL/6 (N1). N1 mice with varying number of 163 hACE2 transgene copies per genome ( Figure 3A ) were sampled and inoculated i.n. with SARS-CoV-2 164 ( Figure 3B ). At 3 dpi, the mean ± SD of lung viral loads was as high as (3.3 ± 1.6) × 10 10 copies of SARS-165

CoV-2 RNA/lung in permissive mice ( Figure 3B ). SARS-CoV-2 RNA copies per lung <1 × 10 7 correspond 166 to the genetic material derived from the input in the absence of viral replication (Ku et al., 2021) . We also 7 noted that the lung viral loads ( Figure 3B) were not proportional to the hACE2 transgene copy number per 168 genome ( Figure 3A ). Remarkably, substantial viral loads, i.e., (5.7 ± 7.1) × 10 10 copies of SARS-CoV-2 169 RNA, were also detected in the brain of the permissive mice ( Figure 3B ). Virus replication/dissemination 170 was also observed, although to a lower extent, in the heart and kidneys. 171

We further compared the replication of SARS-CoV-2 in lungs and brain and the viral dissemination to 172 various organs in B6.K18-hACE2 IP-THV and B6.K18-ACE2 2Prlmn/JAX mice (McCray et al., 2007) ( Figure  173 3C). The lung viral loads were slightly lower in B6.K18-hACE2 IP-THV compared to B6.K18-ACE2 2Prlmn/JAX 174 mice. However, viral loads in the brain of B6.K18-hACE2 IP-THV mice were substantially higher compared 175 to their B6.K18-ACE2 2Prlmn/JAX counterparts ( Figure 3C ). Measurement of brain viral loads by Esg qRT-176 PCR detected (7.55 ± 7.74) × 10 9 copies of SARS-CoV-2 RNA in B6.K18-hACE2 IP-THV mice and no copies 177 of this replication-related RNA in 4 out of 5 B6.K18-ACE2 2Prlmn/JAX mice. This substantial difference of 178 SARS-CoV-2 replication in the brain of both transgenic strains was corroborated with significantly higher 179 hACE2 mRNA expression in the brain of B6.K18-hACE2 IP-THV mice ( Figure 3D ). However, hACE2 mRNA 180 expression in the lungs of B6.K18-hACE2 IP-THV mice was also higher than in B6.K18-ACE2 2Prlmn/JAX mice, 181 which does not account for the lower viral replication in the lungs of the former. A trend towards higher 182 viral loads was also observed in the kidneys and heart of B6.K18-hACE2 IP-ThV compared to B6.K18-183 ACE2 2Prlmn/JAX mice ( Figure 3C ). 184

In concordance with the lung viral loads, as evaluated by qRT-PCR applied to total lung homogenates, 185 B6.K18-hACE2 IP-THV mice displayed less pulmonary inflammation than B6.K18-ACE2 2Prlmn/JAX mice 186 ( Figure 3E ). Remarkably, this assay applied to total brain homogenates detected substantial degrees of 187 inflammation in B6.K18-hACE2 IP-THV -but not in B6.K18-ACE2 2Prlmn/JAX -mice ( Figure 3E ). In 188 addition, B6.K18-hACE2 IP-THV mice reached the humane endpoint between 3 and 4 dpi and therefore 189 displayed a lethal SARS-CoV-2-mediated disease more rapidly than their B6.K18-ACE2 2Prlmn/JAX 190 counterparts (Winkler et al., 2020) . 191 Therefore, the large permissibility to SARS-CoV-2 replication at both lung and CNS, marked brain 192 inflammation and rapid development of a lethal disease are major distinctive features offered by this new 193 B6.K18-hACE2 IP-THV transgenic model. 194

Full protection of lungs and brain in LV::SDF2P-immunized B6.K18-hACE2 IP-THV mice 195

We then evaluated the vaccine efficacy of LV::SDF2P in B6.K18-hACE2 IP-THV mice. In a first 196 experiment with these mice, we used an integrative version of the vector. Individuals (n = 6/group) were 197 primed i.m. with 1 × 10 7 TU/mouse of ILV::SDF2P or an empty LV (sham) at wk 0 and then boosted i.n. 198 at wk 3 with the same dose of the same vectors ( Figure 4A ). Mice were then challenged with SARS-CoV-199 2 at wk 5. A high serum neutralizing activity, i.e., EC50 mean ± SD of 5466 ± 6792, was detected in 200 ILV::SDF2P-vaccinated mice Figure 4B ). This vaccination conferred substantial degrees of protection 201 against SARS-CoV-2 replication, not only in the lungs, but also in the brain ( Figure 4C ). Notably, 202 quantitation of brain viral loads by Esg qRT-PCR detected no copies of this replication-related SARS-203

CoV-2 RNA in ILV::SDF2P-vaccinated mice versus (7.55 ± 7.84) × 10 9 copies in the brain of the sham-204 vaccinated controls. 205

At 3 dpi, cytometric investigation of the lung innate immune cell subsets ( Figure "AC70" C3H × C57BL/6 mice, in which hACE2 mRNA is expressed in various organs including lungs and 281 brain (Tseng et al., 2007) . Comparison of AC70 and B6.K18-hACE2 IP-THV mice could yield information to 282 assess the similarities and distinctions of these two models. However, here we report much higher brain 283 permissibility of B6.K18-hACE2 IP-THV mice to SARS-CoV-2 replication, compared to B6.K18-284 ACE2 2Prlmn/JAX mice. The B6.K18-hACE2 IP-THV murine model not only has broad applications in COVID-285 19 vaccine studies, but also provides a unique rodent model for exploration of COVID-19-derived 286 neuropathology. Based on the substantial permissibility of the brain to SARS-CoV-2 replication and 287 development of a lethal disease, this pre-clinical model can be considered as a far more stringent than the 288 golden hamster model. CoV-2-infected immune cells crossing the hemato-encephalic barrier or direct viral entry pathway via CNS 303 vascular endothelium (Meinhardt et al., 2020) . Although at steady state, viruses cannot penetrate to the 304 brain through an intact blood-brain barrier (Berth, 2009), inflammation mediators which are massively 305 produced during cytokine/chemokine storm, notably TNF-α and CCL2, can disrupt the integrity of blood-306 brain barrier or increase its permeability, allowing paracellular blood-to-brain transport of the virus or virus-307 In addition, substantial reduction in the inflammatory mediators was also found in the brain of the i.m.-i.n. 314 vaccinated and protected mice, as well as decreased proportions of neutrophils and inflammatory 315 monocytes respectively in the olfactory bulbs and brain. Regardless of the mechanism of the SARS-CoV-316 2 entry to the brain, we provide evidence of the full protection of the CNS against SARS-CoV-2 by i.n. 317 booster immunization with NILV::SDF2P. 318

We recently demonstrated the strong prophylactic capacity of LV::SFL at inducing sterilizing protection 319 in the lungs against SARS-CoV-2 infection (Ku et al., 2021) . In the present study, moving toward a human 320 clinical trial, we used LV encoding stabilized prefusion SDF2P forms of SCov-2. The choice of SDF2P in this 321 study was based on data indicating that stabilization of viral envelop glycoproteins at their prefusion forms 322 improve the yield of their production as recombinant proteins in industrial manufacturing of subunit 323 vaccines, and the efficacy of nucleic acid-based vaccines by raising availability of the antigen under its 324 optimal immunogenic shape (Hsieh et al., 2020) . The prefusion stabilization approach has been so far 325 applied to S protein of several coronaviruses, including HKU1-CoV, SARS-CoV, and MERS-CoV. 326

Stabilized SMERS-CoV has been shown to elicit much higher NAb responses and protection in pre-clinical 327 animal models (Hsieh et al., 2020) . We detected no difference between the capacity of SFL and SDF2P at 328 inducing anti-SCoV-2 IgG or IgA Ab responses in the sera or lung homogenates of LV-immunized animals. 329

However, the possibility that the yield of LV::SDF2P production at industrial level is higher is likely. 330

The sterilizing protection of the lungs conferred by a single i.n. administration and the full protection of 331 CNS conferred by i.n. boost is an asset of primary importance. The non-cytopathic and non-inflammatory 332 LV encoding either full length, or stabilized forms of SCoV-2, from either ancestral or emerging variants of 333 SARS-CoV-2 provides a promising COVID-19 vaccine candidate of second generation. Protection of the 334 brain, so far not directly addressed by other vaccine strategies, has to be taken into the account, considering 335 the multiple and sometimes severe neuropathological manifestations associated with COVID-19. in the lungs or brain of B6.K18-hACE2 IP-THV and B6.K18-ACE2 2Prlmn/JAX transgenic mice at 3 dpi. Data 399 were normalized versus untreated controls. Statistical significance of the difference was evaluated by Mann-400 Whitney test (*= p < 0.05, **= p <0.01). 401 

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Construction and production of LV::SDF2P 425 A codon-optimized SDF2P sequence (1-1262) (Table S1 ) was amplified from pMK-RQ_S-2019-nCoV 426 and inserted into pFlap by restriction/ligation between BamHI and XhoI sites, between the native human 427 ieCMV promoter and a mutated Woodchuck Posttranscriptional Regulatory Element (WPRE) sequence. 428The atg starting codon of WPRE was mutated (mWPRE) to avoid transcription of the downstream truncated 429 "X" protein of Woodchuck Hepatitis Virus for safety concerns ( Figure S4 ). Plasmids were amplified and 430 used to produce LV as previously described (Ku et al., 2021) . 431Hamsters 432Male Mesocricetus auratus golden hamsters (Janvier, Le Genest Saint Isle, France) were purchased 433 mature and weighed between 100 to 120 gr at the beginning of the experiments. Hamsters were housed in 434 individually-ventilated cages under specific pathogen-free conditions during the immunization period. For 435 SARS-CoV-2 infection they were transferred into individually filtered cages placed inside isolators in the 436 animal facility of Institut Pasteur. Prior to i.m. or i.n. injections, hamsters were anesthetized by isoflurane 437 inhalation or i.p. injection of Ketamine (Imalgene, 80 mg/kg) and Xylazine (Rompun, 5 mg/kg). 438

Female C57BL/6JRj mice (Janvier, Le Genest Saint Isle, France) were used between the age of 7 and 440 12 wks. Transgenic B6.K18-ACE2 2Prlmn/JAX mice (JAX stock #034860) were from Jackson Laboratories 441 and were a kind gift of Dr Jean Jaubert and Dr Xavier Montagutelli (Institut Pasteur). Transgenic B6.K18-442 hACE2 IP-THV mice were generated and bred, as detailed below at the CIGM of Institut Pasteur. During the 443 immunization period female or male transgenic mice were housed in individually-ventilated cages under 444 specific pathogen-free conditions. Mice were transferred into individually filtered cages in isolator for 445 SARS-CoV-2 inoculation at the Institut Pasteur animal facilities. Prior to i.n. injections, mice were 446 anesthetized by i.p. injection of Ketamine (Imalgene, 80 mg/kg) and Xylazine (Rompun, 5 mg/kg). 447

The human K18 promoter (GenBank: AF179904.1 nucleotide 90 to 2579) was amplified by nested PCR 449 from A549 cell lysate, as described previously (Chow et al., 1997; Koehler et al., 2000) . The "i6x7" intron 450 The i6x7 intronic part was modified to introduce a consensus 5' splicing donor and a 3' donor site sequence. 457The AAGGGG donor site was further modified for the AAGTGG consensus site. Based on a consensus 458 sequence logo (Dogan et al., 2007) , the poly-pyrimidine tract preceding splicing acceptor site 459 (TACAATCCCTC in original sequence GenBank: AF179904.1 and TTTTTTTTTTT in K18 JAX ) was 460 replaced by CTTTTTCCTTCC to limit incompatibility with the reverse transcription step during 461 transduction. Moreover, original splicing acceptor site CAGAT was modified to correspond to the 462 consensus sequence CAGGT. As a construction facility, a ClaI restriction site was introduced between the 463 promoter and the intron. The construct was inserted into a pFLAP plasmid between the MluI and BamHI 464 sites. hACE2 gene cDNA was introduced between the BamHI and XhoI sites by restriction/ligation. 465Integrative LV::K18-hACE2 was produced as described in ( Genomic DNA (gDNA) from transgenic mice was prepared from the tail biopsies by phenol-chloroform 476 extraction. Sixty ng of gDNA were used as a template of qPCR with SYBR Green using specific primers 477 listed in Table S2 . Using the same template and in the similar reaction plate, mouse pkd1 (Polycystic Kidney 478Disease 1) and gapdh were also quantified. All samples were run in quadruplicate in 10 µl reaction as 479 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 480 number, the 2 −ΔΔCt method was applied using the pkd1 as a calibrator and gapdh as an endogenous control. 481The 2 −ΔΔCt provides the fold change in copy number of the hACE2 gene relative to pkd1 gene. SuperScriptTM III Platinum One-Step qRT-PCR System (Invitrogen) and specific primers and probe 523 (Eurofins) ( Table S3 ). The standard curve of Esg mRNA assay was performed using in vitro transcribed 524 19 RNA derived from PCR fragment of "T7 SARS-CoV-2 Esg mRNA". The in vitro transcribed RNA was 525 synthesized using T7 RiboMAX Express Large Scale RNA production system (Promega) and purified by 526 phenol/chloroform extraction and two successive precipitations with isopropanol and ethanol. 527Concentration of RNA was determined by optical density measurement, diluted to 10 9 genome 528 equivalents/μL in RNAse-free water containing 100μg/mL tRNA carrier, and stored at -80°C. Serial 529 dilutions of this in vitro transcribed RNA were prepared in RNAse-free water containing 10μg/ml tRNA 530 carrier to build a standard curve for each assay. PCR conditions were: (i) reverse transcription at 55°C for 531 10 min, (ii) enzyme inactivation at 95°C for 3 min, and (iii) 45 cycles of denaturation/amplification at 95°C 532 for 15 s, 58°C for 30 s. PCR products were analyzed on an ABI 7500 Fast real-time PCR system (Applied 533 Biosystems). 534

Isolation and staining of lung innate immune cells were largely detailed recently (Ku et al., 2021) . 536Cervical lymph nodes, olfactory bulb and brain from each group of mice were pooled and treated with 400 537 U/ml type IV collagenase and DNase I (Roche) for a 30-minute incubation at 37°C. Cervical lymph nodes 538 and olfactory bulbs were then homogenized with glass homogenizer while brains were homogenized by 539 use of GentleMacs (Miltenyi Biotech). Cell suspensions were then filtered through 100 μm-pore filters, 540washed and centrifuged at 1200 rpm during 8 minutes. Cell suspensions from brain were enriched in 541 immune cells on Percoll gradient after 25 min centrifugation at 1360 g at RT. The recovered cells from 542 lungs were stained as recently described elsewhere (Ku et al., 2021) . The recovered cells from brain were 543 stained by appropriate mAb mixture as follows. Lung Histopathology 558 20 Samples from the lungs or brain of hamsters or transgenic mice were fixed in formalin for 7 days and 559 embedded in paraffin. Paraffin sections (5-µm thick) were stained with Hematoxylin and Eosin (H&E). 560 Histopathological lesions were qualitatively described and when possible scored, using: (i) distribution 561 qualifiers (i.e., focal, multifocal, locally extensive or diffuse), and (ii) a five-scale severity grade, i.e., 1: 562 minimal, 2: mild, 3: moderate, 4: marked and 5: severe. 563