A New hACE2 Transgenic Animal with Remarkable Permissiveness of Lung and Central Nervous System to replication of viruses targeting hACE2 - An Experimental Model for vaccine, drug and Neuro/Immune/Physio-Pathoiogy of COVID-19 and other pathologies linked to viruses or coronaviruses using hACE2 as a cellular receptor

The invention relates to a new hACE2 (human Angiotensin-Converting Enzyme 2) transgenic (Tg) animal, especially murine model with remarkable permissiveness of lung and central nervous system to SARS-CoV-2 replication. The invention also relates in particular to the use of such hACE2 Tg animal, especially murine, model as an experimental model for elucidation of neuro/immune/physio-pathology of pathologies linked to viruses or coronaviruses using hACE2 as a cellular receptor, especially COVID-19, and for assessing the efficacy of therapeutics and vaccine candidates against pathologies linked to viruses or coronaviruses using hACE2 as a cellular receptor, especially COVID-19 associated with infection by SARS-CoV-2.

Conventional laboratory mice are not permissive to SARS-CoV-2 replication. Mice Tg for hACE2, the main receptor allowing host cell invasion by SARS-CoV-2 (Hoffmann et al., 2020), were unavailable in Europe until September 2020.

To date several Tg mice of different strains expressing the hACE2 gene under distinct transcription and expression control sequences have been provided, some of them originating from developments performed to fulfil needs that arose when on emergence of SARS-CoV epidemic in 2003. These earlier developed Tg mice and further models have been assessed for the study and understanding of the pathogenesis of SARS-CoV and have shown to be permissible to viral replication and sometimes to some degree of disease symptom or clinical illness but the observed various clinical profiles in Tg mice inoculated with SARS-CoV-2 have not yet provided proved suitable to reproduce all aspects of the outcome of the infection, in particular have not adequately shown virus spread as observed in human patients, in particular spread beyond the airways and the pulmonary tract, such as spread to the brain. Also the available Tg mice have not shown consistent disease symptoms that would reproduce the symptoms observed in human patients. In particular, available Tg animal models to date include the following: Murine strains Promoter hACE2 expression in brain

Genetic background

K18-hACE2 tg hK18 Yes but the amounts of infection 2Prlmn/J (JAX) Originally SJL, backcrossed to is 4 loglO C57BL/6 less than in our TP-ThV Tg mice

AC70, (AC22, under “CAG” mixed promoter mRNA expression in brain but AC36) back-crossed on BALB/c not in lungs

Hepatocyte nuclear The transgenic mice (HFH4-hACE2 Lung and brain factor- 3/forkhead mice) expressing hACE2 under Jiang et al., 2020, Celli 82, 50-58 homologue 4 mixed genetic backgrounds July 9, 2020 (HFH4-ACE-2) (C3H,C57BL/6) were obtained from https ://doi.org/ 10.1016/j . Ralph S. Baric’s lab (Menachery et cell.2020.05.027 al., 2016).

Murine ACE2 promoter apparently

Murine ACE2 not in brain promoter

A need remains for non-human animal models, especially small animals to elucidate the pathogenesis associated with SARS-CoV-2 and to design or evaluate therapeutic and protective vaccine approaches against the virus infection or the COVID-19 disease.

To set up a laboratory mouse model permissive to replication of SARS-CoV-2 clinical isolates and to allow rapid evaluation of the efficacy of COVID-19 vaccine candidates or therapeutics, or to study the neuro- immune- or physio-pathology of SARS-CoV-2, the inventors generated C57BL/6 Tg mice carrying hACE2 gene under the human keratin 18 promoter, namely B6.K18-hACE2 ^{IP THV} (Institut Pasteur-TheraVectys), using a lentiviral vector (LV)-based transgenesis method (Park, 2007). The inventors have obtained a non-human animal that surprisingly exhibits an unexpected phenotype, especially when compared to mice known as K18-hACE2 tg 2Primm/J (Jax). The Tg non-human animal, especially Tg mice of the invention provide accordingly a suitable animal model of interest to study virus infection when the virus targets the hACE2 receptor for infection of their host, especially human host. In particular, the assays performed using the Tg mice illustrated in the present invention when infection is caused by SARS-CoV-2 may be similarly carried out with other viruses or coronaviruses using hACE2 cellular receptor such as SARS-CoV virus also designated SARS- CoV-1 that was epidemic in 2003.

Although lung is the main site of SARS-CoV-2 infection, the virus can infect the central nervous system leading to headache, myalgia, smell loss and taste impairment and reduced consciousness, with possible long-term consequences. In a particular aspect of the invention, the inventors designed a relevant model, suitable to evaluate the protective effects of current COVID-19 vaccines on the brain.

Accordingly, the inventors designed a transgene construct suitable for the efficient expression of the hACE2 in a non-human transgenic animal after transfer with a gene transfer vector, especially a lentiviral gene transfer vector which is a HIV- 1 -based vector. In a preferred embodiment, the polynucleotide encoding the hACE2 is the CDS having the sequence of SEQ ID No.5. In a particular embodiment the expressed transgene is the protein of SEQ ID No.6.

The invention accordingly relates to a transgene construct which comprises a polynucleotide coding for the human angiotensin-converting enzyme 2 (hACE2) (hACE2 coding sequence) under the control of a transcription and expression control nucleic acid comprising, (i) located 5’ of said hACE2 coding sequence, the human keratin 18 promoter region (KI 8 promoter) and, (ii) located 3’ of the hACE2 coding sequence, a wild-type Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), in particular the WPRE of SEQ ID No.4

In a particular embodiment, the nucleic acid for control of transcription and expression located 5’ of the hACE2 coding sequence in the transgene construct, contains the 2.5kb upstream genomic sequence, the promoter and the first intron of the human keratin 18 gene and the nucleic acid for control of transcription and expression, located 3 ’of the hACE2 coding sequence contains the WPRE post-transcriptional element.

The transgene is a DNA molecule in particular a cDNA molecule.

In a particular embodiment, the transgene construct according to the invention is such that the polynucleotide for control of a transcription and expression is obtained from the sequence of the human keratin 18 gene corresponding to nucleotides 90 to 2579 in GenBank:AF179904.1 providing the region located 5’ from the hACE2 coding sequence.

In a particular embodiment, the transgene construct is such that the nucleic acid for control of transcription and expression located 3 ’ of the hACE2 coding sequence is derived from the human keratin 18 gene by modification of the original sequence of the KI 8 genomic sequence in order to contain 5’ consensus donor site and 3’ consensus acceptor site, in particular wherein said nucleic acid is the polynucleotide of SEQ ID No.1 ,

In a particular embodiment in the transgene construct of the invention, the polynucleotide of the coding sequence for the hACE2 consists of the sequence of SEQ ID No.5.

A particular transgene construct of the invention is the polynucleotide spanning from nucleotide position 2453 to nucleotide position 8753 in the sequence of SEQ ID No. 7 or a nucleic acid molecule which is a variant thereof with the proviso that it retains the functional feature of encoding the hACE2 protein (in particular the protein of SEQ ID No. 6), in a nonhuman animal when said nucleic acid molecule is provided by a lentiviral particle as defined in the present invention and integrated in the genome of the cells of said non-human animal. Such a particular variant may be a variant of the hACE2 coding sequence of SEQ ID No. 5 and is especially a nucleotide construct wherein the variant hACE2 coding sequence exhibits at least 95%, preferably at least 98% or 99% identity in the hACE2 coding sequence with respect to the sequence of SEQ ID No.5. In particular, the nucleic acid variant encodes the protein of SEQ ID No. 6.

The invention is also directed to a lentiviral vector genome, in a particular a HIV-1 based vector genome, which is recombined with the transgene construct of any of the embodiments defined herein. A lentiviral genome, in particular a HIV-1 based genome is obtained according to techniques and methods well known from the person skilled in the art starting from a cDNA sequence of a selected lentivirus, especially HIV-1 such as described in Zennou et al, 2000; Firat H. et al, 2002; VandenDriessche T. et al, 2002.

In a particular embodiment of the invention, the transgene or the lentiviral vector genome, in particular the HIV-1 based vector genome is contained or comprised in a recombinant plasmid, in particular plasmid pFLAP K18-hACE2 WPRE of SEQ ID No.7. Plasmid pFLAP K18-hACE2 WPRE of SEQ ID No.7 is as such a recombinant plasmid of the invention.

The invention also relates to a recombinant lentiviral vector particle, in particular a peudotyped HIV-1 vector particle, which comprises a lentiviral vector genome or a plasmid according to the invention.

In a preferred embodiment, the recombinant lentiviral vector particle is integrative, i.e. is suitable for integration of the transgene of the invention in the genome of the cells of the non- human animal after transduction of the host cells, thereby enabling the expression of the hACE2 enzyme by the transgenic non-human animal.

The recombinant lentiviral vector particle of the invention is advantageously pseudotyped with a non-lentiviral virus envelope protein, in particular with a vesiculovirus VSV-G envelope protein.

Using the transgenic construct and the obtained lentiviral, especially HIV-1 based recombinant viral particles of the invention for transfer into the zona pellucida of fertilized eggs of a non-human animal of the hACE2 coding sequence, the inventors succeeded in providing transgenic non-human animals, especially transgenic mice, exhibiting remarkable permissiveness to the SARS-CoV-2 infection including for replication of the SARS-CoV-2 viral particles in the lungs and in the brain, thereby providing an animal model that may reproduce the severity of the clinical symptoms observed in some human patients infected with SARS-CoV-2, especially patients affected with ARDS (Acute Respiratory Distress Syndrome) or with severe neurological outcomes associated with the infection.

The invention hence relates to a transgenic non-human animal which is permissible to replication of SARS-CoV-2 virus, in particular to SARS-CoV-2 clinical isolate, and which is transgenic for hACE2 and carries the transgene construct of any of the embodiments of the present invention. Advantageously this transgenic non-human animal obtained after transgenesis of a non-human animal strain with the lentiviral vector particles, especially the HIV-1 vector particles carrying the transgenic construct of the invention, when infected with SARS-CoV-2, is capable of reproducing the pathology observed in human patients infected with SARS-CoV-2, including reproducing the severe form of the pathology. The phenotype observed with the Tg non-human animal thus obtained as a result of the use of the particular transcriptional unit (transgene construct) of the invention, using lentiviral vector, especially HIV-1 based vector for the transgenesis was not foreseeable from results reported using available mice models for SARS-CoV-2.

The transgenic non-human animal according to the invention is especially a non-human mammal, in particular a rodent, a murine such as a mouse or a rat. In a preferred embodiment, it is a transgenic mouse.

The transgenic mouse according to the invention is in particular a transgenic C57BL/6 mouse such as described in the Examples below. The transgenic mouse according to the invention is in particular obtained as the N 1 , N2 or later generation of mice after inter-crossing or crossing a transgenic mouse of the invention with a wild type C57BL/6 mouse or with a mouse humanized for Major Histocompatibility Complex (CMH) of class-I and/or -II, or deficient for one or several genes involved in innate or adaptive immune responses including:

- pMT (transmembrane domain of heavy chain of IgM) resulting in B-cell deficiency, in particular for use to evaluate the role of B-cell compartment and antibody responses in the immune control of SARS-CoV and SARS-CoV-2,

- IFNAR (Receptor for type-I interferons), in particular for use to evaluate the role of type- I interferons in SARS-CoV and SARS-CoV-2 infections or to evaluate the efficacy of measles vaccinal vectors against SARS-CoV and SARS-CoV-2, as the measles vectors actively replicate only in the absence of the action of type-I interferons in mice

- Both Rag2 (“Recombination Activating Gene”) and yc (“common cytokine receptor y chain”) and therefore lacking B, T and NK functionality, in particular for use to generate mice humanized for immune system, useful in the study of immune control of SARS-CoV and SARS-CoV-2.

As examples of strains of laboratory mice on which transgenesis with the lentiviral vector of the invention expressing the hACE2 protein (LV : :hACE2) could be carried out, the following strains are cited: AKR, BALB/c, SJL, C3H, DBA/2, CBA, SV129.

The Tg mouse expresses the hACE2, in particular expresses the hACE2 protein from a transgene of the invention integrated in its genome.

The invention also relates to a process for detection of the hACE2 coding sequence in the genome of a transgenic non-human animal according to the invention which comprises performing amplification of the DNA of a sequence of the hACE2 coding sequence on a tissue sample previously obtained from said Tg non-human animal with primers specific for said coding sequence, in particular forward primer of SEQ ID No 49 (5 ’-TCC TAA CCA GCC CCC TGT T-3’) and reverse primer of SEQ ID No. 50 (5’-TGA CAA TGC CAA CCA CTA TCA CT-3’) and detecting the amplification product wherein a sample positive for the presence of the amplification product shows that the transgenic non-human animal providing the tissue sample is Tg for the hACE2 coding sequence.

The invention is also directed to the use of a Tg non-human animal, in particular a transgenic rodent, Tg murine or Tg mouse or rat as defined and illustrated herein, as a preclinical or co- clinical animal model for evaluating a therapeutic candidate or a prophylactic vaccine candidate or drugs against infection by SARS-CoV-2 or against the onset or the development of the COVID-19 disease or symptoms associated with infection by SARS-CoV-2 or with COVID- 19 disease or with any virus or coronavirus which would use the same hACE2 cellular receptor, including SARS-CoV (also designated SARS-CoV-1).

The invention is in particular directed to the use of a Tg non-human animal, in particular a Tg rodent, Tg murine or Tg mouse or rat as defined and illustrated herein for the screening, the selection, the characterization, the validation, the development or the control of a therapeutic candidate or a prophylactic vaccine candidate or drugs against infection by SARS-CoV-2 or other viruses or coronaviruses for which studies and testing performed in the non-human animal model of the invention may be beneficial or against the onset or the development of the COVID- 19 disease or other disease caused by viruses or coronaviruses or symptoms associated with infection by SARS-CoV, SARS-CoV-2, or with COVID-19 disease or other disease caused by viruses or coronaviruses which would use the hACE2 cellular receptor.

The clinical status of non-human animal infected with SARS-CoV-2 developing the associated disease COVID-19 may be characterised by a variety of symptoms including weight loss arising as soon as a few days (3 days or less) after infection, reduced activity, lethargy, eye closure, appearance of fur and posture, and respiration change such as laborious breathing.

The damage to the organs may be determined from tissue biopsies of various organs in particular airways or lungs biopsies enabling in particular assessing the contents of the alveolar space or the septal thickening in lungs, presence of infiltrating neutrophils, granulocytes and inflammatory monocytes in the fluids. Importantly, and in a distinctive manner compared to other hACE2 Tg mice, the non-human animals of the invention also enable the assessment of damage made to the central nervous system after infection with SARS-CoV-2, on brain tissue biopsies.

The viral load may be determined in tissue biopsies as disclosed in detail in the Examples below.

The invention also relates to a method of assessing a therapeutic candidate or a drug which comprises the steps of: a. Inoculating a Tg non-human animal according to the invention with a strain of SARS- CoV-2, in particular a clinical strain of SARS-CoV-2 and allowing said SARS-CoV-2 to replicate in the non-human animal, in particular in the lungs or in the brain or both and/or allowing the non-human animal to develop symptoms associated with Acute Respiratory Distress Syndrome (ARDS); b. Administering the therapeutic candidate or the drug to the non-human animal of step a.; c. Assessing the therapeutic effect of the administered therapeutic candidate by determining the viral loads, inflammation status or the histopathological status in tissue biopsies, in particular in the lungs or in the brain or both, of the non-human animal or determining the clinical status of the non-human animal.

Assessment of the viral loads is illustrated in the example by determination of the RNA, especially the RNA originating from replication (subgenomic RNA), in particular by assessing the copy number of SARS-CoV-2 RNA. Inflammation may be assessed by measurement of expression of inflammatory cytokines or chemokines as disclosed in the Examples.

In another embodiment, the invention relates to a method of assessing efficiency of a prophylactic vaccine candidate which comprises the steps of: a. Administering the prophylactic vaccine candidate to the non-human Tg animal, according to the invention in particular in a prime/boost administration regimen; b. Inoculating the Tg non-human animal of step a. with a strain of SARS-CoV-2, in particular a clinical strain of SARS-CoV-2 and allowing said SARS-CoV-2 to replicate in the Tg non-human animal, in particular in the lungs or in the brain or both and/or allowing the non-human animal to develop symptoms associated with Acute Respiratory Distress Syndrome (ARDS); c. assessing the protective effect of the administered prophylactic vaccine candidate by determining the viral load, inflammatory status, or the histopathological status in tissue biopsies, in particular in the lungs or in the brain or both, of the non-human animal or determining the clinical status of the non-human animal.

Assessment of the viral loads is illustrated in the example by determination of the RNA, especially the RNA originating from replication (subgenomic RNA). Inflammation may be assessed by measurement of expression of inflammatory cytokines or chemokines as disclosed in the Examples.

The methods hence provided may include inoculation with a strain of SARS-CoV-2 performed via intranasal route or by aerosol.

In a particular embodiment of the methods herein disclosed, the administration of the therapeutic candidate or drug to the non-human animal is performed less than three days following SARS-CoV-2 virus inoculation. In the methods of the invention disclosed herein, the SARS-CoV-2 virus may be replaced by a different virus, in particular coronavirus, especially SAR-CoV-1, when such virus or coronavirus uses hACE2 as a cellular receptor.

In an aspect of the method of assessing the suitability or the efficacy of the vaccine candidate this candidate is administered in a prime/boost regimen through different routes in the prime and in the boost, in particular is administered by systemic immunization in the prime and by mucosal immunization in the boost, in particular is administered intramuscularly in the prime and intranasally in the boost or vice versa. Compared to the systemic routes of immunization, the intranasal route of immunization can have a particular advantage in the protection of the central nervous system, through induction of local immunity in the cervical lymph nodes, olfactive bulb and/or within the brain. The Tg mouse model provides a unique tool for the demonstration and characterization of the involved mechanisms.

In another aspect, the invention also relates to the use of a Tg non-human animal, in particular a Tg rodent, transgenic murine or Tg mouse according to any one of the embodiments disclosed herein as a preclinical or co-clinical animal model for elucidating immune and neurophysiopathology of COVID-19 or other pathologies linked to viruses or coronaviruses using hACE2 as a cellular receptor.

In a further aspect, the invention also relates to a method of making a therapeutic composition or a protective prophylactic composition which comprises the steps of: a. Performing the method of assessing efficiency of a therapeutic or a prophylactic vaccine candidate and determining the condition of the treated Tg non-human animal after administration of the therapeutic or respectively the prophylactic candidate and inoculation of the SARS-CoV-2, improvement or stabilization of the condition of the treated non-human animal being indicative of suitability of the therapeutic or the prophylactic candidate for the treatment or respectively the prevention of the infection by SARS-CoV-2 or of the outcome of the infection, b. Combining the therapeutic or the prophylactic candidate as an active ingredient with a suitable pharmaceutical carrier in a formulation for administration to a human patient. In the methods of the invention disclosed herein, the SARS-CoV-2 virus may be replaced by a different virus, in particular coronavirus, especially SAR-CoV-1, when such virus or coronavirus uses hACE2 as a cellular receptor.

Additional aspects and characteristics of the invention are disclosed in the following detailed description, in the Examples and the figures.

Brief description of the drawings

The patent or application file contains at least one drawing executed in color.

Figure 1. Permissiveness of B6.K18-hACE2 ^{IP THV} (also designated as K18- hACE2 ^{IP THV} ) transgenic mice to SARS-CoV-2 replication: large permissiveness is shown in the lungs and brain

(A) Representative genotyping results from 15 N1 B6.K18-hACE2 ^IP-THV mice as performed by qPCR to determine their hACE2 gene copy number per genome. Dots represent individual mice.

(B) Phenotyping of the same mice, presented in the same order, inoculated i.n. with 0.3 x 10 ⁵ TCID50 of SARS-CoV-2 at the age of 5-7 wks and viral RNA content was determination in the indicated organs at 3 dpi by conventional E-specific qRT-PCR. Red lines indicate the qRT- PCR limits of detection.

C, D) Quantification (C) and images (D) of hACE2 protein expression level by Western blot from the lungs of the same mice as in panels A and B, presented in the same order.

(E, F) Kinetics of SARS-CoV-2 replication in the lungs and brain of B6.K18-hACE2 ^{IP THV} mice as followed by measuring viral RNA contents by conventional E-specific qRT-PCR (E) or by PFU counting (F).

(G) Percentage of initial weight measured from the mice of panels E and F.

Figure 2. Histology of the lungs and brain of B6.K18-hACE2 ^{IP THV} transgenic mice after SARS-CoV-2 inoculation

(A) Representative H&E whole-lung section at 3 dpi in B6.K18-hACE2 ^{IP THV} transgenic mice inoculated i.n. with 0.3 x 10 ⁵ TCID50 of SARS-CoV-2, compared to non-infected controls (NI). In the infected lung, less transparent, purple-red areas resulted from inflammatory lesions of the lung parenchyma. Scale bar: 500 pm. (B-E) Examples of lesions observed in the lungs of infected mice at higher magnification. The two top panels depict mild (B) (scale bar: 100 pm) and mild to moderate (C) interstitium thickening accompanied by dense inflammatory infiltrates predominantly localized in the vicinity of bronchioles (yellow stars) and also present around blood vessels (green stars) (scale bar: 200 pm). (D) Alveoli filled with a proteinaceous exudate containing a few cells (green arrows) (scale bar: 50 pm). (E) Discrete degenerative lesions of the bronchiolar epithelium, such as perinuclear clear spaces (blue arrows), hyper-eosinophilic cells with condensed nuclei (orange arrow) and some intraluminal fibrinous and cell debris (black arrow) in an overall well- preserved epithelium (scale bar: 20 pm).

(F) Heatmap representing the histological scores for various parameters in infected mice at 3 dpi, compared to NI controls (n = 3/group). Statistical significance was evaluated by Mann- Whitney test (*= p < 0.05, **=p <0.01, ****=p < 0.0001, ns = not significant).

(G) Representative Ncov-2-specific IHC image of the brain in NI control or SARS-CoV-2 - infected mice at 3 dpi. Scale bar: 500 pm.

(H) Closer view of a Ncov-2 positive area from an infected mouse. Scale bar: 500 pm.

Figure 3. Comparison of SARS-CoV-2 replication and infection-derived inflammation in B6.K18-hACE2 ^{IP THV} and B6.K18-hACE2 ^2prlmn/Jax transgenic mice

(A) Comparative permissiveness of organs from B6.K18-hACE2 ^{IP THV} and B6.K18- ACE2 ^2Prlnm/JAX transgenic mice to SARS-CoV-2 replication. Female B6.K18-hACE2 ^IP-THV and B6.K18-ACE2 ^2Prlnm/JAX transgenic mice were inoculated i.n. with 0.3 x 10 ⁵ TCIDso/mouse. The viral loads, as determined by qRT-PCR specific to total E RNA or subgenomic R (Esg) RNA, were assessed in diverse organs at 3dpi, as described above. The red line indicates the qRT- PCR detection limit.

(B) Expression of hACE2 mRNA and comparative quantitation in the lungs and brain of B6.K18-hACE2 ^IP-THV and B6.K18-ACE2 ^2Prlnm/JAX mice. Statistical significance of the difference was evaluated by Mann-Whitney test (*= p < 0.01, **=p <0.001). (n = 5-6/group)

(C) Expression of inflammatory cytokines or chemokines in the lungs or brain of individual mice of each group, as determined at 3 dpi by qRT-PCR: Heatmaps represent log2 fold change in cytokine and chemokine mRNA expression, (n = 5-6/group). Data were normalized versus untreated controls.

Data information: Statistical significance was evaluated by Mann- Whitney test (*= p < 0.05, **=/? <0.01, ns = not significant).

Figure 4. Use of B6.K18-hACE2 ^{IP THV} transgenic mice to demonstrate the interest of i.n. vaccination with a LV::S- vaccine candidate against SARS-CoV2: Vaccination with LV::S protects both lungs and central nervous system from SARS-CoV-2 infection in B6.K18-hACE2 ^{IP THV} transgenic mice

(A) Timeline of the prime-boost strategy based on LV::S followed by SARS-CoV-2 challenge in B6.K18-hACE2 ^IP-THV mice, (n = 6/group). The LVs used in this experiment were integrative.

(B) Serum neutralization capacity of anti-Scov-2 Abs in LV::S-vaccinated mice compared to sham mice (n = 6/group).

(C) Viral loads as determined by qRT-PCR specific to total E RNA or subgenomic R (Esg) RNA in diverse organs at 3dpi (n = 6/group), by use of conventional E-specific or subgenomic Esg-specific qRT-PCR. The red line indicates the qRT-PCR detection limit.

(D) Percentages of NK cells or neutrophils in the lungs of LV::S- or sham- vaccinated and SARS-CoV-2-challenged B6.K18-hACE2 ^IP-THV transgenic mice at 3 dpi (n = 6/group). Percentages were calculated versus total lung live CD45 ⁺ cells.

(E) Expression of inflammatory cytokines or chemokines in the lungs of individual mice of each group, as determined at 3 dpi by qRT-PCR. Statistical significance of the difference was evaluated by Mann-Whitney test (*= p < 0.01, **= p <0.001)

Figure 5. Cellular and humoral immunity in LV::S-vaccinated B6.K18-hACE2 ^{IP THV} mice B6.K18-hACE2 ^{IP THV} mice were primed (i.m.) at wk 0 and boosted (i.n.) at wk 5 (n = 5) with non-integrative LV::S. Control mice were injected with an empty LV (sham).

(A) Representative IFN-y response by lung CD8 ⁺ T cells of as studied at wk 7 after in vitro stimulation with the indicated Scov-2-derived peptides. (B, C) Cytometric strategy to detect lung CD8 ⁺ T central memory (Tcm, CD44 ⁺ CD62L ⁺ CD69 ), T effector memory (Tern, CD44 ⁺ CD62L CD69 ) and T resident memory (Trm, CD44 ⁺ CD62L CD69 ⁺ CD103 ⁺ ) and (C) percentages of these subsets among CD8 ⁺ T-cells in LV::S-vaccinated (n = 9) or sham (n = 5) mice.

(D) Cytometric strategy to detect Scov-2-specific CD8 ⁺ T cells by use of the H-2D ^b -Scov-2:538- 546 dextramer in the lungs of LV::S or sham- vaccinated mice. Inside CD8 ⁺ dextramer ⁺ T-cell subset, Tcm, Tern and Trm have been distinguished.

(E) Percentages of dextramer ⁺ cells were calculated versus CD8 ⁺ T cells in both mouse groups and those of Tcm, Tern and Trm were calculated versus dextramer ⁺ CD44 ⁺ cells in LV::S-vaccinated mice (n = 4/group). N/A= not applicable.

(F, G) Anti-Scov-2 IgG or IgA titers (F) and neutralizing activity (EC50) (G) in the sera or lung homogenates at 3 dpi. Samples from individual mice (n = 4/group) were studied.

Data information: Statistical significance of the difference between the two groups was evaluated by Mann-Whitney test (*= p < 0.05, **=p <0.01).

Figure 6. Comparison of vaccination routes in the protective efficacy of LV::S. Comparative histopathology of lungs from unprotected and LV::S-vaccinated and protected mice

(A) B6.K18-hACE2 ^{IP THV} mice were immunized with LV::S via i.m. or i.n. at wk 0 and boosted via i.m. or i.n. at wk 5 (n = 5/group) with non-integrative LV::S. Control mice were injected i.m. i.n. with an empty LV (sham). Viral RNA contents were determined by conventional E-specific RT-PCR at 3 dpi, in the brain, lung and nasal washes.

(B) Lung histology in B6.K18-hACE2 ^{IP THV} mice, vaccinated with LV::S after SARS-CoV- 2 inoculation. H&E (rows 1 and 3) (scale bar: 500 pm) and Ncov-2-specific IHC (rows 2 and 4) (scale bar: 50 pm) staining of whole lung sections (scale bar: 50 pm) from the primed (i.m.), boosted (i.n.) and challenged B6.K18-hACE2 ^{IP THV} mice compared with their sham controls. H&E and Ncov-2-specific IHC were performed on contiguous sections. The IHC fields correspond to the rectangles in the corresponding H&E images above them. A representative Ncov-2-specific IHC on a lung section from a non-infected (NI) mouse is also shown.

(C) Heatmap representing the histological scores for various parameters in LV::S- vaccinated or sham mice at 3 dpi (n = 6/group). Data information: Statistical significance was evaluated by Mann- Whitney test (*=p < 0.05, **= p <0.01, ****= p < 0.0001, ns = not significant).

Figure 7. Features of olfactive bulbs or brains in the protected LV::S- or unprotected sham-vaccinated B6.K18-hACE2 ^{IP THV} mice

Mice are those detailed in the Figure 6.

(A) Example of CD3-positive cells in an olfactory bulb from an LV::S i.m.-i.n. vaccinated and protected mice and representative results from this group versus sham-vaccinated and unprotected mice at 3 dpi (n = 7-9/group). Scale bar: 50 pm. Statistical significance was evaluated by Mann- Whitney test (**=/? <0.01).

(B-D) Cytometric analysis of cells extracted from pooled olfactory bulbs from the same groups. (C, D) Innate immune cells in the olfactory bulbs (C) or brain (D). The CD1 lb ⁺ Ly6C ⁺ Ly6G ⁺ population in the olfactory bulbs are neutrophils and the CD1 lb ⁺ Ly6C ⁺ Ly6G“ cells of the brain are inflammatory monocytes.

(E) Brain H&E histology at 3 dpi. The top right and both bottom panels show examples, in two different mice, of leukocyte clusters (arrows) alongside the ventricular wall. No such clusters were detected in the LV::S i.m.-i.n. vaccinated mice (top left panel). Scale bar: 200 pm. The close up view (bottom right panel) highlights the thickened, disorganized ependymal lining, compared to the normal ependymal cells and cilia of an LV : :S i.m.-i.n. vaccinated mouse (top left panel). Scale bar: 50 pm.

Figure 8. Full protective capacity of LV::S against the SARS-CoV-2 Gamma variant

(A) Timeline of LV::S i.m.-i.n. immunization and challenge with SARS-CoV-2 Gamma in B6.K18-hACE2 ^IP-THV mice (n = 5/group). The LVs used in this experiment were non- integrative. Olfactory bulbs, brains and lungs were collected at 3 dpi.

(B) Brain or lung viral RNA contents, determined by conventional E-specific or sub- genomic Esg-specific qRT-PCR at 3 dpi. Two mice out of the 5 sham- vaccinated mice did not have detectable viral RNA in the lungs despite high viral RNA content in the brain and hACE2 mRNA expression levels comparable to that of the other mice in the same group.

(C) Neutralizing activity (EC50) of sera from individual LV::S-vaccinated mice against pseudo-viruses harboring Scov-2 from the ancestral strain or D614G, Alpha, Beta or Gamma variants. Symbol T| indicates significance versus ancestral, Symbol * indicates significance versus D614G variant, while symbol //indicates significance versus Alpha variant. Statistical comparisons were made at the respective boosting timepoint. In homologous settings, sera from mice immunized with LV::SBet _a or LVzSoamma, fully inhibited pseudo viruses bearing S from Beta or Gamma, validating the assay for all pseudo-viruses used.

(D) Cytometric analysis of CD8 ⁺ T cells in pooled olfactory bulbs of LV::S i.m.-i.n. vaccinated and protected mice versus sham-vaccinated and unprotected mice.

(E) Wild type or 11MT KO mice (n = 5-9/group) were injected by LV::S or sham following the time line shown in (A), then pretreated with Ad5::hACE2 4 days before challenge with the ancestral SARS-CoV-2 strain. Lung viral RNA contents were determined at 3 dpi.

(F) T-splenocyte responses in LV::S-primed and -boosted C57BL/6 WT mice or sham controls (n = 3-5/group), evaluated by IFN-y ELISPOT using 15-mer peptides encompassing Scov-2 MHC-I -restricted epitopes.

Data information: Statistical significance was evaluated by Mann- Whitney test (* or # = p < 0.05, 0.01, ****= p < 0.0001).

Figure 9 Inflammation status of the lungs

(A) Cytometric analysis of innate cell population and qRT-PCR analysis of cytokines and chemokines in the lungs of LV::S- or sham-vaccinated and SARS-CoV-2 -challenged B6.K18- hACE2 ^IP-THV transgenic mice. Cytometric gating strategy to quantify various lung innate immune cells at 3 dpi. Cells were first gated on hematopoietic CD45 ⁺ cells and then by sequential gates, through three distinct paths.

(B) Percentages of selected innate immune subsets versus total lung CD45 ⁺ cells were determined in individual mice (n = 6/group).

(C) Heatmap representing log2 fold change in cytokine and chemokine mRNA expression in the lungs of LV::S- or sham-vaccinated mice at 3 dpi (n = 6/group). Data were normalized versus untreated controls. Statistical significance was evaluated by Mann-Whitney test (*= p < 0.05, **= p <0.01, ns = not significant).

Figure 10. Lung and brain histology in B6.K18-hACE2 ^{IP THV} mice, vaccinated with LV::S and challenged with SARS-CoV-2 Gamma

(A, B) H&E (rows 1 and 3) and Ncov-2-specific IHC (rows 2 and 4) staining of 3 dpi wholelung sections from B6.K18-hACE2 ^{IP THV} mice, LV::S- or sham-vaccinated and challenged, following the time line in Figure 7A. H&E and IHC were performed on contiguous sections. Scale bar: 500 pm. The boxed area in the IHC images harbor Ncov-2-specific labeling, as exemplified at higher magnification in (B) Scale bar: 100 pm. They correspond to inflammatory infiltrates seen in the corresponding H&E-stained sections.

(C) Representative images of Ncov-2-specific IHC staining of whole-brain section in sham or LV::S-vaccinated mice. Boxes highlight clusters of N ^CoV-2 positive cells. Scale bar: 500 pm.

(D) An example of brain IHC signal at higher magnification and frequency of NcoV2 ⁺ cells in brains from vaccinated and sham animals. Scale bar: 100 pm. Numbers of Ncov2 ⁺ cells per mm2 of brain were determined in individual mice (n = 5/group). Statistical significance was evaluated by Mann- Whitney test (**=/? <0.01).

Figure 11. Comparative description of the hACE2 constructs used to generate B6.K18-hACE2 ^{IP THV} and B6.K18- ACE2 ^Prlmn/JAX transgenic mice and their features after inoculation of SARS-CoV-2

The characteristics of B6.K18-ACE2 ^Prlmn/JAX mice are based on the previous description (McCray et al., 2007; Winkler et al., 2020). The characteristics of B6.K18-hACE2 ^{IP THV} mice are based on the results in the present work. K18 = human cytokeratin 18 promoter, CDS = Coding DNA Sequence, TE = Translational Enhancer from alfalfa mosaic virus, WPRE = Woodchuck Posttranscriptional Regulatory Element translational enhancer.

Figure 12. Wild-type and prefusion forms of Scov-2 protein and comparative antibody responses of mice immunized with LV encoding each of these forms

(A) Schematic representation of wild type or prefusion forms of Scov-2 encoded by LV. RBD, S1/S2 and S2’ cleavage sites, Fusion Peptide (FP), TransMembrane domain (TM) and short internal tail (T), 675 ^QTQTNSPRRAR 685 sequence encompassing RRAR furin cleavage site, and K ⁹⁸⁶ P and V ⁹⁸⁷ P consecutive substitutions are indicated. (B-C) C57BL/6 mice were primed i.m. at wkO with 1 x 10 ⁷ TU and boosted i.n. at wk5 with 3 x 10 ⁷ TU of either of LV or a control LV (sham). The LVs used in this experiment were integrative. (B) Sera were collected at 3, 5 and 7 wks post immunization and anti-Scov-2 (TriS) IgG responses were evaluated by ELISA. Results are expressed as mean endpoint dilution titers. (C) Lung homogenates were studied at 7 wks post immunization for anti-Scov-2 (TriS) IgG or IgA responses. (D) Comparative serum neutralization capacity of anti-Scov-2 sera induced by LV immunization, determined as 50% Effective Concentration (EC50) neutralizing titers. Shown are Mean ± SD.

Figure 13. Additional immune features of B6.K18-hACE2 ^{IP THV} mice primed (i.m.) and boosted (i.n.) with LV::S.

(A) IFN-y T splenocyte responses quantified by ELISPOT in the LV::S-vaccinated B6.K18- hACE2 ^IP-THV mice, using various 15-mer peptides encompassing SC0V-2MHC-I- (left) or MHC- II- (right) restricted T-cell epitopes. (B) Representative results of intracellular staining of IFN- y to measure lung CD4 ⁺ T cells, after in vitro stimulation with Scov-2::61-75 peptide containing an MHC-II- restricted T-cell epitope. (C) Cytometric analysis of CD69 and CCR7 expression on CD3 ⁺ CD4 ⁺ T cells gated on cells extracted from pooled olfactory bulbs of B6.K18- hACE2 ^{IP THV} mice vaccinated with LV::S or sham at 3 dpi after SARS-CoV-2 challenge. (D) Expression of CCL19 or CCL21 chemo-attractants in the brain, as evaluated by qRT-PCR at 3 dpi. (E) Heatmap representing log2 fold change in cytokine and chemokine mRNA expression in the olfactory bulbs, from LV::S-vaccinated (n = 4) and sham (n = 5) individuals, at 3 dpi. Data were normalized versus untreated, non-infected and age-matched controls. Statistical significance was evaluated by Mann- Whitney test (*= p < 0.05, ns = not significant). The scale of the heatmap is identical to that of Figure 3C and Figure 9 to facilitate comparison of the degrees of inflammation in various organs studied.

Figure 14. Map of lentiviral plasmid encoding for Scov-2.

The Full-length ancestral Scov-2 sequence is depleted for 675 ^QTQTNSPRRAR 685 sequence, encompassing the RRAR furin cleavage site, and harbors K ⁹⁸⁶ P and V ⁹⁸⁷ P consecutive substitutions, as indicated on the map. WPREm = mutated Woodchuck Posttranscriptional Regulatory Element translational enhancer.

Figure 15. Nucleic acid sequence of the transfer vector pFLAP K18-HACE2 WPRE - SEQ ID No.7 and of functional regions thereof - Amino acid sequence of the expressed polypeptides, including hACE2

Figure 15A: SEQ ID No.7

FOCUS Exported 11342 bp ds-DNA circular SYN

DEFINITION synthetic circular DNA

ACCESSION pFLAP CMV EGFP WPRE.xdna

SOURCE synthetic DNA construct

ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 11342) COMMENT Serial Cloner Genbank Format S erialC loner_T ype=DN A SerialCloner_Comments= SerialCloner_Ends=0,0„0,

FEATURES Location/ Qualifiers source 1..11342

/organism- 'synthetic DNA construct" /mol_type="other DNA" misc feature complement(87..164) /label=SV40 ORI misc_feature 233..868 /label=HIVl-5LTR misc feature 870..1086

/label=HIV-l psi pack misc_feature 1533..1766 /label=RRE misc_feature 2288..2411 /label=cPPT-CTS primer_bind 2446..2476 /label=Primer 1 misc_feature 2453..5701

/label=K18 promoter misc feature 4961..4966

/label=Modified Splicing Donor Site misc feature 5686..5697

/label=Modified Poly-pyrimidine tract misc feature 5697..5701

/label=Modified Splicing acceptor site CDS 5714..8131

/codon_start=l /label=hACE2 /note=7type=CDS" /note="/vntifkey=4" /translation (SEQ ID No. 6) misc_feature 8149..8753

/label=WPRE

/label=WPRE-WT misc feature 8838..9099 /label=LTR terminator 9136..9363 /label=bGH PA CDS 9539..10330 /codon_start=l /label=Kan/neoR /translation (SEQ ID No. 10) rep origin 10634..11262

/label=ColEl origin

Figure 15B: transcriptional control region (encompassing promoter): KI 8 promoter (SEQ ID No. 1) (BOLD UNDERLINED = modified splicing donor (SEQ ID No. 3) and acceptor sites (SEQ ID No. 4)), corresponds to nucleotides 2453 to 3249 in SEQ ID No. 7

Figure 15C: hACE2 coding sequence- SEQ ID No.5, corresponds to nucleotides 5714 to 8131 in SEQ ID No.7

Figure 15D: Amino acid sequence of the hACE2 protein (SEQ ID No.6)

Figure 15E: nucleotide sequence of WPRE wild type - SEQ ID No.4, corresponds to nucleotides 8149 to 8753 in SEQ ID No.7

Figure 15F: polypeptide encoded by of Kan/neoR gene (SEQ ID No. 8)

Figure 16. Restriction map of the transfer vector pFLAP K18-HACE2 WPRE

Preparation of the lentiviral vectors

The recombinant lentiviral vector (i.e., lentiviral vectors particles or lentiviral-based vector particles) defined in the present invention are pseudotyped lentiviral vectors consisting of vector particles bearing envelope protein or envelope proteins which originate from a virus different from the particular lentivirus (especially a virus different from HIV, in particular HIV-1), which provides the vector genome of the lentiviral vector particles. Accordingly, said envelope protein or envelope proteins, are “heterologous” viral envelope protein or viral envelope proteins with respect to the vector genome of the particles. In the following pages, reference will also be made to “envelope protein(s)” to encompass any type of envelope protein or envelope proteins suitable to perform the invention.

When reference is made to “lentiviral vectors” (lentiviral-based vectors) in the application, it relates in particular to HIV-based vectors, and especially HIV-l-based vectors.

The lentiviral vectors suitable to perform the invention are so-called replacement vectors, meaning that the sequences of the original lentivirus encoding the lentiviral proteins are essentially deleted in the genome of the vector or, when present, are modified, and especially mutated, especially truncated, to prevent expression of biologically active lentiviral proteins, in particular, in the case of HIV, to prevent the expression by said transfer vector providing the genome of the recombinant lentiviral vector particles, of functional ENV, GAG, and POL proteins and optionally of further structural and/or accessory and/or regulatory proteins of the lentivirus, especially of HIV.

The “vector genome” of the vector particles is a recombinant nucleic acid which also comprises as a recombined sequence the transgene of the invention encoding the hACE2 protein according to the invention. The lentiviral-based sequence and polynucleotide/transgene of the vector genome are borne by a plasmid vector such as pFLAP K18-hACE2 WPRE thus giving rise to the “transfer vector” also referred to as “sequence vector” such as pFLAP K18-hACE2 WPRE. This specific plasmid has the backbone of the pTRIP vector as disclosed in Zennou et al (2000) with the modification of the sequence coding for the resistance to antibiotic which corresponds to the kanamycin resistance gene instead of the ampicillin resistance gene in pTRIP . According to a particular embodiment, a vector genome prepared for the invention comprises a transgene construct corresponding to the sequence from nucleotide position 2453 to nucleotide position 8753 in the sequence of SEQ ID No.7.

The vector genome as defined herein accordingly contains, apart from the recombinant polynucleotide(s) (transgenic construct) encoding the hACE2 protein according to the invention under control of the herein defined proper regulatory sequences for its transcription and expression, the sequences of the original lentiviral genome which are non-coding regions of said genome and are necessary to provide recognition signals for DNA or RNA synthesis and processing (mini-viral genome). These sequences are cis-acting sequences necessary for packaging (\|/), reverse transcription (LTRs possibly mutated with respect to the original ones, in particular in the U3 region, especially to provide a delta U3 LTR) and transcription and optionally integration (RRE) and furthermore for the particular purpose of the invention, they contain a functional sequence favouring nuclear import in cells and accordingly transgene transfer efficiency in said cells, which element is described as a DNA Flap element that contains or consists of the so-called central cPPT-CTS nucleotidic domain present in lentiviral genome sequences especially in HIV-1 or in some retroelements such as those of yeasts. Additionally, the cis-acting sequence include a sequence which is a wild-type Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) and is brought as part of the transgene construct.

The structure and composition of the vector genome used to prepare the lentiviral vectors of the invention are based on the principles described in the art and on examples of such lentiviral vectors primarily disclosed in (Zennou et al, 2000; Firat H. et al, 2002; VandenDriessche T. et al, 2002). Constructs of this type have been deposited at the CNCM (Institut Pasteur, France) as will be referred to herein. In this respect reference is also made to the disclosure, including to the deposited biological material, in patent applications WO 99/55892, WO 01/27300 and WO 01/27304.

According to a particular embodiment of the invention, a vector genome may be a replacement vector in which all the viral protein coding sequences between the 2 long terminal repeats (LTRs) have been replaced by the recombinant polynucleotide encoding the hACE2 protein according to the invention and wherein the DNA-Flap element has been re-inserted in association with the required cis-acting sequences described herein. Further features relating to the composition of the vector genome and plasmid containing the same are disclosed in relation to the preparation of the particles and in figure 16.

The vector genome herein disclosed is included in the backbone or a plasmid in order to provide a transfer vector. In particular, the transfer vector obtained is a plasmid of SEQ ID No. 7.

According to the invention, the lentiviral vector particles are pseudotyped with a heterologous viral envelope protein or viral polyprotein of envelope originating from an RNA virus which is not the lentivirus providing the lentiviral sequences of the genome of the lentiviral particles.

As examples of typing envelope proteins for the preparation of the lentiviral vector, the invention relates to viral transmembrane glycosylated (so-called G proteins) envelope protein(s) of a Vesicular Stomatitis Virus (VSV), which is(are) for example chosen in the group of VSV-G protein(s) of the Indiana strain and VSV-G protein(s) of the New Jersey strain.

The VSV-G protein presents an N-terminal ectodomain, a transmembrane region and a C- terminal cytoplasmic tail. It is exported to the cell surface via the trans-Golgi network (endoplasmic reticulum and Golgi apparatus).

Vesicular stomatitis Indiana virus (VSIV) and Vesicular stomatitis New Jersey virus (VSNJV) are examples of strains known to pseudotype the lentiviral vectors of the invention, or to design recombinant envelope protein(s) to pseudotype the lentiviral vectors. Their VSV- G proteins are disclosed in GenBank, where several strains are presented. For VSV-G New Jersey strain reference is especially made to the sequence having accession number V01214. For VSV-G of the Indiana strain, reference is made to the sequence having accession number AAA48370.1 in Genbank corresponding to strain JO2428.

In a particular embodiment, the lentiviral vector is built from a first-generation vector, in particular a first-generation of a HIV-based vector which is characterized in that it is obtained using separate plasmids to provide (i) the packaging construct, (ii) the envelope and (iii) the transfer vector genome. Alternatively, it may be built from a second-generation vector, in particular a second-generation of a HIV-based vector which in addition, is devoid of viral accessory proteins (such as in the case of HIV-1, Vif, Vpu, Vpr or Nef) and therefore includes only four out of nine HIV full genes: gag, pol, tat and rev. In another embodiment, the vector is built from a third-generation vector, in particular a third-generation of a HIV-based vector which is furthermore devoid of said viral accessory proteins and also is Tat-independent; these third-generation vectors may be obtained using 4 plasmids to provide the functional elements of the vector, including one plasmid encoding the Rev protein of HIV when the vector is based on HIV-1. Such vector system comprises only three of the nine genes of HIV- 1. The structure and design of such generations of HIV-based vectors is well known in the art.

According to the invention, the lentiviral vectors are hence the product recovered from cotransfection of mammalian cells, with:

- a vector plasmid comprising (i) lentiviral, especially HIV-1, cis-active sequences necessary for packaging, reverse transcription, and transcription and further comprising a functional lentiviral, especially derived from HIV-1, DNA flap element and (ii) a transgene construct of the invention, and optionally comprising sequences for integration into the genome of the host cell; an example of such vector is disclosed in figure 15.

- an expression plasmid encoding a pseudotyping envelope derived from an RNA virus, said expression plasmid comprising a polynucleotide encoding an envelope protein or proteins for pseudotyping, wherein said envelope pseudotyping protein is advantageously from a VSV.

- an encapsidation plasmid, which comprises lentiviral, especially HIV-1, gag-pol packaging sequences suitable for the production of integration-competent vector particles.

The invention thus also concerns lentiviral vector particles as described above, which are the product recovered from a stable cell line transfected with:

- a vector plasmid comprising (i) lentiviral, especially HIV-1, cis-active sequences necessary for packaging, reverse transcription, and transcription and further comprising a functional lentiviral, especially HIV-1, DNA flap element and optionally comprising cis-active sequences necessary for integration, said vector plasmid further comprising, (ii) a transgene construct of the invention;

- a VSV-G envelope expression plasmid comprising a polynucleotide encoding a VSV-G envelope protein, wherein said polynucleotide is under the control of regulating expression sequences, in particular regulatory expression sequences comprising a promoter, and;

- an encapsidation plasmid, wherein the encapsidation plasmid comprises lentiviral, especially HIV-1, gag-pol coding sequences suitable for the production of integration-competent vector particles wherein said gag-pol sequences are from the same lentivirus sub-family as the DNA flap element, wherein said lentiviral gag-pol is under the control of regulating expression sequences.

The stable cell lines expressing the vector particles of the invention are in particular obtained by transfection of the plasmids.

The particular embodiments may be carried out when preparing the lentiviral vector based on human lentivirus, and especially based on HIV-1 virus.

According to a preferred embodiment of the invention, the genome of the lentiviral vector is derived from a human lentivirus, especially from the HIV lentivirus. In particular, the pseudotyped lentiviral vector is an HIV-based vector, such as an HIV-1, or HIV-2 based vector, in particular is derived from HIV-1M, for example from the BRU, LAI or NDK HIV-1 isolates.

The invention accordingly involves lentiviral vectors which are recombinant lentiviral particles (i.e. recombinant vector particles), and which may be replication-competent lentiviral vectors, especially replication-competent HIV-1 based vectors or alternatively may be replication-incompetent lentiviral vectors, especially replication-incompetent HIV-1 based vectors characterized in that: (i) they are pseudotyped with a determined heterologous viral envelope protein or viral envelope proteins originating from a RNA virus which is not HIV, and (ii) they comprise in their genome the transgene construct of the invention..

According to a particular embodiment of the invention, the lentiviral vectors are designed to express proficient (i.e., integrative-competent) particles. According to a particular embodiment of the invention, the recombinant lentiviral vector particles are integration-competent and replication-incompetent. The preparation of the lentiviral vectors is well known from the skilled person and has been extensively disclosed in the literature (confer for review Sakuma T. et al (Biochem. J. (2012) 443, 603-618). The preparation of such vectors is also illustrated herein. In a particular embodiment, the lentiviral vector particles are produced as described in the Examples.

EXAMPLES

A B6.K18-ACE2 ^2Prlnm/JAX mouse strain has been previously deposited at JAX Laboratories (Jackson Laboratories, Bar Harbor, ME). However, the new B6.K18-hACE2 ^{IP THV} transgenic mice that the inventors generated according to the present invention display distinctive characteristics identified following SARS-CoV-2 intranasal (i.n.) inoculation. In fact, in addition to the large permissiveness of their lungs to SARS-CoV-2 replication and viral dissemination to peripheral organs, B6.K18-hACE2 ^{IP THV} mice surprisingly allow substantial viral replication in the brain, which is ~ 4 logic higher than the replication range observed in the previously available B6.K18-ACE2 ^2Prlnm/JAX strain (Yang et al., 2007). This new mouse model, not only has broad applications in the study of COVID-19 vaccine or COVID-19 therapeutics efficacy, but also provides an experimental model to elucidate COVID-19 immune/neuro-physiopathology. Neurotropism of SARS-CoV-2 has been demonstrated and some COVID-19 human patients present symptoms like headache, confusion, anosmia, dysgeusia, nausea, and vomiting (Bourgonje et al., 2020). Olfactory transmucosal SARS-CoV- 2 invasion is also very recently described as a port of central nervous system entry in human individuals with COVID-19 (https://doi.org/10.1038/s41593-020-00758-5). Since coronaviruses can infect the central nervous system (Bergmann et al., 2006), the B6.K18- hACE2 ^{IP THV} small rodent experimental model represents an invaluable pre-clinical or co- clinical animal model of major interest for: (i) investigation of immune protection of the brain and (ii) exploration of COVID-19-derived neuropathology.

Methods

I. Construction and production of LV

A codon-optimized prefusion S sequence (1-1262) (Table S2) was amplified from pMK- RQ_S-2019-nCoV and inserted into pFlap by restriction/ligation between BamHI and Xhol sites, between the native human ieCMV promoter and a mutated Woodchuck Posttranscriptional Regulatory Element (WPRE) sequence. The atg starting codon of WPRE was mutated (mWPRE) to avoid transcription of the downstream truncated “X” protein of Woodchuck Hepatitis Virus for safety concerns (Figure 145). Plasmids were amplified and used to produce LV as previously described (Ku et al., 2021a).

II. Mice

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

III. Mouse transgenesis

1. Construction of the human keratin 18 promoter

The human K18 promoter (GenBank: AF179904.1 nucleotides 90 to 2579) was amplified by nested PCR from A549 cell lysate, as described previously (Chow et al., 1997; Koehler et al., 2000). The “i6x7” intron (GenBank: AF 179904.1 nucleotides 2988 to 3740) was synthesized by Genscript. The “K18i6x7PA” promoter (also designated K18 ^JAX promoter), previously used to generate B6.K18-ACE2 ^2Prlnm/JAX strain, includes the KI 8 promoter, the “i6x7” intron at 5' and an enhancer/polyA sequence (PA) at 3’ of the hACE2 gene. The ^{K18 IP-Thv} promoter used here contains, instead of PA, the stronger wild-type Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) at 3 ’of the hACE2 gene. In contrast to KI 8i6x7PA construct which harbors the 3’ regulatory region containing a polyA sequence, the K18 ^IP-Thv construct takes benefice of the polyA sequence already present within the 3’ Long Terminal Repeats (LTR) of the lentiviral (pFLAP LV) plasmid, used for transgenesis. The i6x7 intronic part was modified to introduce a consensus 5’ splicing donor and a 3’ donor site sequence. The AAGGGG (SEQ ID No. 75) donor site was further modified for the AAGTGG (SEQ ID No. 76) consensus site in SEQ ID No. 2. Based on a consensus sequence logo (Dogan et al., 2007), the poly-pyrimidine tract preceding splicing acceptor site (TACAATCCCTC (SEQ ID No. 77) in original sequence GenBank AF 179904.1 and _TTTTTTTTTTT ( _SE Q _ID No. 78) in K18 ^JAX ) was replaced by CTTTTTCCTTCC (SEQ ID No. 79) to limit incompatibility with the reverse transcription step during transduction. Moreover, original splicing acceptor site CAGAT was modified to correspond to the consensus sequence CAGGT. As a construction facilitator, a Clal restriction site was introduced between the promoter and the intron. The construct was inserted into a pFLAP plasmid between the Mlul and BamHI sites. The hACE2 cDNA was introduced between the BamHI and Xhol sites by restriction/ligation. Integrative LV::K18-hACE2 ^IP-THV was produced as described elsewhere (Sayes et al., 2018) and concentrated by two cycles of ultracentrifugation at 22,000 rpm for Ih at 4°C.

2. Transgenesis

High tittered (4.16 ^X 10 ⁹ TU/ml) integrative LV::K18-hACE2 ^IP-THV was micro-injected into the pellucid area of fertilized eggs which were transplanted into pseudo-pregnant B6CBAF1 females (Charles Rivers). The NO mice were investigated for integration and copy number of hACE2 gene per genome by using hACE2-forward: 5’-TCC TAA CCA GCC CCC TGT T-3’ (SEQ ID No. 65) and hACE2-reverse: 5’-TGA CAA TGC CAA CCA CTA TCA CT-3’ (SEQ ID No. 66) primers in PCR applied on genomic DNA prepared from the tail biopsies. Toward stabilization of the progeny, transgene positive males were then crossed to WT C57BL/6 females (Charles Rivers). Transgene transfer by microinjection of integrative LV::K18- hACE2 ^IP-THV into the nucleus of fertilized eggs was particularly efficient (Nakagawa & Hougenraad, 2011). At the NO generation, ~ 11% of the mice obtained, i.e., 15 out of 139, had at least one copy of the transgene per genome. Eight NO males carrying the transgene were crossed with female C57BL/6 WT mice. At the N1 generation, ~ 62% of the mice obtained, i.e., 91 out of 147, had at least one copy of the transgene per genome. 10 N1 males carrying the transgene were further crossed with female C57BL/6 WT mice.

In the B6.K18-hACE2 ^IP-THV Tg descendent mice, as analyzed atN4 (4 back cross generations to wild type C57BL/6 mice), one vector copy has integrated on mouse chromosome 16. Seven sequence variants and no structural variants were detected in the integrated vector sequence. The lentiviral vector has integrated at mouse chrl6:95, 981, 235-95, 981, 236, which is in intron 6 of Psmgl (NM_019537.2).

Genotyping and quantitation of hACE2 gene copy number/genome in transgenic mice Genomic DNA (gDNA) from transgenic mice was prepared from the tail biopsies by phenolchloroform extraction. Sixty ng of gDNA were used as a template of qPCR with SYBR Green using specific primers listed in Table S3. Using the same template and in the same reaction plate, mouse pkdl (Polycystic Kidney Disease 1) and gapdh were also quantified. All samples were run in quadruplicate in 10 pl reaction as 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 number, the 2 ^AACt method was applied using the pkdl as a calibrator and gapdh as an endogenous control. The 2 ^AACt provides the fold change in copy number of the hACE2 gene relative to pkdl gene.

Western blot

Levels of hACE2 in the lungs of transgenic mice were assessed by western blotting. Lung cell suspensions were resolved on 4%-12% NuPAGE Bis-Tris protein gels (Thermo Fisher Scientific), then transferred onto a nitrocellulose membrane (Biorad, France). The nitrocellulose membrane was blocked in 5% non-fat milk in PBS-T for 2 h at room temperature and probed overnight with goat anti-hACE2 primary Ab at 1 mg/mL (AF933, R&D systems). Following three wash intervals of 10 min with PBS-T, the membrane was incubated for 1 h at room temperature with HRP-conjugated anti-goat secondary Ab and HRP-conjugated anti-0- actin (ab 197277, Abeam). The membrane was washed with PBS-T thrice before visualization with enhanced chemiluminescence via the super signal west femto maximum sensitivity substrate (Thermo Fisher Scientific) on ChemiDoc XRS+ (Biorad, France). PageRuler Plus prestained protein ladder was used as size reference. Relative quantification of western blots was performed using ImageJ program. Images from the same blot were taken with the same exposure time and were inverted before measuring the protein band intensity. The ratio of hACE2 to 0-actin was calculated to indicate the relative expression of hACE2 in each sample.

Ethical Approval of Animal Studies

Experimentation on mice was realized in accordance with the European and French guidelines (Directive 86/609/CEE and Decree 87-848 of 19 October 1987) subsequent to approval by the Institut Pasteur Safety, Animal Care and Use Committee, protocol agreement delivered by local ethical committee (CETEA DAP20007, CETEA #DAP200058) and Ministry of High Education and Research APAFIS#24627-2020031117362508 vl, APAFIS#28755-2020122110238379 vl.

Humoral and T-cell immunity, Inflammation As recently detailed elsewhere (Ku et al., 2021a), T-splenocyte responses were quantitated by IFN-y ELISPOT and anti-S IgG or IgA Abs were detected by ELISA by use of recombinant stabilized Scov-2. NAb quantitation was performed by use of LV particles pseudo-typed with Scov-2 from the diverse variants, as previously described (Anna et al., 2020; Sterlin et al., 2020). The qRT-PCR quantification of inflammatory mediators in the lungs, brain and olfactory bulbs was performed as recently detailed (Ku et al., 2021a) on total RNA extracted by TRIzol reagent (Invitrogen) and immediately stored at -80°C. The RNA quality was assessed using a Bioanalyzer 2100 (Agilent Technologies). RNA samples were quantitated using a NanoDrop Spectrophotometer (Thermo Scientific NanoDrop). The RNA Integrity Number (RIN) was 7.5- 10.0. CCL19 and CCL21 expression were verified using the following primer pairs: forward primers were 5’-CTG CCT CAG ATT ATC TGC CAT-3’ for CCL19 (SEQ ID No. 80) and 5’- AAG GCA GTG ATG GAG GGG-3’ for CCL21(SEQ ID No. 82); reverse primers were 5’- AGG TAG CGG AAG GCT TTC AC -3’ for CCL19 (SEQ ID No. 81) and 5’- CGG GGT AAG AAC AGG ATT G -3’ for CCL21(SEQ ID No. 83).

3. B6.K18-hACE2 ^{IP THV} permissiveness to SARS-CoV-2 replication

The permissiveness of N1 transgenic B6.K18-hACE2 ^IP-THV mice to SARS-CoV-2 replication was evaluated in the sampled individuals. N1 females with varying number of transgene copies per genome were sampled (Figure 1 A) and evaluated for their permissiveness to SARS-CoV-2 replication (Figure IB). B6.K18-ACE2 ^2Prlnm/JAX mice were treated similarly. To do so, the selected transgenic B6.K18-hACE2 ^IP-THV or B6.K18-ACE2 ^2Prlnm/JAX mice were anesthetized by i.p. injection of Ketamine and Xylazine mixture, transferred into a level 3 biosafety cabinet and were inoculated i.n. under general anesthesia with 0.3 x 10 ⁵ TCID50 of the BetaCoV/France/IDF0372/2020 or Gamma (P.I) SARS-CoV-2 clinical isolate (Lescure et al., 2020), supplied by the National Reference Centre for Respiratory Viruses hosted by Institut Pasteur (Paris, France). The viral inoculum was contained in 20 pl for mice. Animals were then housed in an isolator in BioSafety Level 3 animal facilities of Institut Pasteur.

The organs recovered from the animals infected with live SARS-CoV-2 were manipulated following the approved standard operating procedures of these facilities.

Ad5::hACE2 pretreatment of WT of 11MT KO mice before SARS-CoV-2 inoculation was performed as previously described (Ku et al., 2021a). All experiments with SARS-CoV-2 were performed in a biosafety level 3 laboratory and with approval from the department of hygiene and security of Institut Pasteur, under the protocol agreement # 20.070 A-B. Determination of viral RNA content in the organs

Organs from mice were removed aseptically and immediately frozen at -80°C. RNA from circulating SARS-CoV-2 was prepared from lungs as recently described (Ku et al., 2021a). Briefly, lung homogenates were prepared by thawing and homogenizing of the organs in lysing matrix M (MP Biomedical) with 500 pl of ice-cold PBS using a MP Biomedical Fastprep 24 Tissue Homogenizer. RNA was extracted from the supernatants of lung homogenates centrifuged during 10 min at 2000g, using the Qiagen Rneasy kit according to the manufacturer instructions, except that the neutralization step with AVL buffer/carrier RNA was omitted. These RNA preparations were used to determine viral RNA content by E-specific qRT-PCR. Alternatively, total RNA was prepared from lungs or other organs using lysing matrix D (MP Biomedical) containing 1 mL of TRIzol reagent and homogenization at 30 s at 6.0 m/s twice using MP Biomedical Fastprep 24 Tissue Homogenizer. Total RNA was extracted using TRIzol reagent (ThermoFisher). These RNA preparations were used to determine viral RNA content by Esg-specific qRT-PCR, hACE2 expression level or inflammatory mediators. RNA was isolated from nasal washes using QIAamp Viral RNA Mini Kit (Qiagen).

SARS-CoV-2 E gene (Corman et al., 2020) or E sub-genomic mRNA (Esg RNA) (Wolfel et al., 2020), was quantitated following reverse transcription and real-time quantitative TaqMan® PCR, using SuperScriptTM III Platinum One-Step qRT-PCR System (Invitrogen) and specific primers and probe (Euro fins) (Table S4). The standard curve of Esg mRNA assay was performed using in vitro transcribed RNA derived from PCR fragment of “T7 SARS-CoV- 2 Esg mRNA”. The in vitro transcribed RNA was synthesized using T7 RiboMAX Express Large Scale RNA production system (Promega) and purified by pheno 1/chloro form extraction and two successive precipitations with isopropanol and ethanol. Concentration of RNA was determined by optical density measurement, diluted to 10 ⁹ genome equivalents/pL in RNAse- free water containing lOOpg/mL tRNA carrier, and stored at -80°C. Serial dilutions of this in vitro transcribed RNA were prepared in RNAse-free water containing lOpg/ml tRNA carrier to build a standard curve for each assay. PCR conditions were: (i) reverse transcription at 55°C for 10 min, (ii) enzyme inactivation at 95°C for 3 min, and (iii) 45 cycles of denaturation/ amplification at 95°C for 15 s, 58°C for 30 s. PCR products were analyzed on an ABI 7500 Fast real-time PCR system (Applied Biosystems). PFU assay was performed as previously described (Ku et al., 2021a).

Cytometric analysis of immune lung and brain cells Isolation and staining of lung innate immune cells were largely detailed recently (Ku et al., 2021a). Cervical lymph nodes, olfactory bulb and brain from each group of mice were pooled and treated with 400 U/ml type IV collagenase and DNase I (Roche) for a 30-minute incubation at 37°C. Cervical lymph nodes and olfactory bulbs were then homogenized with glass homogenizer while brains were homogenized by use of GentleMacs (Miltenyi Biotech). Cell suspensions were then filtered through 100 pm-pore filters, washed and centrifuged at 1200 rpm during 8 minutes. Cell suspensions from brain were enriched in immune cells on Percoll gradient after 25 min centrifugation at 1360 g at RT, without brakes. The recovered cells from lungs were stained as recently described elsewhere (Ku et al., 2021a). The recovered cells from brain were stained by appropriate mAb mixture as follows, (i) To detect innate immune cells, Near IR Live/Dead (Invitrogen), Fcyll/III receptor blocking anti-CD16/CD32 (BD Biosciences), BV605-anti-CD45 (BD Biosciences), PE-anti-CDl lb (eBioscience), and PE- Cy7-antiCDl lc (eBioscience) were used, (ii) To detect NK, neutrophils, Ly-6C ^+/_ monocytes and macrophages, Near IR DL (Invitrogen), Fcyll/III receptor blocking anti-CD16/CD32 (BD Biosciences), BV605-anti-CD45 (BD Biosciences), PE-anti-CDl lb (eBioscience), PE-Cy7- antiCDl 1c (eBioscience), APC-anti-Ly6G (Miltenyi), BV711-anti-Siglec-F (BD), AF700-anti- NKp46 (BD Biosciences), and FITC-anti-Ly6C (ab25025, Abeam), were used, (iii) To detect adaptive immune cells, Near IR Live/Dead (Invitrogen), Fcyll/III receptor blocking antiCD 16/CD32 (BD Biosciences), APC-anti-CD45 (BD), PerCP-Cy5.5-anti-CD3 (eBioscience), FITC-anti-CD4 (BD Pharmingen), BV71 l-anti-CD8 (BD Horizon), BV605-anti-CD69 (Biolegend), PE-anti-CCR7 (eBioscience) and VioBlue-Anti-B220 (Miltenyi), were used, (iv) To identify lung memory CD8 ⁺ T-cell subsets, PerCP-Vio700-anti-CD3, BV510-anti-CD8, PE- anti-CD62L, APC-anti-CD69, APC-Cy7-anti-CD44 and FITC-anti-CD103 were used, (v) To identify Scov-2-specific CD8 ⁺ T-cells, a dextramer of H2-D ^b combined to Scov-2:538-546 (CVNFNFNGL) epitope (Zhuang et al., 2021) was used (Immudex, Danmark). Lung cells were first stained with the PE-conjugated dextramer for 30 min in dark at room temperature prior at the addition of a cocktail of Yellow Live/Dead (Invitrogen) and PerCP-Vio700-anti-CD3, BV510-anti-CD8, BV421-anti-CD62L, APC-anti-CD69, APC-Cy7-anti-CD44 and FITC-anti- CD103 mAbs. Cells were incubated with appropriate mixtures for 25 minutes at 4°C, washed in PBS containing 3% FCS and fixed with Paraformaldehyde 4% by an overnight incubation at 4°C. Samples were acquired in an Attune NxT cytometer (Invitrogen) and data analyzed by FlowJo software (Treestar, OR, USA). Histopathology

Samples from the lungs or brain of transgenic mice were fixed in formalin for 7 days and embedded in paraffin. Paraffin sections (5 -pm thick) were stained with Hematoxylin and Eosin (H&E). In some cases, serial sections were prepared for IHC analyses. Slides were scanned using the AxioScan Z1 (Zeiss) system and images were analyzed with the Zen 2.6 software. Histopathological lesions were qualitatively described and when possible scored, using: (i) distribution qualifiers (i.e., focal, multifocal, locally extensive or diffuse), and (ii) a five-scale severity grade, i.e., 1 : minimal, 2: mild, 3: moderate, 4: marked and 5: severe. For the histological heatmaps, the scores were determined as follows: the percentage of abnormal zone was estimated from low magnification images of scanned slides. All other scores were established at higher magnification (20 to 40x in the Zen program); the interstitial and alveolar syndrome scores reflected the extent of the syndrome, while the inflammation seriousness represented an evaluation of the intensity of the inflammatory reaction, i.e., abundance of inflammatory cells and exudate, conservation or disruption of the lung architecture; the bronchiolar epithelium alteration score was derived from both the extent and the severity of the lesions. IHC was performed as described elsewhere. Rabbit anti-Ncov-2 antibody (NB1 GO- 56576, Novus Biologicals, France) and biotinylated goat anti-rabbit Ig secondary antibody (E0432, Dako, Agilent, France) were used in IHC.

Statistical analyses

Experiments were performed with numbers of animals previously determined as sufficient for a correct statistical assessment, based on biostatistical prediction. The Mann- Whitney statistical test was applied to the results, using Graph Pad Prism8 software.

Results

Generation of new hACE2 transgenic mice with high brain permissiveness to SARS- CoV-2 replication

To set up a mouse model permissive to SARS-CoV-2 replication allowing assessment of our vaccine candidates we generated C57BE/6 transgenic mice with an EV (Nakagawa and Hoogenraad, 2011) carrying the hACE2 gene under the human cytokeratin 18 promoter, namely “B6.K18-hACE2 ^IP-THV ” (Figure 11). The permissiveness of these mice to SARS-CoV-2 replication was evaluated after one generation backcross to WT C57BL/6 (Nl). N1 mice with varying number of hACE2 transgene copies per genome (Figure 1A) were sampled and inoculated i.n. with the ancestral SARS-CoV-2. At day 3 post-inoculation (3 dpi), the mean ± SD of lung viral RNA content was as high as (3.3 ± 1.6) x IO ¹⁰ copies of SARS-CoV-2 RNA/lung in permissive mice (Figure IB). SARS-CoV-2 RNA copies per lung <1 x 10 ⁷ correspond to the genetic material derived from the input in the absence of viral replication (Ku et al., 2021a). We also noted that the lung viral RNA content (Figure IB) was not proportional to the hACE2 transgene copy number per genome (Figure 1A) or to the amount of hACE2 protein expression in the lungs (Figure 1C, D). Remarkably, viral RNA content, as high as (5.7 ± 7.1) x IO ¹⁰ copies of SARS-CoV-2 RNA, were also detected in the brain of the permissive mice (Figure IB). Virus replication/dissemination was also observed, although to a lesser extent, in the heart and kidneys. In another set of experiment with mice from our B6.K18- hACE2 ^{IP THV} colony, we also established the replication kinetics of ancestral SARS-CoV-2 in the lungs and brain by measuring viral RNA contents (Figure IE) and viral loads determined by PFU counting (Figure IF) over time. Viral RNA contents reached a plateau at 1 dpi in the lungs, while they increased between 1 and 3 dpi in the brain. We observed a 5-10% weight loss at 2-3dpi (Figure 1G). At this time point, mice started to be lethargic, with hunched posture and ruffled hair coat, reaching the humane endpoint. Comparatively, B6.K18-hACE2 ^JAX transgenic mice reportedly experienced significant weight loss from 4 dpi on, with an average of 20% weight loss recorded between 5 and 7 dpi (Winkler et al., 2020).

At 3 dpi, lung histological sections of SARS-CoV-2 -inoculated B6.K18-hACE2 ^{IP THV} mice displayed significant interstitial inflammation (Figure 2A-F) and alveolar exudates (Figure 2D and F), accompanied by peribronchiolar and perivascular infiltration (Figure 2C) and minimal to moderate alterations of the bronchiolar epithelium (Figure 2E). Thus, SARS-CoV-2 infection essentially induced an alveolo-interstitial syndrome in B6.K18-hACE2 ^{IP THV} mice, similarly to what was reported in the B6.K18-ACE2 ^2Prlnm/JAX transgenic mouse model (Winkler et al., 2020). At the same time-point, immunohistochemistry (IHC) analysis of the brain of infected B6.K18-hACE2 ^IP-THV mice, by use of a SARS-CoV-2 nucleocapsid protein (Ncov-2)-specific polyclonal antibody, revealed multiple clusters of Ncov-2 ⁺ cells (Figure 2G, H).

4. Comparison of B6.K18-ACE2 ^2Prlmn/JAX and B6.K18-hACE2 ^{IP THV} strains in terms of permissiveness to SARS-CoV-2 replication

We compared the replication of SARS-CoV-2 in lungs and brain and the viral dissemination to various organs in B6.K18-hACE2 ^IP-THV and B6.K18-ACE2 ^2Prlnm/JAX mice (McCray et al., 2007) (Figure 3A). The lung viral RNA contents were slightly lower (i.e., (2.1 ± 2.2) x 10 ¹⁰ copies of SARS-CoV-2 RNA/mouse in B6.K18-hACE2 ^IP-THV compared to (18.3 ± 13.3) x IO ¹⁰ copies in B6.K18-ACE2 ^2Prlnm/JAX mice. However, viral RNA contents in the brain of B6.K18- hACE2 ^IP-THV mice i.e. (7.4 ± 7.9) x IO ¹⁰ copies of SARS-CoV-2 RNA/mouse, were ~ 4 log higher compared to (1.9 ± 74.3) x 10 ⁸ copies in their B6.K18-ACE2 ^2Prlnm/JAX counterparts (Figure 3 A). Viral RNA contents were also assessed by a sub-genomic Ecov-2 RNA (Esg) qRT- PCR, which is an indicator of active viral replication (Chandrashekar et al., 2020; Tostanoski et al., 2020; Wolfel et al., 2020). Measurement of brain RNA contents by Esg qRT-PCR detected (7.55 ± 7.74) x 10 ⁹ copies of SARS-CoV-2 RNA in B6.K18-hACE2 ^IP-THV mice and no copies of this replication-related RNA in 4 out of 5 B6.K18-ACE2 ^2Prlnm/JAX mice. This dramatic difference of SARS-CoV-2 replication in the brain of the two transgenic strains was associated with significantly higher hACE2 mRNA expression in the brain of B6.K18-hACE2 ^{Ip_ THV} mice (Figure 3B). However, hACE2 mRNA expression in the lungs of B6.K18-hACE2 ^{Ip_ THV} mice was also higher than in B6.K18-ACE2 ^2Prlnm/JAX mice, despite of the lower viral replication rate in the lungs of the former. A trend towards higher viral RNA contents was also observed in the kidneys and heart of B6.K18-hACE2 ^{IP Thv} compared with B6.K18- ACE2 ^2Prlnm/JAX mice (Figure 3 A).

In accordance with the lower lung viral RNA contents, B6.K18-hACE2 ^{IP THV} mice displayed less pulmonary inflammation than B6.K18-ACE2 ^2Prlnm/JAX mice, as evaluated by qRT-PCR study of 20 inflammatory analytes, applied to RNA extracted from total lung homogenates (Figure 3C). This same assay applied to RNA extracted from total brain homogenates detected robust inflammation in B6.K18-hACE2 ^{IP THV} — but not B6.K18-ACE2 ^2Prlnm/JAX — mice (Figure 3C).

Also, as mentioned above, B6.K18-hACE2 ^{IP THV} mice generally reached the humane endpoint between 3 and 4 dpi and therefore displayed a more rapidly lethal SARS-CoV-2 - mediated disease than their B6.K18-ACE2 ^2Prlnm/JAX counterparts (Winkler et al., 2020) (Figure 11). Therefore, large permissiveness to SARS-CoV-2 replication in both lung and CNS, marked brain inflammation and rapid development of a lethal disease are major distinctive features offered by this new B6.K18-hACE2 ^{IP THV} transgenic model. Difference in pathology between B6.K18-hACE2 ^IP-THV and B6.K18-ACE2 ^2Prlnm/JAX mice suggests that some future results can be model dependent. 5. First application: Use of B6.K18-hACE2 ^{IP THV} transgenic mice to demonstrate the interest of i.n. vaccination with a LV-based vaccine candidate against SARS-CoV2;

Substantial protection of the brain from viral dissemination/replication

We previously showed that vaccination by systemic prime followed by nasal boost with an LV encoding the full length Spike protein of SARS-CoV-2 confers almost sterilizing protection against SARS-CoV-2 in a mouse model in which the expression of hACE2 was induced in the respiratory tracts by instillation Ad5::hACE2, as well as in Outbred Mesocricetus auratus golden hamsters, which are naturally permissive to SARS-CoV-2 replication and restitute the human COVID-19 physiopathology.

More recently, we set up a new generation of this LV vaccine encoding for a stabilized prefusion of the Scov-2. This prefusion Scov-2 variant: (i) is deleted of 675-QTQTNSPRRAR- 685 sequence encompassing the RRAR furin cleavage site at the boundary of S1/S2 subunits, in order to limit its conformational dynamics and to maintain better exposure of the SI B-cell epitopes (McCallum et al., 2020) and (ii) harbors K986P and V987P consecutive proline substitutions in S2, within the hinge loop between the central helix and heptad repeat 1 (HR1) (“SAF2P”) for an improved half-life, the sequence also harbors the K ⁹⁸⁶ P and V ⁹⁸⁷ P consecutive proline substitutions in S2 (Figure 12A) (Walls et al., 2020). C57BL/6 mice primed (i.m.) and boosted (i.n.) with LV encoding the wild type or prefusion Scov-2 possessed high serum titers of anti-Scov-2 IgG (Figure 12B), high titers of anti-Scov-2 IgG and IgA in the lung extracts (Figure 12C), and comparable sero-neutralizing activity (Figure 12D). These results indicate that the modifications in the prefusion form does not impact positively or negatively its capacity to induce Ab responses against native Scov-2.

We then evaluated the vaccine efficacy of LV::S in B6.K18-hACE2 ^{IP THV} mice. In a first set of experiments with these mice, we used an integrative version of the vector. Individuals (n = 6/group) were primed i.m. with 1 x 10 ⁷ TU/mouse of LV::S or an empty LV (sham) at wk 0 and then boosted i.n. at wk 3 with the same dose of the same vectors (Figure 4A). Mice were then challenged with 0.3 x 10 ⁵ TCIDso/mouse the ancestral SARS-CoV-2 at wk 5. A high serum neutralizing activity was detected in LV::S-vaccinated mice (Figure 4B). Many studies use PFU counting to determine viral loads in vaccine efficacy studies. We noticed that large amounts of NAbs in the lungs of intranasally vaccinated individuals, although not necessarily spatially in contact with circulating viral particles in live animals, can come to contact with and neutralize viral particles in the lung homogenates in vitro, causing the PFU assay to underestimate the amounts of cultivable viral particles. Therefore, in the following studies, we evaluated the viral contents/replication by use of E or Esg qRT-PCR. In the lungs, but also in the brain, vaccination conferred complete protection against SARS-CoV2-2 replication, maintaining the viral RNA content close to the input level (Figure 4C top). Lung viral RNA content assessed by Esg qRT-PCR, did not detect any viral replication in vaccinated mice (Figure 4C bottom). Remarkably, Esg qRT-PCR quantitation of viral RNA contents in brain detected no copies of this replication-related SARS-CoV-2 RNA in LV::S- vaccinated mice versus J .55 ± 7.84) x 10 ⁹ copies in the brain of the sham-vaccinated controls (Figure 4C bottom).

At 3 dpi, cytometric investigation of the lung innate immune cell subsets (Figure 9A) detected significantly lower proportions of NK cells (CDl lb ^mt NKp46 ⁺ ) and neutrophils (CDl lb ⁺ CD24 ⁺ SiglecF’ Ly6G ⁺ ) among the lung CD45 ⁺ cells in the LV::S-vaccinated and protected B6.K18-hACE2 ^{IP THV} mice, than in the sham-vaccinated controls (Figure 4D). Both cell populations have been associated with enhanced lung inflammation and poor outcome in the context of COVID-19 (Cavalcante-Silva et al., 2021; Masselli et al., 2020). Frequencies of the other lung innate immune cell subsets were not significantly distinct in the protected and unprotected groups (Figure 9B). This protective anti-inflammatory effect of the vaccine was also recorded in the brain, as expression levels of the inflammatory mediators IFN-a, TNF-a, IL-5, IL-6, IL- 10, IL-12p40, CCL2, CCL3, CXCL9 and CXCL10 were significantly lower in LV::S-immunized animals than in the sham group (Figure 4E). In the lungs, where SARS-CoV- 2 infection in non- or sham-vaccinated animals does not induce strong cytokine and chemokine expression (Figures 3C and Figure 9C), qRT-PCR analysis rather detected a modest increase in the level of factors classically produced during T-cell responses, such as TNF-a and IL-2 (Figure 9C), which probably results from the vaccine immunogenicity. Sham-vaccinated and challenged B6.K18-hACE2 ^{IP THV} mice reached the humane endpoint, being hunched and lethargic with ruffled hair coat, at 3 dpi while the LV::S-vaccinated counterparts had no detectable symptoms. Therefore, an i.m.-i.n. prime-boost with LV::S prevents SARS-CoV-2 replication in both lung and CNS and inhibits virus-mediated lung infiltration, as well as neuroinflammation.

Immune response and protection in LV::S-vaccinated B6.K18-hACE2 ^{IP THV} mice

For further characterization of the protective properties of LV::S in B6.K18-hACE2 ^{IP THV} mice, we used the safe and non-integrative version of LV (Ku et al., 2021a). Thus, “LV::S” hereafter refers to this non-integrative version. B6.K18-hACE2 ^{IP THV} mice were primed i.m. at wk 0 and boosted i.n. at wk 5 with LV::S. Sham-vaccinated controls received an empty LV following the same regimen. At wk 7, IFN-y-producing CD8 ⁺ T cells, specific to several Scov- 2 epitopes, were detected in the lungs (Figure 5 A) and spleen (Figure 13A left) of FV::S- vaccinated mice. Small numbers of IFN-y-producing CD4 ⁺ T cells were also detected in the spleen (Figure 133 A right) and lungs (Figure 13B) of these mice. The proportion of effector memory (Tern) and resident memory (Trm) cells among CD8 ⁺ T cells of the lung was higher in FV::S i.m.-i.n.-vaccinated mice than in their sham counterparts (Figure 5B, C). By use of a H-2D ^b -Scov-2:538-546 dextramer, we further focused on a fraction of Scov-2-specific CD8 ⁺ T cells in the lungs of FV::S- or sham- vaccinated mice (Figure 5D, E). In contrast to EV::S- vaccinated mice, no dextramer ⁺ cells were detected in lung CD8 ⁺ T cells of the sham group. Inside this specific CD8 ⁺ T-cell subset, the proportions of central memory (Tcm) and Tern were comparable and a Trm subset was identifiable. High titers of serum and lung anti-Scov- 2 IgG and IgA (Figure 5F), and notable serum and lung SARS-CoV-2 neutralizing activity (Figure 5G) were detected in EV::S-vaccinated mice.

To assess the impact of EV::S vaccination route on brain or lung protection in this murine model, B6.K18-hACE2 ^{IP THV} mice were vaccinated by the i.m. or i.n. route at wk 0 and then left untreated or boosted by the i.m. or i.n. route at wk 5. Mice were challenged with SARS- CoV-2 at wk 7. At 3 dpi, the highest brain protection was observed in mice that were primed i.m. or i.n. and boosted i.n. (Figure 6A). An i.m.-i.m. prime-boost or a single i.m. or i.n. immunization with EV::S was not sufficient to significantly reduce the viral RNA content in the brain. In the lungs, a single i.m. or i.n. administration of EV::S failed to confer protection in the lungs of these highly susceptible B6.K18-hACE2 ^{IP THV} model (Figure 6A). The primeboost vaccination regimen led to the highest levels of lung protection, regardless of the immunization route tested. In nasal washes from the EV::S i.m.-i.n. immunized group, viral RNA contents were lower than in the sham group, although the difference did not reach statistical significance (Figure 6A). This result is consistent with the observation that systemic or mucosal immune responses significantly reduces viral loads and tissue damage in the lungs of hamsters intranasally challenged with SARS-CoV-2, but not in their nasal turbinate (Zhou et al., 2021). Administration of a single i.n. dose of the chimpanzee adenovirus-vectorized SARS-CoV-2 (ChAd-SARS-CoV-2-S) vaccine to wild-type C57BE/6 mice, pretreated with a hACE2-encoding serotype 5 adenoviral vector (Ad5::hACE2) prior to SARS-CoV-2 challenge, resulted in complete elimination of viral RNA from nasal washes, measured at 4-8 dpi (Hassan et al., 2020). The discrepancy between these results in Ad5::hACE2-pretreated mice and those observed here in B6.K18-hACE2 ^{IP THV} mice, may be explained by the differences in the characteristics of the murine models used and the time points studied. I.n. immunization of hamsters with ChAd-SARS-CoV-2-S also resulted in minimal or no viral RNA content in nasal swabs and nasal olfactory neuroepithelium (Bricker et al., 2021). However, in rhesus monkeys, ChAd-SARS-CoV-2-S i.n. vaccination did not result in significant reduction of the viral RNA contents in nasal swabs at 3 and 5 dpi, although statistical significance was reached at 7 dpi (Hassan et al., 2021). Thus, the differences in pre- clinical models and the kinetics studied appear to well impact the reduction of viral loads in the upper respiratory tract.

At 3 dpi, H&E analysis of the lung sections in the sham group showed the same kind of lesions detailed in Figure 2B-E. Compared to the sham group, inflammation seriousness and interstitial syndrome were reduced in the LV::S-vaccinated mice, even if some degree of inflammation was present (Figure 6B, C). The inflamed zones from LV::S- and sham- vaccinated controls contained Ncov-2 antigen detected by IHC study of contiguous lung sections (Figure 6B), indicating that, even if the virus replication has been largely reduced in the i.m.- i.n. vaccinated mice (Figure 6A), the infiltration and virus remnants have not yet been completely resorbed at the early time point of 3 dpi.

We also detected higher density of CD3 ⁺ T cells in the olfactory bulbs of EV::S vaccinated and protected mice than in the sham individuals (Figure 7A). As expected with this EV vaccine, the T-cell response was polarized towards the CD8 ⁺ compartment, as evidenced by the higher proportion of CD8 ⁺ T cells in the olfactory bulbs of protected animals (Figure 7B) and by the presence of high amounts of anti-Scov-2 CD8 ⁺ T-cell responses in the spleen, while specific CD4 ⁺ T cells were few (Figure 13 A). A very few specifically reacting CD4 ⁺ T cells was found in the lungs (Figure 13B), and in the olfactory bulb, CD4 ⁺ T cells had no distinctive activated or migratory phenotype, as assessed by their surface expression of CD69 or CCR7 (Figure 13C). In line with the absence of CCR7 expression on these T cells, and unlike Murine Hepatitis Virus (MHV) infection (Cupovic et al., 2016), we saw no up-regulation of CCE19 and CCE21 (CCR7 ligands) in the brain, regardless of the protected status of the mice (Figure 13D). At 3 dpi, qRT-PCR analysis of olfactory bulbs detected very low levels of inflammation, ranging from -2 to +2 log2 fold change compared with untreated negative controls, with no significant difference between the EV::S and sham groups (Figure 13E). Compared to the EV::S- vaccinated and protected group, there were more neutrophils (CDl lb ⁺ Ly6C ⁺ Ly6G ⁺ ) in the olfactory bulbs (Figure 7C) and inflammatory monocytes (CD1 lb ⁺ Ly6C ⁺ Ly6G ) in the brain (Figure 7D) of unprotected sham mice, reflecting a higher level of neuroinflammation in these mice. Histological examination of brains did not reveal gross alterations of the organ. However, in each of the three infected sham-vaccinated mice studied, periventricular alterations were visible. In two out of three mice studied, there were infiltrates of predominately mononuclear leukocytes (Figure 7E), and in the third mouse, a small periventricular hemorrhage was observed (not shown). Such alterations were not detected in any of the four LV::S-vaccinated and SARS -Co V-2 -challenged mice studied.

Complete cross-protection induced by LV::S against the genetically distant SARS- CoV-2 Gamma variant

A critical issue regarding the COVID-19 vaccines currently in use is the protective potency against emerging variants. To assess this question with the vaccine candidate developed here, B6.K18-hACE2 ^{IP THV} mice were primed i.m. (wk 0) and boosted i.n. (wk 5) with LV::S or sham (Figure 8A). Mice were then challenged at wk 7 with the SARS -Co V-2 Gamma strain which is among the most genetically distant SARS-CoV-2 variants so far described (Buss et al., 2021). Determination of the brain and lung viral loads at 3 dpi demonstrated that prime-boost vaccination with LV encoding the Scov-2 from the ancestral sequence induced full protection of the brain and lungs against SARS-CoV-2 Gamma (Figure 8B). Studies involving H&E and IHC staining of serial lung sections were performed to visualize the Ncov-2 antigen in the tissue and to localize it with respect to the inflammatory foci (Figure 10A, B). H&E images did not reveal significant differences in the extent and severity of pulmonary inflammatory lesions between LV::S- and sham- vaccinated mice (Figure 10, rows 1 and 3). However, within the inflammatory areas, as inferred from the contiguous H&E-stained sections, Ncov-2 ⁺ patches were readily discernable in lungs of sham mice, even at low magnification, while they were less frequent in LV::S-vaccinated mice (Figure 10A, rows 2 and 4). Moreover, the brains of infected sham controls contained multiple areas positive for Ncov-2 staining and enumeration at the single cell level revealed significantly less Ncov-2 ⁺ cells in the brains of LV::S-vaccinated mice (Figure 10C, D).

The markedly decreased ability of the sera of LV::S-vaccinated mice to neutralize SBeta Or So mm pseudo-viruses, compared to SAncestrai, SD614G or SAipha pseudo-viruses, (Figure 8C), raised the possibility of T-cell involvement in this total protection. To evaluate this possibility, we vaccinated C57BL/6 WT or 11MT KO mice following the same i.m.-i.n. protocol as above (Figure 14A). 11MT KO are deficient in mature B-cell compartment and therefore lack Ig/antibody response (Kitamura et al., 1991). To make these non-transgenic mice permissive to SARS-CoV-2 replication, they were pre-treated 4 days before the SARS-CoV-2 challenge with 3 x 10 ⁸ IGU of Ad5::hACE2 (Ku et al., 2021a). Determination of lung viral loads at 3 dpi showed complete protection of the lungs in vaccinated WT mice as well as a highly significant protection in vaccinated iiMT KO mice (Figure 8E). This observation determined that B-cell independent and antigen-specific cellular immunity, specifically the T-cell response, plays a major role in LV-mediated protection. This is consistent with the strong T- cell responses induced by LV::S at the systemic level (Figure 8F) and in the lungs (Figure 5A- E), and the recruitment of CD8 ⁺ T cells in the olfactory bulbs, detectable in vaccinated and challenged mice (Figure 7A, B, Figure 8D). Importantly, all murine and human CD8 ⁺ T-cell epitopes identified on the ancestral Scov-2 sequence are preserved in the mutated Scov-2 Gamma (Table SI). These observations indicate the strong potential of LV 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 (Hoffmann et al., 2021), the so far identified T-cell epitopes have not been impacted by mutations accumulated in the Scov-2 of the emerging variants.

Discussion LV-based platforms emerged recently as a powerful vaccination approach against COVID-19, notably when used as a systemic prime followed by mucosal i.n. boost, inducing sterilizing immunity against lung SARS-CoV-2 infection in preclinical animal models (Ku et al., 2021a). In the present study, to investigate the efficacy of vaccine candidates, we generated a new transgenic mouse model, using the LV-based transgenesis approach (Nakagawa and Hoogenraad, 2011). The ILV used in this strategy encodes for hACE2 under the control of the cytokeratin KI 8 promoter, i.e., the same promoter as previously used by Perlman’s team to generate B6.K18-ACE2 ^2Prlnm/JAX mice (McCray et al., 2007), with a few adaptations to the lentiviral FLAP transfer plasmid. However, the new B6.K18-hACE2 ^{IP THV} mice have certain distinctive features, as they express much higher levels of hACE2 mRNA in the brain and display markedly increased brain permissiveness to SARS-CoV-2 replication, in parallel with a substantial brain inflammation and development of a lethal disease in <4 days post infection. These distinctive characteristics can arise from differences in the hACE2 expression profile due to: (i) alternative insertion sites of ILV into the chromosome compared to naked DNA, and/or (ii) different effect of the Woodchuck Posttranscriptional Regulatory Element (WPRE) versus the alfalfa virus translational enhancer (McCray et al., 2007), in B6.K18-hACE2 ^IP-THV and B6.K18-ACE2 ^2Prlnm/JAX animals, respectively (Figure 11). Other reported hACE2 humanized mice express the transgene under: (i) murine ACE2 promoter, without reported hACE2 mRNA expression in the brain (Yang et al., 2007), (ii) “hepatocyte nuclear factor-3/forkhead homologue 4” (HFH4) promoter, i.e., “HFH4-hACE2” C3B6 mice, in which lung is the principal site of infection and pathology (Jiang et al., 2020; Menachery et al., 2016), and (iii) “CAG” mixed promoter, i.e. “AC70” C3H x C57BL/6 mice, in which hACE2 mRNA is expressed in various organs including lungs and brain (Tseng et al., 2007). Comparison of AC70 and B6.K18-hACE2 ^{IP THV} mice could yield information to assess the similarities and distinctions of these two models. The B6.K18-hACE2 ^{IP THV} murine model not only has broad applications in COVID-19 vaccine studies, but also provides a unique rodent model for exploration of COVID-19-derived neuropathology. Based on the substantial permissiveness of the brain to SARS-CoV-2 replication and development of a lethal disease, this pre-clinical model can be considered as even more stringent than the golden hamster model. The report of a new transgenic mouse to provide a model to assess the efficiency of vaccine candidates against the highly critical neurological component of the disease is an important breakthrough. This new and unique model will also be beneficial for the research community to have an accelerated understanding on the immune protection against neural COVID-19 disease, so far a neglected niche due to the lack of a model.

The source of neurological manifestations associated with COVID-19 in patients with comorbid conditions can be: (i) direct impact of SARS-CoV-2 on CNS, (ii) infection of brain vascular endothelium and, (iii) uncontrolled anti-viral immune reaction inside CNS. ACE2 is expressed in human neurons, astrocytes and oligodendrocytes, located in middle temporal gyrus and posterior cingulate cortex, which may explain the brain permissiveness to SARS-CoV-2 in patients (Song et al., 2020). Previous reports have demonstrated that respiratory viruses can invade the brain through neural dissemination or hematogenous route (Desforges et al., 2014). Besides that, the direct connection of olfactory system to the CNS via the frontal cortex also represents a plausible route for brain invasion (Mori et al., 2005). Neural transmission of viruses to the CNS can occur as a result of direct neuron invasion through axonal transport in the olfactory mucosa. Subsequent to intraneuronal replication, the virus spreads to synapses and disseminate to anatomical CNS zones receiving olfactory tract projections (Berth et al., 2009; Koyuncu et al., 2013; Roman et al., 2020; Zubair et al., 2020). However, the detection of viral RNA in CNS regions without connection with olfactory mucosa suggests the existence of another viral entry into the CNS, including migration of SARS -Co V-2 -infected immune cells crossing the hemato-encephalic barrier or direct viral entry pathway via CNS vascular endothelium (Meinhardt et al., 2021). Although at steady state, viruses cannot penetrate into the brain through an intact blood-brain barrier (Berth et al., 2009), inflammation mediators which are massively produced during cytokine/chemokine storm, notably TNF-a and CCL2, can disrupt the integrity of blood-brain barrier or increase its permeability, allowing paracellular blood-to-brain transport of the virus or virus-infected leukocytes (Aghagoli et al., 2020; Hu et al., 2011). The use of the highly stringent B6.K18-hACE2 ^{IP THV} mice demonstrated the importance of i.n. booster immunization for inducing sterilizing protection of CNS by our LV- based vaccine candidate developed against SARS-CoV-2. Olfactory bulb may control viral CNS infection through the action of local innate and adaptive immunity (Durrant et al., 2016). In line with these observations, we detected increased frequencies of CD8 ⁺ T cells at this anatomically strategic area in i.m.-i.n. vaccinated and protected mice. In addition, substantial reduction in the inflammatory mediators was also found in the brain of the i.m.-i.n. vaccinated and protected mice, as well as decreased proportions of neutrophils and inflammatory monocytes respectively in the olfactory bulbs and brain. Regardless of the mechanism of the SARS-CoV-2 entry into the brain, we provide evidence of the full protection of the CNS against SARS-CoV-2 by i.n. booster immunization with LV::S.

Table SI. Scov-2-derived murine and human T-cell epitopes.

Murine CD8 ⁺ -T cell epitopes have been previously identified (Ku et al., 2021b). Human T-cell epitopes are from the updated Immudex data base (https://www.immudex.com/media/ 1535/tfl 19203 -published- list-of-covid- 19-t- cell-epitopes.pdf). The a.a. indicated in bold are those substituted or deleted by mutations occurred in the Scov-2 of Alpha, Beta, Gamma, Delta or Omicron SARS-CoV-2 variants of concern. Highlighted a.a. represent (the most probable) anchoring residues. Table S2. Sequences of prefusion Scov-2, as encoded by LV vaccinal vectors (SEQ ID No.64)

Prefusion a.a. sequence

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLP FFS NVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIV NNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMD LEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QT LLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSET KCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRIS N CVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIA DYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGST PCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVWLSFELLHAPATVCGPKKSTNLVKN KCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVS VITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEH VNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIP T NFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDK NTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQ Y GDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIP FAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDWNQN AQALNTLVKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRA A EIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGWFLHVTYVPAQEKN FTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDWIGIVN NTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASWNIQKEIDRLNEVAKNLN ESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCC KFDEDDSEPVLKGVKLHYT

The deleted sequence encompassing the furin cleavage site and double proline substitution in S2 are indicated in bold.

Table S3. Sequences of primers used to genotype B6.K18-hACE2 ^{IP THV} transgenic mice.

Primers SEQ ID No. hACE2 Fw TCCTAACCAGCCCCCTGTT 65 hACE2 Rv TGACAATGCCAACCA CTATCACT 66

PKD1 Fw GGCTGCTGAGCGTCTGGTA 67

PKD1 Rv CCAGGTCCTGCGTGTCTGA 68

GAPDH-ACE2 69

Fw GCCCAGAACATCATCCCTGC

GAPDH-ACE2 Rv CCGTTCAGCTCTGGGATGACC 70

Table S4. Sequences of primers used to quantitate SARS-CoV-2 RNA content by qRT-PCR.

Primer/Probe DNA Sequence SEQ ID No.

“E-Sarbeco” Fw 5’-ACAGGTACGTTAATAGTTAATAGCGT- 71

3’

“E-Sarbeco” Rv 5’-ATATTGCAGCAGTACGCACACA-3’ 72

“E-Sarbeco” 5’-FAM- 73

ACACTAGCCATCCTTACTGCGCTTCG- BHQ-1-3’

“E-sgmRNA” 5’-CGATCTCTTGTAGATCTGTTCTC-3’ 74

Fw 7. References

Aghagoli G, Gallo Marin B, Katchur NJ, Chaves-Sell F, Asaad WF, Murphy SA (2020) Neurological Involvement in COVID-19 and Potential Mechanisms: A Review. Neurocrit Care

Ali Awan H, Najmuddin Diwan M, Aamir A, Ali M, Di Giannantonio M, Ullah I, Shoib S, De Berardis D (2021) SARS-CoV-2 and the Brain: What Do We Know about the Causality of 'Cognitive COVID? J Clin Med 10

Anna F, Goyard S, Lalanne Al, Nevo F, Gransagne M, Souque P, Louis D, Gillon V, Turbiez

I, Bidard FC et al (2020) High seroprevalence but short-lived immune response to SARS- CoV-2 infection in Paris. Eur J Immunol

Arce F, Rowe HM, Chain B, Lopes L, Collins MK (2009) Lentiviral vectors transduce proliferating dendritic cell precursors leading to persistent antigen presentation and immunization. Mol Ther 17: 1643-1650

Berth SH, Leopold PL, Morfini GN (2009) Virus-induced neuronal dysfunction and degeneration. Front Biosci (Landmark Ed) 14: 5239-5259

Bourgonje AR, Abdulle AE, Timens W, Hillebrands JL, Navis GJ, Gordijn SJ, Bolling MC, Dijkstra G, Voors AA, Osterhaus AD et al (2020) Angiotensin-converting enzyme 2 (ACE2), SARS-CoV-2 and the pathophysiology of coronavirus disease 2019 (COVID-19). J Pathol 251 : 228-248

Bricker TL, Darling TL, Hassan AO, Harastani HH, Soung A, Jiang X, Dai YN, Zhao H, Adams LJ, Holtzman MJ et al (2021) A single intranasal or intramuscular immunization with chimpanzee adenovirus-vectored SARS-CoV-2 vaccine protects against pneumonia in hamsters. Cell Rep 36: 109400

Brown EL, Essigmann HT (2021) Original Antigenic Sin: the Downside of Immunological Memory and Implications for COVID- 19. mSphere 6

Buss LF, Prete CA, Jr., Abrahim CMM, Mendrone A, Jr., Salomon T, de Almeida-Neto C, Franca RFO, Belotti MC, Carvalho M, Costa AG et al (2021) Three-quarters attack rate of SARS-CoV-2 in the Brazilian Amazon during a largely unmitigated epidemic. Science 371 : 288-292

Cavalcante-Silva LHA, Carvalho DCM, Lima EA, Galvao J, da Silva JSF, Sales-Neto JM, Rodrigues-Mascarenhas S (2021) Neutrophils and COVID-19: The road so far. Int Immunopharmacol 90: 107233

Chandrashekar A, Liu J, Martinet AJ, McMahan K, Mercado NB, Peter L, Tostanoski LH, Yu

J, Maliga Z, Nekorchuk M et al (2020) SARS-CoV-2 infection protects against rechallenge in rhesus macaques. Science May 2020:eabc4776 doi: 101126/scienceabc4776 PMID: 32434946

Chen R, Wang K, Yu J, Howard D, French L, Chen Z, Wen C, Xu Z (2020) The spatial and cell-type distribution of SARS-CoV-2 receptor ACE2 in human and mouse brain. BioRxiv Chow YH, O'Brodovich H, Plumb J, Wen Y, Sohn KJ, Lu Z, Zhang F, Lukacs GL, Tanswell AK, Hui CC et al (1997) Development of an epithelium-specific expression cassette with human DNA regulatory elements for transgene expression in lung airways. Proc Natl Acad Sci USA 94: 14695-14700

Corman V, Bleicker T, Brunink S, Drosten C (2020) Diagnostic detection of 2019-nCoV by real-time RT-PCR. https: //www'whoint/docs/default-source/coronaviruse/protocol-v2-lp df Cousin C, Oberkampf M, Felix T, Rosenbaum P, Weil R, Fabrega S, Morante V, Negri D, Cara A, Dadaglio G et al (2019) Persistence of Integrase-Deficient Lentiviral Vectors Correlates with the Induction of STING-Independent CD8(+) T Cell Responses. Cell Rep 26: 1242-1257 el247

Cupovic J, Onder L, Gil-Cruz C, Weiler E, Caviezel-Fimer S, Perez-Shibayama C, Rulicke T, Bechmann I, Ludewig B (2016) Central Nervous System Stromal Cells Control Local CD8(+) T Cell Responses during Virus-Induced Neuroinflammation. Immunity 44: 622-633 Desforges M, Le Coupanec A, Stodola JK, Meessen-Pinard M, Talbot PJ (2014) Human coronaviruses: viral and cellular factors involved in neuroinvasiveness and neuropathogenesis. Virus Res 194: 145-158

Di Nunzio L, Lelix T, Arhel NJ, Nisole S, Chameau P, Beignon AS (2012) HIV-derived vectors for therapy and vaccination against HIV. Vaccine 30: 2499-2509

Dogan RI, Getoor L, Wilbur WJ, Mount SM (2007) Leatures generated for computational splice-site prediction correspond to functional elements. BMC Bioinformatics 8: 410

Durrant DM, Ghosh S, Klein RS (2016) The Olfactory Bulb: An Immunosensory Effector Organ during Neurotropic Viral Infections. ACS Chem Neurosci 7: 464-469

Eotuhi M, Mian A, Meysami S, Raji CA (2020) Neurobiology of COVID-19. J Alzheimer s Dis 76: 3-19

Glass WG, Subbarao K, Murphy B, Murphy PM (2004) Mechanisms of host defense following severe acute respiratory syndrome-coronavirus (SARS-CoV) pulmonary infection of mice. J Immunol 173: 4030-4039

Hassan AO, Case JB, Winkler ES, Thackray LB, Kafai NM, Bailey AL, McCune BT, Fox JM, Chen RE, Alsoussi WB et al (2020) A SARS-CoV-2 Infection Model in Mice Demonstrates Protection by Neutralizing Antibodies. Cell 182: 744-753 e744

Hassan AO, Feldmann F, Zhao H, Curiel DT, Okumura A, Tang-Huau TL, Case JB, Meade- White K, Callison J, Chen RE et al (2021) A single intranasal dose of chimpanzee adenovirus-vectored vaccine protects against SARS-CoV-2 infection in rhesus macaques. Cell Rep Med 2: 100230

Hoffmann M, Arora P, Gross R, Seidel A, Homich BF, Hahn AS, Kruger N, Graichen L, Hofmann-Winkler H, Kempf A et al (2021) SARS-CoV-2 variants B.1.351 and P.l escape from neutralizing antibodies. Cell

Hoffmann M, Kleine-Weber H, Schroeder S, Kruger N, Herrler T, Erichsen S, Schiergens TS, Herrler G, Wu NH, Nitsche A et al (2020) SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell 181 : 271-280 e278 https://www.who.int/en/activities/tracking-SARS-CoV-2-varian ts/

Hu B, Tai A, Wang P (2011) Immunization delivered by lentiviral vectors for cancer and infectious diseases. Immunol Rev 239: 45-61

Hu J, Jolkkonen J, Zhao C (2020) Neurotropism of SARS-CoV-2 and its neuropathological alterations: Similarities with other coronaviruses. Neurosci Biobehav Rev 119: 184-193

Jiang RD, Liu MQ, Chen Y, Shan C, Zhou YW, Shen XR, Li Q, Zhang L, Zhu Y, Si HR et al (2020) Pathogenesis of SARS-CoV-2 in Transgenic Mice Expressing Human Angiotensin- Converting Enzyme 2. Cell 182: 50-58 e58 Kitamura D, Roes J, Kuhn R, Rajewsky K (1991) A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin mu chain gene. Nature 350: 423-426 Koehler DR, Chow YH, Plumb J, Wen Y, Rafii B, Belcastro R, Haardt M, Lukacs GL, Post M, Tanswell AK et al (2000) A human epithelium-specific vector optimized in rat pneumocytes for lung gene therapy. Pediatr Res 48: 184-190

Koyuncu OO, Hogue IB, Enquist LW (2013) Virus infections in the nervous system. Cell Host Microbe 13: 379-393

Ku MW, Anna F, Souque P, Petres S, Prot M, Simon-Loriere E, Chameau P, Bourgine M (2020) A Single Dose of NILV-Based Vaccine Provides Rapid and Durable Protection against Zika Virus. Mol Ther 28: 1772-1782

Ku MW, Bourgine M, Authie P, Lopez J, Nemirov K, Moncoq F, Noirat A, Vesin B, Nevo F, Blanc C et al (2021) Intranasal vaccination with a lentiviral vector protects against SARS- CoV-2 in preclinical animal models. Cell Host Microbe 29: 236-249 e236

Lazarevic I, Pravica V, Miljanovic D, Cupic M (2021) Immune Evasion of SARS-CoV-2 Emerging Variants: What Have We Learnt So Far? Viruses 13

Lescure FX, Bouadma L, Nguyen D, Parisey M, Wicky PH, Behillil S, Gaymard A, Bouscambert-Duchamp M, Donati F, Le Hingrat Q et al (2020) Clinical and virological data of the first cases of COVID-19 in Europe: a case series. Lancet Infect Dis 20: 697-706 Li K, Wohlford-Lenane C, Perlman S, Zhao J, Jewell AK, Reznikov LR, Gibson-Corley KN, Meyerholz DK, McCray PB, Jr. (2016) Middle East Respiratory Syndrome Coronavirus Causes Multiple Organ Damage and Lethal Disease in Mice Transgenic for Human Dipeptidyl Peptidase 4. J Infect Dis 213: 712-722

Lopez J, Anna F, Authie P, Pawlik A, Ku MW, Blanc C, Souque P, Moncoq F, Noirat A, Hardy D et al An optimized lentiviral vector induces CD4+ T-cell immunity and predicts a booster vaccine against tuberculosis. Submitted

Lopez J, Anna F, Authie P, Pawlik A, Ku MW, Blanc C, Souque P, Moncoq F, Noirat A, Sougakoff W et al. An Optimized Poly-antigenic Lentiviral Vector Induces Protective CD4+ T-Cell Immunity and Predicts a Booster Vaccine against Mycobacterium tuberculosis. (Submitted).

Lund FE, Randall TD (2021) Scent of a vaccine. Science 373: 397-399

Mao L, Jin H, Wang M, Hu Y, Chen S, He Q, Chang J, Hong C, Zhou Y, Wang D et al (2020) Neurologic Manifestations of Hospitalized Patients With Coronavirus Disease 2019 in Wuhan, China. JAMA Neurol 77: 683-690

Masselli E, Vaccarezza M, Carubbi C, Pozzi G, Presta V, Mirandola P, Vitale M (2020) NK cells: A double edge sword against SARS-CoV-2. Adv Biol Regul 77: 100737

McCallum M, Walls AC, Bowen JE, Corti D, Veesler D (2020) Structure-guided covalent stabilization of coronavirus spike glycoprotein trimers in the closed conformation. Nat Struct Mol Biol 27: 942-949

McCray PB, Jr., Pewe L, Wohlford-Lenane C, Hickey M, Manzel L, Shi L, Netland J, Jia HP, Halabi C, Sigmund CD et al (2007) Lethal infection of K18-hACE2 mice infected with severe acute respiratory syndrome coronavirus. J Virol 81 : 813-821

Meinhardt J, Radke J, Dittmayer C, Franz J, Thomas C, Mothes R, Laue M, Schneider J, Brunink S, Greuel S et al (2021) Olfactory transmucosal SARS-CoV-2 invasion as a port of central nervous system entry in individuals with COVID-19. Nat Neurosci 24: 168-175 Menachery VD, Yount BL, Jr., Sims AC, Debbink K, Agnihothram SS, Gralinski LE, Graham RL, Scobey T, Plante JA, Royal SR et al (2016) SARS-like WIVl-CoV poised for human emergence. Proc Natl Acad Sci USA 113: 3048-3053

Moore JP, Offit PA (2021) SARS-CoV-2 Vaccines and the Growing Threat of Viral Variants. JAMA 325: 821-822 Mori I, Nishiyama Y, Yokochi T, Kimura Y (2005) Olfactory transmission of neurotropic viruses. J Neurovirol 11 : 129-137

Nakagawa T, Hoogenraad CC (2011) Lentiviral transgenesis. Methods Mol Biol 693: 117-142 Netland J, Meyerholz DK, Moore S, Cassell M, Perlman S (2008) Severe acute respiratory syndrome coronavirus infection causes neuronal death in the absence of encephalitis in mice transgenic for human ACE2. J Virol 82: 7264-7275

Politi LS, Salsano E, Grimaldi M (2020) Magnetic Resonance Imaging Alteration of the Brain in a Patient With Coronavirus Disease 2019 (CO VID-19) and Anosmia. JAMA Neurol T. 1028-1029

Roman GC, Spencer PS, Reis J, Buguet A, Faris MEA, Katrak SM, Lainez M, Medina MT, Meshram C, Mizusawa H et al (2020) The neurology of COVID-19 revisited: A proposal from the Environmental Neurology Specialty Group of the World Federation of Neurology to implement international neurological registries. J Neurol Sci 414: 116884

Rosenberg SA, Zhai Y, Yang JC, Schwartzentruber DJ, Hwu P, Marincola FM, Topalian SL, Restifo NP, Seipp CA, Einhorn JH et al (1998) Immunizing patients with metastatic melanoma using recombinant adenoviruses encoding MART-1 or gplOO melanoma antigens. J Natl Cancer Inst 90: 1894-1900

Schirmbeck R, Reimann J, Kochanek S, Kreppel F (2008) The immunogenicity of adenovirus vectors limits the multispecificity of CD8 T-cell responses to vector-encoded transgenic antigens. Mol Ther 16: 1609-1616

Shang J, Ye G, Shi K, Wan Y, Luo C, Aihara H, Geng Q, Auerbach A, Li F (2020) Structural basis of receptor recognition by SARS-CoV-2. Nature 581 : 221-224

Song E, Zhang C, Israelow B, Lu-Culligan A, Prado AV, Skriabine S, Lu P, Weizman OE, Liu F, Dai Y et al (2020) Neuroinvasion of SARS-CoV-2 in human and mouse brain. bioRxiv Sterlin D, Mathian A, Miyara M, Mohr A, Anna F, Claer L, Quentric P, Fadlallah J, Devilliers H, Ghillani P et al (2020) IgA dominates the early neutralizing antibody response to SARS-CoV-2. Sci Transl Med

Tarke A, Sidney J, Methot N, Zhang Y, Dan JM, Goodwin B, Rubiro P, Sutherland A, da Silva Antunes R, Frazier A et al (2021) Negligible impact of SARS-CoV-2 variants on CD4 (+) and CD8 (+) T cell reactivity in COVID-19 exposed donors and vaccinees. bioRxiv Tostanoski LH, Wegmann F, Martinet AJ, Loos C, McMahan K, Mercado NB, Yu J, Chan CN, Bondoc S, Starke CE et al (2020) Ad26 vaccine protects against SARS-CoV-2 severe clinical disease in hamsters. Nat Med 26: 1694-1700

Tseng CT, Huang C, Newman P, Wang N, Narayanan K, Watts DM, Makino S, Packard MM, Zaki SR, Chan TS et al (2007) Severe acute respiratory syndrome coronavirus infection of mice transgenic for the human Angiotensin-converting enzyme 2 virus receptor. J Virol 81 : 1162-1173 van Doremalen N, Purushotham JN, Schulz JE, Holbrook MG, Bushmaker T, Carmody A, Port JR, Yinda CK, Okumura A, Saturday G et al (2021) Intranasal ChAdOxl nCoV- 19/AZD1222 vaccination reduces viral shedding after SARS-CoV-2 D614G challenge in preclinical models. Sci Transl Med 13 von Weyhem CH, Kaufmann I, Neff F, Kremer M (2020) Early evidence of pronounced brain involvement in fatal COVID-19 outcomes. Lancet 395: el09

Walls AC, Park YJ, Tortorici MA, Wall A, McGuire AT, Veesler D (2020) Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein. Cell 181 : 281-292 e286 Whittaker A, Anson M, Harky A (2020) Neurological Manifestations of COVID-19: A systematic review and current update. Acta Neurol Scand 142: 14-22

Wijeratne T, Crewther S (2020) Post-COVID 19 Neurological Syndrome (PCNS); a novel syndrome with challenges for the global neurology community. J Neurol Sci 419: 117179 Winkler ES, Bailey AL, Kafai NM, Nair S, McCune BT, Yu J, Fox JM, Chen RE, Earnest JT, Keeler SP et al (2020) SARS-CoV-2 infection of human ACE2-transgenic mice causes severe lung inflammation and impaired function. Nat Immunol 21 : 1327-1335

Wolfel R, Corman VM, Guggemos W, Seilmaier M, Zange S, Muller MA, Niemeyer D, Jones TC, Vollmar P, Rothe C et al (2020) Virological assessment of hospitalized patients with COVID-2019. Nature 581 : 465-469

Xu J, Lazartigues E (2020) Expression of ACE2 in Human Neurons Supports the Neuro- Invasive Potential of COVID-19 Virus. Cell Mol Neurobiol

Yang XH, Deng W, Tong Z, Liu YX, Zhang LF, Zhu H, Gao H, Huang L, Liu YL, Ma CM et al (2007) Mice transgenic for human angiotensin-converting enzyme 2 provide a model for SARS coronavirus infection. Comp Med 57: 450-459

Zennou V, Petit C, Guetard D, Nerhbass U, Montagnier L, Chameau P (2000) HIV-1 genome nuclear import is mediated by a central DNA flap. Cell 101 : 173-185

Zhou D, Chan JF, Zhou B, Zhou R, Li S, Shan S, Liu L, Zhang AJ, Chen SJ, Chan CC et al (2021) Robust SARS-CoV-2 infection in nasal turbinates after treatment with systemic neutralizing antibodies. Cell Host Microbe 29: 551-563 e555

Zhuang Z, Lai X, Sun J, Chen Z, Zhang Z, Dai J, Liu D, Li Y, Li F, Wang Y et al (2021) Mapping and role of T cell response in SARS-CoV-2 -infected mice. J Exp Med 218

Zubair AS, McAlpine LS, Gardin T, Farhadian S, Kuruvilla DE, Spudich S (2020)

Neuropathogenesis and Neurologic