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  • A61P31/12—Antivirals
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  • C12N15/86—Viral vectors
• • A—HUMAN NECESSITIES
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  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
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  • A61K2039/5256—Virus expressing foreign proteins
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  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/53—DNA (RNA) vaccination
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
  • A61K2039/541—Mucosal route
  • A61K2039/543—Mucosal route intranasal
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  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
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  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
  • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
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  • C12N2770/00011—Details
  • C12N2770/20011—Coronaviridae
  • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

• the invention relates to the field of immunity against coronaviruses.
• the invention provides a lentiviral-based immunogenic agent that is suitable for use in boost or target immunization treatment in a subject, in particular a human subject, who had previously developed an immunity against Severe Acute Respiratory Syndrome coronavirus 2 (SARS- CoV-2) based on: (i) vaccination with a first generation of vaccines against SARS-CoV-2 infection or disease such as a protein, an mRNA, an adenovirus, an inactivated virus or a protein subunit vaccine composition against SARS-CoV-2 infection or disease, in particular a protein- or an mRNA-based vaccine, or (ii) SARS-CoV-2-induced or correlated disease.
• SARS- CoV-2 Severe Acute Respiratory Syndrome coronavirus 2
• the invention accordingly concerns a lentiviral-based immunogenic agent that in particular may help overcome the deficiencies of available vaccines against SARS-CoV-2, especially may be efficient in overcoming the waning immune response or insufficient cellular memory response observed after immunization with available first generation of vaccines such as a protein, an mRNA, an adenovirus, an inactivated virus or a protein subunit vaccine, in particular protein or mRNA vaccine, by triggering a mucosal humoral and cellular immune response against coronaviruses, including a long-lasting immune response.
• vaccines such as a protein, an mRNA, an adenovirus, an inactivated virus or a protein subunit vaccine, in particular protein or mRNA vaccine
• VOCs new viral Variants of Concerns
• new effective vaccine platforms can be critical for the future primary or booster vaccines
• the inventors recently demonstrated the strong performance of a lentiviral vaccination vector (LV) encoding the full-length sequence of Spike glycoprotein (S) from the ancestral SARS-CoV-2 (LV::S), when used in systemic prime followed by intranasal (i.n.) boost in multiple preclinical models (Ku MW, et al. Cell Host Microbe, 29(2), 236-249 e236, 2021).
• LV::S ensures complete (cross) protection of the respiratory tract against ancestral SARS- CoV-2 and VOCs (Ku MW, etal. EMBO Mol Med, e14459, 2021).
• hACE2 human Angiotensin Converting Enzyme 2
• LV::S boost with LV::S is required for full protection of the central nervous system (Ku MW, et al. EMBO Mol Med, e14459, 2021).
• LV::S is intended to be used as a primary vaccine or a booster to reinforce and broaden protection against emerging VOCs with immune evasion potential (Juno JA, Wheatley AK. Nat Med, 27(11), 1874-1875, 2021).
• the duration of the protection conferred by the first generation COVID-19 vaccines is not yet well established, hardly predictable with serological laboratory tests and variable in diverse individuals and against distinct VOCs.
• the current exacerbation of the world-wide pandemic indicates that repeated booster immunizations will be needed to ensure individual and collective immunity against COVID-19.
• the LV::S vaccine candidate has a serious potential for prophylactic use against COVID-19, mainly based on its strong capacity to induce, not only strong neutralizing humoral responses, but also robust protective T-cell responses which are not impacted by the escape mutations accumulated in the SARS-CoV-2 VOCs (Ku MW, et al. EMBO Mol Med, e14459, 2021).
• heterologous prime-boost strategies may reinforce better the specific adaptive immune responses and long-term protection, without triggering/reinforcing vector-specific immunity or the risk of aggravation of possible reactogenicity to the vaccines themselves or excipients.
• sequence of the Spike antigen has to be adapted according to the dynamics of SARS-CoV-2 VOC emergence in order to induce the greatest neutralization breadth.
• protection against symptomatic SARS-CoV-2 infection is mainly related to sero-neutralizing activity
• protection against severe COVID-19 involves CD8 + T-cell immunity.
• an appropriate B- and T-cell vaccine platform including an adapted Spike sequence, is of utmost interest at the current step of the pandemic.
• LV::S could be remarkably suitable to be used as a heterologous i.n. booster vaccine, to reinforce and broaden protection against the SARS-CoV- 2 in particular against its known and emerging VOCs (including but not limited to Alpha, Beta, Gamma, Delta and Omicron variants of SARS-CoV-2), while collective immunity in early vaccinated nations is waning only a few months after completion of the initial immunization, and while new waves of infections are on the rise (Juno JA, Wheatley AK. Nat Med, 27(11), 1874- 1875, 2021).
• VOCs including but not limited to Alpha, Beta, Gamma, Delta and Omicron variants of SARS-CoV-2
• LVs for use in the present invention are in particular non-integrating, non-replicative, non-cytopathic and negligibly inflammatory (Hu B, Tai A, Wang P. Immunol Rev, 239(1), 45- 61, 2011; Ku MW, Charneau P, Majlessi L. Expert Rev Vaccines, 1-16, 2021).
• VSV-G Vesicular Stomatitis Virus
• the latter are mainly non-dividing cells and thus barely permissive to gene transfer.
• LVs possess the central property to efficiently transfer genes to the nuclei of non-dividing cells, which therefore renders possible efficient transduction of dendritic cells.
• the resulting endogenous antigen expression in these cells with unique ability to activate naive T cells correlates with outstanding ability of LV at inducing high-quality effector and memory T cells (Ku MW, et al. Commun Biol, 4(1), 713, 2021).
• VSV-G pseudo-typing also avoids LVs to be targets of preexisting vector-specific immunity in humans which is key in vaccine development (Hu B, Tai A, Wang P.
• the i.n. administration route presents well-recognized advantages of triggering mucosal IgA responses, as well as resident memory B and T lymphocytes in the respiratory tract (Lund FE, Randall TD. Science, 373(6553), 397- 399, 2021). This route has also been shown to be the most effective at reducing SARS-CoV- 2 transmission in both hamster and macaque preclinical models (van Doremalen N, et al. Sci Transl Med, 13(607), 2021). Induction of mucosal immunity by i.n.
• the inventors generated an LV encoding the down-selected Scov-2 of the Beta variant, stabilized by K 986 P and V 987 P substitutions in the S2 domain of Scov-2 (LV::S B eta- 2 p).
• mice primed and boosted intramuscularly (i.m.) with mRNA-1273 (Moderna) vaccine, and in which the (cross) sero-neutralization potential was progressively turning down
• the inventors compared the systemic and mucosal immune responses and the protective potential of an i.n. LV::SBeta-2P heterologous boost vs an i.m. mRNA-1273 (Moderna) (Jackson LA, etal.
• the invention hence relates to a pseudotyped lentiviral vector particle encoding a Spike (S) protein of a Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) or a derivative thereof for use as a heterologous boost or target immunization agent in a vaccine regimen for administration to the upper respiratory tract of a subject, in particular a human subject, who received a prime immunization with a vaccine composition against SARS-CoV-2 infection or disease selected from the group consisting of a protein, an mRNA, an adenovirus, an inactivated virus and a protein subunit vaccine composition against SARS-CoV-2 infection or disease, in particular a protein or an mRNA vaccine composition against SARS-CoV-2 infection or disease.
• SARS-CoV-2 Severe Acute Respiratory Syndrome coronavirus 2
• Spike (S) protein of SARS-CoV-2 virus is well identified in the art as an envelop- anchored glycoprotein (Walls et al, 2020, Structure, Function, and Antigenicity of the SARS- CoV-2 Spike Glycoprotein. Cell 181 :281-292 e286). More precisely, the SARS-CoV-2 S (Scov- 2) is a (180 kDa)s homotrimeric class I viral fusion protein, which engages the carboxypeptidase Angiotensin-Converting Enzyme 2 (ACE2), expressed on host cells. The monomer of Scov-2 protein possesses an ecto-domain, a transmembrane anchor domain, and a short internal tail.
• Scov-2 is activated by a two-step sequential proteolytic cleavage to initiate fusion with the host cell membrane. Subsequent to Sc o v-2-ACE2 interaction, which leads to a conformational reorganization, the extracellular domain of Scov-2 is first cleaved at the highly specific furin 682 RRAR 685 (SEQ ID NO: 21 ) site (Guo et al., 2020, The origin, transmission and clinical therapies on coronavirus disease 2019 (COVID-19) outbreak - an update on the status.
• the resulted subunits are constituted of: (i) S1 , which harbors the ACE2 Receptor Binding Domain (RBD), with the atomic contacts restricted to the ACE2 protease domain and also harbors main B-cell epitopes, targeted of neutralizing antibodies (NAbs) (Walls et al., 2020), and (ii) S2, which bears the membranefusion elements.
• S1 which harbors the ACE2 Receptor Binding Domain (RBD)
• NAbs neutralizing antibodies
• S2 which bears the membranefusion elements.
• the shedding of S1 renders accessible on S2 the second proteolytic cleavage site 797 R , namely S2' (Belouzard et al., 2009, Activation of the SARS coronavirus spike protein via sequential proteolytic cleavage at two distinct sites.
• TMPRSS TransMembrane Protease Serine Protease
• the S protein for expression by the lentiviral particles of the invention may originate from a SARS-CoV-2 strain and accordingly maybe characterized by an amino acid sequence that is the native sequence of the viral protein.
• the invention is performed using the S protein of known SARS-CoV-2 strains such as the S protein of the Ancestral strain (wherein the amino acid sequence is SEQ ID NO: 1 ), or of variant strains discovered later such as the Alpha, Beta, Gamma, Delta or Omicron strain (all regarded as variant strains with respect to one another).
• the invention may alternatively be performed with a derivative of the S protein, i.e., a derivative of a native S protein obtained by mutation in the amino acid sequence of the S protein, as will be disclosed herein.
• the nucleic acid encoding the S protein may have the sequence of the gene present in the viral strain of origin or may be a codon-optimized acid nucleic suitable for expression in mammalian cells, in particular in human cells.
• the nucleic acid encoding the derivative of the S protein may have the sequence deduced from the sequence of the gene of the S protein present in a viral strain and may be a codon-optimized acid nucleic suitable for expression in mammalian cells.
• the recombinant lentiviral particles (LV) used in the invention are HIV-1 -based lentiviral particles. Accordingly, where the expressions “lentiviral particle” of "LV” are used herein it is in particular directed to the HIV-1 based lentiviral particles especially LV particles pseudotyped with VSV-G protein, in particular LV as illustrated in the examples.
• boost or “boost immunization” or “boost administration” or “target immunization” refer according to the invention to an administration of the immunogenic agent that comes after a first administration of a heterologous immunization agent, in particular a heterologous vaccine, or after a second or later administration of such heterologous immunization agent or vaccine. Otherwise stated the immunization agent used according to the invention is administered to a subject who previously received a prime administration, or a prime and further one or multiple administration doses, of a heterologous immunization agent or vaccine against the same SARS-CoV-2 or against a variant strain thereof.
• the boost or target immunization is achieved through administration to the upper respiratory tract, in particular as an intranasal administration, that accordingly distinguish over administration route of a first generation of vaccines against SARS-CoV-2 infection or disease such as a protein, an mRNA, an adenovirus, an inactivated virus or a protein subunit vaccine composition against SARS-CoV-2 infection or disease, in particular protein or mRNA vaccines that most often make use of systemic, including intramuscular, intradermal or subcutaneous administration route.
• the boost or target immunization is intended to enhance, improve or lengthen the immune response previously raised and possibly to broaden such response to elicit cross-neutralization against multiple SARS-CoV-2 viruses.
• Improvement of the response may arise from the capability of the immunization agent used in the invention to elicit a mucosal response and to accordingly protect, not only the systemic sites, but also the upper and lower respiratory tracts and the central nervous system that may not have been successfully targeted or protected with heterologous vaccines against SARS-CoV-2 infection or disease such as a protein, an mRNA, an adenovirus, an inactivated virus or a protein subunit vaccine composition against SARS- CoV-2 infection or disease, in particular protein or mRNA vaccines injected via systemic routes.
• the boost administration is intended to raise crossneutralizing immune response in the subject against emerging stains of the virus.
• the boost or target immunization may be administered to a subject who received a heterologous immunization agent as disclosed herein and who had and recovered from infection by SARS-CoV-2 or disease related to such infection such as COVID-19. Additional features relating to the use of the immunization agent and to the treatment course of the subject will be disclosed in the following description.
• Administration “to the upper respiratory tract” includes any type of administration that results in delivery to the mucosa lining of the upper respiratory tract and includes in particular nasal administration.
• Administration to the upper respiratory tract includes without limitation, aerosol inhalation, nasal instillation, nasal insufflation, and all combinations thereof.
• the administration is by aerosol inhalation.
• the administration is by nasal instillation.
• the administration is by nasal insufflation.
• the pseudotyped lentiviral vector particle encoding a SARS-CoV-2 S protein or a derivative thereof is for administration as intranasal mucosal boost or target immunization in a subject who received a prime administration with a vaccine composition against SARS-CoV-2 infection or disease selected from the group consisting of a protein, an mRNA, an adenovirus, an inactivated virus and a protein subunit vaccine composition against SARS-CoV-2 infection or disease, in particular a protein or an mRNA vaccine composition against SARS-CoV-2 infection or disease.
• a vaccine composition against SARS-CoV-2 infection or disease selected from the group consisting of a protein, an mRNA, an adenovirus, an inactivated virus and a protein subunit vaccine composition against SARS-CoV-2 infection or disease, in particular a protein or an mRNA vaccine composition against SARS-CoV-2 infection or disease.
• the pseudotyped lentiviral vector particle encoding a S protein of a SARS-CoV-2 or a derivative thereof for use in accordance with the embodiments disclosed herein is further characterized by the following features: the S protein is from a SARS-CoV-2 virus which is pathogenic for a human host, in particular (i) a S protein from a SARS-CoV-2 virus selected from the group consisting of SARS-CoV-2 Ancestral strain, D614G strain, Alpha strain, Beta strain, Gamma strain, Delta strain and Omicron strain, preferably from Beta strain or Omicron strain, more preferably from Beta strain, or (ii) a S protein from a variant of said Ancestral, D614G strain, Alpha, Beta, Gamma, Delta or Omicron strains wherein such variants encode a S protein with an amino acid sequence at least 90% identical to SEQ ID NO: 1 or, the S protein is a derivative of the native S protein of one of the Ancestral, D614G
• the S protein of the Ancestral strain of SARS-CoV-2 has an amino acid sequence of SEQ ID NO: 1 and the native sequence of the polynucleotide encoding the S protein of the Ancestral strain of SARS-CoV-2 is defined in SEQ ID NO: 2.
• the S protein of the D614G strain of SARS-CoV-2 comprising said mutation 2P (SD614G-2P) has an amino acid sequence of SEQ ID NO: 4.
• the S protein of the Alpha strain of SARS-CoV-2 comprising said mutation 2P (SAi P ha-2p) has an amino acid sequence of SEQ ID NO: 6.
• the native sequence of the polynucleotide encoding the S protein of the Beta strain of SARS-CoV-2 is defined in SEQ ID NO: 7 (SBeta).
• the S protein of the Beta strain of SARS-CoV-2 (SBeta) has an amino acid sequence of SEQ ID NO: 8.
• the S protein of the Beta strain of SARS-CoV-2 comprising said mutation 2P (SBeta-2p) has an amino acid sequence of SEQ ID NO: 10.
• the S protein of the Gamma strain of SARS-CoV-2 comprising said mutation 2P (SGamma-2p) has an amino acid sequence of SEQ ID NO: 12.
• the S protein of the Delta strain of SARS-CoV-2 comprising said mutation 2P (SDeita-2p) has an amino acid sequence of SEQ ID NO: 14.
• the native sequence of the polynucleotide encoding the S protein of the Omicron strain of SARS-CoV-2 is defined in SEQ ID NO: 15 (Somicron).
• the S protein of the Omicron strain of SARS-CoV-2 (Somicron) has an amino acid sequence of SEQ ID NO: 16.
• the S protein of the Omicron strain of SARS-CoV-2 comprising said mutation 2P has an amino acid sequence of SEQ ID NO: 18.
• the S protein of the Ancestral strain of SARS-CoV-2 comprising said mutation 2P (S2P) has an amino acid sequence of SEQ ID NO: 20.
• the pseudotyped lentiviral vector particle encodes the S protein of the Beta strain of SARS-CoV-2 comprising the mutation 2P (SBeta-2p) that is encoded by the vector pFlap-ieCMV-S-B351 -2P-WPREm that has been deposited at the COLLECTION NATIONALE DE CULTURES DE MICROORGANISMES (CNCM) located at Institut Pasteur, 25-28 rue du Dondel Roux, 75724 Paris Cedex 15 FRANCE, on July 6, 2021 under N°CNCM 1-5710.
• SBeta-2p the mutation 2P
• CNCM COLLECTION NATIONALE DE CULTURES DE MICROORGANISMES
• vector pFlap-ieCMV-S-B351-2P-WPREm (CNCM 1-5710).
• the nucleotide sequence of pFlap-ieCMV-S-B351-2P-WPREm is defined in SEQ ID NO: 22.
• Also provided is a host cell comprising the vector pFlap-ieCMV-S-B351-2P-WPREm (CNCM 1-5710 or SEQ ID NO: 22).
• pseudotyped lentiviral vector particle encoding the S protein of the Beta strain of SARS-CoV-2 comprising the mutation 2P (SBeta-2p), wherein the pseudotyped lentiviral vector particle is made by a method comprising co-transfection of a host cell with the vector pFlap-ieCMV-S-B351-2P-WPREm (CNCM 1-5710 or SEQ ID NO: 22).
• the pseudotyped lentiviral vector particle encoding a S protein of a SARS-CoV-2 or a derivative thereof for use in accordance with the embodiments disclosed herein is further characterized by the following features: the amino acid sequence of the S protein is SEQ ID NO: 1 or is a derivative thereof having an amino acid sequence at least 90% identical to SEQ ID NO: 1 , and the derivative of the S protein of SARS-CoV-2 comprises at least five amino acid mutations including (i) a mutation of the lysine residue to the asparagine residue at position 417 of the amino acid sequence of SEQ ID NO: 1 (K417N), (ii) a mutation of the glutamic acid residue to the lysine residue at position 484 of the amino acid sequence of SEQ ID NO: 1 (E484K) or a mutation of the glutamic acid residue to the alanine residue at position 484 of the amino acid sequence of SEQ ID NO: 1 (E484A), (iii) a mutation of
• the pseudotyped lentiviral vector particle encoding a SS protein of a SARS-CoV-2 or a derivative thereof for use according to the invention is such that the S protein of SARS-CoV-2 further comprises amino acid mutations selected from the group consisting of (vi) a mutation of the glycine residue to the serine residue at position 446 of the amino acid sequence of SEQ ID NO: 1 (G446S), (vii) a mutation of the threonine residue to the lysine residue at position 478 of the amino acid sequence of SEQ ID NO: 1 (T478K), (viii) a mutation of the glutamine residue to the arginine residue at position 493 of the amino acid sequence of SEQ ID NO: 1 (Q493R) and (ix) a mutation of the glutamine residue to the arginine residue at position 498 of the amino acid sequence of SEQ ID NO: 1 (Q498R).
• amino acid mutations selected from the group consisting of (vi) a mutation of the glycine
• the pseudotyped lentiviral vector particle encoding a S protein of a SARS-CoV-2 or a derivative thereof for use according to the invention is such that the encoded mutated S protein of SARS-CoV-2 has the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 18, preferably of SEQ ID NO: 10.
• the pseudotyped lentiviral vector particle encoding a S protein of a SARS-CoV-2 or a derivative thereof for use according to the invention is pseudotyped with a vesicular stomatitis virus glycoprotein G (VSV-G) protein.
• VSV-G vesicular stomatitis virus glycoprotein G
• VSV-G protein is advantageously provided by a VS virus of the Indiana strain or the New-Jersey strain.
• the pseudotyped lentiviral vector particle encoding a S protein of a SARS-CoV-2 or a derivative thereof for use according to the invention is such that the pseudotyped lentiviral vector particle is non-integrative, non-cytopathic and non-replicative.
• the immunogenic agent or composition comprising the agent is for use in a method of prevention of infection of a human subject by SARS-CoV-2.
• the immunogenic agent or composition is for use in a method of protection against SARS-CoV-2 replication in a human subject at risk of being exposed to SARS-CoV-2 or infected by SARS-CoV-2.
• the immunogenic composition is for use in a method of preventing development of symptoms or development of a disease associated with infection by SARS-CoV-2, such as COVID-19 in a human subject at risk of being exposed to SARS-CoV-2 or infected by SARS-CoV-2.
• the immunogenic composition is for use in a method of preventing the onset of neurological outcome associated with infection by SARS-CoV-2 in a human subject at risk of being exposed to SARS-CoV-2 or infected by SARS-CoV-2.
• the immunogenic composition is for use in a method of protecting the Central Nervous System (CNS) of a human subject at risk of being exposed to SARS-CoV-2 or infected by SARS-CoV-2.
• the vaccine provides protection against the infection by SARS-CoV-2, especially sterilizing protection.
• the immunogenic agent or composition is to be administered to the subject as a prophylactic agent in a boost or target administration step in an effective amount for administration to the upper respiratory tract in order to elicit an immune response against SARS-CoV-2.
• the immunogenic composition is for use in a method of protection of a human subject against SARS-CoV-2 infection or against development of the symptoms or the COVID-19 disease associated with SARS-CoV-2 infection, wherein the subject is at risk of developing lung and/or CNS pathology.
• the human subject is in need of immune protection of CNS from SARS-CoV-2 replication because he/she is affected with comorbid conditions, in particular comorbid conditions affecting the CNS.
• the pseudotyped lentiviral vector particle encoding a S protein of a SARS-CoV-2 or a derivative thereof for use according to any one of the embodiments disclosed herein is administered in a subject selected from the group consisting of (a) a subject that has previously received a vaccine composition against SARS-CoV-2 infection or disease selected from the group consisting of a protein, an mRNA, an adenovirus, an inactivated virus and a protein subunit vaccine composition against SARS-CoV-2 infection or disease, in particular a protein- or an mRNA-based vaccine against SARS-CoV-2 infection or disease as a systemic prime and/or boost administration(s) such as intramuscular, intradermal or sub-cutaneous administration(s), in particular an intramuscular prime and/or boost administration(s), (b) a subject that has received a systemic prime administration such as intramuscular, intradermal or sub-cutaneous administration(s), in particular an intramuscular prime administration, of a vaccine composition against SARS-Co
• the pseudotyped lentiviral vector particle encoding a S protein of a SARS-CoV-2 or a derivative thereof is for use in a prime/boost or a target immunization regimen for elicitation of a long-lasting protective mucosal humoral immune response and/or a long-lasting mucosal cellular immune response against SARS-CoV-2 infection or disease, wherein said response protects the respiratory system and/or the CNS of the subject.
• the pseudotyped lentiviral vector particle encoding a S protein of a SARS-CoV-2 or a derivative thereof is for use in an immunization regimen wherein the pseudotyped lentiviral vector particle elicits a CD8 + T-cell response against SARS-CoV-2.
• the pseudotyped lentiviral vector particle encoding a S protein of a SARS-CoV-2 or a derivative thereof is for use in an immunization regimen wherein the pseudotyped lentiviral vector particle elicits lung-resident memory CD8 + T cells (Trm) and/or effector CD8 + T cells (Tc1 ) specific to Spike and able to produce Interferon-gamma (IFN-y)/Tumor Necrosis Factor (TNF)/lnterleukin-2 (IL-2) cytokines.
• IFN-y Interferon-gamma
• TNF Tumor Necrosis Factor
• IL-2 lnterleukin-2
• the pseudotyped lentiviral vector particle encoding a S protein of a ARS-CoV-2 or a derivative thereof for use according to the invention is used in an immunization regimen wherein the subject has a waning immunity from week 12 after the first injection of the initial vaccination with a vaccine composition against SARS-CoV-2 infection or disease selected from the group consisting of a protein, an mRNA, an adenovirus, an inactivated virus and a protein subunit vaccine composition against SARS-CoV-2 infection or disease, in particular a protein- or an mRNA-based vaccine against SARS-CoV-2 infection or disease or post SARS- CoV-2 disease recovery, in particular post-COVID-19 recovery.
• a vaccine composition against SARS-CoV-2 infection or disease selected from the group consisting of a protein, an mRNA, an adenovirus, an inactivated virus and a protein subunit vaccine composition against SARS-CoV-2 infection or disease, in particular a protein- or an mRNA-based vaccine against S
• the pseudotyped lentiviral vector particle encoding a S protein of a SARS-CoV-2 ora derivative thereof for use according to the invention is formulated as a liquid composition or a dry powder for an administration as intranasal aerosols, intranasal drops or intranasal insufflations.
• the pseudotyped lentiviral vector particle encoding a S protein of a SARS-CoV-2 or a derivative thereof for use according to the invention is used in an immunization regimen wherein the administration regimen comprises administration of one or more dosage form(s) of the pseudotyped lentiviral vector particle wherein the dose of each dosage form is from 10 7 to 10 9 Transduction Unit (TU).
• TU Transduction Unit
• the invention also concerns an immunogenic composition
• a pseudotyped lentiviral vector particle encoding a S protein of a SARS-CoV-2 or a derivative thereof and a pharmaceutically acceptable carrier
• the pseudotyped derivative of the S protein of SARS-CoV-2 comprises at least nine amino acid mutations including (i) a mutation of the lysine residue to the asparagine residue at position 417 of the amino acid sequence of SEQ ID NO: 1 (K417N), (ii) a mutation of the glutamic acid residue to the alanine residue at position 484 of the amino acid sequence of SEQ ID NO: 1 (E484A), (iii) a mutation of the asparagine residue to the tyrosine residue at position 501 of the amino acid sequence of SEQ ID NO: 1 (N501 Y), (iv) a mutation of the lysine residue to the proline residue at position 986 of the amino acid sequence of SEQ ID NO: 1 (K986P), (v)
• This immunogenic composition may be such that the pseudotyped lentiviral vector particle encodes mutated S protein of SARS-CoV-2 the amino acid sequence of which is SEQ ID NO: 18.
• the immunogenic composition is formulated for intranasal administration as disclosed in the embodiments herein.
• kits suitable for use in practicing a use or a method disclosed herein comprises a dosage form for administration to the upper respiratory tract of a subject of the pseudotyped lentiviral vector particle encoding a SARS-CoV-2 S protein or a derivative thereof according to this disclosure, and an applicator.
• the applicator is an applicator for aerosol inhalation.
• the applicator is an applicator for nasal instillation.
• the applicator is an applicator for nasal insufflation. Suitable examples of each are known in the art and may be used.
• Preparation of recombinant LV particles is known in the art, including to obtain non- integrative, non-replicative recombinant LV particles.
• Polynucleotide constructs may be adapted with the sequence encoding the selected Spike protein or derivative thereof.
• the lentiviral vector particle comprises HIV-1 Gag and Pol proteins. In some embodiments, the lentiviral vector particle comprises subtype D, especially HIV-1 NDK, Gag and Pol proteins.
• the lentivector particles are obtained in a host cell transformed with a DNA plasmid.
• Such a DNA plasmid can comprise:
• plIC ori - bacterial origin of replication
• - antibiotic resistance gene (ex: AmpiR or KanR) for selection; and more particularly: - a lentiviral vector comprising at least one nucleic acid encoding a SARS-CoV-2 S protein or a derivative thereof, transcriptionally linked to a promoter, for example a CMV promoter.
• Such a method allows producing a recombinant vector particle for use according to the invention, comprising the following steps of: i) transfecting a suitable host cell with a lentiviral vector; ii) transfecting said host cell with a packaging plasmid vector, containing viral DNA sequences encoding at least structural and polymerase (+ integrase) activities of a retrovirus (preferably lentivirus);
• packaging plasmids are described in the art (Dull et al., 1998, J Virol, 72(11 ):8463-71 ; Zufferey et al., 1998, J Wro/ 72(12):9873-80); iii) culturing said transfected host cell in order to obtain expression and packaging of said lentiviral vector into lentiviral vector particles; and iv) harvesting the lentiviral vector particles resulting from the expression and packaging of step iii) in said cultured host cells.
• An appropriate host cell is preferably a human cultured cell line as, for example, a HEK cell line, such as a HEK293T line.
• the method for producing the vector particle is carried out in a host cell, which genome has been stably transformed with one or more of the following components: a lentiviral vector DNA sequence, the packaging genes, and the envelope gene.
• a lentiviral vector DNA sequence may be regarded as being similar to a proviral vector according to the invention, comprising an additional promoter to allow the transcription of the vector sequence and improve the particle production rate.
• the host cell is further modified to be able to produce viral particle in a culture medium in a continuous manner, without the entire cells swelling or dying.
• a culture medium in a continuous manner, without the entire cells swelling or dying.
• One may refer to Strang et al. , 2005, J Virol 79(3): 1165-71 ; Relander et al. , 2005, Mol Ther 11 (3):452-9; Stewart et al., 2009, Gene Ther, 16(6):805-14; and Stuart et al., 2011 , Hum gene Ther, with respect to such techniques for producing viral particles.
• the lentiviral particle vectors can comprise the following elements, as previously defined:
• MHC-I Major Histocompatibility Complex classe I
• Figure 1 Down-selection of a Scov-2 variant with the highest potential to induce cross sero-neutralizing antibodies.
• B Half maximal Effective Concentration (EC50) of neutralizing activity of sera from vaccinated mice was evaluated before and after the boost, against pseudo-viruses carrying Scov-2 from D614G, Alpha, Beta or Gamma variants.
• FIG. 2 Anti-Scov-2 humoral responses in mRNA-1273-vaccinated mice which were further intranasally boosted with l_V::SBeta-2P.
• B Serum EC50 determined at the indicated time points against pseudo-viruses carrying Scov-2 from D614G, Alpha, Beta, Gamma, Delta or Delta+ variants.
• FIG. 3 Lung resident memory B-cell subsets in mRNA-1273-vaccinated mice which were further intranasally boosted with LV::SBeta-2P. The mice are those detailed in Figure 2. Mucosal immune cells were studied two weeks after LV::SBeta-2P i.n. boost.
• A Cytometric gating strategy to detect lung B resident memory cells
• B percentages of Brm among surface IgM/lgD' B cells in various groups.
• FIG 4 Systemic CD8 + T-cell responses to Scov-2 in mRNA-1273-vaccinated mice which were further intranasally boosted with LV::SBeta-2P.
• the mice are those detailed in Figure 2.
• T-splenocyte responses were evaluated two weeks after LV::SBeta-2P i.n. boost by IFN-y ELISPOT after stimulation with S:256-275, S:536-550 or S:576-590 synthetic 15-mer peptides encompassing Scov-2 MHC-l-restricted epitopes, pertinent in C57BL/6 (H-2 b ) mice.
• FIG. 5 Mucosal CD8 + T-cell responses to Scov-2 in mRNA-1273-vaccinated mice which were further intranasally boosted with l_V::SBeta-2P. The mice are those detailed in Figure 2.
• ICS IntraCellular Cytokine Staining
• FIG. 6 Lung resident memory T-cell subset in mRNA-1273-vaccinated mice which were further intranasally boosted with LV::SBeta-2P. The mice are those detailed in Figures 2 and 3. Mucosal immune cells were studied two weeks after LV::SBeta-2p i.n. boost.
• A Cytometric gating strategy to detect lung CD8 + T resident memory cells (Trm, CD44 + CD62L' CD69 + CD103 + ), and
• Figure 7 Full protective potential of a late LV::SBeta-2P i.n. boost in mRNA-1273- primed and-boosted mice.
• FIG. 8 Anti-Scov-2 humoral responses in mRNA-1273-vaccinated mice which were further intranasally boosted with LV::SBeta-2P.
• A Follow-up of anti-Scov-2 (left) and anti-RBD (right) IgG in the sera of mice initially primed and boosted i.m. with mRNA-1273.
• B Anti-Scov-2 IgG (top), anti-RBD IgG (middle), and anti-Scov-2 IgA (bottom) in the total lung extracts of mice initially primed and boosted i.m. with mRNA-1273 and then boosted later with a third i.m. dose of mRNA-1273 or an i.n. boost of LV::SBeta-2P.
• FIG. 9 Absence of mucosal CD8 + Tc2 responses to Scov-2 in mRNA-1273- vaccinated mice which were further intranasally boosted with LV::SBeta-2P. The mice are those detailed in Figure 2. Absence of IL-4, IL-5, IL-10 and IL-13 production by lung CD8 + T cells after in vitro stimulation with a pool of S:256-275, S:536-550 and S:576-590 peptides, studied by ICS in parallel to the assay performed to detect IFN-y/TNF/IL-2 (see Figure 5). Cells are gated on alive CD45 + CD8 + T cells.
• FIG. 10 Maps of plasmids used for production of LV encoding
• A SD614G-2P
• B SAIpha-2P
• C SGamma-2P
• D Soelta-2P
• E SBeta-2P
• F Somicron-2P antigens.
• the LV-based strategy which is highly productive, not only in inducing humoral responses but also and particularly in establishing high quality and memory T-cell responses (Ku MW, etal. Commun Biol, 4(1), 713, 2021), is a favorable platform for a heterologous boost, even if it is also largely efficacious by its own as a primary COVID-19 vaccine candidate Ku MW, et al. Cell Host Microbe, 29(2), 236-249 e236, 2021; Ku MW, et al. EMBO Mol Med, e14459, 2021). Furthermore, and importantly, LV is non-cytopathic, non-replicative and scarcely inflammatory and thus can be used to perform non-invasive i.n.
• LV-based immunization Another major advantage of LV-based immunization is the induction of strong T-cell immune responses with high cross-reactivity of T-cell epitopes from Spike of diverse VOCs. Therefore, when the neutralizing antibody fails or wanes, the T-cell arm remains largely protective, as the inventors recently described in antibody-deficient, B-cell compromised pMT KO mice (Ku MW, et al. EMBO Mol Med, e14459, 2021). This property is relative to a high- quality and long-lasting T-cell immunity induced against multiple preserved T-cell epitopes, despite the mutation accumulated in the Spike of the emerging VOCs (Ku MW, et al. EMBO Mol Med, e14459, 2021).
• the inventors first down-selected Seeta antigen which induced the greatest neutralization breadth against the VOCs and designed a non-integrating LV encoding a stabilized version of this antigen.
• the inventors used escalating doses of LV::SBeta- 2p in i.n. boost.
• the inventors demonstrated a dose-dependent increase in anti-Scov-2 IgG and IgA titers, and a broadened sero-neutralization potential both in the sera and lung homogenates against VOCs. No anti-Scov-2 IgA was detected in the lungs of mice injected i.m.
• the systemic CD8 + T-cell responses against various immunogenic regions of Scov-2 were also increased with 1 x 10 8 or 1 x 10 9 Til of LV::SBeta-2P i.n. boost in initially mRNA-1273-primed and boosted mice.
• the highest i.n. dose of LV::SBeta-2p was comparable to the additional i.m. dose of mRNA-1273.
• the fact that the i.n. administration of LV::SBeta-2p has a boost effect on the systemic T-cell immunity represents another advantage of this vaccination regimen.
• mice primed and boosted with mRNA-1273 showed that 20 wks after the first injection of mRNA- 1273, there was no detectable protective capacity left against the Delta variant of SARS-CoV- 2.
• an i.n. booster injection of suboptimal dose i.e., 1 x 10 8 Til of LV::SBeta-2P completely inhibited SARS-CoV-2 replication in the lungs.
• a third late i.m. booster injection of mRNA-1273 reduced SARS-CoV-2 RNA content in the lungs in a similar manner, but did not completely inhibit viral replication in all mice.
• LV:: SBeta-2P i.n. boost can be used to induce robust systemic and mucosal adaptive immunity, to broaden the specificity of the protective response.
• the LV::SBeta-2P i.n. boost strengthen the intensity, broaden the VOC cross-recognition, and targets B-and T-cell immune responses to the principal entry point of SARS-CoV-2 to the mucosal respiratory of the host organism and avoid the infection of main anatomical sites.
• a phase l/lla clinical trial is currently in preparation for the use of i.n. boost by LV::SBeta-2P in previously vaccinated persons or in COVID- convalescents.
• mice Female C57BL/6JRj mice were purchased from Janvier (Le Genest Saint Isle, France), housed in individually-ventilated cages under specific pathogen-free conditions at the Institut Pasteur animal facilities and used at the age of 7 wks. Mice were immunized i.m. with 1 pg/mouse of mRNA-1273 (Moderna) vaccine. For i.n. injections with LV, mice were anesthetized by i.p. injection of Ketamine (Imalgene, 80255mg/kg) and Xylazine (Rompun, 5 mg/kg). For protection experiments against SARS-CoV-2, mice were transferred into filtered cages in isolator.
• mice Four days before SARS-CoV-2 inoculation, mice were pretreated with 3 x 10 8 IGU of Ad5::hACE2 as previously described (Ku MW, et al. Cell Host Microbe, 29(2), 236- 249 e236, 2021). Mice were then transferred into a level 3 biosafety cabinet and inoculated i.n. with 0.3 x 10 5 TCID50 of the Delta SARS-CoV-2 clinical isolate (Lescure FX, et al. Lancet Infect Dis, 20(6), 697-706, 2020) contained in 20 pl. Mice were then housed in filtered cages in an isolator in BioSafety Level 3 animal facilities. The organs recovered from the infected animals were manipulated according to the approved standard procedures of these facilities.
• Anti-Scov-2 IgG and IgA antibody titers were determined by ELISA by use of recombinant stabilized Scov-2 or RBD fragment for coating. Neutralization potential of clarified and decomplemented sera or lung homogenates was quantitated by use of lentiviral particles pseudo-typed with Scov-2 from diverse variants, as previously described Ku MW, et al. Cell Host Microbe, 29(2), 236-249 e236, 2021; Sterlin D, et al. Sci Transl Med, 13(577), 2021).
• T-splenocyte responses were quantitated by IFN-y ELISPOT after in vitro stimulation with S:256-275, S:536-550 or S:576-590 synthetic 15-mer peptides which contain Scov-2 MHC- l-restricted epitopes in H-2 d mice ⁇ Ku MW, et al. Cell Host Microbe, 29(2), 236-249 e236, 2021). Spots were quantified in a CTL I mmunospot S6 ultimate-V Analyser by use of CTL Immunocapture 7.0.8.1 program.
• Enrichment and staining of lung immune cells were performed as detailed elsewhere ⁇ Ku MW, etal. Cell Host Microbe, 29(2), 236-249 e236, 2021; Ku MW, etal. EMBO Mol Med, e14459, 2021) after treatment with 400 U/ml type IV collagenase and DNase I (Roche) for a 30- minute incubation at 37°C and homogenization by use of GentleMacs (Miltenyi Biotech). Cell suspensions were then filtered through 100pm-pore filters, centrifuged at 1200 rpm and enriched on Ficoll gradient after 20 min centrifugation at 3000 rpm at RT, without brakes.
• the recovered cells were co-cultured with syngeneic bone-marrow derived dendritic cells loaded with a pool of A, B, C peptides, each at 1 pg/ml or negative control peptide at x290pg/ml.
• the following mixture was used to detect lung Tc1 cells: PerCP-Cy5.5-anti-CD3 (45-0031- 82, eBioScience), eF450-anti-CD4 (48-0042-82, eBioScience) and APC-anti-CD8 (17-0081-82, eBioScience) for surface staining and BV650-anti- IFN-g (563854, BD), FITC-anti-TNF (554418, BD) and PE-anti-IL- 2 (561061 , BD) for intracellular staining.
• the following mixture was used to detect lung Tc2 cells: PerCP-Cy5.5-anti-CD3 (45-0031 -82, eBioScience), eF450-anti-CD4 (48-0042-82, eBioScience), BV711 -anti-CD8 (563046, BD Biosciences), for surface staining and BV605-anti-IL-4 (504125, BioLegend Europe BV), APC-anti-IL-5 (504306, BioLegend Europe BV), FITC-anti-IL-10 (505006, BioLegend Europe BV), PE-anti- IL-13 (12-7133-81 , eBioScience) for intracellular staining.
• the intracellular staining was performed by use of the Fix Perm kit (BD), following the manufacturer's protocol. Dead cells were excluded by use of Near IR Live/Dead (Invitrogen). Staining was performed in the presence of Fcyll/lll receptor blocking anti-CD16/CD32 (BD).
• BD Fix Perm kit
• Lung B cells were studied by surface staining with a mixture of PerCP Vio700-anti-lgM (130-106-012, Miltenyi), and PerCP Vio700-anti-lgD (130-103-797, Miltenyi), APC-H7-anti-CD19 (560143, BD Biosciences), PE-anti-CD38 (102708, BioLegend Europe BV), PE-Cy7-anti-CD62L (ab25569, AbCam), BV711-anti-CD69 (740664, BD Biosciences), BV421-anti-CD73 (127217, BioLegend Europe BV), FITC-anti-CD80 (104705, BioLegend Europe BV) and yellow Live/Dead (Invitrogen).
• RNA from circulating SARS-CoV-2 was prepared from lungs as described elsewhere Ku MW, et al. Cell Host Microbe, 29(2), 236-249 e236, 2021). Lung homogenates were prepared by thawing and homogenizing in lysing matrix M (MP Biomedical) with 500 pl of PBS using a MP Biomedical Fastprep 24 Tissue Homogenizer. RNA was extracted from the supernatants of organ homogenates centrifuged during 10 min at 2000g, using the Qiagen Rneasy kit, except that the neutralization step with AVL buffer/carrier RNA was omitted.
• RNA samples were then used to determine viral RNA content by E-specific qRT-PCR.
• total RNA was prepared using lysing matrix D (MP Biomedical) containing 1 mL of TRIzol reagent (ThermoFisher) and homogenization at 30 s at 6.0 m/s twice using MP Biomedical Fastprep 24 Tissue Homogenizer.
• the quality of RNA samples was assessed by use of a Bioanalyzer 2100 (Agilent Technologies). Viral RNA contents were quantitated using a NanoDrop Spectrophotometer (Thermo Scientific NanoDrop).
• the RNA Integrity Number (RIN) was 7.5-10.0.
• SARS-CoV-2 E or E sub-genomic mRNA were 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 (Eurofins), as recently described (Ku MW, et al. EMBO Mol Med, e14459, 2021).
• the inventors generated LVs encoding the full length Scov-2 from the Alpha, Beta or Gamma SARS- CoV-2 VOCs.
• mice which have been initially primed and boosted with mRNA-1273 and in which the (cross) sero-neutralization potential is decreasing.
• C57BL/6 mice were primed i.m. at wk 0 and boosted i.m. at wk 3 with 1 pg/mouse of mRNA-1273, defined as the optimal dose of this vaccine in mice (Nature, 2020, Vol. 586, 567- 571) ( Figure 2A).
• mice received i.n. escalating doses of 1 x 1O 6 ,1 x 10 7 , 1 x 10 8 , or 1 x 10 9 TU/mouse of LV::S Beta-2P ( Figure 2A).
• Control mRNA-1273-primed and -boosted mice received i.n. 1 x 10 9 Til of an empty LV (LV Ctrl).
• mRNA-1273-primed and -boosted mice were injected i.m. with 1 pg of mRNA-1273 or PBS.
• age-matched mice received i.n. 1 x 10 9 TU of LV: :SBeta-2P or PBS.
• the titers of anti-Scov-2 IgA were higher in the mice injected with 1 x 10 9 TU of LV::SBeta-2pthan those injected with a third dose of mRNA-1273 ( Figure 2C).
• titers of anti-Scov-2 and anti-RBD IgG in the total lung extracts increased in a dosedependent manner in LV::SBeta-2P-injected mice, with the highest dose of LV::SBeta-2P was comparable to the third i.m. dose of mRNA-1273 ( Figure 8B).
• Systemic anti-Scov-2 T-cell immunity was assessed by IFN-y-specific ELISPOT in the spleen of individual mice immunized following the above-mentioned regimen (Figure 2A) after in vitro stimulation with individual S:256-275, S:536-550 or S:576-590 peptide, encompassing immunodominant Scov-2 regions for CD8 + T cells in H-2 b mice Ku MW, et al. Cell Host Microbe, 29(2), 236-249 e236, 2021).
• the weak anti-S CD8 + T-cell immunity detectable in the spleen of mRNA-1273 primed-boosted mice at wk 17, largely increased following i.n.
• Tc1 responses were not detected in any experimental group ( Figure 9).
• Trm lung resident memory CD8 + T cells
• mice received i.n. the suboptimal dose of 1 x 10 8 TU of LV::SBeta-2p or control empty LV ( Figure 7A).
• the choice of such suboptimal dose was based on numerous previous observations from the inventors with this dose which was effective in protection in a homologous LV prime-boost experiment (Ku MW, et al.
• mice received mRNA-1273 i.m. or PBS. Unvaccinated, age-and sex-matched controls were left unimmunized.
• mice Four weeks after the late boost, i.e. wk 20, all mice were pre-treated with 3 x 10 8 Infectious Genome Units (IGU) of an adenoviral vector serotype 5 encoding hACE2164(Ad5::hACE2) Ku MW, et al.
• IGU Infectious Genome Units
• mice were challenged with SARS-CoV-2 Delta variant, which, at the time of the present invention, i.e., November 2021 , was the most expanded SARS- CoV-2 variant worldwide.
• mice In mice initially primed and boosted with mRNA-1273, and then injected i.n. with the control LV or i.m. with PBS alone, no significant protection potential was detectable against the challenge with SARS-CoV-2 Delta variant.
• the LV::SBeta-2P i.n. boost drastically reduced the total Ecov-2 RNA content of SARS-CoV-2 and no copies of the replication-related Esg Ecov-2 RNA were detected in this group ( Figure 7B).
• the content of total Ecov-2 RNA was also significantly reduced in the group which received a late mRNA-1273 i.m. boost.
• the content of Esg Ecov-2 RNA was undetected in 3 out of 5 in this group.

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