Boosting SARS-CoV-2 immunity with a lentivirus-based nasal vaccine

Abstract

The present invention relates to the field of immunization against coronaviruses. In this regard, the present invention provides lentivirus-based immunogenic agents suitable for use in (i) vaccination with first generation vaccines, in particular protein or mRNA-based vaccines, against SARS-CoV-2 infection or disease, such as protein, mRNA, adenovirus, inactivated virus or protein subunit vaccine compositions against SARS-CoV-2 infection or disease, or (ii) boosting or targeted immunization treatments in subjects, in particular human subjects, previously immunized against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), based on SARS-CoV-2-induced or correlated diseases. The present invention therefore relates in particular to a lentivirus-based immunogenic agent that may help to overcome the shortcomings of the available vaccines against SARS-CoV-2 and in particular be efficient in overcoming the fading immune response or insufficient cellular memory response observed after immunization with the available first generation vaccines, such as protein, mRNA, adenovirus, inactivated virus or protein subunit vaccines, in particular protein or mRNA vaccines, by inducing a mucosal humoral and cellular immune response against coronaviruses, including a long-lasting immune response.

Description

[Technical field]

[0001] The present invention relates to the field of immunization against coronaviruses. In this regard, the present invention provides lentivirus-based immunogenic agents suitable for use in (i) vaccination with first generation vaccines, in particular protein or mRNA-based vaccines, against SARS-CoV-2 infection or disease, such as protein, mRNA, adenovirus, inactivated virus or protein subunit vaccine compositions against SARS-CoV-2 infection or disease, or (ii) boosting or targeted immunization treatments in subjects, in particular human subjects, previously immunized against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), based on SARS-CoV-2-induced or correlated diseases. The present invention therefore relates in particular to a lentivirus-based immunogenic agent that may help to overcome the shortcomings of the available vaccines against SARS-CoV-2 and in particular be efficient in overcoming the fading immune response or insufficient cellular memory response observed after immunization with the available first generation vaccines, such as protein, mRNA, adenovirus, inactivated virus or protein subunit vaccines, in particular protein or mRNA vaccines, by inducing a mucosal humoral and cellular immune response against coronaviruses, including a long-lasting immune response. [Background technology]

[0002] Given (i) the persistence of the coronavirus disease 2019 (COVID-19) pandemic, (ii) the weakening protective capacity of first-generation vaccines against SARS-CoV-2, and (iii) the constant emergence of new viral variants of concern (VOCs), new effective vaccine platforms may be crucial for future primary or booster vaccines ("Global COVID-19 Vaccination - Strategic Vision for 2022", World Health Organization, SAGE meeting October 2021). We recently demonstrated the robust performance of a lentiviral vaccine vector (LV) (LV::S) encoding the full-length sequence of the spike glycoprotein (S) from the ancestral SARS-CoV-2 when used in a systemic prime followed by an intranasal (in) boost in multiple preclinical models (Ku MW et al., Cell Host Microbe, 29(2), 236-249 e236, 2021). LV::S provides robust and complete respiratory protection against ancestral SARS-CoV-2 and VOCs (Ku MW et al., EMBO Mol Med, e14459, 2021). Furthermore, in new transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) and with unprecedented brain permissiveness to SARS-CoV-2 replication, in-boosting with LV::S is required to fully protect 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 booster to strengthen and extend protection against emerging VOCs with immune evasion potential (Juno JA, Wheatley AK. Nat Med, 27(11), 1874-1875, 2021).

[0003] The duration of protection conferred by first-generation COVID-19 vaccines is not yet well established, is difficult to predict by serological tests, and is variable in diverse individuals and against different VOCs. The current worsening of the global pandemic, despite high vaccination rates, points to the need for repeated booster vaccinations to ensure individual and population immunity against COVID-19. In this context, the safety and potential side effects of multiple homologous booster doses of first-generation COVID-19 vaccines must be considered, for example those related to allergic reactions to polyethylene glycol (PEG) contained in mRNA vaccines (Castells MC, Phillips EJ.N Engl J Med, 384(7), 643-649, 2021). Importantly, non-matched vaccine delivery methods, i.e., heterologous prime-boost formats, have proven to be more successful strategies than homologous prime-boost approaches in many preclinical models of various infectious diseases (He Q et al., Emerg Microbes Infect, 10(1), 629-637, 2021; Lu S. Curr Opin Immunol, 21(3), 346-351, 2009; Nordstrom P et al., Lancet Reg Health Eur, 100249, 2021). Therefore, new efficient vaccination platforms are of particular interest to develop heterologous boosters against COVID-19. The LV::S vaccine candidate has great potential for prophylactic use against COVID-19, based primarily on its robust ability to induce not only potent neutralizing humoral responses but also robust protective T cell responses that are not affected by escape mutations accumulated in SARS-CoV-2 VOCs (Ku MW et al., EMBO Mol Med, e14459, 2021).

[0004] Moreover, it has been observed that the protective capacity of the immunity initially induced by first generation vaccines, especially against novel VOCs, is declining, making booster vaccination essential ("Global COVID-19 Vaccination - Strategic Vision for 2022", World Health Organization, SAGE meeting October 2021). As an alternative to booster doses of the same vaccine, combining vaccine platforms in a heterologous prime-boost regime may provide a protective effect (Barros-Martins J et al., Nat Med, 27(9), 1525-1529, 2021). Compared to homologous vaccination, a heterologous prime-boost strategy may better boost specific adaptive immune responses and long-term protection without inducing / enhancing vector-specific immunity or the risk of exacerbating possible reactogenicity against the vaccine itself or excipients. Moreover, to induce maximum neutralization breadth, the sequence of the spike antigen needs to be adapted according to the dynamics of SARS-CoV-2 VOC emergence. In addition, protection against symptomatic SARS-CoV-2 infection is primarily related to serum neutralizing activity, whereas protection against severe COVID-19 is related to CD8 + It is related to T cell immunity. These cells, with their ability to lyse virus-infected cells, specifically control viral replication and result in the resolution of SARS-CoV-2 infection (Sette A, Crotty S. Cell, 184(4), 861-880, 2021). Therefore, appropriate B and T cell vaccine platforms containing adapted spike sequences are of utmost interest at the current stage of the pandemic. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] “Global COVID-19 Vaccination - Strategic Vision for 2022”, World Health Organization, SAGE meeting October 2021 [Non-licensed document 2] Ku MWら, Cell Host Microbe, 29(2), 236-249 e236, 2021 [Non-licensed document 3] Ku MWら、EMBO Mol Med、e14459、2021

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[0006] The inventors considered that LV::S may be highly suitable for use as a heterologous in-booster vaccine to strengthen and extend protection against SARS-CoV-2, particularly its known and emerging VOCs, including but not limited to alpha, beta, gamma, delta, and omicron variants of SARS-CoV-2. Early vaccinated countries are experiencing increasing waves of new infections, with population immunity waning just a few months after the completion of the first round of immunization (Juno JA, Wheatley AK. Nat Med, 27(11), 1874-1875, 2021).

[0007] The LVs used in the present invention are in particular non-integrating, non-replicating, 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). These vectors are pseudotyped with a heterologous glycoprotein from the vesicular stomatitis virus (VSV-G), which confers on them a broad tropism for a variety of cell types, including in particular dendritic cells. The latter are mainly non-dividing cells and therefore poorly permissive for gene transfer. Thus, LVs have the central property of efficiently transferring genes to the nuclei of non-dividing cells, thereby allowing efficient transduction of dendritic cells. The resulting endogenous antigen expression in these cells with their unique ability to activate naive T cells (Guermonprez P et al., J. Int Rev Cell Mol Biol, 349, 1-54, 2019) correlates with the exceptional ability of LV to induce high-quality effector and memory T cells (Ku MW et al., Commun Biol, 4(1), 713, 2021). Importantly, pseudo-typing of VSV-G also avoids LV being targeted by pre-existing vector-specific immunity in humans, which is key for vaccine development (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). The safety of LVs has been established in a phase I / IIa human immunodeficiency virus-1 therapeutic vaccine trial in humans (Clinical Trial No. 2011-006260-52, EU Clinical Trials Registry). Their non-cytopathic and non-inflammatory properties (Cousin C et al., Cell Rep, 26(5), 1242-1257 e1247, 2019; Lopez J et al., "An optimized lentiviral vector induces CD4 +LV is well suited for mucosal vaccination because of the in route of administration, which has the well-recognized advantage of inducing mucosal IgA responses, as well as airway-resident memory B and T lymphocytes (Lund FE, Randall TD. Science, 373(6553), 397-399, 2021). This route has also been shown to be the most effective in reducing SARS-CoV-2 infection in preclinical models of both hamsters and macaques (van Doremalen N et al., Sci Transl Med, 13(607), 2021). Induction of mucosal immunity by in immunization allows neutralizing SARS-CoV-2 directly at the point of entry into the host organism, before it reaches the main anatomical sites where it can infect (Ku MW et al., Cell Host Microbe, 29(2), 236-249 e236, 2021).

[0008] In the present invention, the inventors CoV-2 K in the S2 domain of 986 P and V 987 Downselected S of beta variant stabilized by P substitution CoV-2 We generated an LV encoding the Beta-2P In mice that were primed and boosted intramuscularly (im) with the mRNA-1273 (Moderna) vaccine and showed a gradual decline in serum neutralization, we demonstrated that inLV::S Beta-2P We compared the systemic and mucosal immune responses and protective capacity of a heterologous boost with an allogeneic boost using immRNA-1273 (Moderna) (Jackson LA et al., N Engl J Med, 383(20), 1920-1931, 2020; Wang F et al., Med Sci Monit, 26, e924700, 2020). We observed multiple advantages over previous regimens due to improved antigen design, a new vaccine delivery LV platform, and an alternative in administration route.

[0009] According to a first aspect, the present invention therefore relates to a pseudotyped lentiviral vector particle encoding the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a derivative thereof for use as a heterologous boost or targeted immunizing agent in a vaccine regimen for administration to the upper respiratory tract of a subject, in particular a human subject, that has received a prime immunization with a vaccine composition against SARS-CoV-2 infection or disease selected from the group consisting of a protein, mRNA, adenovirus, inactivated virus and protein subunit vaccine composition against SARS-CoV-2 infection or disease. Non-limiting examples of protein subunit vaccine compositions against SARS-CoV-2 infection or disease according to the present invention may include a vaccine based on adjuvanted recombinant spike protein or a vaccine based on recombinant spike protein packaged in nanoparticles.

[0010] The spike (S) protein of the SARS-CoV-2 virus is well-identified in the art as an envelope-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 (S CoV-2 ) is a (180 kDa) homotrimeric class I viral fusion protein that binds to the carboxypeptidase angiotensin-converting enzyme 2 (ACE2) expressed on host cells. CoV-2 The protein monomer has an ectodomain, a transmembrane anchor domain, and a short internal tail. CoV-2 is activated by two sequential proteolytic cleavages to initiate fusion with the host cell membrane. CoV-2 -After ACE2 interaction, S CoV-2 First, the extracellular domain of RRAR685 (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." Mil Med Res 7, 11; Walls et al., 2020), which is a key factor determining the pathological characteristics of the virus associated with ubiquitous furin expression (Wang et al., 2020, "A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility." Virol Sin 2020 Jun;35(3):337-339. doi: 10.1007 / s12250-020-00212-7. Epub 2020 Mar 20). The resulting subunits consist of (i) S1, which contains the ACE2 receptor-binding domain (RBD) with atomic contacts restricted to the ACE2 protease domain and also contains the major B cell epitopes targeted by neutralizing antibodies (NAbs) (Walls et al., 2020), and (ii) S2, which contains the membrane fusion element. CoV-1 As in the case of R, i.e., S2' becomes accessible on S2 (Belouzard et al., 2009, "Activation of the SARS coronavirus spike protein via sequential proteolytic cleavage at two distinct sites", Proc Natl Acad Sci USA 106:5871-5876). Depending on the cell or tissue type, one or several host proteases, including furin, trypsin, cathepsin, TMPRSS (TransMembrane Protease Serine Protease)-2 or -4, may be involved in this second cleavage step (Coutard et al., 2020, "The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent in CoV of the same clade." Antiviral Res 176:104742). The resulting "fusogenic" conformational change of S leads to the formation of S CoV-2 This results in a highly stable post-fusion form of S2', which initiates the fusion reaction with the host cell membrane (Sternberg and Naujokat, 2020, "Structural features of coronavirus SARS-CoV-2 spike protein: Targets for vaccination." Life Sci 257, 118056) and leads to the exposure of the fusion peptide (FP) adjacent to S2'. Insertion of the FP into the host cell / vesicle membrane stimulates the fusion reaction, which releases viral RNA into the host cytoplasm (Lai et al., 2017, "The SARS-CoV Fusion Peptide Forms an Extended Bipartite Fusion Platform that Perturbs Membrane Order in a Calcium-Dependent Manner." J Mol Biol 429:3875-3892). CoV-2The fact that the RBD contains multiple conformational B cell epitopes ( Walls et al., 2020 ) points to this viral envelope glycoprotein as a primary target for neutralizing antibodies (NAbs).

[0011] The S protein for expression by the lentiviral particles of the invention may be derived from a SARS-CoV-2 strain and therefore may be characterized by an amino acid sequence that is the native sequence of the viral protein. In a particular embodiment, the invention is carried out with the S protein of a known SARS-CoV-2 strain, such as the S protein of the ancestral strain (whose amino acid sequence is SEQ ID NO: 1) or the S protein of a later discovered variant strain, such as the alpha, beta, gamma, delta or omicron strains (all considered variant strains with respect to each other).

[0012] Alternatively, the invention may be practiced with derivatives of the S protein, i.e. derivatives of the native S protein obtained by mutation of the amino acid sequence of the S protein, as disclosed herein. To express LV::S recombinant particles, the nucleic acid encoding the S protein may have the sequence of the gene present in the original viral strain or may be a codon-optimized nucleic acid suitable for expression in mammalian cells, in particular human cells. To express LV recombinant particles expressing a derivative of the S protein, the nucleic acid encoding the derivative of the S protein may have a sequence derived from the sequence of the gene for the S protein present in the viral strain or may be a codon-optimized nucleic acid suitable for expression in mammalian cells.

[0013] In a specific embodiment, the recombinant lentiviral particle (LV) used in the present invention is an HIV-1-based lentiviral particle.Therefore, when the expression "LV" of "lentiviral particle" is used in this specification, it is particularly directed to HIV-1-based lentiviral particles, especially LV particles pseudotyped with VSV-G protein, especially the LV shown in the examples.

[0014] The expressions "boost" or "boost immunization" or "boost administration" or "targeted immunization" refer according to the present invention to the administration of an immunogenic substance following a first administration of a heterologous immunizing agent, in particular a heterologous vaccine, or following a second or subsequent administration of such a heterologous immunizing agent or vaccine. In other words, the immunizing agent used according to the present invention is administered to a subject who has previously received a prime administration, or a prime administration and one or more further administrations, of a heterologous immunizing agent or vaccine against the same SARS-CoV-2 or variant strain thereof. The boost or targeted immunization is achieved by administration to the upper respiratory tract, in particular intranasal administration, which is correspondingly different from the administration route of first generation vaccines against SARS-CoV-2 infection or disease, such as protein, mRNA, adenovirus, inactivated virus or protein subunit vaccine compositions against SARS-CoV-2 infection or disease, in particular protein or mRNA vaccines, which most often utilize systemic administration routes, including intramuscular, intradermal or subcutaneous administration routes. Boosting or targeted immunization is intended to enhance, improve or extend a previously raised immune response, and in some cases to expand such response to induce cross-neutralization against multiple SARS-CoV-2 viruses. Improved response may result from the ability of the immunizing agents used in the present invention to induce a mucosal response and accordingly protect not only systemic sites, but also the upper and lower respiratory tract, the central nervous system, which may not be well targeted or protected by heterologous vaccines against SARS-CoV-2 infection or disease, such as protein, mRNA, adenovirus, inactivated virus or protein subunit vaccine compositions, particularly protein or mRNA vaccines injected by systemic route, against SARS-CoV-2 infection or disease. In certain embodiments, boosting administration is intended to enhance the subject's cross-neutralizing immune response against newly emerged strains of the virus. In certain embodiments, boosting or targeted immunization may be administered to subjects who have been administered a heterologous immunizing agent as disclosed herein and have suffered and recovered from infection with SARS-CoV-2 or disease associated with such infection, such as COVID-19.Additional features regarding the use of the immunizing agent and the course of treatment of a subject are disclosed in the following description.

[0015] Administration "to the upper respiratory tract" includes any type of administration that results in delivery to the mucous membranes of the upper respiratory tract, and particularly includes administration to the nasal passages. Administration to the upper respiratory tract includes, but is not limited to, aerosol inhalation, nasal instillation, nasal inhalation, and any combination thereof. In some embodiments, administration is by aerosol inhalation. In some embodiments, administration is by nasal administration. In some embodiments, administration is by nasal inhalation.

[0016] According to certain embodiments, pseudotyped lentiviral vector particles encoding the SARS-CoV-2 S protein or derivatives thereof are for administration as an intranasal mucosal boost or targeted immunization in a subject who has been primed with a vaccine composition against SARS-CoV-2 infection or disease selected from the group consisting of a protein, mRNA, adenovirus, inactivated virus and protein subunit vaccine compositions against SARS-CoV-2 infection or disease, in particular a protein or mRNA vaccine composition against SARS-CoV-2 infection or disease.

[0017] According to certain embodiments, pseudotyped lentiviral vector particles encoding the S protein of SARS-CoV-2 or a derivative thereof for use according to the embodiments disclosed herein are further characterized by the following features: - the S protein is derived from a SARS-CoV-2 virus pathogenic for a human host, in particular (i) an S protein from a SARS-CoV-2 virus selected from the group consisting of the SARS-CoV-2 ancestral, D614G, alpha, beta, gamma, delta and Omicron strains, preferably an S protein from a SARS-CoV-2 virus selected from the beta or Omicron strain, more preferably from the beta strain, or (ii) an S protein from a variant of said ancestral, D614G, alpha, beta, gamma, delta or Omicron strain, such variant encoding an S protein having 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 any of the ancestral, D614G, alpha, beta, gamma, delta or Omicron strains by mutation, in particular substitution and / or deletion of 1 to 12, in particular 1 to 6, amino acid residues, in particular 1 to 12, in particular 1 to 6 amino acid residues, in particular (a) a stabilized form of the S protein characterized by the substitution of two consecutive amino acid residues in the S2 domain of the S protein at the positions provided as residues 986 and 987 with reference to SEQ ID NO: 1 for a proline residue, or (b) a pre-fusion form of the S protein by deletion of the furin site located from residue 675 to residue 685 in the amino acid sequence of the S protein with reference to SEQ ID NO: 1, or (c) a stabilized pre-fusion form of the S protein by deletion of the furin site and substitution of two consecutive amino acid residues in the S2 domain at the positions provided as residues 986 and 987 with reference to SEQ ID NO: 1 for a proline residue.

[0018] In a specific embodiment, the S protein of an ancestral strain of SARS-CoV-2 has the amino acid sequence of SEQ ID NO:1, and the native sequence of the polynucleotide encoding the S protein of an ancestral strain of SARS-CoV-2 is defined in SEQ ID NO:2.

[0019] In another particular embodiment, the native sequence of a polynucleotide encoding the S protein of the D614G strain of SARS-CoV-2 comprises a mutation of an aspartic acid residue at position 614 of the amino acid sequence of SEQ ID NO:1 to a glycine residue (D614G), a mutation of a lysine residue at position 986 of the amino acid sequence of SEQ ID NO:1 to a proline residue (K986P), and a mutation of a valine residue at position 987 of the amino acid sequence of SEQ ID NO:1 to a proline residue (V987P), i.e., mutation 2P is SEQ ID NO:3 (S D614G-2P ) The S protein of the D614G strain of SARS-CoV-2 containing the 2P mutation (S D614G-2P ) has the amino acid sequence of SEQ ID NO:4.

[0020] In another particular embodiment, the native sequence of a polynucleotide encoding the S protein of the alpha strain of SARS-CoV-2, comprising the mutation of the lysine residue at position 986 of the amino acid sequence of SEQ ID NO:1 to a proline residue (K986P) and the mutation of the valine residue at position 987 of the amino acid sequence of SEQ ID NO:1 to a proline residue (V987P), i.e., the mutation 2P, is represented by SEQ ID NO:5 (S Alpha-2P ) The S protein (S) of the alpha strain of SARS-CoV-2 containing the mutation 2P is Alpha-2P ) has the amino acid sequence of SEQ ID NO:6.

[0021] In another specific embodiment, the native sequence of the polynucleotide encoding the S protein of the beta strain of SARS-CoV-2 is SEQ ID NO: 7 (S Beta ) as defined in the SARS-CoV-2 beta strain (S Beta ) S protein has the amino acid sequence of SEQ ID NO:8.

[0022] In another particular embodiment, the native sequence of a polynucleotide encoding the S protein of the beta strain of SARS-CoV-2, comprising the mutation of the lysine residue at position 986 of the amino acid sequence of SEQ ID NO:1 to a proline residue (K986P) and the mutation of the valine residue at position 987 of the amino acid sequence of SEQ ID NO:1 to a proline residue (V987P), i.e., the mutation 2P, is represented by SEQ ID NO:9 (S Beta-2P ) The S protein (S) of the beta strain of SARS-CoV-2 containing the mutation 2P is Beta-2P ) has the amino acid sequence of SEQ ID NO:10.

[0023] In another particular embodiment, the native sequence of a polynucleotide encoding the S protein of the gamma strain of SARS-CoV-2, comprising the mutation of the lysine residue at position 986 of the amino acid sequence of SEQ ID NO:1 to a proline residue (K986P) and the mutation of the valine residue at position 987 of the amino acid sequence of SEQ ID NO:1 to a proline residue (V987P), i.e., the mutation 2P, is represented by SEQ ID NO:11 (S Gamma-2P ) The S protein of the gamma strain of SARS-CoV-2, which contains the mutated 2P (S Gamma-2P ) has the amino acid sequence of SEQ ID NO:12.

[0024] In another particular embodiment, the native sequence of a polynucleotide encoding the S protein of the Delta strain of SARS-CoV-2, comprising the mutation of the lysine residue at position 986 of the amino acid sequence of SEQ ID NO:1 to a proline residue (K986P) and the mutation of the valine residue at position 987 of the amino acid sequence of SEQ ID NO:1 to a proline residue (V987P), i.e., the mutation 2P, is represented by SEQ ID NO:13 (S Delta-2P ) The S protein of the delta strain of SARS-CoV-2 containing the mutation 2P (S Delta-2P ) has the amino acid sequence of SEQ ID NO:14.

[0025] In another specific embodiment, the native sequence of a polynucleotide encoding the S protein of the Omicron strain of SARS-CoV-2 is SEQ ID NO: 15 (S Omicron) as defined in the Omicron strain of SARS-CoV-2 (S Omicron ) S protein has the amino acid sequence of SEQ ID NO:16.

[0026] In another particular embodiment, the native sequence of a polynucleotide encoding the S protein of the Omicron strain of SARS-CoV-2, comprising the mutation of the lysine residue at position 986 of the amino acid sequence of SEQ ID NO:1 to a proline residue (K986P) and the mutation of the valine residue at position 987 of the amino acid sequence of SEQ ID NO:1 to a proline residue (V987P), i.e., the mutation 2P, is SEQ ID NO:17 (S Omicron-2P ) The S protein of the Omicron strain of SARS-CoV-2 containing the mutated 2P (S Omicron-2P ) has the amino acid sequence of SEQ ID NO:18.

[0027] In another specific embodiment, the native sequence of a polynucleotide encoding the S protein of the Omicron-BA.1 strain of SARS-CoV-2 is SEQ ID NO: 23 (S Omicron-BA.1 ) as defined in the Omicron strain of SARS-CoV-2 (S Omicron-BA.1 ) S protein has the amino acid sequence of SEQ ID NO:24.

[0028] In another specific embodiment, the native sequence of a polynucleotide encoding the S protein of the Omicron-BA.4 or BA.5 strain of SARS-CoV-2 is SEQ ID NO: 25 (S Omicron-BA.4 / 5 ) as defined in the Omicron-BA.4 or BA.5 strains of SARS-CoV-2 (S Omicron-BA.4 / 5 ) S protein has the amino acid sequence of SEQ ID NO:26.

[0029] In another particular embodiment, the native sequence of a polynucleotide encoding the S protein of an ancestral strain of SARS-CoV-2 comprising the mutation of the lysine residue at position 986 of the amino acid sequence of SEQ ID NO:1 to a proline residue (K986P) and the mutation of the valine residue at position 987 of the amino acid sequence of SEQ ID NO:1 to a proline residue (V987P), i.e., the mutation 2P, is defined in SEQ ID NO:19 (S2P). 2P ) has the amino acid sequence of SEQ ID NO:20.

[0030] In a particular embodiment, the pseudotyped lentiviral vector particles are encoded by the mutated 2P (S) encoded by the vector pFlap-ieCMV-S-B351-2P-WPREm, deposited on July 6, 2021 at the COLLECTION NATIONALE DE CULTURES DE MICROORGANISMES (CNCM), Institute Pasteur, 25-28 rue du Docteur Roux, 75724 Paris Cedex 15, FRANCE, under the number CNCM I-5710. Beta-2P ) that encodes the S protein of beta strains of SARS-CoV-2.

[0031] Also provided is the vector pFlap-ieCMV-S-B351-2P-WPREm (CNCM I-5710), the nucleotide sequence of which is defined in SEQ ID NO:22.

[0032] Also provided is a host cell containing the vector pFlap-ieCMV-S-B351-2P-WPREm (CNCM I-5710 or SEQ ID NO:22).

[0033] In addition, the mutation 2P(S Beta-2PAlso provided is a pseudotyped lentiviral vector particle encoding the S protein of the beta strain of SARS-CoV-2 comprising the vector pFlap-ieCMV-S-B351-2P-WPREm (CNCM I-5710 or SEQ ID NO: 22), wherein the pseudotyped lentiviral vector particle is produced by a method comprising co-transfection of a host cell with the vector pFlap-ieCMV-S-B351-2P-WPREm (CNCM I-5710 or SEQ ID NO: 22).

[0034] In certain embodiments, pseudotyped lentiviral vector particles encoding the S protein of SARS-CoV-2 or a derivative thereof for use according to the embodiments disclosed herein are further characterized by the following features: the amino acid sequence of the S protein is SEQ ID NO:1 or 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 has (i) a mutation at position 417 of the amino acid sequence of SEQ ID NO:1 to an asparagine residue (K417N); (ii) a mutation at position 420 of the amino acid sequence of SEQ ID NO:1 to an asparagine residue (K417N); 1 (E484K), or a glutamic acid residue at position 484 of the amino acid sequence of SEQ ID NO:1 to an alanine residue (E484A); (iii) a mutation of an asparagine residue at position 501 of the amino acid sequence of SEQ ID NO:1 to a tyrosine residue (N501Y); (iv) a mutation of a lysine residue at position 986 of the amino acid sequence of SEQ ID NO:1 to a proline residue (K986P); and (v) a mutation of a valine residue at position 987 of the amino acid sequence of SEQ ID NO:1 to a proline residue (V987P).

[0035] In a particular embodiment, the pseudotyped lentiviral vector particle encoding the SS protein of 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 an amino acid mutation selected from the group consisting of: (vi) a mutation of a glycine residue at position 446 of the amino acid sequence of SEQ ID NO:1 to a serine residue (G446S); (vii) a mutation of a threonine residue at position 478 of the amino acid sequence of SEQ ID NO:1 to a lysine residue (T478K); (viii) a mutation of a glutamine residue at position 493 of the amino acid sequence of SEQ ID NO:1 to an arginine residue (Q493R); and (ix) a mutation of a glutamine residue at position 498 of the amino acid sequence of SEQ ID NO:1 to an arginine residue (Q498R).

[0036] In a specific embodiment, the pseudotyped lentiviral vector particle encoding the S protein of 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 SEQ ID NO: 10.

[0037] In another specific embodiment, the pseudotyped lentiviral vector particle encoding the S protein of 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:24 or SEQ ID NO:26.

[0038] In a specific embodiment, pseudotyped lentiviral vector particles encoding the S protein of SARS-CoV-2 or a derivative thereof for use according to the invention are pseudotyped with the vesicular stomatitis virus glycoprotein G (VSV-G) protein.

[0039] In particular, the VSV-G protein is advantageously provided by the Indiana or New Jersey strain of VS virus.

[0040] In certain embodiments, the pseudotyped lentiviral vector particles encoding the S protein of SARS-CoV-2 or derivatives thereof for use according to the present invention are such that the pseudotyped lentiviral vector particles are non-integrating, non-cytopathic and non-replicating.

[0041] In some embodiments of the uses of the immunogenic agents disclosed herein according to the invention, the immunogenic agent or a composition comprising the agent is for use in a method of preventing infection of a human subject with SARS-CoV-2. In some embodiments, the immunogenic agent or composition is for use in a method of protecting against replication of SARS-CoV-2 in a human subject at risk of exposure to or infection with SARS-CoV-2. In some embodiments, the immunogenic composition is for use in a method of preventing the onset of symptoms or disease associated with infection by SARS-CoV-2, such as COVID-19, in a human subject at risk of exposure to or infection with SARS-CoV-2. In some embodiments, the immunogenic composition is for use in a method of preventing the onset of neurological outcomes associated with infection by SARS-CoV-2 in a human subject at risk of exposure to or infection with SARS-CoV-2. In some embodiments, the immunogenic composition is for use in a method of protecting the central nervous system (CNS) of a human subject exposed to or at risk of infection with SARS-CoV-2. In some embodiments, the vaccine provides protection, particularly sterilizing protection, against infection by SARS-CoV-2.

[0042] In any of these applications for use in the disclosed methods, the immunogenic agent or composition is administered to a subject as a prophylactic agent in a boost or targeted administration phase in an amount effective for administration to the upper respiratory tract to elicit an immune response against SARS-CoV-2.

[0043] In some embodiments, the immunogenic composition is for use in a method for protecting a human subject against SARS-CoV-2 infection or against developing symptoms associated with SARS-CoV-2 infection or COVID-19 disease, where the subject is at risk of developing pulmonary and / or CNS pathology, particularly where the human subject suffers from a comorbidity, particularly a comorbidity affecting the CNS, and therefore there is a need to immunologically protect the CNS from SARS-CoV-2 replication.

[0044] In a particular embodiment, the pseudotyped lentiviral vector particle encoding the S protein of SARS-CoV-2 or a derivative thereof for use according to any one of the embodiments disclosed herein is selected from the group consisting of: (a) a vaccine composition against SARS-CoV-2 infection or disease selected from the group consisting of protein, mRNA, adenovirus, inactivated virus and protein subunit vaccine compositions against SARS-CoV-2 infection or disease, in particular a protein or mRNA based vaccine against SARS-CoV-2 infection or disease, as a systemic prime and / or boost administration, such as intramuscular, intradermal or subcutaneous administration, in particular an intramuscular prime and / or boost administration; (b) a vaccine composition against SARS-CoV-2 infection or disease selected from the group consisting of protein, mRNA, adenovirus, inactivated virus and protein subunit vaccine compositions against SARS-CoV-2 infection or disease, in particular a protein or mRNA based vaccine against SARS-CoV-2 infection or disease, in particular an intramuscular prime and / or boost administration; (c) a subject who has first recovered from a coronavirus disease, such as COVID-19, and has subsequently received a systemic prime such as an intramuscular, intradermal or subcutaneous administration, particularly an intramuscular administration, of a vaccine composition against SARS-CoV-2 infection or disease, particularly a protein or mRNA based vaccine against SARS-CoV-2 infection or disease, and has subsequently recovered from a coronavirus disease, such as coronavirus disease 2019 (COVID-19); (d) a subject who has received more than two, particularly more than three, systemic administrations such as intramuscular, intradermal or subcutaneous, particularly an intramuscular administration, of a protein or mRNA based vaccine against SARS-CoV-2 infection or disease.

[0045] In certain embodiments, pseudotyped lentiviral vector particles encoding the S protein of SARS-CoV-2 or derivatives thereof are for use in prime / boost or targeted immunization regimens to induce a long-lasting protective mucosal humoral and / or long-lasting mucosal cellular immune response against SARS-CoV-2 infection or disease, wherein said response protects the respiratory system and / or CNS of a subject.

[0046] In certain embodiments, pseudotyped lentiviral vector particles encoding the S protein of SARS-CoV-2 or a derivative thereof are for use in an immunization regimen, wherein the pseudotyped lentiviral vector particles encode CD4A or a derivative thereof against SARS-CoV-2. 8+ Induce a T cell response. In certain embodiments, pseudotyped lentiviral vector particles encoding the S protein of SARS-CoV-2 or derivatives thereof are for use in immunization regimens, where the pseudotyped lentiviral vector particles are spike-specific and target lung-resident memory CD8+ cells capable of producing interferon-gamma (IFN-γ) / tumor necrosis factor (TNF) / interleukin-2 (IL-2) cytokines. + T cells (Trm) and / or effector CD8 + Induces T cells (Tc1). In a particular embodiment, pseudotyped lentiviral vector particles encoding the S protein of ARS-CoV-2 or a derivative thereof for use according to the invention are used in an immunization regimen in which a subject shows waning immunity from week 12 after the first injection of a primary vaccination with a vaccine composition against SARS-CoV-2 infection or disease selected from the group consisting of protein, mRNA, adenovirus, inactivated virus and protein subunit vaccine compositions against SARS-CoV-2 infection or disease, in particular a protein or mRNA based vaccine against SARS-CoV-2 infection or disease, or after SARS-CoV-2 disease recovery, in particular after COVID-19 recovery.

[0047] In preclinical mouse studies, the inventors showed that 4-5 months after the last administration of an mRNA-based vaccine expressing the spike antigen, protective immunity against SARS-CoV-2 was no longer present (Vesin et al., 2022, Mol Ther 30, 2984-2997). Moreover, it is now well established that 3-10 months after the last administration of an mRNA-based vaccine expressing the spike antigen, neutralizing anti-spike antibody levels in the serum of vaccine recipients are significantly reduced (Decru et al., 2022, Front Immunol. 13, 909-910).

[0048] It is also widely established that memory immunity is commonly induced in the B and T cell compartments in the context of vaccination, independent of vaccination strategies, or even in the context of infection, after prior exposure of the immune system to antigens containing the SARS-CoV-2 spike (Valyi-Nagy et al., 2022, Int J Mol Sci, 23.10.3390). As far as currently assessed, such memory immunity is expected to last on average for at least one year in the case of anti-spike immunity induced by vaccination or infection (Gallais et al., 2021, EBioMedicine, 71, 103561). This memory immunity can be boosted at least up to one year after the last dose of the mRNA vaccine.

[0049] In a particular embodiment, pseudotyped lentiviral vector particles encoding the S protein of SARS-CoV-2 or a derivative thereof for use according to the invention are administered to a subject according to the invention as an intranasal mucosal boost or targeted immunization at least 3 months, in particular 3 to 24 months, preferably 3 to 12 months after last contact with SARS-CoV-2, following administration of a vaccine composition against SARS-CoV-2 infection or disease selected from the group consisting of a protein, mRNA, adenovirus, inactivated virus and protein subunit vaccine composition against SARS-CoV-2 infection or disease, in particular a protein or mRNA vaccine composition against SARS-CoV-2 infection or disease.

[0050] In certain embodiments, pseudotyped lentiviral vector particles encoding the S protein or derivatives thereof of SARS-CoV-2 for use according to the invention are formulated as a liquid composition or dry powder for administration as an intranasal aerosol, intranasal drops or intranasal inhalation. ... 7 ~10 9 The transduction units (TU) used in the immunization regimen.

[0051] According to another aspect, the present invention also relates to an immunogenic composition comprising a pseudotyped lentiviral vector particle encoding the S protein of SARS-CoV-2 or a derivative thereof, and a pharma- ceutically acceptable carrier, wherein the pseudotyped derivative of the S protein of SARS-CoV-2 has (i) a mutation at position 417 of the amino acid sequence of SEQ ID NO:1 from a lysine residue to an asparagine residue (K417N), (ii) a mutation at position 484 of the amino acid sequence of SEQ ID NO:1 from a glutamic acid residue to an alanine residue (E484A), (iii) a mutation at position 501 of the amino acid sequence of SEQ ID NO:1 from an asparagine residue to a tyrosine residue (N501Y), (iv) a mutation at position 986 of the amino acid sequence of SEQ ID NO:1 from a lysine residue to an asparagine residue (K417N), or (v) a mutation at position 986 of the amino acid sequence of SEQ ID NO:1 from a glutamic acid residue to an alanine residue (E484A). (v) a mutation at position 987 of the amino acid sequence of SEQ ID NO:1 from a valine residue to a proline residue (V987P), (vi) a mutation at position 446 of the amino acid sequence of SEQ ID NO:1 from a glycine residue to a serine residue (G446S), (vii) a mutation at position 478 of the amino acid sequence of SEQ ID NO:1 from a threonine residue to a lysine residue (T478K), (viii) a mutation at position 493 of the amino acid sequence of SEQ ID NO:1 from a glutamine residue to an arginine residue (Q493R), and (ix) a mutation at position 498 of the amino acid sequence of SEQ ID NO:1 from a glutamine residue to an arginine residue (Q498R).

[0052] The immunogenic composition may be such that the pseudotyped lentiviral vector particles encode a mutated S protein of SARS-CoV-2 having the amino acid sequence SEQ ID NO:18.

[0053] According to another embodiment, the immunogenic composition may be such that the pseudotyped lentiviral vector particles encode a mutated S protein of SARS-CoV-2 having the amino acid sequence SEQ ID NO:24 or SEQ ID NO:26.

[0054] According to further embodiments, the immunogenic composition, as disclosed in the embodiments herein, is formulated for intranasal administration.

[0055] The present invention also relates to kits suitable for use in carrying out the uses or methods disclosed herein. In some embodiments, the kit includes a dosage form for administering a pseudotyped lentiviral vector particle encoding a SARS-CoV-2 S protein according to the present disclosure or a derivative thereof to the upper respiratory tract of a subject, and an applicator. In some embodiments, the applicator is an applicator for aerosol inhalation. In some embodiments, the applicator is an applicator for nasal instillation. In some embodiments, the applicator is an applicator for nasal inhalation. Suitable examples of each are known in the art and can be used.

[0056] Preparation of recombinant LV particles is known in the art, including obtaining non-integrating, non-replicating recombinant LV particles. See in particular the disclosures of Ku MW et al., Cell Host Microbe, 29(2), 236-249 e236, 2021; Ku MW et al., EMBO Mol Med, e14459, 2021; Ku MW, Charneau P, Majlessi L. Expert Rev Vaccines, 1-16, 2021. The polynucleotide construct may be adapted with a sequence encoding the selected spike protein or a derivative thereof.

[0057] In some embodiments, the lentiviral vector particle comprises HIV-1 Gag and Pol proteins. In some embodiments, the lentiviral vector particle comprises HIV-1 subtype D, particularly HIV-1 NDK , Gag and Pol proteins.

[0058] According to some embodiments, the lentivector particles are obtained in host cells transformed with a DNA plasmid.

[0059] Such a DNA plasmid may include: - a bacterial origin of replication (e.g., pUC ori); - an antibiotic resistance gene for selection (e.g. AmpiR or KanR); and more specifically: - 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, e.g., a CMV promoter.

[0060] Such a method comprises the following steps and makes it possible to produce recombinant vector particles for use according to the invention: i) transfecting a suitable host cell with a lentiviral vector; ii) transfecting said host cells with a packaging plasmid vector comprising viral DNA sequences encoding at least the structural and polymerase (+ integrase) activities of a retrovirus, preferably a lentivirus; such packaging plasmids have been described in the art (Dull et al., 1998, J Virol, 72(11):8463-71; Zufferey et al., 1998, J Virol 72(12):9873-80); iii) culturing the transfected host cells to obtain expression and packaging of the lentiviral vector into lentiviral vector particles; and iv) harvesting the lentiviral vector particles resulting from expression and packaging of step iii) in said cultured host cells.

[0061] Suitable host cells are preferably human cultured cell lines, for example HEK cell lines such as the HEK293T line.

[0062] Alternatively, the method of producing vector particles is carried out in a host cell whose genome has been stably transformed with one or more of the following components: lentiviral vector DNA sequences, packaging genes, and envelope genes. Such DNA sequences can be considered similar to the proviral vectors of the present invention, containing an additional promoter that allows transcription of the vector sequences and enhances the rate of particle production.

[0063] In a preferred embodiment, the host cell is further modified to be able to continuously produce viral particles in the culture medium without the whole cell swelling or dying. For such techniques for producing viral particles, see 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.

[0064] A lentiviral particle vector, as defined above, may comprise the following elements: - a cPPT / CTS polynucleotide sequence; and - a nucleic acid encoding a CAR under the control of a β2m or major histocompatibility complex class I (MHC-I) promoter, and optionally one of the additional elements listed above.

[0065] Other characteristics and advantages of the invention will become apparent from the examples which follow and are illustrated in the drawings. [Brief description of the drawings]

[0066] [Figure 1]Down-selection of SCoV-2 variants most likely to induce cross-serum neutralizing antibodies. (A) Timeline of prime-boost vaccination with LV::SAlpha, LV::SBeta or LV::SGamma and (cross-)serum neutralization assay in C57BL / 6 mice (n=4-5 / group). (B) The half maximal effective concentration (EC50) of neutralizing activity of sera from vaccinated mice against pseudotyped viruses carrying SCoV-2 derived from D614G, alpha, beta or gamma variants was assessed before and after boost. (C) EC50 of sera from C57BL / 6 mice vaccinated according to the regimen detailed in (A) with LV encoding SD614G (WT or with K986P-V987P substitutions in the S2 domain). EC50 was assessed before and after boost as shown in (B). [Diagram 2] Anti-SCoV-2 humoral responses in mRNA-1273 vaccinated mice further boosted intranasally with LV::SBeta-2P. (A) Timeline of intramuscular (im)-im prime-boost vaccination and (cross-)seroneutralization follow-up of mRNA-1273 in C57BL / 6 mice subsequently immunized in with increasing doses of LV::SBeta-2P (n=4-5 / group). (B) Serum EC50s measured at the indicated time points against pseudotyped viruses harboring D614G, alpha, beta, gamma, delta or delta+ variants of SCoV-2. (C) Anti-SCoV-2 IgG or IgA in serum 2 weeks after late LV::SBeta-2P in or mRNA-1273 (Moderna) im boost. Statistical significance was determined by the Mann-Whitney test (*=p<0.05, **=p<0.01, ***=p<0.001). [Diagram 3]Lung-resident memory B cell subsets in mRNA-1273 vaccinated mice further boosted intranasally with LV::SBeta-2P. Mice are detailed in Figure 2. Mucosal immune cells were examined 2 weeks after LV::SBeta-2P in boost. (A) Cytometric gating strategy to detect lung B resident memory cells and (B) percentage of Brm among surface IgM / IgD-B cells in various groups. [Figure 4] Systemic CD8+ T cell responses to SCoV-2 in mRNA-1273 vaccinated mice further boosted intranasally with LV::SBeta-2P. Mice are detailed in FIG. 2. T splenocyte responses were assessed by IFN-γ ELISPOT in C57BL / 6 (H-2b) mice after stimulation with synthetic 15mer peptides S:256-275, S:536-550 or S:576-590 encompassing relevant SCoV-2 MHC-I restricted epitopes, 2 weeks after in boost with LV::SBeta-2P. Statistical significance was assessed by Mann-Whitney test (*=p<0.05). [Diagram 5] Mucosal CD8+ T cell responses to SCoV-2 in mRNA-1273 vaccinated mice further boosted intranasally with LV::SBeta-2P. Mice are detailed in Figure 2. (A) Representative IFN-γ responses by pulmonary CD8+ T cells detected by intracellular cytokine staining (ICS) after in vitro stimulation with a pool of S:256-275, S:536-550 and S:576-590 peptides. Cells are gated for viable CD45+CD8+ T cells. [Figure 6]Lung-resident memory T cell subsets in mRNA-1273 vaccinated mice further boosted intranasally with LV::SBeta-2P. Mice were detailed in Figures 2 and 3. Mucosal immune cells were examined 2 weeks after LV::SBeta-2P in boost. (A) Cytometric gating strategy to detect lung CD8+ T resident memory cells (Trm, CD44+CD62L-CD69+CD103+) and (B) percentage of Trm among CD8+CD44+ T cells in different groups. [Figure 7] Full protection by late LV::SBeta-2P in boost in mice primed and boosted with mRNA-1273. (A) Timeline of mRNA-1273 im-im prime-boost vaccination in C57BL / 6 mice immunized in with a suboptimal dose of LV::SBeta-2P (n=4–5 / group) of 1×108TU / mouse and pretreated in with Ad5::hACE-2 4 days before in challenge with 0.3×105TCID50 (50% tissue culture infectious dose) of SARS-CoV-2 delta variant. (B) Comparative quantification of hACE-2 mRNA in lungs of different groups of mice pretreated in with Ad5::hACE-2. (C) Viral RNA content assessed at 3 dpi by conventional ECoV-2-specific (top) or subgenomic Esg-specific (bottom) qRT-PCR. The bottom line indicates the limit of detection. Statistical significance was assessed by the Mann-Whitney test (*=p<0.05, **=p<0.01). [Figure 8]Anti-SCoV-2 humoral responses in mRNA-1273 vaccinated mice further boosted intranasally with LV::SBeta-2P. (A) Follow-up anti-SCoV-2 (left) and anti-RBD (right) IgG in serum from mice first primed and boosted im with mRNA-1273. (B) Anti-SCoV-2 IgG (top), anti-RBD IgG (middle), and anti-SCoV-2 IgA (bottom) in whole lung extracts from mice first primed and boosted im with mRNA-1273, followed by a third dose of mRNA-1273 im or an in boost with LV::SBeta-2P. [Figure 9] Lack of mucosal CD8+ Tc2 responses to SCoV-2 in mRNA-1273 vaccinated mice further boosted intranasally with LV::SBeta-2P. Mice are detailed in Figure 2. Lack of IL-4, IL-5, IL-10 and IL-13 production by pulmonary CD8+ T cells after in vitro stimulation with pools of S:256-275, S:536-550 and S:576-590 peptides examined by ICS in parallel with assays performed to detect IFN-γ / TNF / IL-2 (see Figure 5). Cells are gated for viable CD45+CD8+ T cells. [Figure 10A] (A) Map of the plasmid used to produce LV encoding the SD614G-2P antigen. [Figure 10B] (B) Map of the plasmid used to produce LV encoding the SAlpha-2P antigen. [Figure 10C] (C) Map of the plasmid used to produce LV encoding the SGamma-2P antigen. [Figure 10D] (D) Map of the plasmid used to produce LV encoding the SDelta-2P antigen. [Figure 10E] (E) Map of the plasmid used to produce LV encoding the SBeta-2P antigen. [Figure 10F] (F) Map of the plasmid used to produce LV encoding the SOmicron-2P antigen. [Figure 11A] (A) Amino acid sequence of spike from OmicronBA.1 (top) subvariant. Sequences in bold and underlined are mouse MHC-I restricted T cell epitopes in H-2b mice (Ku MW et al., EMBO Mol Med, e14459, 2021). Sequences highlighted in grey are MHC-I or II restricted human T cell epitopes identified in HHD-DR1 MHC humanized mice. [Figure 11B] (B) Amino acid sequence of spike from Omicron BA 4 / 5 (bottom) subvariant. Sequences in bold and underlined are mouse MHC-I restricted T cell epitopes in H-2b mice (Ku MW et al., EMBO Mol Med, e14459, 2021). Sequences highlighted in grey are MHC-I or II restricted human T cell epitopes identified in HHD-DR1 MHC humanized mice. [Figure 12]Humoral immunity in hamsters im immunized with various LV::S. (A) Schematic diagram of LV encoding SCoV-2 proteins from the ancestral WA1 or beta SARS-CoV-2 strains. Codon-optimized sequences encoding SCoV-2 were cloned into a pFLAP lentiviral vector plasmid under the control of the human PCMVie promoter; RRE, rev response element; cPPT, central polypurine tract. LV::SWA1 contains the entire sequence of SCoV-2. The 675QTQTNSPRRAR685 sequence encompassing the RBD, S1 / S2, S2' cleavage site, RRAR furin cleavage site of SEQ ID NO: 27, as well as the K986P, V987P, K417N, E484K and N501Y substitutions are indicated. (B) Western blot analysis to detect the expression of SWA1, SWA1-2P, SWA1-ΔF-2P and SBeta2-P in LV-transduced 293T cells. Total cell lysates were analyzed under non-reducing conditions with anti-S2 rabbit polyclonal antibody. LV::GFP was included as a negative control. Full-length spike (S) and S2 subunits are indicated. (C) Golden hamsters (n=4 / group) were immunized im with 1×108 TU of LV::SWA1, LV::SWA1-2P, LV::SWA1ΔF-2P, or LV::SBeta-2P. Five weeks later, anti-SWA1 responses in serum were measured by ELISA and expressed as mean end-point dilution titers. Error bars represent standard error of the mean (SEM). Statistical significance of differences was determined by Kruskal-Wallis test followed by Dunn's multiple comparison test and was found to be non-significant. Dotted lines indicate the limit of detection (LOD). [Figure 13]Humoral immunity after LV::S administration in hamsters. (A) Timeline of LV::S prime-boost vaccination regimen and WA1 SARS-CoV-2 challenge in hamsters (n=6 / group). (B) Serum anti-SWA1 or anti-RBDWA1 IgG responses are presented as mean end-point dilution titers measured by ELISA. (C) Neutralizing activity (EC50) of sera collected before WA1 SARS-CoV-2 challenge or lung homogenates collected 4 dpi measured with pseudotyped viruses carrying SCoV-2 from the D614G SARS-CoV-2 variant. Data are shown as mean ± SEM. Asterisks indicate the significance of differences between groups. p values ​​were determined using the Kruskal-Wallis test followed by Dunn's multiple comparison test; *p<0.05, **p<0.01. Only significant differences are shown. Dotted lines indicate LOD. [Figure 14] A single inLV::S injection fully protects hamsters against WA1 SARS-CoV-2. Hamsters are as described in the legend of Figure 13. (A) Lung viral load quantified by total E (left) or Esg qRT-PCR (right) at 4 dpi. Bars represent geometric mean values. (B) Percentage of body weight loss in LV::S- or LV ctrl-vaccinated hamsters at 4 dpi. (C) Expression of inflammatory cytokines in lung tissue after challenge. Heatmaps summarize the relative log2 fold change in expression of inflammation-related mediators in LV::S-vaccinated or LV ctrl-treated individuals analyzed at 4 dpi using RNA extracted from total lung homogenates and normalized to samples from naive controls. Six hamsters from each group are shown in the heatmap. Statistical differences between the LV::S and LV ctrl groups were determined by Kruskal-Wallis test followed by Dunn's multiple comparison test and are indicated by asterisks; *p<0.05; **p<0.01; ***p<0.001. Comparisons were made between vaccinated groups and LV ctrl. [Figure 15]A single in LV::S injection significantly reduced lung histopathology. (A) Histological H&E analysis of lungs examined at 4 dpi. Heatmap summarizing 1) inflammation score and 2) interstitial syndrome histopathological score. (B) Representative alveolar-interstitial syndrome and (C) severe inflammation in LV ctrl-injected and infected hamsters, showing a large loss of organ architecture but remnants of alveolar and bronchiolar spaces. (D-F) Bronchial lesions in LV ctrl-immunized animals, showing epithelial cells and cellular debris (black arrows) within the bronchial lumen (D), papillary projections (stars) into the lumen of the bronchial epithelium (E), and degenerative lesions with loss of epithelium (green arrows) (F). (G) Mild alveolar infiltrates in vaccinated hamsters. Some alveoli (arrows) are partially or completely filled with cells and eosinophilic exudate. (H) Representative NCoV-2-specific IHC images performed on lungs from SARS-CoV-2-infected hamsters. The lower panels show enlarged views of the upper panels. Scale bars are 1 mm (upper) and 25 μm (lower). [Figure 16]Reduction of SARSCoV-2 omicron infectious virus in lungs and nasal turbinates by single or booster in LV::SBeta-2P administration. (A) Timeline of single or prime-boost vaccination and omicron SARS-CoV-2 challenge. Hamsters (n=4-5 / group) were administered 1x108TU of LV::SBeta-2P in or im. Three weeks later, they were boosted in with the same amount of LV::SBeta-2P or LV ctrl. Serum samples were taken at weeks 3 and 7 for serological analysis. (B) Serum or anti-SOmicron (upper panel) or anti-RBDOmicron (lower panel) IgG responses measured by ELISA and expressed as mean end-point dilution titers. Data are shown as mean ± SEM. Percentage of weight loss post-challenge (C) and 4 dpi (D) in LV::SBeta-2P- or LV ctrl-vaccinated hamsters. Viral load in (E) lungs and (F) NT was quantified by Esg qRT-PCR at 4 dpi. Bars represent geometric mean values. Statistical differences were determined by Kruskal-Wallis test followed by Dunn's multiple comparison test and are indicated by asterisks. *p<0.05, **p<0.01, ***p<0.001. Dotted lines indicate LOD. [Figure 17] Immunodetection of NCoV-2 antigens in the lungs of Omicron SARS-CoV-2 infected hamsters. Hamsters are as described in Figure 16. (A) An example of each vaccination regimen is shown at low magnification. Solid arrows indicate foci of inflammatory infiltrates and dotted arrows indicate areas where immunodetection signals are discernible even at this low magnification. (B) Magnification shows concentrations of viral antigens (brown) within inflammatory foci (bottom) and only faint staining in areas with no or little inflammation (top). [Figure 18]Robust humoral responses in hamsters vaccinated with LV::SWA1-2P or LV::SBeta-2P prime (im)-LV::SBeta-2P boost (in). (A) Timeline of prime-boost vaccination. Hamsters (n=4 / group) were primed im with 1x108TU of LV::SWA1-2P or LV::SBeta-2P. Five weeks later, hamsters were boosted in with the same amount of LV::SBeta-2P. (B) Serum anti-SWA1 or anti-RBDWA1 (left panel) or anti-SOmicron or anti-RBDOmicron (right panel) IgG responses measured by ELISA and expressed as mean endpoint dilution titers ± SEM. Statistical differences are indicated with an asterisk. *p<0.05. [Figure 19] Imprinting of anti-SCoV-2 antibodies in LV::S prime (im)-LV::SBeta-2P boost (in) vaccinated hamsters. Hamsters are as described in the legend of Figure 18. (A-C) EC50s determined using pseudotyped viruses carrying SCoV-2 from D614G, alpha, beta, gamma, delta or omicron variants. EC50s 5 weeks after prime in serum (A) or 2 weeks after boost in serum (B) and in lung homogenates (C). Data are presented as geometric mean EC50s. Statistical significance was analyzed using two-way ANOVA followed by Sidak's multiple comparison test; **p<0.01; ****p<0.0001. Dotted lines indicate the lower limit of detection (LOD). [Figure 20]Full protection against the omicron variant of LV::SBeta-2P used in a prime (im) boost (in) regimen. (A) Timeline of prime-boost vaccination and in challenge with 0.3x105TCID50 of SARS-CoV-2 omicron variant in B6.K18-hACE2 IP-THV transgenic mice (n=5 / group). (B) Lung viral RNA content assessed by subgenomic Esg-specific qRT-PCR at 5 dpi. The bottom line indicates the limit of detection. Statistical significance was assessed by Mann-Whitney test (*=p<0.05, **=p<0.01). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0067] Highly productive not only in inducing humoral responses but also in establishing particularly high-quality memory T cell responses (Ku MW et al., Commun Biol, 4(1), 713, 2021), the LV-based strategy is an advantageous platform for heterologous boosting, even as it is highly effective in itself as a leading candidate for a COVID-19 vaccine (Ku MW et al., Cell Host Microbe, 29(2), 236-249 e236, 2021; Ku MW et al., EMBO Mol Med, e14459, 2021). Moreover, importantly, LV is non-cytopathic, non-replicating, and largely non-inflammatory, and can therefore be used to perform a non-invasive in boost to effectively induce sterile mucosal immunity that protects the respiratory system as well as the central nervous system (Ku MW et al., Cell Host Microbe, 29(2), 236-249 e236, 2021; Ku MW et al., EMBO Mol Med, e14459, 2021). The in vaccination route has been shown by several teams to be the most effective in reducing viral load in nasal swabs and in the nasal olfactory neuroepithelium (Bricker TL, Cell Rep, 36(3), 109400, 2021; Hassan AO et al., Cell Rep Med, 2(4), 100230, 2021). Thus, we hypothesized that in vaccination could be effective in blocking / reducing the respiratory chain of SARS-CoV-2 infection.

[0068] Another major advantage of LV-based immunization is the induction of a strong T cell immune response due to the high cross-reactivity of T cell epitopes from the spikes of diverse VOCs. Thus, when neutralizing antibodies collapse or fade, the T cell arm remains largely protective, as we have recently described in antibody-deficient and B cell-impaired μMT KO mice (Ku MW et al., EMBO Mol Med, e14459, 2021). This property is associated with high-quality and long-lasting T cell immunity induced against multiple conserved T cell epitopes despite accumulated mutations in the spikes of emerged VOCs (Ku MW et al., EMBO Mol Med, e14459, 2021).

[0069] In the present invention, the inventors first identified S that induces the maximum neutralization width against VOCs. Beta We down-selected the antigen and engineered a non-integrating LV encoding a stabilized version of this antigen. In mice primed and boosted with mRNA-1273, which has a reduced (cross)seroneutralizing capacity, we demonstrated that LV::S Beta-2P Increasing doses of anti-S CoV-2 We demonstrated that IgG and IgA titers increased in a dose-dependent manner and serum neutralizing capacity against VOCs expanded in both serum and lung homogenates. In the lungs of mice injected im with the third dose of mRNA-1273, anti-S CoV-2 No IgA was detected. These mice express CD38, which is associated with resident memory properties. + CD73 + CD62L + CD69 + CD80 + Lung surface IgM / D with phenotype - A dose-dependent increase in the proportion of B cells was detected, whereas no increase in such B cell subsets was detected in mice boosted im with mRNA-1273.

[0070] Spike-specific effector lung CD8 +Tc1 cells were first primed and boosted with mRNA-1273 and then in LV::S Beta-2P These lung CD8+ T cells did not show a Tc2 phenotype. + CD44 + CD69 + CD103 + An increase in the proportion of Trm also Beta-2P was detected in a dose-dependent manner only in mice in-boosted with S, but not in mice im-boosted with mRNA-1273. CoV-2 Systemic CD8 against various immunogenic regions of + T cell responses were measured using 1 × 10 8 Or 1×10 9 TU's LV::S Beta-2P Increased by in boost. LV::S Beta-2P The highest in dose of LV::S was comparable to an additional im dose of mRNA-1273. Beta-2P The fact that in administration of the vaccine provides a boosting effect on systemic T cell immunity is another advantage of this vaccination regimen.

[0071] We assessed lung protection in mice primed and boosted with mRNA-1273 and found that without a boost, 20 weeks after the first injection of mRNA-1273, there was no detectable protection against the delta variant of SARS-CoV-2. 8 TU's LV::S Beta-2P An in booster injection of mRNA-1273 completely inhibited SARS-CoV-2 replication in the lungs. A third late im booster injection of mRNA-1273 similarly reduced SARS-CoV-2 RNA content in the lungs but did not completely inhibit viral replication in all mice.

[0072] The lack of protection against delta variants 4 months after prime-boost with mRNA-1273 may be explained by the hypothesis that adaptive immune memory is likely located in secondary lymphoid organs, i.e., anatomical sites far from the respiratory tract. In this context, the very rapid replication of new VOCs such as delta and omicron variants at the site of infection does not allow enough time for reactivation of immune memory fast enough to prevent local mucosal infection, replication, and viral infection.

[0073] In the present invention, the inventors have Beta-2P These results provide mounting evidence that in boosting with LV::S can be used to induce robust systemic and mucosal adaptive immunity and to broaden the specificity of protective responses. Beta-2P The in-boost of LV::S enhances the strength and broadens the cross-recognition of VOCs, targets B and T cell immune responses to the primary entry point of SARS-CoV-2 into the mucosal respiratory tract of the host organism, and avoids infection at the primary anatomical site. Beta-2P A Phase I / IIa clinical trial is underway for the use of in boosting with vaccinia in previously vaccinated people or convalescent COVID patients.

[0074] We established that B cell-independent and antigen-specific T cell immunity plays a major role in LV-mediated protection against SARS-CoV-2 infection (Ku MW et al., EMBO Mol Med, e14459, 2021). This was consistent with the strong T cell responses induced by LV::SBeta-2P at the systemic level and in the lungs and detectable in vaccinated mice (Vesin, B. et al., Mol Ther 30, 2984-2997, 2022). Importantly, the ancestral S CoV-2 All or most of the mouse and human T cell epitopes identified in the sequence and contained in SBeta-2P were conserved in mutant spikes from the newly emerged variants, including the Omicron BA.1 and BA.4 / 5 subvariants (Figure 11).

[0075] These observations support the role of LVs expressing the spike protein of SARS-CoV-2, particularly LV::S, in inducing complete protection against ancestral as well as recently emerged SARS-CoV-2 variants by eliciting robust T cell responses. Beta-2P This indicates the powerful capabilities of

[0076] In contrast to the B cell epitopes targeted by neutralizing antibodies, the T cell epitopes identified so far are not specific to the newly emerged variants of S CoV-2 were not or only slightly affected by the mutations accumulated in the .

[0077] This observation indicates why the LV platform, which induces potent T cell immunity, is robust and fully protective against emerging variants.

[0078] Materials and Methods Mouse immunization and SARS-CoV-2 infection Female C57BL / 6JRj mice were purchased from Janvier (Le Genest Saint Isle, France), housed in individual ventilated cages under specific pathogen-free conditions in the animal facility of the Pasteur Institute, and used at 7 weeks of age. Mice were immunized im with mRNA-1273 (Moderna) vaccine 1 μg / mouse. For in-injection of LV, mice were anesthetized by i.p. injection of ketamine (Imalgene, 80255 mg / kg) and xylazine (Rompun, 5 mg / kg). For protection experiments against SARS-CoV-2, mice were transferred to filtered cages in isolators. Four days before SARS-CoV-2 inoculation, mice were inoculated with 3 × 10 8 Mice were pretreated with IGU of Ad5::hACE2 as previously described (Ku MW et al., Cell Host Microbe, 29(2), 236-249 e236, 2021). Mice were then transferred to a level 3 biosafety cabinet and treated with 0.3 × 10 5 TCID 50Mice were inoculated in situ with a delta SARS-CoV-2 clinical isolate (Lescure FX et al., Lancet Infect Dis, 20(6), 697-706, 2020). Mice were then housed in filtered cages in isolators in biosafety level 3 animal facilities. Organs harvested from infected animals were processed according to standard procedures approved in these facilities.

[0079] Ethics approval for animal experiments Animal experiments were performed according to European and French guidelines (Directive 86 / 609 / CEE and Decree 87-848 of 19 October 1987), after approval by the Institut Pasteur Animal Care and Use Committee and protocol agreement by the local ethical committee (CETEA #DAP20007, CETEA #DAP200058, and Ministry of Higher Education and Research APAFIS#24627-2020031117362508 v1, APAFIS#28755-2020122110238379 v1).

[0080] Construction and production of vaccine LV First, the codon-optimized sequences of spikes from the ancestral l, D614G, alpha, beta or gamma VOCs were synthesized and inserted into the pMK-RQ_S-2019-nCoV_S501YV2 plasmid. The S sequences were then extracted by BamHI / XhoI digestion and ligated between the BamHI and XhoI restriction sites located between the native human ieCMV promoter and the mutated atg start codon sequence of the woodchuck posttranscriptional regulatory element (WPRE) sequence in the pFlap lentiviral plasmid (see plasmid map, Figure 10). D614G Or S Beta To 986 PV 987 To introduce the P "2P" double mutation, the corresponding pFlap plasmids were subjected to directional mutagenesis using the Takara In-Fusion kit. 2PThe -WPREm plasmid was amplified and used to produce non-integrating vaccinal LV as described elsewhere (Ku MW et al., Cell Host Microbe, 29(2), 236-249 e236, 2021; Ku MW, Charneau P, Majlessi L. Expert Rev Vaccines, 1-16, 2021).

[0081] Analysis of humoral and systemic T cell immunity anti-S CoV-2 IgG and IgA antibody titers were measured using recombinant stabilized S CoV-2 Neutralizing capacity of clarified and decomplemented serum or lung homogenates was measured by ELISA using S or RBD fragments as coating. CoV-2 Quantification was performed using lentiviral particles pseudotyped with (Ku MW et al., Cell Host Microbe, 29(2), 236-249 e236, 2021; Sterlin D et al., Sci Transl Med, 13(577), 2021).

[0082] H-2 d In mice, S CoV-2 T-splenocyte responses were quantified by IFN-γ ELISPOT after in vitro stimulation with S:256-275, S:536-550 or S:576-590 synthetic 15mer peptides containing MHC-I restricted epitopes (Ku MW et al., Cell Host Microbe, 29(2), 236-249 e236, 2021). Spots were quantified with a CTL Immunospot S6 ultimate-V Analyser using the CTL Immunocapture 7.0.8.1 program.

[0083] Phenotypic and functional cellular analysis of pulmonary immune cells Enrichment and staining of pulmonary immune cells was performed as detailed elsewhere after treatment with 400U / ml collagenase type IV and DNase I (Roche), incubation at 37°C for 30 min, and homogenization using GentleMacs (Miltenyi Biotech) (Ku MW et al., Cell Host Microbe, 29(2), 236-249 e236, 2021; Ku MW et al., EMBO Mol Med, e14459, 2021). The cell suspension was then filtered through a 100 μm pore filter, centrifuged at 1200 rpm, and enriched on a Ficoll gradient after centrifugation at 3000 rpm, RT, 20 min, without brake. The harvested cells were co-cultured with syncytial bone marrow-derived dendritic cells loaded with A, B, C peptide pools (1 μg / ml each) or negative control peptide (x290 μg / ml). The following mixtures were used for detection of pulmonary 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 mixtures were used to detect pulmonary Tc2 cells: PerCP-Cy5.5-anti-CD3 (45-0031-82, eBioScience), eF450-anti-CD4 (48-0042-82, eBioScience), and 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), and PE-anti-IL-13 (12-7133-81, eBioScience) for intracellular staining. Intracellular staining was performed using the Fix Perm kit (BD) according to the manufacturer's protocol. Dead cells were excluded using Near IR Live / Dead (Invitrogen).Staining was performed in the presence of anti-CD16 / CD32 (BD), which blocks FcγII / III receptors.

[0084] Lung-resident memory CD8 + To identify T cell subsets, PerCP-Vio700-anti-CD3 (130-119-656, Miltenyi Biotec), PECy7-CD4 (552775, BD Biosciences), BV510-anti-CD8 (100752, BioLegend), PE-anti-CD62L (553151, BD Biosciences), APC-anti-CD69 (560689, BD Biosciences), APC-Cy7-anti-CD44 (560568, BD Biosciences), FITC-anti-CD103 (11-1031-82, eBiosciences) and yellow Live / Dead (Invitrogen) were used. Lung B cells were treated with PerCP Vio700-anti-IgM (130-106-012, Miltenyi), PerCP Vio700-anti-IgD (130-103-797, Miltenyi), APC-H7-anti-CD19 (560143, BD Biosciences), and 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) The cells were examined by surface staining with a mixture of Live / Dead (BV) and yellow (Invitrogen).

[0085] Cells were incubated with the appropriate mixtures for 25 min at 4°C, washed with PBS containing 3% FCS, incubated overnight at 4°C, and then fixed with 4% paraformaldehyde. Samples were acquired on an Attune NxT cytometer (Invitrogen) and data were analyzed with FlowJo software (Treestar, OR, USA).

[0086] Measurement of viral RNA content in organs Organs were removed from mice and immediately frozen at -80°C on dry ice. 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 500 μl lysis matrix M (MP Biomedical) plus PBS using an MP Biomedical Fastprep 24 Tissue Homogenizer. RNA was extracted from the supernatant of organ homogenates centrifuged at 2000g for 10 min using the Qiagen Rneasy kit, except that the neutralization step with AVL buffer / carrier RNA was omitted. This RNA sample was then used to determine viral RNA content by E-specific qRT-PCR. To measure viral RNA content by Esg-specific qRT-PCR, total RNA was prepared using lysis matrix D (MP Biomedical) with 1 mL of TRIzol reagent (ThermoFisher) and homogenized twice for 30 s at 6.0 m / s using an MP Biomedical Fastprep 24 Tissue Homogenizer. RNA sample quality was assessed using a Bioanalyzer 2100 (Agilent Technologies). Viral RNA content was quantified using a NanoDrop Spectrophotometer (Thermo Scientific NanoDrop). RNA Integrity Numbers (RIN) ranged from 7.5 to 10.0. SARS-CoV-2 E or E subgenomic mRNA was quantified after reverse transcription and real-time quantitative TaqMan® PCR using the SuperScript™ III Platinum One-Step qRT-PCR System (Invitrogen) with specific primers and probes (Eurofins) as described recently (Ku MW et al., EMBO Mol Med, e14459, 2021). EXAMPLES

[0087] Antigen design and down-selection of lead candidates The optimal S that can take into account the kinetics of viral spread of known variants and induce maximum neutralization breadth can be determined. CoV-2 To select variants, we selected full-length S sequences from alpha, beta or gamma SARS-CoV-2 VOCs. CoV-2 LVs encoding the following were produced: 1 × 10 8 TU / mouse were primed im (week 0) and boosted im (week 3), and various S CoV-2 The (cross)neutralizing ability of the sera against pseudotyped viruses carrying LV::S was evaluated (Fig. 1A). Alpha Immunity by S. D614G and S Alpha Although it produced adequate neutralizing activity against S. Beta and S Gamma No effect was observed in the LV::S mice (Fig. 1B). Beta and LV::S Gamma Between the two, the former is S D614G , S Alpha and S Gamma Consistent with previous observations using other vaccine strategies in the context of immunization with LV, the K in the S2 domain of SCoV-2 produced the highest cross-seroneutralization potential against the variants. 986 PV 987 The P substitution improved the (cross)seroneutralization potential (Fig. 1C), possibly due to the CoV-2-2P This is due to an extension of the half-life of (Walls AC et al., Cell, 181(2), 281-292 e286, 2020).

[0088] Taken together, these data suggest that LV (LV::S) may be used to boost the waning immunity previously induced by first-generation COVID-19 vaccines such as mRNA-1273. Beta-2P ) as the best cross-reactive antigen candidate for use in the context of Beta-2PI was able to downselect.

[0089] Humoral immunity follow-up and LV::S in mice primed and boosted with mRNA-1273 Beta-2P The effect of in boost The present inventors have Beta-2P We analyzed the possibility that in boost vaccination with mRNA-1273 could enhance and expand immune responses in mice that were first primed and boosted with mRNA-1273 and have reduced (cross-)seroneutralizing capacity. C57BL / 6 mice were primed im at week 0 and boosted im at week 3 with 1 μg / mouse, defined as the optimal dose of mRNA-1273 in mice (Nature, 2020, Vol. 586, 567-571) (Figure 2A). Long-term serological follow-up showed that at 3 weeks post-prime, S D614G and S. Alpha We demonstrated that cross-neutralizing activity against both S. cerevisiae and S. cerevisiae was readily detectable (Fig. 2B). Cross-neutralization was also observed, to a lesser extent, against S. cerevisiae. Gamma It was also detected against S Beta , S Delta , S Delta+ At week 6, i.e., 3 weeks after the boost, all S CoV-2 Although cross-serum neutralizing activity against the variants was detectable, S Beta , S Delta and S Delta+ From the 6th to the 10th week, the activity against S Beta , S Delta , or S Delta+ The cross-neutralization of serum against IgG decreased significantly over time.

[0090] At week 15, mRNA-1273 primed and boosted mice were treated with 1 × 10 6 , 1×10 7 , 1×10 8 , or 1 × 10 9 TU / Mouse LV::S Beta-2PControl mRNA-1273 primed and boosted mice received 1 × 10 empty LV (LV Ctrl) infusions (Figure 2A). 9 In parallel, mice primed and boosted with mRNA-1273 at this time point were injected im with 1 μg of mRNA-1273 or PBS. Unprimed, age-matched mice were injected im with 1 × 10 9 TU's LV::S Beta-2P Or PBS was administered in.

[0091] In mice primed with mRNA-1273, serum anti-S CoV-2 and anti-RBD IgG were detected at week 3, increased after the mRNA-1273 boost as examined at weeks 6 and 10, and then declined at week 17 in the absence of additional boosts (Fig. 8A). In the mRNA-1273 primed and boosted mice boosted at week 15, 1 × 10 8 Or 1×10 9 TU's LV::S Beta-2P or a third dose of mRNA-1273 injected into mice with anti-S CoV-2 A significant increase in IgG titers was observed (Figure 2C). CoV-2 The IgA titer is 1 × 10 9 TU's LV::S Beta-2P At the mucosal level, at this time point, anti-S in whole lung extracts was significantly higher in mice injected with the first dose than in mice injected with the third dose of mRNA-1273 (Figure 2C). CoV-2 and anti-RBD IgG titers in LV::S Beta-2P In mice injected with LV::S, the levels increased in a dose-dependent manner. Beta-2P The highest dose of was comparable to the third im dose of mRNA-1273 (Fig. 8B). Importantly, the pulmonary anti-S CoV-2 Significant IgA titers were observed in LV::S Beta-2P was only detected in mice boosted im late with mRNA-1273, but was barely detectable in mice boosted im late with mRNA-1273 ( Fig. 8B ).

[0092] At the mucosal cell level, IgM in the lungs - / IgD - CD19 + (Ig switch) Within the B cell population, CD38 may constitute resident memory B cells + CD62L + CD69 + CD73 + CD80 + An increased proportion of cells were identified as LV::S Beta-2P It was detected in a dose-dependent manner in mice in-boosted with mRNA-1273, but not in mice im-boosted with mRNA-1273 (Figures 3A and 3B).

[0093] LV::S in mice previously primed and boosted with mRNA-1273 Beta-2P of systemic and mucosal T cell immunity after in boost of influenza A vaccine Systemic anti-S CoV-2 T cell immunity is H-2 b Mouse CD8 + S immunodominant to T cells CoV-2 After in vitro stimulation with individual S:256-275, S:536-550 or S:576-590 peptides encompassing the region, IFN-γ-specific ELISPOT was assessed in the spleens of individual mice immunized according to the above regimen (Figure 2A) (Ku MW et al., Cell Host Microbe, 29(2), 236-249 e236, 2021). Importantly, weak anti-S CD8 expression was detected in the spleens of mice primed and boosted with mRNA-1273 at week 17. + T cell immunity is 1 x 10 8 and 1×10 9 TU / Mouse LV::S Beta-2P In-boosting with 1 × 10 9 TU dose LV::S Beta-2P The booster effect of in administration of tended to be equal to or greater than that of im booster administration of mRNA-1273.

[0094] In parallel, in the same animals, mucosal anti-S:CD11 expression was confirmed by intracellular Tc1 and Tc2 cytokine staining in T cell-enriched fractions from individual mice after in vitro stimulation with autologous bone marrow dendritic cells loaded with a pool of S:256-275, S:536-550 and S:576-590 peptides. CoV-2 T cell immunity was assessed (Figure 5A). Mice previously primed and boosted with mRNA-1273 showed minimal S in the lungs. CoV-2 Specific IFN-γ / TNF / IL-2 CD8 + Only T cell responses were detected (Figure 5A). Beta-2P Intravenous administration of 1 × 10 8 Or 1×10 9 A significant proportion of Tc1 cells was induced at the TU dose. Im administration of mRNA-1273 had a much lower boosting effect on mucosal T cells (Fig. 5A, B). Tc2 responses (IL-4, IL-5, IL-10, IL-13) were not detected in any of the experimental groups (Fig. 9).

[0095] Importantly, LV::S Beta-2P Mice boosted in with increasing doses of + The proportion of T cells (Trm) was significantly increased, in stark contrast to the corresponding mice that received the third dose of mRNA-1273 im, in which the latter did not in fact show a significant proportion of this Trm cell population that very often correlates with protection in many infections (Figure 6A,B).

[0096] Late LV::S in mice primed and boosted with mRNA-1273 Beta-2P Complete defensive capability with in boost. Next, we investigated the expression of LV::S in mice primed and boosted with mRNA-1273. Beta-2P The protective vaccine efficacy of in boosting with 1 × 10 IgG was evaluated following a vaccination regimen equivalent to that described above. At week 15, mRNA-1273 primed and boosted mice were vaccinated with a suboptimal dose of 1 × 10 IgG.8 TU's LV::S Beta-2P or control empty LV in (Figure 7A). The choice of such a suboptimal dose was based on our numerous previous observations at this dose that was effective for protection in allogeneic LV prime-boost experiments (Ku MW et al., Cell Host Microbe, 29(2), 236-249 e236, 2021; Ku MW et al., EMBO Mol Med, e14459, 2021). Control mRNA-1273-immunized mice were administered mRNA-1273 im or PBS. Unvaccinated age- and sex-matched controls remained unimmunized. Four weeks after the late boost, i.e. at week 20, all mice were immunized with 3 × 10 8 Mice were pretreated with an adenoviral vector serotype 5 encoding the infectious genomic unit (IGU) hACE2164 (Ad5::hACE2) (Ku MW et al., Cell Host Microbe, 29(2), 236-249 e236, 2021) to render their lungs permissive for SARS-CoV-2 replication (Figure 7B). Four days later, mice were challenged with the SARS-CoV-2 delta variant, which was the most widespread SARS-CoV-2 variant worldwide at the time of this invention, i.e., November 2021.

[0097] At 3 days postinfection (dpi), analysis of lung total RNA first demonstrated that hACE-2 was uniformly expressed in all mice after in vivo transduction with Ad5::hACE2 (Fig. 7B). Then, at 3 dpi, total E RNA and subgenomic (Esg)E RNA were significantly increased. CoV-2Pulmonary viral load was determined by assessing RNA qRT-PCR, the latter being an indicator of active viral replication (Chandrashekar A et al., Science, 369(6505), 812-817, 2020; Tostanoski LH et al., Nat Med, 26(11), 1694-1700, 2020; Wolfel R, Corman VM, Guggemos W et al., "Virological assessment of hospitalized patients with COVID-2019." Nature, 581(7809), 465-469, 2020). No significant protection against challenge with SARS-CoV-2 delta variants was detected in mice first primed and boosted with mRNA-1273 and then injected in with control LV or im with PBS alone. In sharp contrast, LV::S Beta-2P The in boost of SARS-CoV-2 E CoV-2 Dramatically reduces total RNA content and reduces replication-associated Esg E CoV-2 No copies of RNA were detected in this group (Figure 7B). In the group that received the im boost of late mRNA-1273, E CoV-2 Total RNA content was also significantly reduced. In this group, Esg E was detected in 3 of 5 cases. CoV-2 No RNA content was detected.

[0098] Complete pulmonary protection against SARS-CoV-2 by one-shot or booster intranasal lentiviral vaccination in Syrian golden hamsters We demonstrated that a single intranasal administration of a vaccine lentiviral vector encoding a stabilized form of the original SARS-CoV-2 spike glycoprotein induces complete respiratory lung protection and strongly reduces lung inflammation in a prototypic SARS-CoV-2 susceptible Syrian golden hamster model. Furthermore, we demonstrated that a lentiviral vector encoding the stabilized spike of the SARS-CoV-2 beta variant (LV::S Beta-2P) prevented pathology in the lungs and nasal turbinates and reduced infectious viral load following challenge with the SARS-CoV-2 omicron variant. Importantly, LV::S Beta-2P When intranasally boosted with LV::S Beta-2P Hamsters primed with LV showed much improved cross-seroneutralization compared to their counterparts primed with LV encoding the spike from the ancestral SARS-CoV-2. These results strongly suggested that immune imprinting by the original spike sequence negatively impacts cross-protection against the new variant. Our results address the issue of vaccine efficacy in already vaccinated and immunocompromised individuals and show that LV-based intranasal vaccination is efficient either as a single dose or as a booster.

[0099] Construction and production of LV expressing S protein LV::S WA1 and LV::S WA1-ΔF2P The construction of was previously described (Ku MW et al., Cell Host Microbe, 29(2), 236-249 e236, 2021; Ku MW et al., EMBO Mol Med, e14459, 2021).

[0100] LV production and titration Lentiviral particles are prepared using the vector plasmid pFlap / S for the production of integration-deficient vectors. CoV-2LV was produced by transient calcium phosphate co-transfection of HEK293T cells with pFLAP, vesicular stomatitis virus G Indiana envelope plasmid, and encapsidation plasmid pD64V. Supernatants were harvested 48 hours after transfection and clarified by centrifugation at 2500 rpm for 6 min at 4°C. LV was aliquoted and stored at -80°C. Vector titers were determined by transducing aphidicolin-treated HEK293T cells. Titers, which are proportional to the efficiency of nuclear gene transfer, were determined as Transduction Units (TU) / mL by qPCR on total lysates 3 days after transduction using forward and reverse primers specific for the pFLAP plasmid and forward and reverse primers specific for the host housekeeping gene gadph (Iglesias et al., J. Gene Med., 2006, 8, 265-274).

[0101] SDS-PAGE and Western blotting HEK293T cells (2 × 10 6Cells / well) were seeded and grown overnight and then transduced with LV encoding the SARS-CoV-2 S transgene at a multiplicity of infection of 10. Cell lysates were collected and quantified 48 hours after transduction. After heating at 95°C for 5 min with Volt sample buffer, samples were loaded onto precast Volt 4-12% Bis-Tris gels (Invitrogen). Proteins were transferred to nitrocellulose membranes using the iBlot2 dry blotting system (Invitrogen), and the membranes were blocked with TBST blocker (Tris-buffered saline (TBS) containing 0.2% Tween 20 and 5% milk). After 1 h of blocking, the membranes were incubated overnight with anti-SARS-CoV-2 S2 rabbit polyclonal antibody (SinoBiological 40590-T62) in TBST blocker. The membrane was then washed three times for 10 min with TBST, followed by incubation with DyLight 800-labeled goat anti-rabbit IgG (H+L) secondary antibody (Invitrogen, Cat # SA5-35571) at 1:2,500 in TBST blocker for 1 h. Finally, the membrane was washed three times for 10 min with TBST and developed using an ODYSSEY CLx Infrared Imaging System (Li-COR). E-PAGE SeeBlue Pre-stained Standard (Invitrogen) was used as a ladder.

[0102] hamster Male Mesocricetus auratus golden hamsters (Le Genest Saint Isle, France) were purchased at maturity and weighed 80–100 grams at the start of the experiment. Hamsters were housed in individual ventilated cages under specific pathogen-free conditions during the immunization period. For SARS-CoV-2 infection, these hamsters were transferred to individual filtered cages installed in an isolator in the animal facility of the Pasteur Institute. Prior to im or in injections, hamsters were sedated with isoflurane inhalation or ip injection of ketamine (Imalgene, 100 mg / kg) and xylazine (Rompun, 5 mg / kg).

[0103] Ethics approval for animal experiments Experiments on hamsters were performed according to European and French guidelines (Directive 86 / 609 / CEE and Decree 87-848 of 19 October 1987) after approval by the Institut Pasteur Animal Experimentation Committee, protocol agreement given by the local ethical committee (CETEA #DAP200007) and the Ministry of Higher Education and Research (APAFIS#24627-2020031117362508 v1).

[0104] SARS-CoV-2 spike protein production Codon-optimized nucleotide fragments encoding the WA1 or Omicron BA.1 RBD protein containing a stabilized version of the SARS-CoV-2 WA1 or Omicron BA.1 spike (HexaPro) ectodomain (followed by a foldon trimerization motif) and C-terminal tags (Hisx8-tag, Strep-tag, and AviTag) were synthesized and cloned into the pcDNA3.1 / Zeo(+) expression vector (Thermo Fisher Scientific). Recombinant proteins were produced by transient transfection into exponentially growing Freestyle 293-F suspension cells (Thermo Fisher Scientific, Waltham, MA) using the polyethylenimine (PEI) precipitation method as previously described (PMID: 25910833). Proteins were purified from culture supernatants by high-performance chromatography using Ni Sepharose® Excel Resin (GE Healthcare) according to the manufacturer's instructions, dialyzed against PBS using Slide-A-Lyzer® dialysis cassettes (Thermo Fisher Scientific), quantified using a NanoDrop 2000 instrument (Thermo Fisher Scientific), and purity controlled by SDS-PAGE using NuPAGE 3-8% Tris-acetate gels (Life Technologies) as previously described (PMID: 25910833).

[0105] Humoral response The immunoglobulin G (IgG) antibody was generated using recombinant stabilized SARS-CoV-2 derived from the WA1 or Omicron strains. CoV-2Detection was performed by enzyme-linked immunosorbent assay (ELISA) using the IgG and RBD proteins. Nunc Polysorp ELISA plates (ThermoFisher, 475094) were coated with 1 μg / mL in 50 mM Na2CO3 (pH 9.6) at 4°C overnight. After incubation, the plates were washed with 1×PBS+0.05% Tween-20 (PBST) and blocked with PBST+1% BSA for 2-3 h at 37°C. The plates were incubated with sera serially diluted in PBS-T+1% BSA for 1.5 h at 37°C. After washing, rabbit anti-hamster IgG-horseradish peroxidase conjugate (Jackson Immuno Research, M37470) was used as the secondary antibody, and 3,5,3',5'-tetramethylbenzidine (Eurobio Scientific, 5120-0047) was used as the antibody reaction detection substrate. The reaction was stopped with 50 μL of 2 M sulfuric acid. The endpoint titer was calculated as the highest serum dilution that resulted in an absorbance greater than the mean + 3 SD of pre-immune sera.

[0106] SARS-CoV-2 vaccination Hamsters were anesthetized with an i.p. injection of a mixture of ketamine and xylazine, transferred to biosafety cabinet 3, and incubated with 0.3 × 10 5 TCID 50 The animals were inoculated in situ with 50 μl of virus inoculum containing the Omicron BA.1 variant (Pango lineage BA.1, GISAID: EPI_ISL_6794907 and EPI_ISL_7413964) of SARS-CoV-2 WA1 (Lescure et al., Lancet Infect. Dis., 2020, 20, 697-706) or SARS-CoV-2 clinical isolate (Planas et al., Nature, 2022, 602, 671-675). Animals were housed in isolators in the biosafety level 3 animal facility of the Institut Pasteur. Organs harvested from infected animals were treated according to standard procedures approved in these facilities.

[0107] Pseudotyped virus neutralization assay Quantification of Nabs was performed as previously described (Sterlin et al., Sci. Transl. Med., 2021, 13, eabd2223) in HEK293T cells stably expressing human ACE2 (HEK293T-ACE2) and nonreplicating S harboring a reporter luciferase firefly gene. CoV-2 Host cell invasion by mimicking the fusion step of the native SARS-CoV-2 virus was assessed by an inhibition assay that allows quantification using pseudotyped LV particles. Serum samples or clarified lung homogenates were heat inactivated at 56 °C for 30 min. Serial 4-fold dilutions of samples diluted in 25 μl of DMEM-glutamax (Gibco, 21063-029) containing 10% heat inactivated FCS, 100 U / mL penicillin and 100 mg / mL streptomycin and 1 mM sodium pyruvate (Gibco, 11360-070) were incubated in U-bottom plates with 1 ng S in 25 μl for 30 min at room temperature. CoV-2 The samples were then mixed with an equivalent amount of pseudotyped LV p24. 4 The cells were transferred to clear flat-bottom 96-well black plates (Corning, CLS3603) containing HEK 293T-ACE2 cells. The plates were incubated at 37°C for 72 hours, after which luciferase expression was measured using the ONE-Glo™ Luciferase Assay System (Promega, E6120) on an EnSpire plate reader (PerkinElmer). EC50 was calculated using the indicated S CoV-2 Reported as the reciprocal of the serum dilution resulting in 50% infection of HEK 293T-ACE2 cells with lentiviral vectors harboring the variants.

[0108] Measurement of viral load in organs Lungs and nasal turbinates (NT) were aseptically removed and immediately frozen at -80°C. RNA from circulating SARS-CoV-2 was prepared from lungs as recently described (Ku MW et al., Cell Host Microbe, 29(2), 236-249 e236, 2021). Briefly, lung homogenates were prepared by thawing and homogenizing the organs with Lysis Matrix A (MP Biomedicals, 116913050-CF) in 500 μl ice-cold PBS in an MP Biomedical Fastprep 24 Tissue Homogenizer and used to measure viral load by E-specific qRT-PCR. Alternatively, total RNA was prepared from lungs or NT by adding 1 mL of TRIzol Reagent (ThermoFisher, 15596026) in Lysis Matrix D (MP Biomedical, 116910050-CF) and homogenizing twice for 30 seconds at 6.0 m / s using an MP Biomedical Fastprep 24 Tissue Homogenizer. These RNA preparations were used to measure viral load by Esg-specific qRT-PCR or to measure inflammatory mediators.

[0109] After reverse transcription and real-time quantitative TaqMan® PCR using the SuperScript™ III Platinum™ One-Step qRT-PCR Kit (Invitrogen, 11732020) with specific primers and probes (Eurofins), SARS-CoV-2 E gene or E subgenomic mRNA (Esg RNA) was quantified as previously described (Corman et al., Euro Surveill. 2020, 25(3); Wolfel et al., Nature 2020, 581(7809):465-9). Standard curves for Esg mRNA assays were performed using in vitro transcribed RNA derived from a PCR fragment of “T7 SARS-CoV-2 Esg mRNA”. In vitro transcribed RNA was synthesized using the T7 RiboMAX Express Large Scale RNA production system (Promega, P1320) and purified by phenol / chloroform extraction and two successive precipitations with isopropanol and ethanol. The RNA concentration was determined by optical density measurement and diluted to 10 with RNAse-free water containing 100 μg / mL tRNA carrier. 9 The in vitro transcribed RNA was diluted to genome equivalents / μL and stored at −80°C. Serial dilutions of this in vitro transcribed RNA were prepared in RNAse-free water containing 10 μg / ml tRNA carrier to generate standard curves 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 and 58°C for 30 s. PCR products were analyzed on an ABI 7500 Fast real-time PCR system (Applied Biosystems). RNA copy values ​​were extrapolated from the standard curve and multiplied by the volume to obtain the number of RNA copies per organ. The limit of detection was based on the standard curve and was defined as the amount of RNA that gave a Ct value of 40.

[0110] qRT-PCR quantification of inflammatory mediators in hamster lungs and brain was performed on total RNA extracted with TRIzol reagent as recently detailed (Ku MW et al., Cell Host Microbe, 29(2), 236-249 e236, 2021).

[0111] histopathology Samples from hamster lungs were fixed in formalin for 7 days and embedded in paraffin. Paraffin sections (5 μm thick) were stained with hematoxylin and eosin (H&E). Histopathological lesions were described qualitatively and, when possible, scored using: (i) distribution modifiers (i.e., focal, multifocal, localized widespread, or diffuse); (ii) a 5-point severity grade (i.e., 1: minimal, 2: mild, 3: moderate, 4: marked, 5: severe). In some cases, serial sections were prepared for immunohistochemistry (IHC) analysis. IHC was performed as previously described (Ku MW et al., EMBO Mol Med, e14459, 2021). For IHC, rabbit anti-N CoV-2 Antibody (Novus Biologicals, NB100-56576) and biotinylated goat anti-rabbit Ig secondary antibody (Dako, E0432) were used. Slides were scanned using an AxioScan Z1 (Zeiss) system, and images were analyzed with Zen 2.6 software.

[0112] statistical analysis Statistical significance was assigned at P values ​​<0.05. ELISA titers were multiplied by log 10 The mean mean and mean mean scores were transformed. For comparison of two groups, the nonparametric Mann-Whitney test was used. For comparison of more than two experimental groups, Kruskal-Wallis ANOVA and Dunn's multiple comparison test were applied. Differences in the neutralizing activity of VoCs were analyzed by two-way analysis of variance (two-way ANOVA) and Sidak's test for multiple comparisons. Tests were performed using GraphPad Prism software (version 9, Graphpad Software, La Jolla, CA, USA).

[0113] result Various S CoV-2 Immunogenicity of LV encoding forms of Cytomegalovirus (CMV) immediate early promoter (P CMVie ) under the transcriptional control of S CoV-2 A non-integrated LV was constructed that encodes a stabilized conformer of the first two S CoV-2 The conformer is the human codon-optimized full-length membrane-anchored ancestral WA1 S CoV-2 (Ku MW et al., Cell Host Microbe, 29(2), 236-249 e236, 2021). LV::S WA1-2P has two stabilizing K 986 P and V 987 S with P substitution WA1 LV::S WA1ΔF-2P is two K 986 P and V 987 In addition to the P substitution, the loop encompassing the S1 / S2 furin cleavage site (675-QTQTNSPRRAR-685 of SEQ ID NO:27) was deleted for further stability in the prefusion state. WA1 (McCallum et al., Nat. Struct. Mol. Biol., 2020, 27, 942-949; Launay et al., EBioMedicine 2022, 75, 103810). Beta-2P is derived from Beta (B.1.351) VoC, and K 986 P and V 987 Contains two substitutions of P. S Beta is S WA1 Unlike N located in the RBD, 501 Y / K 417 N / E 484 K mutations (Tegally et al., Nature 2021, 592, 438-443). WA1Pseudoviruses carrying these RBD mutations are neutralized by sera from individuals vaccinated with currently approved vaccines, whereas those exhibiting these RBD mutations are moderately to strongly resistant to neutralization (Kuzmina et al., iScience 2021, 24, 103467). This observation provided a rationale for adapting the S sequence variants for further vaccination. CoV-2 Expression of the immunogen was confirmed by Western blot on total cell lysates (Figure 12B). As expected, the S2 furin cleavage product was expressed as S WA1 , S WA1-2P Or S Beta-2P was only detected in cells transduced with LV encoding

[0114] LV::S WA1 , LV::S WA1-2P , LV::S WA1ΔF-2P and S Beta2P To compare the immunogenicity of 8 After 5 weeks (wk), high serum titers of anti-S WA1 IgG antibodies were induced by all LVs examined (Figure 12C). Since no significant differences in immunogenicity were observed among these LVs, hereafter referred to as "LV::S" WA1ΔF-2P were selected for evaluation of protection against homologous SARS-CoV-2.

[0115] Induction of robust humoral responses against SARS-CoV-2 by a single infusion of LV::S We recently showed that LV::S used in a prime (im)-boost (in) protocol significantly improved protection against SARS-CoV-2 compared to a single im injection in a hamster model (Ku MW et al., Cell Host Microbe, 29(2), 236-249 e236, 2021). Here, we evaluated the protective ability of a single in dose of LV::S against ancestral WA1 SARS-CoV-2. Hamsters (n=6 / group) were immunized at week 0 or week 5 with 1 × 10 8 As a positive control, a group of hamsters was immunized in week 0 with a single injection of 1 × 10 8 Hamsters were primed im with TU LV::S and boosted in with the same amount of LV::S on week 5. Control hamsters followed the same regimen and received the same amount of LV expressing green fluorescent protein (LV ctrl) as an irrelevant antigen. On week 7, all animals received 0.3 × 10 5 TCID 50 The WA1 mice were challenged in with SARS-CoV-2 (Figure 13A). Pre-challenge, pre-immune sera and sera from the LV ctrl group were positive for anti-S WA1 Antibodies and anti-RBD WA1 After a single in-jection of LV::S, all animals were negative for anti-S antibodies (Fig. 13B). WA1 IgG and anti-RBD WA1 These antibody titers were obtained as early as 2 weeks after immunization, as shown by hamsters vaccinated at week 5. Serum IgG titers remained stable until week 7. At week 7, there was a significantly lower anti-S antibody titer in the in-treated group compared to the im-in group. WA1 and anti-RBD WA1 IgG titers were detected. Serum neutralizing activity was WA1Anti-RBD IgG titers were assessed using pseudotyped viruses carrying SARS-CoV-2. Consistent with anti-RBD IgG titers, serum neutralizing activity was lower in hamsters immunized with a single in injection compared to the im-in group (Figure 13C). Despite comparable anti-S and anti-RBD IgG titers at 2 or 7 weeks after in-injection, sera from hamsters vaccinated earlier showed slightly higher neutralizing capacity, suggesting that antibodies require time to mature for efficient neutralizing capacity. However, in whole lung homogenates 4 days post-inoculation (4 dpi) after SARS-CoV-2 inoculation, all vaccinated groups had comparable neutralizing capacity (Figure 13C). Virus neutralizing activity in the lung may be more relevant for protection than activity detectable in serum.

[0116] A single in-dose of LV::S induces protection against homologous SARS-CoV-2 challenge In the lungs of LV::S vaccinated individuals in either the im-in or single i-n groups, there was an approximately 2-4 log decrease in viral load compared to the LV ctrl group, as determined by qRT-PCR detection of SARS-CoV-2 envelope (E) RNA at 4 dpi. 10 A reduction in SARS-CoV-2 Esg RNA was observed (Figure 14A, left panel). Lung viral load, measured by subgenomic E RNA (Esg) qRT-PCR, is an indicator of active viral replication (Wolfel et al., Nature 2020, 581(7809):465-9). This analysis demonstrated a complete absence of viral replication in the three vaccinated groups, whereas the geometric mean ± SD of SARS-CoV-2 Esg RNA per lung was (5.4 ± 6.8) × 10 8At 4 dpi, consistent with the observed protection, only 2-3% weight loss was detected in hamsters vaccinated with either in alone or in the im-in prime-boost regimen, whereas 12% weight loss was detected in hamsters administered LV ctrl (Figure 14B). In whole lung homogenates of LV:.S vaccinated and SARS-CoV-2 challenged hamsters, a significant decrease in the expression of pro-inflammatory IFN-γ, TGF-β, IL-6 cytokines, anti-inflammatory IL-10 cytokines, and CCL2, CCL3, CCL5 and CXCL10 chemokines, and FoxP3 was detected compared to corresponding hamsters injected and challenged with LV ctrl, as assessed by qRT-PCR at 4 dpi (Figure 14C). Changes in inflammatory markers were particularly prominent in the group administered in 2 weeks prior to challenge. Consistent with these results, a positive correlation was observed between viral load and inflammation (r=0.46, p<0.05), whereas body weight was inversely correlated with viral load and inflammation, respectively (r=-0.6842, p<0.001 and r=-0.56, p<0.01, Spearman test).

[0117] A single in dose of LV::S reduces infection-induced inflammation in vaccinated hamsters. Lung histopathology showed that vaccinated controls exhibited pulmonary infiltrates (Figure 15A) and severe alveolar-interstitial inflammation (Figure 15B) leading to areas of dense preconsolidation (Figure 15C). These lungs also exhibited bronchial lesions, with images of hyperplastic epithelial proliferation resulting in epithelial desquamation of individual or clustered cells (Figure 15D), papillary projections (Figure 15E) or intraluminal epithelial folds (Figure 15F). In the vaccinated group, interstitial lesions (Figure 15A) and alveolar lesions (Figure 15G) were minimal to moderate. SARS-CoV-2 nucleocapsid protein (N CoV-2 Immunohistochemical analysis of lungs from LVctrl-treated and infected hamsters using a specific polyclonal antibody revealed N-terminal domains in bronchial epithelial cells (not shown) and in the interstitium (Figure 15H, right panel). CoV-2 +In contrast, the severity of inflammation was reduced in LV::S vaccinated animals. When detectable, the inflammatory zones were still N CoV-2 + Cells contained 100% lysate, indicating that although viral replication was controlled (Fig. 14A), invasion and viral debris had not yet been fully absorbed at the early 4 dpi time point.

[0118] Collectively, these data indicated that immunization with a single in dose of LV::S was as efficient as an im priming followed by an in boosting regimen and conferred strong protective immunity against homologous SARS-CoV-2 infection.

[0119] LV::S Beta-2P Prime-boost vaccination confers cross-protection against omicron variants Given the dynamics of the pandemic, a key question is the ability of vaccines to induce cross-protection against emerging VoCs. Based on a series of LVs encoding Ss from various VoCs, we have recently developed LV::S Beta-2P was selected as the best candidate to generate the broadest spectrum of cross-neutralizing capacity (Vesin, B. et al., Mol Ther 30, 2984-2997, 2022). Beta-2P To evaluate the efficacy of 8 TU's LV::S Beta-2P Hamsters (n=4-5 / group) were primed im or in with LV::S at the same dose in the third week. Beta-2P At week 7, 0.3 × 10 5 TCID 50 All groups were challenged with the SARS-CoV-2 BA.1 omicron subvariant (Planas et al., Nature 2022, 602(7898):671:5). Omicron is S WA1BA.1 omicron strain isolated from a patient harbored 32 mutations compared to BA.1. Of these mutations, 15 were located in the RBD. Infection of hamsters with this patient-isolated BA.1 omicron strain resulted in significant weight loss (Figure 16B).

[0120] All LV::S Beta-2P In vaccinated hamsters, S Omicron and RBD Omicron Robust cross-reactive serum IgG titers against S. cerevisiae were detected by ELISA (Figure 16B). Three weeks after priming, no significant differences in antibody titers were observed between groups. Antibody levels remained stable after a single injection, but in animals primed and boosted in, anti-S. cerevisiae antibody levels were significantly higher. Omicron A significant increase in the titers of RBD antibodies was observed in all vaccinated groups. In contrast, anti-RBD antibody titers continued to increase over time in all vaccinated groups (Fig. 16B, bottom).

[0121] After the challenge, LV::S Beta-2P Hamsters that received a single im injection of LV::S or LV ctrl gradually lost weight (Fig. 16C). Beta-2P Hamsters vaccinated with LV showed less than 5% weight loss and no signs of pathology (Figure 16D). At 4 dpi, the lungs and nasal turbinates were analyzed for viral content. High viral loads were detected in both organs of the LV ctrl-injected group (Figure 16E, Figure 16F). In contrast, Esg RNA was not detected in the lungs of the im-in group, and a significant reduction of about 2 logs was observed in the other groups (Figure 16E). Notably, im-vaccinated hamsters, which did not suppress viral replication, showed the greatest weight loss. Also, although significantly reduced, active viral replication was still detectable in the NT of all hamsters, indicating that LV-based in vaccination, although showing strong lung protection, does not completely prevent intranasal infection (Figure 17F). However, regardless of the priming route, in boosting led to a superior efficacy in controlling and spreading infection in airway tissues compared with a single vaccine dose.

[0122] LV:.S Beta-2P Reduction of virus load in vaccinated hamsters as determined by immunohistochemistry At 4 dpi, histopathological analysis of lung sections from the ctrl group showed lesions similar to those in Figure 15H (Figure 17). Immunohistochemistry showed that mice boosted in or im had less N than animals primed only and injected with LV ctrl. CoV-2 Although most of the patients generally showed staining for Esg, the degree of variation within the group was relatively large (Figure 17). In addition, we did not observe a strict correlation between the degree of IHC signal and Esg qRT-PCR quantification, indicating that a portion of the immunostained antigens corresponds to non-replicating viral debris.

[0123] Taken together, these results indicated that a single in immunization with LV was sufficient to control infection, but LV-based in immunization was more suitable for boosting previously induced anti-COVID-19 immunity.

[0124] LV::S WA1-2P Prime with LV::S Beta-2P Induction of cross-reactive antibodies in hamsters boosted with Next, the inventors WA1 In animals previously exposed to LV::S Beta-2P The efficacy of in-boost was evaluated. 8 TU's LV::S WA1-2P Or LV::S Beta-2P Hamsters (n=4 / group) were primed im with 1×10 8 TU's LV::S Beta-2P In all vaccinated hamsters, robust serum IgG titers against the S and RBD proteins were detected at all post-priming time points tested (Figure 18B). Equivalent S protein titers were detected after priming or boosting. WA1 Or S OmicronThe kinetic profile and strength of the specific antibody response was observed (Figure 18B, top). Either homologous or heterologous in boosting modestly increased anti-S antibody titers by 1.8-fold or 2.5-fold, respectively. In contrast, RBD WA1 Approximately 4 to 10 times lower RBD compared to Omicron Serum IgG responses against anti-RBD were measured (Fig. 18B, bottom). Omicron IgG titers were significantly improved by heterologous boosting compared with homologous boosting, 3.8-fold vs. 1.7-fold, respectively.

[0125] Prime with LV::S and LV::S Beta-2P Anti-S in hamsters boosted with CoV-2 Antibody Imprinting Five weeks after the Im injection, LV::S WA1-2P and LV::S Beta-2P Both are S D614G Or S Alpha The serum showed high neutralizing activity against the pseudotyped virus carrying S (Fig. 19A). D614G , S Alpha , and S Delta The cross-neutralizing activity against LV::S was similar in the two groups of immunized hamsters. Notably, after a single im injection, Beta-2P Only hamsters immunized with S Omicron Although the serum neutralizing activity against S variants was weak, it showed serum neutralizing activity against all S variants.

[0126] LV::S Beta-2P In boosting with LV::S increased serum cross-neutralizing activity against all VoCs in both groups (Fig. 19B). WA1-2P Prime with LV::S Beta-2P The serum from hamsters boosted with S Beta The cross-neutralization of pseudotyped viruses carrying S Omicron Lung homogenates were completely unable to cross-neutralize pseudotyped viruses carrying S after heterologous prime-boost. Beta Or SOmicron In stark contrast, LV::S showed a similar profile with no cross-neutralizing activity against Beta-2P Prime with LV::S Beta-2P Serum and lung homogenates from hamsters boosted with S. Beta Or S Gamma were able to cross-neutralize pseudotyped viruses carrying S Omicron Pseudotyped viruses carrying LV::S were also able to cross-neutralize to a lesser extent. However, this prime-boost regimen provided sufficient protection against SARS-CoV-2 Omicron challenge, as observed above (Figure 16). Thus, LV::S Beta-2P Boost is LV::S Beta-2P In hamsters primed with LV::S WA1 These results indicate a clear imprinting effect in anti-S humoral immunity, which was especially evident for the omicron and beta variants.

[0127] Consideration LV-based platforms have recently emerged as a powerful vaccination approach against COVID-19. We have demonstrated its strong preventative ability to induce pulmonary protection against SARS-CoV-2 infection, especially when used as a systemic prime followed by a mucosal in boost (Ku MW et al., Cell Host Microbe, 29(2), 236-249 e236, 2021). In this study, as a further step towards clinical trials, we demonstrate that S WA1 Or S BetaWe used LV encoding a stabilized form of the . This choice was based on data showing that stabilization of the prefusion form of the viral envelope glycoprotein improves the yield when produced as a recombinant protein in the industrial production of subunit vaccines. It also enhances the efficacy of nucleic acid-based vaccines by increasing the availability of the antigen in its optimal immunogenic form (Hsieh et al., Science, 2020, 369(6510):1501-5).

[0128] In the first part of this report, we report WA1 We demonstrated that a single i.m. administration of LV encoding S. cerevisiae resulted in complete lung protection in a highly susceptible hamster model as efficiently as an i.m. prime-boost regimen, as assessed by virological, immunological and histopathological parameters. The hamster ACE2 ortholog protein, S. cerevisiae CoV-2It efficiently interacts with SARS-CoV-2, which easily allows host cell invasion by SARS-CoV-2 with a high replication rate. The rapid weight loss and development of severe lung lesions after SARS-CoV-2 inoculation make outbred hamsters a sensitive model for evaluating the efficacy of drugs or vaccine candidates (Sia et al., Nature 2020, 583(7818):834-8). Hamsters were a more challenging model than rhesus macaques, which only develop mild COVID-19 pathology. Therefore, the strong lung protection against homologous challenge by a single in dose in the hamster model was an asset of major importance. This protection is likely due to the development of mucosal immunity. Induction of antigen-specific secretory dimeric IgA, which can block viral interactions at the mucosal level, has been shown to reduce viral shedding and correlate with protection (Halfmann et al., Cell Rep. 2022;39(3):110688; Munoz-Fontela et al., Nature 2020, 586(7830):509-15; Wang et al., Sci Transl Med. 2021, 13(577)). Although infectious virus was still detected in the nasal turbinates of In-immunized hamsters, a significant reduction in infectious virus titers could lead to reduced infection and transmission, as recently described by Langel SN et al., providing a means of disease control (Langel et al., Sci Transl.Med. 2022, 14(658)). Indeed, we have previously shown in a mouse model that anti-S IgG and secretory IgA antibodies are generated together with lung-resident memory B and T cells after in-administration of LV. The presence of IgA induced by an LV-based SARS-CoV-2 vaccine correlated with complete lung protection against the virus (Vesin, B. et al., Mol Ther 30, 2984-2997, 2022). Unfortunately, the lack of immunological tools, including anti-IgA antibodies and antibodies against activated / memory T cell markers, prevented us from assessing mucosal immunity in the hamster model.On the other hand, there was growing evidence that in immunization provides better protection not only against ancestral strains of SARS-CoV-2 but also against newly emerged VoCs (Afkhami et al., Cell 2022, 185(5):896-915; Bricker et al., Cell Rep. 2021, 36(3):109400). Previous studies exploring this area have used adenovirus vector vaccines in chimpanzees, which are known to be inflammatory and pose risks for use in mucosal vaccination (Coughlan et al., Mol. Ther. 2022, "Adenovirus-based vaccines-a platform for pandemic preparedness against emerging viral pathogens"). In stark contrast to this, LV is non-cytopathic and very weakly inflammatory (Ku et al., Vaccines 2021:1-16, 1988854), making it much more suitable for mucosal vaccination. The fact that a single in situ administration of LV-based vaccines 2 or 7 weeks prior to homologous SARS-CoV-2 challenge induces protection is valuable for setting up clinical trials of LV-based vaccines. This platform, together with the significant benefits of mucosal immunity in reducing viral infection, could provide significant advantages for mass vaccination.

[0129] The continued emergence of SARS-CoV-2 VoCs in terms of anti-S antibody responses is likely due to previous infection or predominantly S. WA1This prompted us to expand our studies by evaluating the protective potential of a heterologous antigen booster that could mimic some aspects of previous vaccination with first-generation vaccines based on S. moniliforme. Numerous breakthrough SARS-CoV-2 infections have been observed in vaccine recipients, indicating that the cross-efficacy of these vaccines is incomplete (Abu-Raddad LJ et al., 2021;385(2):187-9; Kuhlmann C et al., 2022;399(10325):625-6). Recently, it has been reported that mucosal booster vaccination is necessary to establish robust sterilizing immunity in the respiratory tract against SARS-CoV-2 (Tang J et al., "Respiratory mucosal immunity against SARS-CoV-2 following mRNA vaccination.", Sci Immunol. 2022:eadd4853). In LV-immunized hamsters, we found that S. moniliforme was associated with increased protective potential for S. moniliforme. WA1 Antigens and S Beta-2P Antigen S CoV-2 We did not detect any significant differences between the ability of the LVs to induce cross-reactive serum IgG responses against SARS-CoV-2. However, a clear distinction should be made between the protective capacity of vaccines and their ability to induce neutralizing antibodies, since T cell responses are also major effectors against SARS-CoV-2 infection. In particular, the efficacy of LV-based protection depends critically not only on the ability to induce neutralizing antibody responses, but also on T cell immunogenicity. It is noteworthy that near-complete protection of the lungs is achieved in μMT KO mice, which completely lack the mature B cell compartment and antibody responses (Ku MW et al., EMBO Mol Med. 2021:e14459). Moreover, in the lungs of LV::S prime (im) boost (in) mice, mucosal resident memory T cells, as well as IFNγ + IL-2 + TNF + Triple positive CD8 +T cell effectors are easily detected

[26] . Moreover, findings obtained after natural infection suggest that specific T cell immunity, which is generally not affected by mutations occurring in the S antigen of the newly emerged SARS-CoV-2 variants, is largely effective against viral replication (Altmann DM et al., 2021;2(5):100286; Mazzoni A et al., 2022;13:801431). T cell-mediated protection also works reliably in hamsters. However, as mentioned above, the lack of immunological tools hindered the characterization of T cell responses in this study.

[0130] Prime with LV::S and LV::S Beta-2PIn hamsters boosted with SARS-CoV-2, statistically significant reduced cross-neutralizing activity against beta and gamma variants, and no cross-neutralizing activity against omicron variants, was observed, despite enhanced serum neutralizing activity against D614G, alpha, and delta variants. Similarly, in the case of influenza A virus, the first exposure to a serotype can affect future responses to that variant (Gostic KM et al., 2016;354(6313):722-6). This raises concerns regarding the immune imprinting effect of past infection or vaccination on antibody responses, which needs to be taken into account when designing vaccines (Roltgen K et al., "Immune imprinting, breadth of variant recognition, and germinal center response in human SARS-CoV-2 infection and vaccination." Cell. 2022). This study indicates that pre-exposure of the immune system to early S variants negatively impacts neutralizing antibody responses measured after a late boost with a heterologous S variant. Our results support recent data showing that healthcare workers infected with either the ancestral or alpha variants of SARS-CoV-2 exhibit reduced neutralizing immunity to omicron (Reynolds CJ et al., 2022;377(6603):eabq1841). Furthermore, using an mRNA vaccine, Kalnin et al. have also shown that heterologous boosting results in inferior neutralizing antibody titers compared to homologous boosting (Kalnin KV et al., 2022;40(9):1289-98). It can be hypothesized that additional injections of variant S sequences may be required to counteract this negative effect and achieve sufficient levels of cross-neutralization against VoC.

[0131] Collectively, our results demonstrated the capability of LV as an effective vaccine delivery platform. LV was an effective and promising strategy to induce strong protective immunity against SARS-CoV-2 VoC, and had the advantage of being non-inflammatory and therefore suitable for mucosal vaccination. We recently demonstrated that 1 × 10 9 In mice injected with LV at a high dose of TU, ​​LV::S Beta-2P demonstrated the safety of in-administration of (Vesin, B. et al., Mol Ther 30, 2984-2997, 2022). Histopathological analysis of the lungs did not detect any side effects.

[0132] LV::S against SARS-CoV-2 Omicron variant Beta-2P Complete cross-protection The inventors demonstrated that B6.K18-hACE2 predisposes to SARS-CoV-2 infection in the lungs and further demonstrated unprecedented brain permissiveness for SARS-CoV-2 replication. IP-THV In transgenic mice, LV::S Beta-2P The protective effect of B6.K18-hACE 2 was evaluated (Ku MW et al., EMBO Mol Med, e14459, 2021). IP-THV Mice (n=5 / group) were cultured at 1 × 10 8 TU / Mouse LV::S Beta-2P Alternatively, they were primed intramuscularly (im) with empty LV (sham) and boosted intranasally (in) with the same dose of the same vector at week 3 (Figure 20A). 5 TCID 50 Mice were challenged (in) with the SARS-CoV-2 omicron variant.

[0133] Then, at 5 days postinfection, subgenomic E CoV-2Viral RNA content in the lungs and brain was measured using RNA(Esg)qRT-PCR (Chandrashekar et al., 2020, Science, 369, 812-817; Tostanoski et al., 2020, Nat. Med. 26, 1694-1700; Wolfel et al., 2020, Nature, 581, 465-469). Beta-2P Vaccination provided sterile protection against SARS-CoV-2 omicron in the lungs, i.e., Esg viral RNA was undetectable in vaccinated mice compared with (5.83 ± 6.22) × 10 in sham-vaccinated mice. 9 In parallel, Esg qRT-PCR quantification of viral RNA content in the brain detected no viral RNA copies in vaccinated mice, compared with (6.41 ± 1.29) × 10 copies in the brains of sham-vaccinated control mice. 9 We detected 100 copies of viral RNA in the brain (Fig. 20B, right). Of note, although 2 of the 5 mice did not show cervical infection at Omicron, the other 3 mice showed very heavy viral replication in the brain.

[0134] Therefore, LV:S Beta-2P demonstrated complete cross-protection against omicron variants, fully comparable to our previous demonstration of efficiency against the ancestral or delta variants (Ku MW et al., EMBO Mol Med, e14459, 2021; Ku MW et al., Cell Host Microbe, 29(2), 236-249 e236, 2021; Vesin, B. et al., Mol Ther 30, 2984-2997, 2022).

Claims

1. 1. A vaccine composition against SARS-CoV-2 infection or disease selected from the group consisting of protein, mRNA, adenovirus, inactivated virus, and protein subunit vaccine compositions against SARS-CoV-2 infection or disease, particularly a protein or mRNA vaccine composition against SARS-CoV-2 infection or disease, for use as a heterologous boost or targeted immunization agent in a vaccine regimen for administration to the upper respiratory tract of a subject, particularly a human subject, who has received prime immunization with the vaccine composition, the composition comprising pseudotyped lentiviral vector particles encoding the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a derivative thereof.

2. 2. The composition of claim 1, wherein the administration is as an intranasal mucosal boost or targeted immunization in a subject who has been primed with a vaccine composition against SARS-CoV-2 infection or disease selected from the group consisting of protein, mRNA, adenovirus, inactivated virus and protein subunit vaccine compositions against SARS-CoV-2 infection or disease, particularly a protein or mRNA vaccine composition against SARS-CoV-2 infection or disease.

3. - the S protein is derived from a SARS-CoV-2 virus that is pathogenic for a human host, in particular (i) an S protein derived from a SARS-CoV-2 virus selected from the group consisting of the SARS-CoV-2 ancestral, D614G, alpha, beta, gamma, delta and Omicron strains, preferably an S protein derived from a SARS-CoV-2 virus selected from the beta or Omicron strain, more preferably from the beta strain, or (ii) an S protein derived from a variant of said ancestral, D614G, alpha, beta, gamma, delta or Omicron strain, such variant encoding a spike protein having an amino acid sequence at least 90% identical to SEQ ID NO: 1, or 10. The composition of claim 1, wherein the S protein is a derivative of the native S protein of any of the ancestral, D614G, Alpha, Beta, Gamma, Delta or Omicron strains by mutation, particularly substitution and / or deletion of 1 to 12, particularly 1 to 6, amino acid residues, in particular 1 to 12, particularly 1 to 6 amino acid residues, in particular (a) a stabilized form of the S protein characterized by substitution of two consecutive amino acid residues in the S2 domain of the S protein at the positions provided as residues 986 and 987 with reference to SEQ ID NO: 1 for proline residues in the amino acid sequence of the spike protein, or (b) a pre-fusion form of the S protein by deletion of the furin site located from residue 675 to residue 685 with reference to SEQ ID NO: 1 for proline residues in the S2 domain at the positions provided as residues 986 and 987 with reference to SEQ ID NO: 1, or (c) a stabilized pre-fusion form of the S protein by deletion of the furin site and substitution of two consecutive amino acid residues in the S2 domain at the positions provided as residues 986 and 987 with reference to SEQ ID NO: 1 for proline residues.

4. 10. The composition of claim 1, wherein the amino acid sequence of the S protein is SEQ ID NO: 1 or 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 a lysine residue at position 417 of the amino acid sequence of SEQ ID NO: 1 to an asparagine residue (K417N); (ii) a mutation of a glutamic acid residue at position 484 of the amino acid sequence of SEQ ID NO: 1 to a lysine residue (E484K) or a mutation of a glutamic acid residue at position 484 of the amino acid sequence of SEQ ID NO: 1 to an alanine residue (E484A); (iii) a mutation of an asparagine residue at position 501 of the amino acid sequence of SEQ ID NO: 1 to a tyrosine residue (N501Y); (iv) a mutation of a lysine residue at position 986 of the amino acid sequence of SEQ ID NO: 1 to a proline residue (K986P); and (v) a mutation of a valine residue at position 987 of the amino acid sequence of SEQ ID NO: 1 to a proline residue (V987P).

5. The subject to be administered the composition is (a) a subject who has previously received a vaccine composition against SARS-CoV-2 infection or disease selected from the group consisting of protein, mRNA, adenovirus, inactivated virus and protein subunit vaccine compositions against SARS-CoV-2 infection or disease, particularly a protein or mRNA-based vaccine against SARS-CoV-2 infection or disease, as a systemic prime and / or boost administration, such as intramuscular, intradermal or subcutaneous administration, particularly an intramuscular prime and / or boost administration; (b) a vaccine composition against SARS-CoV-2 infection or disease selected from the group consisting of protein, mRNA, adenovirus, inactivated virus and protein subunit vaccine compositions against SARS-CoV-2 infection or disease, particularly a SARS-CoV-based vaccine 10. The composition of claim 1, wherein the subject is selected from the group consisting of: (a) a subject who has received a systemic prime dose, such as an intramuscular, intradermal, or subcutaneous dose, particularly an intramuscular prime dose, of a protein- or mRNA-based vaccine against SARS-CoV-2 infection or disease and has subsequently recovered from a coronavirus disease, such as coronavirus disease 2019 (COVID-19); (b) a subject who has initially recovered from a coronavirus disease, such as COVID-19, and has subsequently received a systemic prime dose, such as an intramuscular, intradermal, or subcutaneous dose, particularly an intramuscular dose, of a protein- or mRNA-based vaccine against SARS-CoV-2 infection or disease; and (c) a subject who has received more than two, particularly more than three, systemic doses, such as an intramuscular, intradermal, or subcutaneous dose, particularly an intramuscular dose, of a protein- or mRNA-based vaccine against SARS-CoV-2 infection or disease.

6. 10. The composition of claim 1, wherein the S protein of SARS-CoV-2 further comprises an amino acid mutation selected from the group consisting of: (vi) a mutation of a glycine residue at position 446 of the amino acid sequence of SEQ ID NO:1 to a serine residue (G446S); (vii) a mutation of a threonine residue at position 478 of the amino acid sequence of SEQ ID NO:1 to a lysine residue (T478K); (viii) a mutation of a glutamine residue at position 493 of the amino acid sequence of SEQ ID NO:1 to an arginine residue (Q493R); and (ix) a mutation of a glutamine residue at position 498 of the amino acid sequence of SEQ ID NO:1 to an arginine residue (Q498R).

7. The composition of claim 1, wherein the encoded SARS-CoV-2 mutant S protein has the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 18, preferably SEQ ID NO:

10.

8. 10. The composition of claim 1, wherein the pseudotyped lentiviral vector particles are pseudotyped with a vesicular stomatitis virus glycoprotein G (VSV-G) protein.

9. 10. The composition of claim 1 in a prime / boost or targeted immunization regimen to induce a long-lasting protective mucosal humoral and / or long-lasting mucosal cellular immune response against SARS-CoV-2 infection or disease, wherein the response protects the subject's respiratory system and / or central nervous system.

10. Pseudotyped lentiviral vector particles inhibit CD8 expression against SARS-CoV-2 + The composition of claim 1, which induces a T cell response.

11. Pseudotyped lentiviral vector particles express spike-specific lung-resident memory CD8 + T cells (Trm) and / or effector CD8 + The composition of claim 1, which induces T cells (Tc1) to produce interferon-gamma (IFN-γ) / tumor necrosis factor (TNF) / interleukin-2 (IL-2).

12. 10. The composition of claim 1, wherein the pseudotyped lentiviral vector particles are non-integrating, non-cytopathic, and non-replicating.

13. 10. The composition of claim 1, wherein the subject shows waning immunity from week 12 after the first injection of primary vaccination with a vaccine composition against SARS-CoV-2 infection or disease selected from the group consisting of protein, mRNA, adenovirus, inactivated virus and protein subunit vaccine compositions against SARS-CoV-2 infection or disease, particularly a protein or mRNA based vaccine against SARS-CoV-2 infection or disease, or after SARS-CoV-2 disease recovery, particularly after COVID-19 recovery.

14. 10. The composition of claim 1, wherein the pseudotyped lentiviral vector particles are formulated as a liquid composition or a dry powder for administration as an intranasal aerosol, intranasal drops, or intranasal inhalation.

15. The administration regimen comprises administering one or more dosage forms of pseudotyped lentiviral vector particles, each dosage form being 10 7 ~10 9 10. The composition of claim 1, which is a transduction unit (TU).

16. 1. An immunogenic composition comprising pseudotyped lentiviral vector particles encoding the S protein of SARS-CoV-2 or a derivative thereof, and a pharmaceutically acceptable carrier, wherein the pseudotyped derivative of the S protein of SARS-CoV-2 has (i) a mutation of a lysine residue at position 417 of the amino acid sequence of SEQ ID NO: 1 to an asparagine residue (K417N), (ii) a mutation of a glutamic acid residue at position 484 of the amino acid sequence of SEQ ID NO: 1 to an alanine residue (E484A), (iii) a mutation of an asparagine residue at position 501 of the amino acid sequence of SEQ ID NO: 1 to a tyrosine residue (N501Y), (iv) a mutation of a lysine residue at position 986 of the amino acid sequence of SEQ ID NO: 1 to a proline residue (N501Y). an immunogenic composition comprising at least nine amino acid mutations, including (i) a mutation from a valine residue to a proline residue at position 987 of the amino acid sequence of SEQ ID NO: 1 (K986P); (ii) a mutation from a valine residue to a proline residue at position 987 of the amino acid sequence of SEQ ID NO: 1 (V987P); (iii) a mutation from a glycine residue to a serine residue at position 446 of the amino acid sequence of SEQ ID NO: 1 (G446S); (iv) a mutation from a threonine residue to a lysine residue at position 478 of the amino acid sequence of SEQ ID NO: 1 (T478K); (viii) a mutation from a glutamine residue to an arginine residue at position 493 of the amino acid sequence of SEQ ID NO: 1 (Q493R); and (ix) a mutation from a glutamine residue to an arginine residue at position 498 of the amino acid sequence of SEQ ID NO: 1 (Q498R).

17. The immunogenic composition of claim 16, wherein the encoded SARS-CoV-2 mutant S protein has the amino acid sequence of SEQ ID NO:

18.

18. 17. The immunogenic composition of claim 16, formulated for intranasal administration.

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