NUCLEIC ACID VACCINE AGAINST THE SARS-CoV-2 CORONAVIRUS

Abstract

The invention relates to an immunogenic or vaccine composition against the 2019 novel coronavirus (SARS-CoV-2), comprising a nucleic acid construct encoding a SARS-CoV-2 coronavirus Spike (S) protein antigen or a fragment thereof comprising the receptor-binding domain, wherein the nucleic acid construct sequence is codon-optimized for expression in human.

Claims

1-24. (canceled)

25. An immunogenic or vaccine composition against SARS-CoV-2 virus comprising a nucleic acid construct encoding a SARS-CoV-2 virus Spike (S) protein antigen having at least 90% identity with the amino acid sequence from positions 19 to 1273 of SEQ ID NO: 2 or a fragment thereof comprising the receptor-binding-domain (RBD), wherein the nucleic acid construct sequence is codon-optimized for expression in human.

26. The immunogenic or vaccine composition according to claim 25, wherein the nucleic acid construct comprises a sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 3, the nucleotide sequences having at least 80% identity with said sequences, and the RNA sequences thereof.

27. The immunogenic or vaccine composition according to claim 25, wherein said RBD fragment comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 4.

28. The immunogenic or vaccine composition according to claim 25, wherein the S antigen or RBD fragment thereof comprises one or more mutations within the RBD selected from the group consisting of: K417N or K417T, N439N, L452R, Y453F, S477N, E484K, F490S, and N501Y, said positions being indicated according to the numbering in the sequence SEQ ID NO: 2.

29. The immunogenic or vaccine composition according to claim 28, wherein the S antigen or RBD fragment thereof comprises at least one mutation close to the pocket of contact with the receptor ACE2 chosen from E484K, F490S, and N501Y.

30. The immunogenic or vaccine composition according to claim 28, wherein the S antigen or RBD fragment thereof comprises the following mutations: N501Y; E484K and N501Y; K417T or K417N, E484K and N501Y; K417N, N439N, Y453F, S477N, E484K, F490S, and N501Y; K417N, N439N, L452R, S477N, E484K, F490S, and N501Y.

31. The immunogenic or vaccine composition according to claim 25, wherein the S antigen further comprises at least one mutation which inactivates the furin cleavage site selected from the group consisting of: the R682G, R683S and R685S substitutions, said positions being indicated according to the numbering in the sequence SEQ ID NO: 2.

32. The immunogenic or vaccine composition according to claim 25, wherein the S antigen comprises a mutation which stabilizes the Spike trimer selected from the group consisting of: the K986P and V987P mutations, and optionally one to four additional proline mutations selected from the group consisting of F817P, A892P, A899P and A942P, said positions being indicated according to the numbering in the sequence SEQ ID NO: 2.

33. The immunogenic or vaccine composition according to claim 25, wherein the S antigen comprises a C-terminal deletion of 1 to 25 or more amino acids, said position being indicated according to the numbering in the sequence SEQ ID NO: 2.

34. The immunogenic or vaccine composition according to claim 33, wherein the S antigen comprises the deletion of the C-terminal residues from position K1255.

35. The immunogenic or vaccine composition according to claim 25, wherein the S antigen comprises one or more mutations selected from the group consisting of: the substitutions L18F, T20N, P26S, D80A, D138Y, R190S, D215G, A570D, D614G, H655Y, P681H, A701V, T7161, S982A, T10271, D1118H and V1176F; and the deletions delta 69-70, delta 144 and delta 242-244.

36. The immunogenic or vaccine composition according to claim 35, wherein the S antigen comprises at least five of said substitutions, and optionally also at least one or two of said deletions.

37. The immunogenic or vaccine composition according to claim 25, wherein said S protein antigen or RBD fragment thereof comprises a signal peptide.

38. The immunogenic or vaccine composition according to claim 37, wherein the signal peptide is selected from the group consisting of the sequences SEQ ID NO: 5, 6 and 7.

39. The immunogenic or vaccine composition according to claim 25, wherein said S protein antigen or RBD fragment thereof further comprises at least an epitope recognized by human T cells.

40. The immunogenic or vaccine composition according to claim 39, wherein the epitope is a Universal Pan HLA-DR Epitope such as PADRE. (SEQ ID NO: 8).

41. The immunogenic or vaccine composition according to claim 39, wherein the S protein antigen or fragment thereof and the epitope are separated by a linker.

42. The immunogenic or vaccine composition according to claim 25, wherein said nucleic acid construct comprises a Kozak sequence.

43. The immunogenic or vaccine composition according to claim 25, wherein said S protein antigen or RBD fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 11, 13, 15, 17, 19, 21, 23, 25, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66 and the amino acid sequences having at least 90% identity with said sequences.

44. The immunogenic or vaccine composition according to claim 25, wherein said nucleic acid construct comprises a sequence selected from the group consisting of SEQ ID NO: 10, 12, 14, 16, 18, 20, 22, 24, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, the nucleotide sequences having at least 80% identity with said sequences, and the RNA sequences thereof.

45. The immunogenic or vaccine composition according to claim 25, wherein said nucleic acid construct is a mammalian expression cassette, wherein the coding sequence of said S protein antigen or RBD fragment thereof is operably linked to appropriate regulatory sequence(s) for their expression in an individual's target cells or tissue(s).

46. The immunogenic or vaccine composition according to claim 45, wherein the regulatory sequence(s) comprise a promoter and optionally further comprise one or more of an enhancer, a terminator or an intron.

47. The immunogenic or vaccine composition according to claim 25, wherein said nucleic construct is RNA or DNA.

48. The immunogenic or vaccine composition according to claim 47, wherein said RNA is non-replicating or self-amplifying mRNA comprising a cap structure, 5′- and 3′-untranslated regions. (UTRs), and a 3′poly(A) tail operably linked to the coding sequence of said S protein antigen or RBD fragment thereof.

49. The immunogenic or vaccine composition according to claim 25, which comprises a vector comprising said nucleic acid selected from the group consisting of: a viral vector, a plasmid, a nucleic acid delivery agent or combination thereof.

50. The immunogenic or vaccine composition according to claim 25, wherein the viral vector is selected from the group consisting of: cytomegalovirus, adenovirus, modified vaccinia virus Ankara, vesicular stomatitis virus and measles virus.

51. The immunogenic or vaccine composition according to claim 25, wherein the nucleic acid delivery agent comprises tetrafunctional non-ionic amphiphilic block copolymers comprising at least one hydrophilic block and at least one hydrophobic block.

52. The immunogenic or vaccine composition according to claim 25, wherein the nucleic acid delivery agent comprises a particle or vesicle, in particular lipid-based micro- or nano-vesicle or particle such as liposome or lipid nanoparticle. (LNP).

53. The immunogenic or vaccine composition according to claim 25, which induces humoral and cellular immune responses against said SARS-CoV-2 virus, wherein the humoral immune response comprises neutralizing antibodies against said SARS-CoV-2 virus and/or the cellular immune response comprises CD4+ and/or CD8+ T-cells against said SARS-CoV-2 virus.

54. A method of preventing or treating SARS-CoV-2 virus infection in an individual in need thereof, comprising administering to the individual an effective amount of the immunogenic or vaccine composition according to claim 25.

Description

FIGURE LEGENDS

[0112] FIG. 1. Phylogenetic analysis of representative Betacoronaviruses and SARS-CoV-2 based on full length genome sequences.

[0113] The tree is midpoint rooted for ease of visualization, and high bootstrap values are indicated at key nodes.

[0114] FIG. 2A-B. Homology modelling of the S protein of SARS-CoV-2 using the Swiss-Model tool (FIG. 2A) and showing the model based on the top-hit (PDB ID: 6ACD) (FIG. 4C B).

[0115] The putative RBD is highlighted with a black box in the alignment. The QMEAN score reflects the modelling quality. Similar results were obtained using Phyre2.

[0116] FIG. 3. Schematic representation of the selected antigens.

[0117] SP: Signal Peptide. RBD: Receptor Binding Domain.

[0118] FIG. 4A-C. SARS-CoV-2 neutralizing antibody titers in immunized BALB/c mice.

[0119] FIG. 4A Immunization scheme. Groups of 5 female Balb/c mice were immunized intra muscularly with 100 g of pVAX vector containing the sequence of either the SARS-CoV-2 spike (pVAX-Spike), the spike with a mutated furin cleavage site (pVAX-Spike-deltaFurin), the receptor binding domain with the signal peptide of the spike (pVAX-RBD), the same RBD antigen with the PADRE sequence in 3′ (pVAX-RBD-PADRE), or an empty vector (pVAX).

[0120] FIG. 4B Neutralizing antibody titers against SARS-CoV-2 at day 27 post immunization (prime), determined by plaque reduction neutralizing test (PRNT.sub.50).

[0121] FIG. 4C Neutralizing antibody titers against SARS-CoV-2 at day 47 post immunization (prime-boost), determined by PRNT.sub.50.

[0122] FIG. 5A-D. Immunogenicity and protective efficacy.

[0123] Groups of 5-8 female Balb/c mice were immunized intra muscularly (i.m.) with 100 μg of pVAX vector containing the sequence of the spike receptor binding domain with the signal peptide of the spike (pVAX-RBD) or an empty vector (pVAX). The immunization route was either i.m., intra nasal (i.n.) or a mix of i.m. for prime then i.n. for boosts, at 7-10 days intervals. At day 42 post initial immunization, mice were challenged i.n. with 1.10.sup.5 PFU of a mouse adapted SARS-CoV-2 strain. Viral load in the lungs was assessed at day 3 post infection.

[0124] FIG. 5A Immunization and challenge scheme.

[0125] FIG. 5B Neutralizing antibody titers against SARS-CoV-2 at day 42 post immunization (prime-boost-boost), determined by plaque reduction neutralizing test (PRNT.sub.50).

[0126] FIG. 5C Viral load (genomes copies as PFU equivalents) measured in the lungs at day 3 post challenge.

[0127] FIG. 5D Viral load (PFU per g of tissue) measure in the lungs at day 3 post challenge.

[0128] FIG. 6. ratio of IgG2a/IgG1 or Th1/Th2 responses.

[0129] The content of sera of Balb/c mice immunized with the receptor binding domain with the signal peptide of the spike (pVAX-RBD) using an i.m. prime-boost protocol were assessed by isotype specific ELISA against the SARS-CoV-2 RBD.

EXAMPLES

Material and Methods

[0130] 1. Design of the Antigens

[0131] Phylogenetic analysis of publicly available SARS-CoV-2 (2019-nCov) full-length sequences (NCBI sequence data base) with representative sequences for the genus Betacoronavirus indicates that SARS-CoV-2 is part of a well-defined Sarbecovirus clade that includes viruses sampled in bats (FIG. 1).

[0132] It is significantly different from the well-known human sarbecovirus SARS-Cov with only 79% identity at the nucleotide level over the full length of the genome. This value drops to 72.7% for S in nucleotides, and 76.2% in amino acids. However structural modelling using the Swiss-Model program (Waterhouse et al., Nucleic Acids Res., 2018 Jul. 2; 46(W1): W296-W303) or Phyre2 (Kelley et al., Nat Protoc. 2015 June; 10(6):845-58) and a representative sequence of the S protein of 2019-nCov (SARS-CoV-2) as query suggest a similar structural organization to the S protein of SARS-Cov, with core sections showing stronger sequence or structure conservation and modeling quality, and variation (with modelling uncertainty) mostly in the surface residues (FIG. 2).

[0133] In particular, a putative RBD of SARS-CoV-2 can be defined with, like for SARS-Cov (SARS-CoV-1), a core and an external subdomain. As it has been shown for other coronaviruses (Embemovirus MHV, HCov-229E or SARS-Cov), the RDB is highly reactive to anti-S neutralizing antibodies, and could comprise the key epitopes of the neutralizing response.

[0134] Based on the state of the art of betacoronaviruses biology, and in particular building on the structural similarity with SARS-Cov, the S protein is the most relevant antigen to include regardless of the delivery strategy. Two antigens have thus been designed (FIG. 3). One corresponds to the complete S protein, and the second, smaller (minimal) antigen, for ease of expression and production, correspond to the SARS-CoV-2 RBD of the S protein. To ensure secretion of the RBD antigen, 3 signal peptides (SP) have been selected.

[0135] Specifically, antigen 1 consists of 1273 amino acids or 3822 nucleotides, and the sequence has been codon-optimized for expression in Homo sapiens. Antigen 2 consists of 194 amino acids or 582 nucleotides, and the sequence has been codon-optimized for expression in Homo sapiens. Antigen 2 is combined with one of 3 SP (from the SARS-CoV-2) S protein; from the human CD5 or from the human IL-2). Other versions of Antigen 2 having SP variants according to the present disclosure are also engineered, one with a SP lacking SA in positions 20-21 of SEQ ID NO: 23; one with a SP lacking RLVA in positions 25 to 28 of SEQ ID NO: 19; and one with a SP lacking A in positions 20 of SEQ ID NO: 15.

[0136] These antigens can be delivered as nucleic acid immunogens, formulated with appropriate non-viral agent such as amphiphilic block copolymer or in a viral vector.

[0137] The antigens were also combined with a universal Pan HLA-DR Epitope termed PADRE. PADRE is a universal synthetic 13 amino acid peptide that activates CD4+ T cells. As PADRE binds with high affinity to 15 of the 16 most common human HLA-DR types, it provides potent CD4+ T cell responses and may overcome problems caused by polymorphism of HLA-DR molecules in human populations.

[0138] 2. Plasmid Construction

[0139] The various cDNA sequences designed from 2019-nCov (SARS-CoV-2 or SARS2) sequences were codon-optimized for Homo sapiens expression, synthesized (Thermo-Fisher Scientific), and cloned into the pVAX-1 plasmid (Thermo-Fisher) under the control of a CMV promoter and containing a Kozak sequence. The cDNA sequences correspond to SEQ ID NO: 10, 12, 14, 16, 18, 20, 22, 24, 29, 31, 33, 35 in the attached sequence listing and encode r a protein antigen corresponding to the amino acid sequences SEQ ID NO: 11, 13, 15, 17, 19, 23, 25, 30, 32, 34 and 36, respectively in the attached sequence listing. pVAX-Spike comprises the cDNA of SEQ ID NO: 10 encoding a Spike of SEQ ID NO: 11. VAX-Spike-deltaFurin comprises the cDNA of SEQ ID NO: 29 encoding a Spike-deltaFurin of SEQ ID NO: 30. pVAX-RBD comprises the cDNA of SEQ ID NO: 14 encoding a RBD of SEQ ID NO: 15. pVAX-RBD-PADRE comprises the cDNA of SEQ ID NO: 16 encoding a RBD-PADRE of SEQ ID NO: 17. All pVAX derived plasmids were amplified in Escherichia coli and plasmid DNA was purified on EndoFree plasmid purification columns using the NucleoBond Xtra Maxi EF Kit (Macherey Nagel). The constructs were verified by enzymatic digestion and by SANGER sequencing.

[0140] 3. Formulation

[0141] The SARS-2 DNA vaccine is formulated by mixing equal volumes of ABC stock solution (Nanotaxi®, provided by In-Cell-Art; disclosed on page 13 to 17 of WO 2019/092002) in water and plasmid DNA solution at the desired concentration in 2× buffer solution, immediately prior to intramuscular injection. The mixing of ABC Nanotaxi® and plasmid DNA is a self-assembly process that results from hydrogen bonding, hydrophobic, and electrostatic interactions between ABC and DNA.

[0142] 4. Antigen Expression/Western Blot Analysis

[0143] 293 cells are transfected with plasmids expressing the antigens. After 24 h, cell lysates and supernatant are harvested. Samples are fractionated by SDS-PAGE and transferred to cellulose membranes to be probed with anti-S antibodies or sera. A goat anti-mouse immunoglobulin G (IgG)-horseradish peroxidase (HRP) conjugate is used as secondary antibody. Peroxidase activity is visualized with an enhanced chemiluminescence detection kit (Thermo Fisher Scientific).

[0144] 5. Animal Vaccination

[0145] Animal experiments are performed according to institutional, French and European ethical guidelines (Directive EEC 86/609/ and Decree 87-848 of 19 Oct. 1987) subsequent to approval by the Institut Pasteur Safety, Animal Care and Use Committee, protocol agreement delivered by the local ethical committee and the Ministry of High Education and Research. Groups of at least 5 female Balb/c, transgenic K18-ACE2 (McCray et al., J. Virol., 2007, 81(2), 813-821), or other mice type, including C57BL/6C mice and interferon deficient mice such as IFNAR mice were housed under specific pathogen-free conditions in individually ventilated cages during the immunization period at the Institut Pasteur animal facilities. Mice were vaccinated with different constructs using a prime/boost regimen. Formulations was injected bilaterally into both tibial anterior muscles using an 8-mm, 30-gauge syringe (intra muscular (i.m.)), or intra-nasally (i.n.) at different time intervals. Mice were anesthetized by isoflurane before injection. A group of five unvaccinated mice, housed alongside the treated mice was used as controls. Sera were collected at various time points post-immunization to monitor the immune responses.

[0146] 6. Cell Culture

[0147] Vero C10008 clone E6 (CRL-1586, ATCC) cells were maintained in Dulbecco's modified Eagle medium (DMEM) complemented with 10% heat-inactivated serum, 100 U/mL penicillin and 100 μg/mL streptomycin and were incubated at 37° C. and 5% C02.

[0148] 7. ELISA

[0149] Measurement of anti-S IgG antibody titers in serum of vaccinated mice is performed using either a commercial kit or an in house assay. Recombinant SARS-CoV-2 RBD were coated on 96-well MAXISORP plates. Coated plates were incubated overnight at 4° C. The plates were washed 3 times with PBS-0.05% Tween, then blocked 1 h at 37° C. with PBS-0.05% Tween-3% BSA. Serum samples from immunized mice were serially diluted and incubated for 1 h at 37° C. on the plates. HRP-conjugated isotype-specific (IgG1 or IgG2a) secondary antibodies were used to reveal the specific and relative amounts of IgG isotypes. Endpoint titers for each individual serum were calculated as the reciprocal of the last dilution giving twice the absorbance of the negative control sera.

[0150] 8. Plaque Reduction Neutralization Test (PRNT)

[0151] For plaque reduction neutralization titer (PRNT) assays, Vero-E6 cells are seeded onto a 24-well plate and incubated at 37° C. for 12-24 h to 90% confluency. Two-fold serial dilutions of heat-inactivated serum samples are mixed with 50 PFU of SARS-CoV-2 for 1 h at 37 C, then added to cells for 2 h at 37° C. Virus/serum mix are then aspirated, and cells washed with PBS and overlaid with 1 mL of DMEM supplemented with 5% fetal calf serum and 1.5% carboxymethylcellulose. The plates were incubated for 3 days at 37° C. with 5% C02. Viruses were then inactivated and cells fixed and stained with a 30% crystal violet solution containing 20% ethanol and 10% formaldehyde. Serum titer was measured as the dilution that reduced SARS-CoV-2 plaques by 50% (PRNT.sub.50). This test was performed on several SARS-CoV-2 lineages as seen in the circulation in human. The SARS-CoV-2 lineages included in particular clade L, clade G (GISAID) and lineages B.1.1.7 (UK variant), B.1.351 (South Africa variant) and P.1 (Brazil variant).

[0152] 9. SARS-CoV-2 Challenge

[0153] Animals were transferred to an isolator in BioSafety Level 3 animal facilities of Institut Pasteur. Mice were anesthetized by intra peritoneal (i.p.) injection of a mixture of Ketamine and Xylazine, transferred into a biosafety cabinet 3 where they were inoculated i.n. with either 1.10.sup.5 PFU of a mouse adapted strain of SARS-CoV-2 (MaCo3) for wild type Balb/C mice or 1.10.sup.4 PFU of a low passage clinical isolate (BetaCoV/France/GES-1973/2020) for the transgenic K18-ACE2 mice. The isolate BetaCoV/France/GES-1973/2020 was supplied by the National Reference Centre for Respiratory Viruses hosted at Institut Pasteur (Paris, France) and headed by Pr. Sylvie van der Werf.

[0154] Three days after challenge, mice were sacrificed and lung samples were collected aseptically, weighted, and mechanically homogenized in ice-cold PBS. The presence of SARS-CoV-2 in the lung was detected by titration on VeroE6 cells and by detecting viral RNA using a RT-qPCR (nCoV_IP4) targeting the RdRp gene, as described on the WHO website (https://www.who.int/docs/default-34source/coronaviruse/real-time-rt-pcr-assays-for-the-detection-of-sars-cov-2-institut-35pasteur-paris.pdf?sfvrsn=3662fcb6_2).

[0155] As SARS-CoV-2 infection is lethal for K18-ACE2 mice, symptoms and weights were monitored for 14 days after challenge.

[0156] 10. Lung Histopathology

[0157] Samples from the lung were fixed in formalin for at least 7 days and embedded in paraffin for histopathological examination.

Results

[0158] A prime-boost protocol with 4 weeks intervals between immunizations was first used to evaluate the immunogenicity of the different constructs. 100 μg of the pVAX plasmid containing either the complete SARS-CoV-2 spike, a spike modified at the furin site (spike delta furin), only the receptor binding domain (RBD) with the native signal peptide of the spike or the RBD with the PADRE sequence in 3′ (RBD-PADRE) was injected intra-muscularly (i.m.) The plasmid DNA was mixed with an amphiphilic bloc copolymer for delivery.

[0159] The neutralizing potential of the sera was evaluated at day 27 (prior to the second immunization), and 20 days later (FIG. 4A). The neutralization plaque reduction neutralizing tests (PRNT.sub.50) on the different constructs revealed that the smallest antigen (RBD) with the native signal peptide of the spike and without the PADRE sequence resulted in an early response already detectable 4 weeks after the prime (FIG. 4B), and which was more homogenously and consistently boosted by the second immunization in comparison to the other constructs (FIG. 4C).

[0160] Using the RBD construct, an accelerated protocol of a prime with two boosts, administered at 7-10 days intervals was next used (FIG. 5A). At day 42, the neutralizing potential of sera elicited using i.m, intra nasal (i.n.) and a mix of i.m. prime followed by boosts using the i.n. route was compared.

[0161] However, the challenge with a mouse adapted strain of SARS-CoV-2 inoculated i.n. revealed that the mixed protocol of i.m. and i.n. resulted in a lower viral load in the lungs of the animals in terms of viral RNA copies (FIG. 5C) and no infectious particles could be detected by titration. As expected from the PRNT results, mice immunized only by the i.n. route presented viral loads comparable to the mock vaccinated (empty vector pVAX) group (FIG. 5D). This shows that an accelerated immunization scheme over a short period of time can lead to strong neutralizing antibody titers.

[0162] As IgG isotype switching can serve as indirect indicators of Th1 and Th2 responses, the SARS-CoV-2 RBD-specific IgG1 and IgG2a isotype titers were determined in the sera of Balc/c mice immunized with the RBD antigen. Significantly higher IgG2a antibody titers than IgG1 were observed, reflecting a predominant Th1-type immune response (FIG. 6).

[0163] In conclusion, this study indicates that the RBD antigen is able to provide protection from a SARS-CoV-2 challenge of immunized animals, correlating with strong neutralizing antibody induction.