WO2025186660A1 - Mutated spike proteins as vaccines against sars-cov-2 - Google Patents

Abstract

The present invention relates to second-generation vaccine against COVID-19 to abrogate or diminish the binding of the SARS-CoV-2 spike (S) protein, or part of it, to the human angiotensin-converting enzyme 2 (hACE2). Such vaccines present several advantages as to avoid pathways of immune dysregulation activated following S protein/hACE2 interaction while maintaining the ability to elicit a strong and robust antibody neutralization response to SARS-CoV-2. The invention relates also to the use of the S protein for therapeutic uses and for vaccines in the prevention or treatment of SARS-CoV-2 infection or conditions or disorders resulting from such infection.

Description

MUTATED SPIKE PROTEINS AS VACCINES AGAINST SARS-COV-2

DESCRIPTION

Technical field of the invention

The present invention relates to second-generation vaccines against COVID-19 to abrogate or diminish the binding of the SARS-CoV-2 spike (S) protein, or part of it, to the human angiotensin-converting enzyme 2 (hACE2). Such vaccines present several advantages as to avoid pathways of immune dysregulation activated following S protein/hACE2 interaction while maintaining the ability to elicit a strong and robust antibody neutralization response to SARS-CoV-2. The invention relates also to the use of such vaccines in the prevention or treatment of SARS-CoV-2 infection or conditions or disorders resulting from such infection.

State of the art

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible and pathogenic coronavirus that emerged in late 2019 and has caused a pandemic of acute respiratory disease, named ‘coronavirus disease 2019’ (CO VID-19), which threatens human health and public safety. Despite the World Health Organization (WHO) has declared the end of the pandemic in May 2023, the virus continued to evolve and evade the human immune response elicited after infection and/or vaccination impacting many lives all over the world. Therefore, novel, safe and highly immunogenic vaccines are needed. SARS-CoV- 2 infection starts with the interaction between the viral glycoprotein spike (S) and the human angiotensin-converting enzyme 2 (hACE2). The S protein attaches hACE2 on the host cell through the receptor binding domain (RBD) positioned in the SI domain. This interaction allows TMPRSS2 to cleave the S protein, leading to activation of the S2 domain and consequent fusion of the viral membrane with the host cell membrane (Larners et al. SARS- CoV-2 pathogenesis. Nature Reviews Microbiology 20, 270-284, 2022). While hACE2 was previously considered only as a component of the renin-angiotensin system, recent reports highlight the widespread distribution of hACE2 in different tissues and organs and its possible contribution to extrapulmonary manifestations of COVID-19 (Baldari et al. Emerging roles of SARS-CoV-2 Spike-ACE2 in immune evasion and pathogenesis. Trends in Immunology 44, 424-434, 2023). Among the different pathways that involve hACE2, recent findings have shown that SARS-CoV-2 S protein exploits hACE2 signaling to suppress immunological synapse assembly and CD8⁺ cytotoxic T lymphocyte-mediated killing (Onnis et al. SARS-CoV-2 Spike protein suppresses CTL-mediated killing by inhibiting immune synapse assembly. Journal of Experimental Medicine 220, e20220906, 2022). Similar effects could also be used to subvert different activities of the innate and adaptive immune response highlighting a key role of the S protein/hACE2 axis in the SARS- CoV-2 immune evasion.

Since all present SARS-CoV-2 vaccines are based on the sequences and structures of Wuhan or variants of concern S proteins, there is the need to develop second-generation vaccines able to avoid the activation of hACE2 signaling and overcome possible drawbacks of current antigens while remaining highly immunogenic.

Summary of the Invention

The present invention relates to second-generation vaccines against COVID-19 to abrogate or diminish the binding of the SARS-CoV-2 S protein, or part of it, to ACE2, in particular the human ACE2. Such vaccines present several advantages as to avoid pathways of immune dysregulation activated following S protein/hACE2 interaction while maintaining the ability to elicit a strong and robust antibody neutralization response to SARS-CoV-2. Indeed, current vaccines are based on the wild-type sequence of SARS-CoV-2 S protein allowing the binding of this protein to hACE2. This interaction has been associated with mechanisms of immune evasion which can exacerbate the severity of CO VID-19. Data of the authors of the present invention have shown that it is possible to introduce on the S protein RBD novel sets of mutations through in silico prediction to reduce the binding activity to hACE2. After in silico evaluation and manual curation of the aminoacidic mutations to introduce in the antigen, a set of twenty-one RBD residues were selected for mutagenesis (amino acidic position 417, 446, 447, 449, 453, 455, 456, 473, 475, 476, 484, 486, 487, 489, 493, 496, 498, 500, 501, 502, and 505).

From these residues, thirty seven unique combinations, which are reported in the sequence listing that is part of the present specification including (1) N487M; Y505K, (2) K417A; N487M; Y505K, (3) N487G; Y489K; Y505K, (4) N487M; Y489K; Y505K, (5) K417L; N487M; Y489K; Y505K, (6) K417Q; N487M; Y489K; Y505K, and (7) K417L; N487M; Y489K; Y505G, were selected as in silico studies predicted the greatest binding reduction to hACE2 while mutating the lowest number of residues. For seven sequences (mut 2S-MK; mut 3S-AMK; mut 3S-GKK; mut 3S-MKK; mut 4S-LMKK; mut 4S-QMKK; mut 4S- LMKG) the in silico prediction data were experimentally validated, while for the remaining thirty sequences only data on the prediction of loss of binding and stability were evaluated. The seven S protein mutants experimentally validated have been all recombinantly expressed through CHO transient transfection for in vitro evaluation. Specifically, the inventors performed two different ELISA assays to characterize the antigens. The first ELISA was used to confirm that the quaternary structure of the newly generated S protein mutants was not modified by the introduction of selected mutations.

To achieve this goal, five different monoclonal antibodies (mAbs) were used. These mAbs targeted three different RBD sites, the NTD and S2 domain of the SARS-CoV-2 S protein. The second ELISA allowed us to evaluate the binding activity of our new mutated S proteins to hACE2. The results obtained by the inventors showed that produced mutated S proteins maintained their quaternary structure while showing up to 3.3 -fold binding reduction to hACE2 measured by ELISA.

Accordingly, in certain aspects, the invention provides an immunogenic polypeptide comprising or consisting of a receptor-binding domain (RBD) of the SARS-CoV-2 spike protein wherein one or more of the residues of said receptor-binding domain (RBD) in positions 417, 449, 477, 487, 489, 493, 494, 500, 502, and 505 are mutated, wherein the numbering of said positions refers to SEQ ID NO: 1.

In certain aspects, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes the immunogenic polypeptide according to any one of the embodiments herein disclosed and a vector comprising said nucleic acid molecule.

In certain aspects, the invention provides an immunogenic composition comprising the immunogenic polypeptide or the nucleic acid molecule or a vector according to any one of the embodiments herein disclosed, optionally comprising one or more adjuvants or excipients.

In certain aspects, the invention provides the immunogenic polypeptides and immunogenic compositions herein disclosed for use in a prophylactic and/or therapeutic treatment of the SARS-CoV-2 infection or conditions or disorders resulting from such infection, in particular COVID-19 disease.

In certain aspects, the invention provides the immunogenic polypeptides and immunogenic compositions herein disclosed for use as a vaccine against SARS-CoV-2 infection.

The invention contemplates combinations of any of the foregoing aspects and embodiments of the invention.

Brief description of the drawings

Fig- 1 Conservation analysis of the RBD domain in the SARS-CoV-2 S protein. Over 11 million sequences were downloaded from GISAID and aligned to identify the frequency of mutation of each residue composing the SARS-CoV-2 RBD domain. hACE2 interacting residues are highlighted in purple, the Receptor Binding Motif (RBM) is highlighted in red. Fig. 2. Prediction results for the binding affinity (A) and the protein stability (B). Binding affinity measures the AAG score by comparing the WT free energy with the mutated one. In the same way, stability is measured by comparing the WT RBD monomer against the mutated monomer.

Fig. 3. Comparison of binding affinity and stability in construct with two, three and four mutations. Pareto-optimal solutions are highlighted in yellow.

Fig. 4. ELISA results. A) Table shows the binding of mAbs to mutated SARS-CoV-2 S proteins. Technical triplicates were performed for the experiments. B) Graph shows the ability of mutated SARS-CoV-2 S-proteins to bind hACE2, and their binding fold-change reduction compared to corresponding WT SARS-CoV-2 S protein. Mean and standard deviation are denoted on the graph.

Fig- 5. Schematic representation of the SARS-CoV-2 S protein RBD. The illustration shows the four main anchors of interactions between SARS-CoV-2 RBD and hACE2 that were manually identified by the experts through three-dimensional visualization.

Fig- 6 Serum virus neutralization against SARS-CoV-2 Wuhan strain. The graph shows the inhibition of virus infectivity in cell culture in the presence of serum neutralizing antibodies. Technical triplicates were performed for the experiment.

Fig. 7 ELISA results of Second-Round mutated SARS-CoV-2 S-proteins. Graph shows the ability of mutated SARS-CoV-2 S-proteins to bind hACE2, and their binding fold-change reduction compared to corresponding WT SARS-CoV-2 S protein. Technical triplicates were performed for the experiment. Mean and standard deviation are denoted on the graph.

Detailed description of the invention Unless otherwise defined herein, scientific, and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art.

Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, second ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and Ausubel et al, Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990), incorporated herein by reference.

The following terms, unless otherwise indicated, shall be understood to have the following meanings:

The terms "polypeptide", "protein" and "amino acid sequence" as used herein generally refer to a polymer of amino acid residues and are not limited to a minimum length of the product. Polypeptides of the invention can be prepared in many wayse.g.by chemical synthesis (at least in part), by digesting longer polypeptides using proteases, by translation from RNA, by purification from cell culture (e.g. from recombinant expression), from the organism itself. Biological methods are in general restricted to the production of polypeptides based on L- amino acids, but manipulation of translation machinery (e.g. of aminoacyl tRNA molecules) can be used to allow the introduction of D-amino acids (or of other non natural amino acids, such as iodotyrosine or methylphenylalanine, azidohomoalanine, etc.) (Ibba (1996) Biotechnol Genet Eng Rev 13: 197-216).

The term "polypeptide" encompasses native or artificial proteins, protein fragments and polypeptide analogues of a protein sequence, i.e. isolated or purified polypeptide. A polypeptide may be monomeric or polymeric. The term "isolated protein", "isolated polypeptide" is a protein, polypeptide that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) is free of other proteins from the same species, (3) is expressed by a cell from a different species, or (4) does not occur in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be "isolated" from its naturally associated components. A protein may also be rendered substantially free of naturally-associated components by isolation, using protein purification techniques well known in the art. A protein or polypeptide is "substantially pure", "substantially homogeneous", or "substantially purified" when at least about 60 to 75% of a sample exhibits a single polypeptide. The polypeptide or protein may be monomeric or multimeric. A substantially pure polypeptide or protein will typically comprise about 50%, 60%, 70%, 80% or 90% W/W of a protein sample, more usually about 95%, and preferably will be over 99% pure. Protein purity or homogeneity may be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single polypeptide band upon staining the gel with a stain well known in the art. For certain purposes, higher resolution may be provided by using HPLC or other means well known in the art for purification.

The term "polypeptide fragment" as used herein refers to a polypeptide that has an aminoterminal and/or carboxy-terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the naturally occurring sequence.

The term “SARS-CoV-2” refers to severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2), the type of coronavirus that causes coronavirus disease 2019 (COVID-19).

The term "polynucleotide" as referred to herein means a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms.

The term "isolated polynucleotide" as used herein means a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the "isolated polynucleotide" (1) is not associated with all or a portion of a polynucleotides with which the "isolated polynucleotide" is found in nature, (2) is operably linked to a polynucleotide to which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence. As used herein, the term "nucleic acid" is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogues of the DNA or RNA generated using nucleotide analogues. The nucleic acid can be single-stranded or doublestranded.

The term "naturally occurring nucleotides" as used herein includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" as used herein includes nucleotides with modified or substituted sugar groups and the like. The term "oligonucleotide linkages" referred to herein includes oligonucleotides linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate, phosphoroamidate, and the like. See e.g., LaPlanche et al., Nucl. Acids Res. 14:9081 (1986); Stec et al, J. Am. Chem. Soc. 106:6077 (1984); Stein et al., Nucl. Acids Res. 16:3209 (1988); Zon et al., Anti-Cancer Drug Design 6:539 (1991); Zon et al.. Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England (1991)); U.S. Patent No. 5,151,510; Uhlmann and Peyman, Chemical Reviews 90:543 (1990), the disclosures of which are hereby incorporated by reference. The term "expression control sequence" as used herein means polynucleotide sequences that are necessary to affect the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, generally, such control sequences include promoters and transcription termination sequence. The term "control sequences" is intended to include, at a minimum, all components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. The term "vector", as used herein, means a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In some embodiments, the vector is a plasmid, i.e., a circular double stranded piece of DNA into which additional DNA segments may be ligated. In some embodiments, the vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. In some embodiments, the vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). In other embodiments, the vectors (e.g., non- episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.

The term "recombinant host cell" (or simply "host cell"), as used herein, means a cell into which a recombinant expression vector has been introduced. It should be understood that "recombinant host cell" and "host cell" mean not only the particular subject cell but also the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

The term "percent sequence identity" in the context of nucleotide or aminoacidic sequences means the residues in two sequences that are the same when aligned for maximum correspondence. The length of sequence identity comparison may be over a stretch of at least about nine nucleotides, usually at least about 18 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36, 48 or more nucleotides. There are a number of different algorithms known in the art which can be used to measure nucleotide sequence identity. For instance, polynucleotide sequences can be compared using FASTA, Gap or Bestfit, which are programs available, provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol. 132: 185-219 (2000); Pearson, Methods Enzymol. 266:227-258 (1996); Pearson, J Mol. Biol 276:71-84 (1998); incorporated herein by reference). The term "substantial similarity" or "substantial sequence similarity," when referring to a nucleic acid or fragment thereof, or aminoacidic means that when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 85%, preferably at least about 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed above. As applied to polypeptides, the term "substantial identity" means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights as supplied with the programs, share at least 70%, 75% or 80% sequence identity, preferably at least 90% or 95% sequence identity, and more preferably at least 97%, 98% or 99% sequence identity. In certain embodiments, residue positions that are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well- known to those of skill in the art. See, e.g., Pearson, Methods Mol. Biol. 243:307-31 (1994). Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic- hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulphur-containing side chains: cysteine and methionine. Conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al, Science 256: 1443-45 (1992), incorporated herein by reference. A "moderately conservative" replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix. Sequence identity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG contains programs such as "Gap" and "Bestfit" which can be used with default parameters as specified by the programs to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and a mutein thereof. The expression “to reduce the binding to ACE2” means that the one or more mutations of the receptor-binding domain (RBD) are designed to diminish or decrease the binding of the polypeptide of the invention comprising or consisting of said mutated RBD, or part of it, to angiotensin-converting enzyme 2 (ACE2), in particular to human ACE2 (hACE2), with respect to a polypeptide comprising or consisting of a non-mutated or native RBD.

The binding activity of a polypeptide to ACE2 can be determined and characterized by means of any of the techniques or assays known to the person skilled in the art, such as, in particular, an ELISA assay. In one aspect, the ELISA assay is performed as disclosed in paragraph 1.7 of the experimental section of the present specification.

The immunogenic polypeptide or nucleic acid molecules or immunogenic compositions of the present invention provide the induction of an immune response in a subject or host (human or non-human) but they avoid the activation of ACE2 signalling thanks to their reduced binding to ACE2.

The selective induction of an immune response in a subject or host (human or non-human animal) by the immunogenic products described herein, may be determined and characterized by methods described herein and routinely practiced in the art. These methods include in vivo assays, such as animal immunization studies. A number of in vitro assays, such as immunochemistry methods for detection and analysis of antibodies, including Western immunoblot analysis, ELISA, immunoprecipitation, radioimmunoassay, and the like, and combinations thereof. Other methods and techniques that may be used to analyse and characterize an immune response include neutralization assays (such as a plaque reduction assay or an assay that measures cytopathic effect (CPE) or any other neutralization assay practiced by persons skilled in the art). These and other assays and methods known in the art can be used to characterize immunogens and variants thereof.

The statistical significance of the results obtained in the various assays may be calculated and understood according to methods routinely practiced by persons skilled in the relevant art.

An "immunogenic composition" or “vaccine” or “vaccine formulation” as used herein refers to a composition that comprises an antigenic molecule, where administration of the composition to a subject results in the development in the subject of a humoral and/or a cellular immune response to the antigenic/immunogenic molecule of interest. The immunogenic composition can be introduced directly into a recipient subject, such as by injection, inhalation, oral, intranasal or any other parenteral, mucosal, or transdermal (e.g., intra-rectally or intra-vaginally) route of administration.

In any point of the present specification or of the claims, the expression “comprising” or “comprise(s)” can be replaced by “consisting of’ or “consist(s) of’.

Immunogenic polypeptides

A first aspect of the present invention provides an immunogenic polypeptide comprising or consisting of a receptor-binding domain (RBD) of the SARS-CoV-2 spike protein wherein one or more of the residues of said RBD in positions 417, 449, 477, 487, 489, 493, 494, 500, 502, and 505 are mutated, wherein the numbering of said positions refers to SEQ ID NO: 1. In certain aspects, said immunogenic polypeptide is mutated in at least 2, 3, or all said positions. In particular, said one or more mutations in the RBD are capable to reduce the binding of said immunogenic polypeptide to ACE2, in particular to human ACE2, with respect to a polypeptide comprising or consisting of a non-mutated or native RBD.

In one preferred embodiment, the invention provides an immunogenic polypeptide comprising or consisting of the SARS-CoV-2 spike protein having SEQ ID NO: 1, wherein one or more residues of the receptor-binding domain (RBD) of said protein in positions 417, 449, 477, 487, 489, 493, 494, 500, 502, and 505 are mutated, wherein the numbering of said positions refers to said SEQ ID NO: 1.

In some embodiments, one or more of said RBD residues are mutated in alanine or leucine or methionine or lysine or glycine or glutamine.

In some embodiments, the invention provides an immunogenic polypeptide comprising or consisting of a RBD of the SARS-CoV-2 spike protein having one or more of the following mutations: K417A, K417L, K417Q, Y449K, Y449N, Y449W, Y449M, S477Q, S477Y, S477N, N487M, N487G, N487A, N487I, N487P, N487S, N487K, N487L, Y489K, Y489V, Y489R, Q493Y, Q493A, Q493W, Q493I, S494I, T500V, T500I, G502T, G502I, G502A, G502S, G502F, Y505K, Y505Q, and Y505G, wherein the numbering of said positions refers to said SEQ ID NO: 1.

Preferably the immunogenic polypeptide comprises a mutated RBD having at least 2, 3, or 4 of such mutations.

According to a particular embodiment, the immunogenic polypeptide comprises or consists of a RBD of the SARS-CoV-2 spike protein having one of the following groups of mutations: (1) N487M and Y505K; or

(2) K417A, N487M, and Y505K; or

(3) N487G, Y489K, and Y505K; or

(4) N487M, Y489K, Y505K; or

(5) K417L, N487M, Y489K, and Y505K; or

(6) K417Q, N487M, Y489K, and Y505K; or

(7) K417L, N487M, Y489K, and Y505G; or

(8) S477Q, N487P, and Y489V; or

(9) S477Q, N487P and Y489R; or

(10) S477Q, N487G, and Y489V; or

(11) S477Q, N487S, and Y489V; or

(12) S477Q, N487K, and Y489V; or

(13) S477Q, N487G, and Y489R; or

(14) S477Q, N487K, and Y489R; or

(15) S477Q, N487S, and Y489R; or

(16) S477Q, N487M, and Y489R; or

(17) S477Q, N487I, and Y489R; or

(18) S477Q, N487P, T500V, and G502T; or

(19) S477Q, N487P, T500I, and G502T; or

(20) S477Q, N487P, T500V, and G502I; or

(21) S477Q, N487P, T500I, and G502I; or

(22) S477Q, N487P, Q493Y, and S494I; or

(23) S477Q, N487P, Q493A, and S494I; or

(24) S477Q, N487P, Q493W, and S494I; or

(25) Q493Y, S494I, T500V, and G502I; or

(26) S477Y, N487L, T500V, and G502A; or

(27) Q493W, S494I, T500V, and G502I; or

(28) N487P, Q493 Y, Y449K, and G502T; or

(29) N487P, Q493 Y, Y449K, and Y505Q; or

(30) N487P, Q493A, Y449N, and G502T; or

(31) N487P, Q493W, Y449W, and Y505Q; or

(32) N487P, Q493 Y, Y449N, and T500I; or (33) N487A, Q493W, Y449M, and G502S; or

(34) N487G, Q493W, Y449N, and T500V; or

(35) S477N, Q493Y, Y449N, and G502F; or

(36) N487M, Q493W, Y449W, and T500I; or

(37) N487I, Q493I, Y449W, and T500I; wherein the numbering of said positions refers to said SEQ ID NO: 1.

In some embodiments the immunogenic polypeptide comprises or consists of a RBD of the SARS-CoV-2 spike protein, which comprises or consists of a sequence selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID N0:8, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, or SEQ ID NO:45.

According to some embodiments of the present invention immunogenic polypeptides and variants thereof may be produced synthetically or recombinantly. For example, the immunogenic polypeptides may be expressed from a polynucleotide that is operably linked to an expression control sequence, such as a promoter, in a nucleic acid expression construct. For example they may be expressed in mammalian cells, yeast, bacteria, insect or other cells under the control of appropriate expression control sequences. Cell-free translation systems may also be employed to produce such coronavirus proteins using nucleic acids, including RNAs, and expression constructs. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are routinely used by persons skilled in the art and are described, for example, by Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, (1989) and Third Edition (2001), and may include plasmids, cosmids, shuttle vectors, viral vectors, and vectors comprising a chromosomal origin of replication as disclosed therein.

Nucleic acid molecules In certain aspects, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes the immunogenic polypeptide according to any one of the embodiments herein disclosed.

In one preferred embodiment, said isolated nucleic acid molecule comprises or consists of a nucleic acid sequence selected from SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

Preferably, said nucleic acid molecules are DNA or mRNA sequences. By using RNA instead of DNA for genetic vaccination, the risk of undesired genomic integration and generation of anti- DNA antibodies is minimized or avoided.

In some embodiments said mRNA is within lipid nanoparticles.

Lipid nanoparticles formed from cationic lipids with other lipid components, such as neutral lipids, cholesterol, PEG, PEGylated lipids, and oligonucleotides have been used to block degradation of the RNAs in plasma and facilitate the cellular uptake of the oligonucleotides. Procedures to manufacture said mRNA vaccine are known in the art see for example in US 10,576,146, US 10,485,884, and US 9,950,065 herein incorporated by reference.

Vectors

In certain aspects, the invention provides a vector comprising a nucleic acid molecule coding the immunogenic polypeptide according to any of the embodiments herein discloses.

In certain aspects, the invention provides a vector for use as vaccine or as expression vector to produce said immunogenic polypeptides.

The vector according to any of the embodiments herein disclosed optionally comprises an expression control sequence operably linked to the nucleic acid molecule.

In some embodiments, said vector is selected from RNA virus vectors, DNA virus vectors, plasmid viral vectors, adenovirus vectors, adenovirus associated virus vectors, herpes virus vectors and retrovirus vectors.

In some embodiments said vectors are Adenoviral vectors, and specifically simian adenoviral vectors, which are generally known in the art.

A number of replication-defective recombinant simian adenoviruses are described for example in WO 2005/071093. Vectors comprising these nucleic acid molecules can be used for transfection or transformation of a suitable mammalian, plant, bacterial or yeast host cell. Transfection or transformation can be by any known method for introducing polynucleotides into a host cell. Methods for introduction of heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, lipid nanoparticles and direct microinjection of the DNA into nuclei. In addition, nucleic acid molecules may be introduced into mammalian cells by viral vectors. Methods of transforming cells are well known in the art (see, e.g., U.S. Patent Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455, incorporated herein by reference).

Immunogenic composition

In certain aspects, the invention provides an immunogenic composition or a vaccine comprising an immunogenic polypeptide or a nucleic acid molecule or a vector according to any one of the embodiments herein disclosed, optionally comprising one or more adjuvants and/or excipients.

The amount of the immunogenic compound in each composition or vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccines. Such amount will vary depending upon which specific immunogen is employed and the type and amount of adjuvant used. An optimal amount for a particular vaccine may be ascertained by standard studies involving observation of antibody titres and other responses in subjects. Generally, it is expected that each dose will comprise l-1000pg of immunogen, for example l-200pg, or 10-100pg. A typical dose will contain 10-50pg, for example 15- 25pg, suitably about 20pg of immunogen. Alternatively, a "dose-sparing" approach may be used, for example in a pandemic situation. This is based on the finding that it is possible to provide the same protective effect using lower doses of antigen, due to the presence of an effective adjuvant. Accordingly, each human dose may contain a significantly lower quantity of immunogen, for example from 0.1 to lOpg, or 0.5 to 5pg, or 1 to 3pg, suitably 2pg protein per dose. By the term "human dose" is meant a dose which is in a volume suitable for human use. Generally, this is between 0.3 and 1.5 ml. In one embodiment, a human dose is 0.5 ml. Following an initial vaccination, subjects typically receive a boost after a 2 to 4 weeks interval, for example a 3 -week interval, optionally followed by repeated boosts for as long as a risk of infection exists. In a specific embodiment of the invention, a single-dose vaccination schedule is provided, whereby one dose of immunogenic compound in combination with adjuvant is sufficient to provide protection against the SARS CoV-2, without the need for any boost after the initial vaccination.

The immunogenic compositions of the invention may be provided by any of a variety of routes such as oral, topical, subcutaneous, mucosal (typically intravaginal), intravenous, intramuscular, intranasal, sublingual, intradermal and via suppository.

The immunogenic compositions according to any of the embodiments disclosed in the present specification can thus be in any form suitable for administration by oral, topical, subcutaneous, mucosal (typically intravaginal), intravenous, intramuscular, intranasal, sublingual, intradermal or suppository route.

Immunisation can be prophylactic or therapeutic. The invention described herein is primarily but not exclusively concerned with prophylactic vaccination against SARS-COV-2. Appropriate pharmaceutically acceptable carriers or excipients for use in the invention are well known in the art and include for example water or buffers. Vaccine preparation is generally described in Pharmaceutical Biotechnology, Vol.61 Vaccine Design - the subunit and adjuvant approach, edited by Powell and Newman, Plenum Press New York, 1995. New Trends and Developments in Vaccines, edited by Voller et al, University Park Press, Baltimore, Maryland, U.S.A. 1978.

In some embodiments the immunogenic compositions of the invention may further comprise one or more adjuvants, such for example oil-in-water emulsion, mineral containing compositions, saponin formulations and other adjuvant known in the state of the art. Methods of producing oil-in-water emulsions are well known to the person skilled in the art. Commonly, the method comprises mixing the oil phase (optionally comprising a tocol) with a surfactant such as a PBS/TWEEN80™ solution, followed by homogenisation using a homogenizer. Mineral containing compositions suitable for use as adjuvants in the disclosure include mineral salts, such as aluminium salts and calcium salts. The disclosure includes mineral salts such as hydroxides (e.g. oxyhydroxides), phosphates (e.g.hydroxyphoshpates, orthophosphates), sulphates, etc. (e.g. See chapters 8 & 9 of Vaccine desigmthe subunit and adjuvant approach (1995) Powell & Newman. ISBN 0-306-44867-X), or mixtures of different mineral compounds, with the compounds taking any suitable form (e.g. Gel, crystalline, amorphous, etc.), and with adsorption being preferred. The mineral containing compositions may also be formulated as a particle of metal salt (See WO00/23105). Medical uses

The polypeptides, the nucleic acid molecules, the vectors, and immunogenic compositions described herein can be used in a prophylactic and/or a therapeutic treatment of the SARS- CoV-2 infection or conditions or disorders resulting from such infection. Immunisation can thus be prophylactic or therapeutic.

Preferably, said polypeptides, nucleic acid molecules, vectors or immunogenic compositions as described herein are used for prophylactic vaccination against SARS-COV-2.

As previously mentioned, the polypeptides, the nucleic acid molecules, the vectors and immunogenic compositions according to any of the embodiments described herein may be provided by any of a variety of routes such as oral, topical, subcutaneous, mucosal (typically intravaginal), intravenous, intramuscular, intranasal, sublingual, intradermal and via suppository.

In some embodiments, the polypeptides, the nucleic acid molecules, the vectors and immunogenic compositions described herein are for use as a vaccine against SARS-CoV-2 infection, in particular for use in a prophylactic or therapeutic treatment of the SARS-CoV- 2 infection or conditions or disorders resulting from such infection, more in particular for use in the prevention of COVID-19 disease.

***

The following experimental section is provided solely by way of illustration and not limitation and does not intend to restrict the scope of the invention as defined in the appended claims. The claims are an integral part of the description.

EXAMPLES

To produce and characterize the new SARS-CoV-2 mutated S proteins different in silico analyses and in vitro experiments were performed. The processes to generate and characterize the novel mutated S proteins are described below.

1.1 Selection of residues for mutagenesis

The objective of this study is to reduce the interaction strength between the RBD domain of the SARS-CoV-2 S-protein and the human hACE2 receptor. The RBD residues interacting with hACE2 were identified by analysing the crystallised structure of RBD-hACE2 complex downloaded from the PDB database (PDB ID: 6M0J) and all residues within 5 A from the protein interface were identified as interacting. As a result, residues 417, 446, 447, 449, 453, 455, 456, 473, 475, 476, 484, 486, 487, 489, 493, 496, 498, 500, 501, 502, and 505 were selected for mutagenesis. For each interacting position, a list of potential substitution was identified by analysing >11 million available SARS-CoV-2 S protein sequences. The rationale was to select amino acid substitutions less likely to occur, which are associated with detrimental fitness for the virus. More than 11 million sequences were downloaded from the GISAID database (Khare, S., et al. GISAID's Role in Pandemic Response. China CDC weekly 3, 1049-1051, 2021) and aligned using an in-house script. Frequency was generated for each position of the RBD, and amino acid substitutions with lower frequency were selected for single point mutation analysis (Figure 1).

1.2 Selection of Single point mutations

The effect of single point mutations was evaluated as a function of two quantitative values: protein stability and binding affinity, given as the difference in predicted energy (AAG) between the modelled wild type (WT) and mutant structures, predicted in kcal/mol. To calculate the protein stability upon mutation, the monomeric form of the RBD mutated and then compared to the WT, while for the binding affinity, the complex RBD-hACE2 was mutated and compared to the original structure. In both analysis, Rosetta Flex was employed to perform calculations, together with OpenMM molecular dynamic software and custom in-house scripts (Eastman, P., et al. OpenMM 8: Molecular Dynamics Simulation with Machine Learning Potentials. The Journal of Physical Chemistry B 128, 109-116, 2024; Barlow, K.A., et al. Flex ddG: Rosetta Ensemble-Based Estimation of Changes in Protein Protein Binding Affinity upon Mutation. The Journal of Physical Chemistry B 122, 5389- 5399, 2018). Rosetta Flex ddG is the current state-of-the art method for predicting changes in protein-protein and protein-ligand binding free energy. Rosetta is a software suite for macromolecular modelling and design that uses all-atom mixed physics- and knowledgebased potentials and provides a diverse set of protocols to perform specific tasks, such as structure prediction, molecular docking and homology modelling. The Flex ddG protocol has been found to perform better than machine learning methods and comparably to molecular dynamics methods when tested on a large dataset of ligand binding free energy changes upon protein mutation. The S protein RBD-hACE2 complex structure was downloaded from PDB database (PDB ID: 6M0J). The structure was relaxed using Rosetta FastRelax Mover (Tyka, M.D., Jung, K. & Baker, D. Efficient sampling of protein conformational space using fast loop building and batch minimization on highly parallel computers. Journal of Computational Chemistry 33, 2483-2491, 2012). Resfiles describing individual point mutation were generated for each selected residue in the relaxed structure, and backrub sampling was applied around the mutation site. The structure was then allowed to repack and relax globally with both the WT and the mutant. The binding energy AG cross was calculated using the InterfaceAnalyzerMover , and the AG cross difference between the mutant and the WT model was taken as AAG. Figure 2 shows the prediction results for the binding affinity (panel A) and the protein stability (panel B), both measured as AAG in comparison with the Wuhan strain (PDBID 6M0J). For each of the previously identified residues, the WT amino acid replacement by a subset of natural amino acids was calculated individually, depending on the observed mutations for that residue. 50 models for each mutant were generated and the AAG score were averaged. In all 200 analysed single point mutations 143 mutants have positive AAG score, which implies potential mutations with reduced affinity. The highest predicted AAG score was 3.2 Kcal/mol from mutation A489G. At the same time, Rosetta Flex ddG was employed to predict the change in stability (AAG) of the monomeric RBD protein caused by single point mutations. The predicted stability change is given as the difference in predicted energy between the modelled wild-type and mutant structures. The predicted substitutions were then analysed by the inventors that based on their expertise and their analysis of the 3D structure of the molecule decided the amino acid substitutions that would be tested experimentally.

1.3 Identification of candidate sequences by combining single point mutations

The list of single point mutations was analysed in order to define the combination of mutations with high overall predicted binding affinity and stability compared to the WT protein. Specifically, all possible combinations of two, three and four mutations were computed and the combined score for affinity and stability was calculated as separated sets. For each set of mutations, the pareto optimal solutions were identified and further analysed, with a final number of pareto optimal solution for two, three and four mutations of 5, 22 and 42 respectively (Figure 3). The list of candidates underwent manual inspection via Pymol (Rigsby, R.E. & Parker, A.B. Using the PyMOL application to reinforce visual understanding of protein structure. Biochemistry and molecular biology education : a bimonthly publication of the International Union of Biochemistry and Molecular Biology 44, 433-437, 2016) by using virtual reality to provide a better understanding of the changes in the RBD-hACE2 interactions. Table 1 summarises the mutations selected for experimental validation, together with their predicted stability and binding affinity.

ID Mutations Affinity Stability # Mutations mut 2S-MK N487M;Y505K 4.15 -0.31 2 mut 3S-AMK K417A;N487M;Y505K 5.61 -0.35 3 mut 3S-GKK N487G;Y489K;Y505K 6.43 -0.96 3 mut 3S-MKK N487M;Y489K;Y505K 6.75 -0.74 3 mut 4S-LMKK K417L;N487M;Y489K;Y505K 7.95 -0.65 4 mut 4S-QMKK K417Q;N487M;Y489K;Y505K 8.12 -0.78 4 mut 4S-LMKG K417L;N487M;Y489K;Y505G 8.96 -1.7 4

Table 1. Predicted affinity and stability of selected mutations for experimental validation.

1.4 Cloning of mutated SARS-CoV-2 S-proteins

GeneArt Strings DNA Fragments were resuspended with water and amplified using Q5® High-Fidelity DNA Polymerase (NEB), with primer forward 5’- CAGAGAGCATCGTACGATTTCCAAAC-3’ and reverse 5’- GGTATTGGTCCCGGGGGTGATCACAG-3’, following manufacturer’s protocol. PCR products were purified using GenElute™ PCR Clean-Up kit (Sigma-Aldrich), according to the manufacturer's instructions. Amplified DNA fragments were digested with BsiWI-HF and Xmal restriction enzymes (NEB) and purified using GenElute™ PCR Clean-Up kit. S protein expressing plasmids were digested with BsiWI-HF and Xmal restriction enzymes and purified from agarose gel, using QIAquick Gel Extraction Kit (QIAGEN). Digested DNA fragments were ligated to S protein expressing plasmid using T4 DNA Ligase (NEB). Ligation products were used to transformation One Shot® TOP 10 Chemically Competent E. coli (Invitrogen™), following manufacturing instructions. Selected colonies were analysed by plasmid isolation/restriction and sequencing. Large-scale isolation of plasmid DNA from recombinant A. Coli cultures was performed using NucleoBond® Xtra Midi/Maxi - High copy plasmid purification (MACHEREY-NAGEL), according to the manufacturer's instructions. Plasmids were quantified by NanoDrop™ and stored at - 20°C prior to use.

1.5 Expression and purification of mutated SARS-CoV-2 S-proteins

Plasmid encoded mutated SARS-CoV-2 S proteins were transiently transfected into ExpiCHO™ Expression System (Gibco™), according to manufacturer’s instructions. Following harvest and clarification by centrifugation, cell culture supernatants were filtered through a 0.22-pm filter and then purified through immobilized metal affinity chromatography using HisTrap™HP His tag protein purification columns (Cytiva). Chromatography was conducted at room temperature using the AKTA go™ chromatography system (Cytiva). Specifically, the culture supernatant of S protein cell culture was applied to a single 5 mL HisTrap™HP column, previously equilibrated in Buffer A (20 mM NaftPCU, 500 mM NaCl + 30 mM imidazole pH 7.4). The column was washed in Buffer A for 3 column volumes (CV) with all 3 CV collected as the column wash. Recombinant proteins were eluted from the column applying an isocratic elution of 5 CV of 60% Buffer B (20mM Na^PCU, 500mM NaCl + 500 mM imidazole pH 7.4). Elution steps were collected in 1 mL fractions. Eluted fractions were analysed by SDS-PAGE and appropriate fractions containing recombinant proteins were pooled. Protein-containing fractions were dialyzed in PBS buffer overnight at 4°C. Final protein concentrations were determined by NanoDrop™. Purified proteins were stored at -80°C prior to use.

1.6 ELISA assay to evaluate the quaternary structure of mutated S proteins

Briefly, 384-well plates (microplate clear, Greiner Bio-one) were coated with 3 pg/ml of streptavidin (Thermo Fisher) diluted in carbonate-bicarbonate buffer (Bethyl Laboratories) and incubated at room temperature overnight. The next day, 3 pg/mL of S proteins diluted in PBS were added and incubated for Ih at RT. Plates were then saturated with 50 pl/well of blocking buffer (phosphate-buffered saline and 1% BSA) for 1 h at 37 °C. After blocking, 25 pl/well of monoclonal antibodies (mAbs) diluted at 3 pg/mL in samples buffer (phosphate-buffered saline, 1% BSA, 0.05% Tween-20) were added to the plates and was incubated at 37 °C. Goat Anti-Human IgG-AP (Thermo Fisher Scientific) diluted 1 :20’000 in sample buffer was then added and incubated for 1 h at 37 °C. Plates were incubated with Alkaline Phosphatase Yellow (pNPP) (Sigma) for 20-30 minutes. The optical density (OD) values were identified using the Varioskan Lux Reader (Thermo Fisher Scientific) at 405 nm. After each incubation step, plates were washed three times with 100 pl per well of washing buffer (phosphate-buffered saline and 0.05% Tween-20). Sample buffer was used as a blank and the threshold for sample positivity was set at twofold the OD of the blank. Each condition was tested in triplicate and samples tested were considered positive if the OD value was twofold the blank. Figure 4A shows the results for the binding of tested mAbs to mutated SARS-CoV-2 S proteins. All mAbs bind to the seven mutated SARS-CoV-2 S proteins, confirming the maintaining of their quaternary structure, except, as expected, for the unrelated mAb.

1.7 ELISA assay to evaluate the mutated S proteins binding to hACE2

The ELISA assay was performed as previously described in 1.6 paragraph, but adding to the plates, after blocking, 25 pl/well of hACE2 diluted at 3 pg/mL in samples buffer. Figure 4B shows the results for the binding of mutated SARS-CoV-2 S-proteins to hACE2. All mutated SARS-CoV-2 S-proteins showed a binding decrease to ACE2 compared to wildtype SARS-CoV-2 S-protein, particularly mut 2S-MK, mut 4S-LMKG and 4S-QMKK, with a fold change reduction of -1.8x, -3. lx and -3.3x, respectively.

1.8 Identification of candidate sequences by combining single point mutations with theoretical loss of binding to hACE2

In addition to the seven sequences listed and experimentally validated in the previous paragraphs, we selected an additional list of thirty new sequences with high overall prediction of loss of binding and stability compared to the WT protein. The prediction for stability and loss of binding to hACE2 was performed as described in paragraph 1.2 and 1.3, where both Machine learning approaches and manual curation of the experts were employed to select the mutations (Table 2). Three different strategies were used to select the residues to be mutated. We manually divided the S protein receptor binding motif (RBM) into four main anchors of binding to hACE2. The four anchors were named top, middle high, middle low and bottom as shown in Figure 5. The top anchor was constituted by residues F456, A475, G476, N477, N487 and Y489. The middle high anchor was formed by residues Y453, Q493 and S494, while the middle low anchor was formed by residues Y449 and R498. Finally, the bottom anchor was constituted by residues T500, Y501, G502 and H505(Figure 5). Based on the position of the four anchors manually identified by the experts through three-dimensional visualization, we defined three main strategies to abrogate the binding of the SARS-CoV-2 S protein to hACE2. The first strategy was based on the mutation of a single residue per each of the four anchors. The second strategy aimed to mutate at least two residues per each anchor. The third strategy had the objective to mutated up to four residues in the same anchor. Each of these strategies has undergone virtual screening, where the results from single point mutations were combined and analyzed in all possible combinations. This process resulted in a total of 1,563,852, 2,607,142, and 137,541 candidates for the first, second, and third strategies, respectively. A score was calculated for each candidate by combining the results of the single point mutations. Pareto-optimal solutions were then identified to select combinations with high stability and decreased affinity.

ID Name Mutations Affinity Stability l_anchor_mpm l_mpm_QPV S477Q;N487P;Y489V 5.49 -1.31 l_anchor_mpm l_mpm_QPR S477Q;N487P;Y489R 5.12 -1.06 l_anchor_mpm l_mpm_QGV S477Q;N487G;Y489V 4.16 -1.05 l_anchor_mpm l_mpm_QSV S477Q;N487S;Y489V 3.84 -1.03 l_anchor_mpm l_mpm_QKV S477Q;N487K;Y489V 3.83 -0.9 l_anchor_mpm l_mpm_QGR S477Q;N487G;Y489R 3.79 -0.8 l_anchor_mpm l_mpm_QKR S477Q;N487K;Y489R 3.46 -0.65 l_anchor_mpm l_mpm_QSR S477Q;N487S;Y489R 3.46 -0.78 l_anchor_mpm l_mpm_QMR S477Q;N487M;Y489R 3.35 -0.6 l_anchor_mpm l_mpm_QIR S477Q;N487I;Y489R 3.27 -0.56

2_anchor_mpm 2_mpm_QPVT S477Q;N487P;T500V;G502T 6.69 -1.37

2_anchor_mpm 2_mpm_QPIT S477Q;N487P;T500l;G502T 6.48 -1.33

2_anchor_mpm 2_mpm_QPVI S477Q;N487P;T500V;G502l 5.46 -0.66

2_anchor_mpm 2_mpm_QPII S477Q;N487P;T500l;G502l 5.25 -0.62

2_anchor_mpm 2_mpm_QPYI S477Q;N487P;Q493Y;S494I 4.78 -0.22

2_anchor_mpm 2_mpm_QPAI S477Q;N487P;Q493A;S494I 4.28 -0.02

2_anchor_mpm 2_mpm_QPWI S477Q;N487P;Q493W;S494I 4.14 0.02

2_anchor_mpm 2_mpm_YIVI Q493Y;S494l;T500V;G502l 3.68 0.02

2_anchor_mpm 2_mpm_YLVA S477Y;N487L;T500V;G502A 3.37 -0.67

2_anchor_mpm 2_mpm_WIVI Q493W;S494l;T500V;G502l 3.04 0.26

4_anchor_spm 4_spm_PYKT N487P;Q493Y;Y449K;G502T 7.82 -1.9 4_anchor_spm 4_spm_PYKQ N487P;Q493Y;Y449K;Y505Q 7.65 -1.84

4_anchor_spm 4_spm_PANT N487P;Q493A;Y449N;G502T 7.4 -1.7

4_anchor_spm 4_spm_PWWQ N487P;Q493W;Y449W;Y505Q 6.7 -1.42

4_anchor_spm 4_spm_PYNI N487P;Q493Y;Y449N;T500l 6.29 -0.88

4_anchor_spm 4_spm_AWMS N487A;Q493W;Y449M;G502S 4.83 -1.35

4_anchor_spm 4_spm_GWNV N487G;Q493W;Y449N;T500V 4.53 -0.42

4_anchor_spm 4_spm_NYNF S477N;Q493Y;Y449N;G502F 3.62 -1.1

4_anchor_spm 4_spm_MWWI N487M;Q493W;Y449W;T500l 3.51 0.0

4_anchor_spm 4_spm_IIWI N487l;Q493l;Y449W;T500l 2.89 0.1

Table 2. Predicted affinity and stability of selected mutations for experimental validation.

1.9 Production of hyperimmune serum in mice immunized with selected mutated S proteins

Four groups of ten Balb/c female mice aged approximately 8-10 weeks were used for this experiment. Two administrations were performed at days 0 and 21 of the study, by intramuscular (IM) injection. At each administration, Balb/c mice were immunized with 25 pL of AddaVax™ (Squalene-based oil-in-water adjuvant, Catalog code: vac-adx-10, InvivoGen) and 5 pg of recombinant protein diluted in PBS (WT, mut 2S-MK, mut 4S- LMKG and mut 4S-QMKK), to reach a final volume of 50 pL. At day 35, all animals underwent blood collection for serum processing. Terminal blood sample were collected via exsanguination in anesthetized animals using isofluorane. Sacrifice was carried out by cervical dislocation. Targeted volume for terminal blood collection was approximately 800 pl. Serum was obtained from the whole blood placed in the tubes by centrifugation. All serum samples were stored in vials at -20°C until usage.

2.0 Serum virus neutralization assay against SARS-CoV-2

Wild type SARS-CoV-2 authentic viruses’ neutralization assays were all performed in the biosafety level 3 (BSL3) laboratories at Toscana Life Sciences in Siena (Italy). BSL3 laboratories are approved by a Certified Biosafety Professional and are inspected every year by local authorities. The humoral immune response was evaluated by a virus neutralization test, where mice sera were all tested in a cytopathic effect-based neutralization assay (CPE- MN). In brief, serum samples were heat-inactivated for 30 minutes at 56°C; two-fold serial dilutions, starting from 1 : 10, were then mixed with an equal volume of viral solution containing 100 median tissue culture infectious dose (lOOTCIDso) of SARS-CoV-2 virus. After Ih of incubation at 37°C, 5% CO2, the mixture was then added to the wells of a 96- well plate containing a sub-confluent Vero E6 cell monolayer. Plates were incubated for 3 days at 37°C in a humidified environment with 5% CO2, then examined for CPE by means of an inverted optical microscope by two independent operators. Four human sera from seropositive subjects were used in the assay as positive controls. Technical triplicates were performed for all samples.

Results of the viral seroneutralization showed all tested sera exhibited neutralizing activity against SARS-CoV-2 Wuhan strain (Figure 6), demonstrating that, despite the introduction of mutations, mut 2S-MK, mut 4S-LMKG and mut 4S-QMKK S proteins maintain the ability to induce a strong and robust antibody neutralization response against the virus, comparable to the WT spike.

2.1 Identification, expression and purification of Second-Round mutated SARS-CoV-2 S-proteins

A second round of selection was performed among the thirty new sequences identified (Table 2), choosing the highest-affinity candidates from each of the three different strategies for further in vitro analysis (Table 3). These ten S-protein were expressed and purified as previously described in E5 paragraph. Purified proteins were stored at -80°C until usage.

ID Name Mutations Affinity Stability l_anchor_mpm l_mpm_QPV S477Q;N487P;Y489V 5.49 -1.31 l_anchor_mpm l_mpm_QPR S477Q;N487P;Y489R 5.12 -1.06 l_anchor_mpm l_mpm_QGV S477Q;N487G;Y489V 4.16 -1.05

2_anchor_mpm 2_mpm_QPVT S477Q;N487P;T500V;G502T 6.69 -1.37

2_anchor_mpm 2_mpm_QPIT S477Q;N487P;T500l;G502T 6.48 -1.33

2_anchor_mpm 2_mpm_QPVI S477Q;N487P;T500V;G502l 5.46 -0.66

4_anchor_spm 4_spm_PYKT N487P;Q493Y;Y449K;G502T 7.82 -1.9

4_anchor_spm 4_spm_PYKQ N487P;Q493Y;Y449K;Y505Q 7.65 -1.84

4_anchor_spm 4_spm_PANT N487P;Q493A;Y449N;G502T 7.4 -1.7

4_anchor_spm 4_spm_PWWQ N487P;Q493W;Y449W;Y505Q 6.7 -1.42

Table 3. Predicted affinity and stability of Second-Round selected mutated S-proteins for experimental validation.

2.2 ELISA assay to evaluate the Second-Round mutated S proteins The ELISA assay was performed as previously described in 1.6 paragraph, using the Second- Round mutated SARS-CoV-2 S-, adding to the plates, after blocking, 25 pl/well of hACE2 diluted at 3 pg/mL in samples buffer.

Figure 7 shows the results for the binding of mutated SARS-CoV-2 S-proteins to hACE2. All mutated SARS-CoV-2 S-proteins showed a complete binding abrogation to hACE2 receptor compared to wildtype SARS-CoV-2 S-protein.

Sequence Listing in the description

>SEQ ID NO: 1 - SARS-CoV-2 spike protein

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFL PFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSK TQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTF EYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALE PLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYN ENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPF GEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFT NVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGN YNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVG YQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKF LPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVN CTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICA SYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVS MTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQV

KQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGD IAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFA MQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQ NAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQ LIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVT YVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFV SGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNI QKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLC CMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT

SEQ ID No: 46 - forward primer

CAGAGAGCATCGTACGATTTCCAAAC

SEQ ID No: 47 - reverse primer

GGTATTGGTCCCGGGGGTGATCACAG

Declaration according to Art. 170bis

In compliance with Art. 170bis of the Italian code of industrial property, the applicant of the present patent application declares that for the biological material, containing microorganisms or genetically modified organisms, object or used in the aforementioned patent application, the obligations deriving from national or Community regulations, and in particular, from the provisions referred to in paragraph 6 of the Legislative Decree of 12 April 2001 n.206 and 8 July 2003 n. 224, 10 concerning these modifications, have been respected.

Claims

1. An immunogenic polypeptide comprising or consisting of a receptor-binding domain (RBD) of the SARS-CoV-2 spike protein wherein one or more of the residues of said receptor-binding domain (RBD) in positions 417, 449, 477, 487, 489, 493, 494, 500, 502, and 505 are mutated, wherein the numbering of said positions refers to SEQ ID NO: 1.

2. The immunogenic polypeptide according to claim 1, wherein said receptor-binding domain (RBD) has one or more of the following mutations: K417A, K417L, K417Q, Y449K, Y449N, Y449W, Y449M, S477Q, S477Y, S477N, N487M, N487G, N487A, N487I, N487P, N487S, N487K, N487L, Y489K, Y489V, Y489R, Q493 Y, Q493 A, Q493W, Q493I, S494I, T500V, T500I, G502T, G502I, G502A, G502S, G502F, Y505K, Y505Q, and Y505G.

3. The immunogenic polypeptide according to claims 1 or 2, wherein said receptor-binding domain (RBD) has one of the following groups of mutations:

(1) N487M and Y505K; or

(2) K417A, N487M, and Y505K; or

(3) N487G, Y489K, and Y505K; or

(4) N487M, Y489K, Y505K; or

(5) K417L, N487M, Y489K, and Y505K; or

(6) K417Q, N487M, Y489K, and Y505K; or

(7) K417L, N487M, Y489K, and Y505G; or

(8) S477Q, N487P, and Y489V; or

(9) S477Q, N487P and Y489R; or

(10) S477Q, N487G, and Y489V; or

(11) S477Q, N487S, and Y489V; or

(12) S477Q, N487K, and Y489V; or

(13) S477Q, N487G, and Y489R; or

(14) S477Q, N487K, and Y489R; or

(15) S477Q, N487S, and Y489R; or

(16) S477Q, N487M, and Y489R; or

(17) S477Q, N487I, and Y489R; or

(18) S477Q, N487P, T500V, and G502T; or

(19) S477Q, N487P, T500I, and G502T; or

(20) S477Q, N487P, T500V, and G502I; or

(21) S477Q, N487P, T500I, and G502I; or

(22) S477Q, N487P, Q493Y, and S494I; or

(23) S477Q, N487P, Q493A, and S494I; or

(24) S477Q, N487P, Q493W, and S494I; or

(25) Q493Y, S494I, T500V, and G502I; or

(26) S477Y, N487L, T500V, and G502A; or

(27) Q493W, S494I, T500V, and G502I; or

(28) N487P, Q493 Y, Y449K, and G502T; or

(29) N487P, Q493 Y, Y449K, and Y505Q; or

(30) N487P, Q493A, Y449N, and G502T; or

(31) N487P, Q493W, Y449W, and Y505Q; or

(32) N487P, Q493 Y, Y449N, and T500I; or

(33) N487A, Q493W, Y449M, and G502S; or

(34) N487G, Q493W, Y449N, and T500V; or

(35) S477N, Q493Y, Y449N, and G502F; or

(36) N487M, Q493W, Y449W, and T500I; or

(37) N487I, Q493I, Y449W, and T500I.

4. The immunogenic polypeptide according to any one of claims 1 to 3, wherein said immunogenic polypeptide comprises or consists of a sequence selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID N0:8, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, or SEQ ID NO:45.

5. An isolated nucleic acid molecule comprising a nucleotide sequence that encodes the immunogenic polypeptide according to any one of the claims from 1 to 4.

6. The isolated nucleic acid molecule according to claim 5, wherein said nucleic acid molecule is a DNA or mRNA sequence.

7. A vector comprising the nucleic acid molecule according to claims 5 or 6, wherein the vector optionally comprises an expression control sequence operably linked to the nucleic acid molecule, preferably said vector is selected from RNA virus vectors, DNA virus vectors, plasmid viral vectors, adenovirus vectors, adenovirus associated vectors, herpes virus vectors and retrovirus vectors.

8. The immunogenic polypeptide according to any one of the claims from 1 to 4 or the nucleic acid molecule according to claims 5 or 6 or a vector according to claim 7, for use in a prophylactic and/or therapeutic treatment of the SARS-CoV-2 infection or conditions or disorders resulting from such infection, in particular COVID-19 disease.

9. An immunogenic composition comprising an immunogenic polypeptide according to any one of the claims from 1 to 4 or a nucleic acid molecule according to claims 5 or 6 or a vector according to claim 7, and optionally one or more adjuvants and/or excipients.

10. The immunogenic composition according to claim 9, for use in a prophylactic and/or therapeutic treatment of the SARS-CoV-2 infection or conditions or disorders resulting from such infection, in particular COVID-19 disease.