WO2023170016A1 - Novel mimetic polypeptides of the hr1 region of the s2 subunit of coronaviruses - Google Patents

Novel mimetic polypeptides of the hr1 region of the s2 subunit of coronaviruses Download PDF

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WO2023170016A1
WO2023170016A1 PCT/EP2023/055655 EP2023055655W WO2023170016A1 WO 2023170016 A1 WO2023170016 A1 WO 2023170016A1 EP 2023055655 W EP2023055655 W EP 2023055655W WO 2023170016 A1 WO2023170016 A1 WO 2023170016A1
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amino acid
seq
helix
acid sequence
polypeptide
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French (fr)
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Francisco CONEJERO LARA
Mario CANO MUÑOZ
Christiane Moog
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Universidad De Granada
Université De Strasbourg
Institut National De La Santé Et De La Recherche Médicale (Inserm)
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/162—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/73—Fusion polypeptide containing domain for protein-protein interaction containing coiled-coiled motif (leucine zippers)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20033—Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention relates to novel mimetic polypeptides of the HR1 region of the S2 subunit of the Spike protein of coronaviruses that are capable of inhibiting host cell infection by a coronavirus.
  • the invention furthermore relates to the use of such polypeptides for the prevention or treatment of an infection caused by coronaviruses.
  • SARS-CoV-2 Since SARS-CoV-2 emerged in late 2019, a huge amount of research has dramatically increased the understanding of the molecular basis of the disease and has provided novel strategies to fight it.
  • S Spike
  • no effective antivirals have been approved to treat the infection. All these problems make it necessary to continue the development of new treatments, as well as new immunization strategies.
  • SARS-CoV-2 Spike (S) protein decorates the virus surface and promotes its entry into the host cells.
  • S protein is a trimer of heterodimers composed of S1 and S2 subunits.
  • the S1 subunit consists of the N-terminal domain (NTD), the receptor-binding domain (RBD) and two C-terminal domains (CTD).
  • NTD N-terminal domain
  • RBD receptor-binding domain
  • CTD C-terminal domains
  • Three S1 subunits cover the S2 trimer and maintain it in its prefusion conformation.
  • S2 contains a fusion peptide (FP) and two heptad-repeat regions (HR1 and HR2) that are essential to promote membrane fusion.
  • FP fusion peptide
  • HR1 and HR2 two heptad-repeat regions
  • the S2 prefusion structure is organized around a coiled-coil trimer formed by its central helix (CH) and the connector domain (CD), located between the HR1 and HR2 regions.
  • CH central helix
  • CD connector domain
  • the protein is embedded in the viral membrane by a transmembrane (TM) segment that is followed by an internal short C-terminal tail (CT).
  • SARS-CoV-2 starts with S1 binding to the angiotensin-converting enzyme 2 receptor (ACE2) using the receptor-binding domain (RBD). Then, proteolysis of the S2 subunit at the S2’ site mediated by host proteases (TMPRSS2 in plasma membrane or Cathepsins in the endosomes) triggers a conformational transition in which HR1 becomes extended to continue the CH trimeric coiled-coil resulting in insertion of the FP into the cell membrane [1].
  • ACE2 angiotensin-converting enzyme 2 receptor
  • RBD receptor-binding domain
  • HR1 and HR2 are potential targets for coronavirus treatment [2,3].
  • HR2 has a particularly high sequence conservation in SARS-CoV-2 [4], as well as between different coronaviruses [3].
  • HR1 -based peptides are much less active inhibitors [5, 6] but stabilized trimeric helical bundles of HIV-1 gp41 HR1 have shown inhibitory activity of HIV-1 [7-9], as well as MERS-CoV, HCoV-OC43 and SARS-CoV-2 [10]. This evidence indicates that both HR1 and HR2 are exposed during coronavirus fusion and susceptible to inhibition.
  • HIV-1 gp41 HR1 trimers can elicit neutralizing antibodies against HIV-1 [11] and HIV-1 infected patients elicit neutralizing antibody B cell responses that target HR1 epitopes [12, 13]. It is conceivable that similar antibody responses may be elicited during coronavirus infection.
  • Epitope mapping has so far detected a few immunogenic epitopes in S2 [19] mostly located at sequence 765-835, comprising the fusion peptide and the S2 cleavage site, and residues 1140-1 160 at the S2 stem region immediately upstream of HR2. Both epitopes have been described as neutralizing [20] and immunodominant in COVID-19 patients [21], Neutralizing monoclonal antibodies (mAbs) targeting the 1140-1160 linear helical epitope has been recently described [22-24], There is however a lack of highly immunogenic epitopes within HR1 and HR2. A possible reason is that HR1 and HR2 are highly protected from the immune system due to their importance in the conformational changes driving membrane fusion. It is also possible that conformational variability of these regions could make sensitive epitopes to be only transiently exposed. Nevertheless, immune responses to conformational epitopes may have gone unnoticed in epitope mapping studies using linear peptides.
  • the present invention therefore relates to a polypeptide capable of inhibiting host cell infection by a coronavirus, preferably a beta coronavirus, most preferably a SARS-CoV-2 virus, wherein said polypeptide comprises three a-helices and wherein
  • a-helix 1 comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO.1 ,
  • a-helix 2 comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO.2, and
  • a-helix 3 comprises an amino acid sequence that is at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO.3.
  • said three a-helices form an antiparallel trimer of helices and, preferably, said polypeptide is a mimetic polypeptide of the heptad-repeat region 1 (HR1 ) of the S2 subunit of the SARS-CoV-2 Spike protein.
  • At least one amino acid in each a-helix at sequence positions “e” and/or “g” in the heptad repeats is substituted by a lysine (K), arginine (R), aspartic acid (D) or glutamic acid (E), preferably lysine (K) or glutamic acid (E).
  • at least two of the amino acids are substituted in different a-helices at “e” and “g” positions close in space, so that their side chains can form mutually favorable charge-charge interactions or hydrogen bonds.
  • a-helix 1 comprises at least one of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.1 : N1 D, Q13E, V38K, A45K, and/or V63K, or a combination thereof,
  • a-helix 2 comprises at least one of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.2: V1 1 K, S13E, A29R, A31 E, V36E, Q38E, S50E, I56E and/or A63R, or a combination thereof,
  • a-helix 3 comprises at least one of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.3: 118E, A29K, A43K, V50E, G58R, L68E, D72Q and/or E75Q, or a combination thereof.
  • a-helix 1 comprises at least one of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.1 : N1 D, Q13K, A31 E, V38E, A45D, A59R and/or V63E, or a combination thereof,
  • a-helix 2 comprises at least one of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.2: L6D, V11 E, S13K, A31 K, V36E, Q38K, A43E, A45R, S50E, D51 K, A63E and/or N68R, or a combination thereof,
  • a-helix 2 comprises at least one of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.3; A1 1 E, A29K, Q36K, A43K, L68K or a combination thereof.
  • At least one glycine residue (G) has been replaced in at least one of the three a-helices, preferably in two, most preferably in all three a- helices by a polar amino acid, preferably a lysine (K), arginine (R), threonine (T), or serine (S), most preferred lysine (K).
  • a polar amino acid preferably a lysine (K), arginine (R), threonine (T), or serine (S), most preferred lysine (K).
  • the at least one glycine residue (G) is located in the middle of the a-helix.
  • a-helix 1 further comprises at least one of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.1 : G19K, G33K, G58K, or a combination thereof
  • a-helix 2 further comprises at least one of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.2: G16K, G41T, G55K, or a combination thereof
  • a-helix 3 further comprises at least one of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.3; G19K, G33K, G58S, or a combination thereof.
  • the a-helix 1 consists of the amino acid sequence of SEQ ID NO.4, and
  • the a-helix 2 consists of the amino acid sequence of SEQ ID NO.5, and
  • the a-helix 3 consists of the amino acid sequence of SEQ ID NO.6.
  • the a-helix 1 consists of the amino acid sequence of SEQ ID NO.7, and
  • the a-helix 2 consists of the amino acid sequence of SEQ ID NO.8, and
  • the a-helix 3 consists of the amino acid sequence of SEQ ID NO.9.
  • the a-helix 1 consists of the amino acid sequence of SEQ ID NO.10, and
  • the a-helix 2 consists of the amino acid sequence of SEQ ID NO.11 .
  • the a-helix 3 consists of the amino acid sequence of SEQ ID NO.12.
  • At least one amino acid in each a-helix at sequence positions “a” and/or “d” in the heptad repeats (as shown in Figure 1) making internal contacts with opposite a-helices is substituted by a non-polar amino acid, preferably a leucine (L), isoleucine (I), valine (V), phenylalanine (F) or alanine (A), mostly preferred leucine (L) or isoleucine (I).
  • a non-polar amino acid preferably a leucine (L), isoleucine (I), valine (V), phenylalanine (F) or alanine (A), mostly preferred leucine (L) or isoleucine (I).
  • a-helix 1 and a-helix 2 are linked via a first linker and a-helix 2 and a-helix 3 are linked via a second linker, and wherein the first and/or second linker is between 3 to 6 amino acids long, preferably 4 or 5 amino acids long, most preferred 4 amino acids long.
  • the amino acids of the linker are selected from glycine (G), alanine (A), serine (S), aspartic acid (D), asparagine (N), lysine (K), arginine (R), proline (P) and glutamic acid (E), or a combination thereof.
  • the amino acid composition of the linker connecting a-helix 1 and a-helix 2 is selected from the sequences GEPA (SEQ ID NO. 16) or GAPA (SEQ ID NO. 17), or GEPC (SEQ ID NO. 18), and the linker connecting a-helix 2 and a-helix 3 is selected from the sequences SGSG (SEQ ID NO. 19), SGDG (SEQ ID NO. 20), KGSG (SEQ ID NO. 21) or KGDG (SEQ ID NO. 22).
  • At least one pair, preferably two pairs, of residues have been replaced by a cysteine (C) in the polypeptide so as to form at least one disulfide bond, preferably two disulfide bonds.
  • C cysteine
  • a-helix 1 further comprises at least one of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.1 : L3C,
  • a-helix 2 further comprises at least one of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.2: Y70C,
  • a-helix 3 further comprises at least one of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.3: L71C .
  • the first linker having the sequence GEPA (SEQ ID NO. 16) or GAPA (SEQ ID NO. 17) comprises the following amino acid substitutions A4C.
  • the a-helix 1 consists of the amino acid sequence of SEQ ID NO.23, and
  • the a-helix 2 consists of the amino acid sequence of SEQ ID NO.24, and
  • the a-helix 3 consists of the amino acid sequence of SEQ ID NO.25.
  • the a-helix 1 consists of the amino acid sequence of SEQ ID NO.27, and
  • the a-helix 2 consists of the amino acid sequence of SEQ ID NO.28, and
  • the a-helix 3 consists of the amino acid sequence of SEQ ID NO.29.
  • the a-helix 1 consists of the amino acid sequence of SEQ ID NO.31 .
  • the a-helix 2 consists of the amino acid sequence of SEQ ID NO.32, and
  • the a-helix 3 consists of the amino acid sequence of SEQ ID NO.33.
  • the polypeptide of present invention is characterized in that it comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO.13.
  • polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO. 26, SEQ ID NO. 30 and SEQ ID NO. 34, preferably wherein the polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO. 26, SEQ ID NO. 30 and SEQ ID NO. 34.
  • polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15, preferably wherein the polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15.
  • the polypeptide of the present invention has a neutralization titer against at least one, at least 2 or at least 3 coronavirus selected from the group consisting of MERS-CoV, SARS-CoV-1 , SARS-CoV-2 of less than 100 pM, preferably of less than 50pM, more preferably of less than 25 pM.
  • the polypeptide of the present invention has a neutralization titer against at least one, at least 2, at least 3 or at least 4 or at least 5 SARS-CoV-2 variants of concern (VOCs) of less than 100 pM, preferably of less than 50pM, more preferably of less than 25 pM.
  • SARS-CoV-2 VOCs may be selected from the group consisting of Alpha or Alpha-like, Beta or Beta-like, Gamma or Gamma-like, Delta or Delta-like and Omicron or Omicron-like, most preferably selected from the group consisting of Alpha, Beta, Gamma, Delta and Omicron
  • the present invention furthermore relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the polypeptides of any one of the preceding claims further comprising pharmaceutically acceptable excipients.
  • the present invention furthermore relates to a polypeptide for use in the treatment or prevention of an infection by a coronavirus, preferably a beta coronavirus, most preferably a SARS-CoV-2 virus, characterized in that said polypeptide is a mimetic polypeptide of the HR1 region of the S2 subunit of the Spike protein of the said coronavirus, wherein the polypeptide comprises three alpha helices forming an antiparallel trimer of helices, wherein helix one and helix two are linked via a first linker and helix two and helix three are linked via a second linker and wherein helix two is inverted.
  • a coronavirus preferably a beta coronavirus, most preferably a SARS-CoV-2 virus
  • the invention relates to the polypeptide or the pharmaceutical composition as described herein for use in the treatment or prevention of an infection caused by a coronavirus, preferably a beta coronavirus, most preferably a SARS-CoV-2 virus.
  • a coronavirus preferably a beta coronavirus, most preferably a SARS-CoV-2 virus.
  • the present invention furthermore relates to the use of the polypeptides as described herein for the in vitro diagnosis of an infection with a coronavirus, preferably a beta coronavirus, most preferably a SARS-CoV-2 virus.
  • a coronavirus preferably a beta coronavirus, most preferably a SARS-CoV-2 virus.
  • the present invention furthermore relates to the use of the polypeptides as described herein for the neutralization of an infection with a coronavirus, preferably a beta coronavirus, most preferably a SARS-CoV-2 virus.
  • a coronavirus preferably a beta coronavirus, most preferably a SARS-CoV-2 virus.
  • the present invention also relates to a vaccine comprising the polypeptides of any one of the preceding claims for treating or preventing an infection with a coronavirus, preferably a beta coronavirus, most preferably a SARS-CoV-2 virus.
  • a coronavirus preferably a beta coronavirus, most preferably a SARS-CoV-2 virus.
  • the polypeptides of present invention can be combined with other immunogens.
  • the present invention further relates to an in vitro method of detecting the presence of antibodies directed against the HR1 region of the S2 subunit of the Spike protein of a coronavirus, preferably a beta coronavirus, most preferably a SARS-CoV-2 virus in a test sample, wherein the method comprises contacting a test sample with the polypeptides as described herein and detecting the presence of a signal, wherein the presence of the signal is indicative of the presence of said antibodies in the test sample.
  • the antibodies are selected from IgG, IgA and/or IgM.
  • Figure 1 Design of HR1 mimetic proteins.
  • the glycine residues engineered in the L3C variant are underlined in D. Amino acids in core positions “a” and “d” in the heptad repeats of the coiled-coil structure are indicated on top of the first helical sequence. The “stutter” regions correspond to discontinuities in the canonical heptad repeats.
  • Figure 2 Helix-wheel representation of the trimeric coiled-coil structure of the CoVS-HR1 L3B protein.
  • the “a b c d e f g” positions of the heptad repeats in each helix wheel of the coiled-coil are indicated with letters.
  • the coiled-coil has been divided in the three regions separated by the stutters for the sake of clarity.
  • the dashed lines indicate the engineered interactions by amino acid substitutions, as indicated in Figure 1 D.
  • Figure 3 Far-UV CD spectra of the CoVS-HR1 proteins L3A, L3B and L3C (Fig.2 A, B, C) in free form and in presence of V39E peptide at different molar ratio. Spectra were recorded at 25°C in 50 mM sodium phosphate buffer pH 7.4 at a protein concentration of 30 pM.
  • Figure 4 Molecular size of the CoVS-HR1 proteins measured by light scattering.
  • FIG. 5 Thermal denaturation of the CoVS-HR1 proteins measured by differential scanning calorimetry (DSC). Experiments were carried out at pH 7.4 in 50 mM sodium phosphate buffer. A) Comparison of the DSC thermograms of the three protein variants. B) Effect of the addition of the V39E peptide at different molar ratios.
  • FIG. 6 Isothermal titration calorimetry (ITC) analysis of the binding of V39E peptide to the CoVS-HR1 proteins.
  • the upper panels show the experimental ITC thermograms.
  • the lower panels show the normalized binding isotherms fitted using a binding model of n identical and independent sites.
  • Figure 7 Binding of the CoVS-HR1 proteins to recombinant trimeric Spike.
  • A) ELISA experiments with immobilized recombinant trimeric Spike with different concentrations of covS- HR1 L3A, L3B and L3C proteins.
  • CovNHR and covNHR-N-ddS correspond to HR1 mimetics of HIV-1 gp41.
  • B) The same experiments as in panel A but in competition with V39 peptide, added at 2:1 peptide:protein ratio.
  • Figure 8 Inhibition by the CoVS-HR1 proteins of SARS-CoV2-infection on Vero 76 cells.
  • Cells were infected by primary viruses (B1 D614G genotype, panel A, and Omicron BA.1 , panel B) in the presence of CoVS-HR1 proteins at different concentrations. The percentage of inhibition was calculated by reduction in the percentage of infected cells treated with the inhibitory proteins compared to untreated control cells. Data correspond to mean ⁇ standard deviations of 4 independent experiments. Continuous lines correspond to nonlinear regression curves using a sigmoidal Hill’s function.
  • FIG. 9 Antibody binding present in infected patients’ sera 3 months after SARS-CoV-2 infection towards CoVS-HR1 molecules or RBD. A) IgG binding towards RBD, L3A, L3B and L3C. B) IgA binding towards RBD and L3C. The antibody binding in sera from healthy donors for each antigen are labeled with C.
  • Figure 10 Crystallographic structure of CoVS-HR1 -L3B in complex with the V39E peptide (dark ribbons) superimposed to the crystallographic structure of the 6HB post-fusion structure of S2 (Protein Data Bank entry: 6LXT) (grey ribbons).
  • the RMSD between the two structures is 0.634 A.
  • Two HR2 regions of 6LXT have been deleted for the sake of clarity.
  • Fig. 1 1 Ribbon representation of the model structure of CoVS-HR1 -L3C-dSS. The locations of the disulfide bonds C3-147 and C77-C225 are indicated with the arrows.
  • Fig. 12 Inhibition by the CoVS-HR1 L3C and L3C-dSS proteins of WT SARS-CoV2-infection on Vero 76 cells.
  • Cells were infected by primary viruses (B1 D614G genotype) in the presence of CoVS-HR1 proteins at different concentrations.
  • the inventors have herein shown the successful design and the production of several mimetic proteins of the HR 1 region of the Spike protein of a coronavirus, specifically SARS-CoV-2.
  • the inventors could show that such mimetic proteins, collectively named CoVS-HR1 , spontaneously acquire the predicted a-helical structure, as depicted in Fig.1 and Fig. 10 and further described in Example 1 and Example 7.
  • these polypeptides showed a very high stability and structural cooperativity as further detailed in Example 3 and shown in Fig. 5.
  • the polypeptides bind HR2-derived synthetic peptides with very high affinity (Fig. 6) and can also bind to trimeric recombinant S protein through its HR2 region (Fig. 7). All three polypeptides of present invention showed strong binding to the Spike and the level of detected binding at sub nM Spike concentration runs in the order L3C > L3B > L3A but rapidly saturates at higher concentration.
  • the inventors could surprisingly show that the protein variants with higher stability of the trimeric helical bundle sequence displayed stronger inhibitory activity against SARS- CoV-2 in vitro (Fig.7 and Example 5).
  • the polypeptide was modeled by using the X-ray crystal structure of the six-helical bundle formed by HR1 and HR2 in the S2 post-fusion structure.
  • HR2 chains were deleted from the model and one of the HR1 helices, helix 2, was upturned, its sequence reversed and then aligned to the original one ensuring the correct core coiled-coil packing. Due to the antiparallel orientation of the reversed helix, the side chain CA-CB bonds have different spatial orientation compared to the native ones and this may perturb interhelical side chain packing in the coiled- coil structure. To compensate this, the alignment was made trying to superimpose the CB atoms of the core residue side chains. Then, side chain clashes were removed by energy minimization.
  • the present invention therefore relates to a polypeptide capable of inhibiting host cell infection by a coronavirus, preferably a beta coronavirus, most preferably a SARS-CoV-2 virus, wherein said polypeptide comprises three a-helices and wherein
  • a-helix 1 comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO.1
  • a-helix 2 comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO.2, and
  • a-helix 3 comprises an amino acid sequence that is at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO.3.
  • SEQ ID NO. 1 refers to the wildtype amino acid sequence of a-helix 1 of HR1 of the S2 subunit of the SARS-CoV-2 Spike protein.
  • SEQ ID NO. 2 refers to the reversed wildtype amino acid sequence of a-helix 2 of HR1 of the S2 subunit of the SARS-CoV-2 Spike protein.
  • SEQ ID NO. 3 refers to the wildtype amino acid sequence of a-helix 3 of HR1 of the S2 subunit of the SARS-CoV-2 Spike protein.
  • said three a-helices form an antiparallel trimer of helices and said polypeptide is a mimetic polypeptide of the heptad-repeat region 1 (HR1 ) of the S2 subunit of the SARS-CoV-2 Spike protein.
  • An heptad repeat is a polypeptide sequence where a pattern of seven amino acid residues is repeated throughout the sequence.
  • the positions of the heptad repeat are typically designated “a b c d e f g”, where “a” and “d” are typically non-polar residues, most frequently lie, Leu or Vai, and “e” and “g” are frequently polar or charged residues, most frequently Lys or Glu.
  • At least one amino acid in each a-helix at sequence positions “e” and/or “g” in the heptad repeats is substituted by a lysine (K), arginine (R), aspartic acid (D) or glutamic acid (E), preferably lysine (K) or glutamic acid (E).
  • at least two of amino acids are substituted in different a-helices at “e” and “g” positions close in space, so that their side chains can form mutually favorable charge-charge interactions or hydrogen bonds.
  • Additional stabilization of the coiled-coil trimeric bundle structure may be achieved by replacing buried polar amino acids at core positions “a” or “d” in the heptad repeats, as previously demonstrated by the inventors in a recent publication related to a highly similar construct based of HIV-1 gp41 HR1 [25].
  • At least one amino acid in each a-helix at sequence positions “a” and/or “d” in the heptad repeats (as shown in Figure 1 ) making internal contacts with opposite a-helices is substituted by a non-polar amino acid, preferably a leucine (L), isoleucine (I), valine (V), phenylalanine (F) or alanine (A), mostly preferred leucine (L) or isoleucine (I).
  • a non-polar amino acid preferably a leucine (L), isoleucine (I), valine (V), phenylalanine (F) or alanine (A), mostly preferred leucine (L) or isoleucine (I).
  • a-helix 1 comprises at least one, at least two, at least three, or at least four of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.1 : N1 D, Q13E, V38K, A45K, and/or V63K, or a combination thereof,
  • a-helix 2 comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.2: V11 K, S13E, A29R, A31 E, V36E, Q38E, S50E, I56E and/or A63R, or a combination thereof,
  • a-helix 2 comprises at least one, at least two, at least three, at least four, at least five, at least six, or at least seven, of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.3: 118E, A29K, A43K, V50E, G58R, L68E, D72Q and/or E75Q, or a combination thereof.
  • a-helix 1 comprises at least one, at least two, at least three, at least four, at least five or at least six of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.1 : N1 D, Q13K, A31 E, V38E, A45D, A59R and/or V63E, or a combination thereof,
  • a-helix 2 comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or at least eleven of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.2: L6D, V1 1 E, S13K, A31 K, V36E, Q38K, A43E, A45R, S50E, D51 K, A63E and/or N68R, or a combination thereof,
  • a-helix 3 comprises at least one, at least two, at least three, or at least four of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.3: A1 1 E, A29K, Q36K, A43K, L68K, or a combination thereof.
  • the a-helix 1 consists of the amino acid sequence of SEQ ID NO.4, and
  • the a-helix 2 consists of the amino acid sequence of SEQ ID NO.5, and
  • the a-helix 3 consists of the amino acid sequence of SEQ ID NO.6.
  • the a-helix 1 consists of the amino acid sequence of SEQ ID NO.7, and
  • the a-helix 2 consists of the amino acid sequence of SEQ ID NO.8, and
  • the a-helix 3 consists of the amino acid sequence of SEQ ID NO.9.
  • glycine residues in the middle of the a-helical regions were mutated to amino acids comprising polar side chains to increase a-helix propensity.
  • the glycine residues in a-helices 1 to 3 can be replaced by polar amino acids, such as lysine (K), arginine (R), threonine (T) or serine (S). These amino acids are solvent-exposed in the model and, opposite to glycine, stabilize the a-helix conformation.
  • At least one glycine residue (G) has been replaced in at least one of the three a-helices, preferably in two, most preferably in all three a-helices by a polar amino acid, preferably a lysine (K), arginine (R), threonine (T) or serine (S), most preferred lysine (K).
  • a polar amino acid preferably a lysine (K), arginine (R), threonine (T) or serine (S), most preferred lysine (K).
  • the at least one glycine residue (G) is located in the middle of the a-helix.
  • all glycine residues that are located in the middle of each of the a-helices are exchanged by polar amino acids, such as lysine (K), arginine (R), threonine (T) or serine (S).
  • polar amino acids such as lysine (K), arginine (R), threonine (T) or serine (S).
  • K lysine
  • R arginine
  • T threonine
  • S serine
  • a-helix 1 further comprises at least one, or at least two of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.1 : G19K, G33K, G58K, or a combination thereof
  • a-helix 2 further comprises at least one, or at least two of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.2: G16K, G41T, G55K, or a combination thereof
  • a-helix 3 further comprises at least one, or at least two of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.3; G19K, G33K, G58S, or a combination thereof.
  • the a-helix 1 consists of the amino acid sequence of SEQ ID NO.10, and
  • the a-helix 2 consists of the amino acid sequence of SEQ ID NO.11 .
  • the a-helix 3 consists of the amino acid sequence of SEQ ID NO.12.
  • Fig 1 B Four-residue loops were manually built to connect each pair of helix termini to create a helix- loop-helix-loop-helix topology (Fig 1 B). It has been found that these linkers are important for keeping the topology of the polypeptide and can contain 3 to 6 amino acids, more preferred is a length of 4 to 5 amino acids, most preferred 4 amino acids.
  • a-helix 1 and a-helix 2 are linked via a first linker and a-helix 2 and a-helix 3 are linked via a second linker.
  • the first linker links the C-terminal end of a-helix 1 to the N-terminal end of the a-helix 2
  • the second linker links the C-terminal end of a-helix 2 to the N-terminal end of the a-helix 3.
  • the first and/or second linker is between 3 to 6 amino acids long, preferably 4 or 5 amino acids long, most preferred 4 amino acids long to ensure correct folding and stability of the polypeptide.
  • the amino acids of the linker are selected from glycine (G), alanine (A), serine (S), aspartic acid (D), asparagine (N), lysine (K), arginine (R), proline (P) or glutamic acid (E). These amino acids can be combined in any order.
  • both, the first and the second linkers are 4 amino acids long.
  • the most preferred sequences of the linkers of present invention are shown here below:
  • polypeptide of present invention is characterized in that it comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO.13.
  • polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO.13, SEQ ID NO.14 or SEQ ID NO.15, preferably wherein the polypeptide consists of the amino acid sequence of SEQ ID NO.13, SEQ ID NO.14 or SEQ ID NO.15.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the polypeptides as described herein further comprising pharmaceutically acceptable excipients.
  • polypeptides of the present invention can be used alone or in combination, or in combination with other agents having coronavirus inhibitory activity.
  • these agents may be agents that have been reported to have inhibitory activity against coronaviruses or have therapeutic effects against coronavirus diseases, such as coronavirus pneumonia, such as Favipiravir, Nelfinavir, Arbidol, Lopinavir, Ritonavir, Chloroquine Phosphate, Darunavir or Remdesivir, etc.
  • the composition may include a suitable carrier, such as a pharmaceutically acceptable carrier.
  • a suitable carrier such as a pharmaceutically acceptable carrier.
  • Such a composition can be used for external use, for example, as an external preparation, a smear preparation for external use, such as a gel for external use or an infiltration preparation for external use.
  • Such compositions can be coated on articles that need to inhibit viruses, such as, but not limited to, masks, paper towels, gloves, clothing, such as protective clothing, and the like.
  • the composition can be added as an active ingredient to hand sanitizers, such as hand sanitizer, shower gel and the like.
  • Such compositions can be used to inhibit coronavirus in vitro to prevent and/or reduce viral infections.
  • Such compositions can be used to prevent or treat coronavirus infections or diseases caused by coronaviruses in subjects.
  • compositions of present invention can be formulated in any known dosage form, such as tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, suppositories, or freeze-dried powder injections.
  • polypeptides of present invention can also be formulated using a nucleic acid vector encoding the polypeptides of the present invention.
  • an immune response may result from expression of the polypeptides in a host following administration of a nucleic acid vector encoding the immunogen to the host.
  • compositions can be administered to a subject by various well known administration methods, such as for example by injection, including subcutaneous, intravenous, intramuscular and intraperitoneal injection, by intracavitary administration, such as for example transrectal, vaginal and sublingual, respiratory administration, such as for example through the nasal cavity, mucosal administration, or topical administration.
  • injection including subcutaneous, intravenous, intramuscular and intraperitoneal injection
  • intracavitary administration such as for example transrectal, vaginal and sublingual
  • respiratory administration such as for example through the nasal cavity, mucosal administration, or topical administration.
  • the present in invention also relates to a polypeptide or pharmaceutical composition as described herein for use in the treatment of an infection caused by a coronavirus, preferably a beta coronavirus, most preferred a SARS-CoV-2 virus.
  • a coronavirus preferably a beta coronavirus, most preferred a SARS-CoV-2 virus.
  • the capacity of the polypeptides of present invention to inhibit infection by SARS-CoV-2 was tested in an in vitro inhibition assay using Vero 76 cells infected with SARS-CoV-2 viruses (see Example 5). It could surprisingly be shown that in presence of the three polypeptides tested the level of infection decreases in a dose-dependent manner (Fig. 8).
  • the inhibitory activity runs in the order L3C > L3B > L3A, consistently with the order in structural stability of the proteins, as well as with their capacity to interact with HR2. It could thus be shown that by interacting with the HR2 region in the Spike protein of the S2 subunit, the HR1 mimetic proteins can block the conformational transition of S2 that promotes membrane fusion and infection.
  • the use of the polypeptides as described herein for the inhibition of infection with a coronavirus preferably a beta coronavirus, most preferred a SARS-CoV-2 virus, is envisaged.
  • the coronavirus is a beta coronavirus selected from the group consisting of SARS-CoV-1 , MERS-CoV and SARS-CoV-2, preferably SARS-CoV-2.
  • the SARS-CoV-2 is preferably a SARS-CoV-2 variant of concern (VOC), more preferably a SARS-CoV-2 VOC selected from the group consisting of Alpha or Alpha-like, Beta or Beta-like, Gamma or Gamma-like, Delta or Delta-like and Omicron or Omicron-like, most preferably selected from the group consisting of Alpha, Beta, Gamma, Delta and Omicron.
  • VOC SARS-CoV-2 variant of concern
  • SARS-CoV-2 VOCs are as defined by World Health Organization (https://www.who.int/en/activities/tracking-SARS-CoV-2-variants/ as well as https://www.pango. network/ and https://cov-lineages.org/ for further details on their lineages).
  • the present in invention also relates to a method of treatment of an infection caused by a coronavirus, preferably a beta coronavirus, most preferably a SARS-CoV-2 virus, in a patient in need thereof, comprising administering a polypeptide or pharmaceutical composition as described herein.
  • a coronavirus preferably a beta coronavirus, most preferably a SARS-CoV-2 virus
  • the present invention also relates to the use of a polypeptide or pharmaceutical composition as described herein in the manufacture of a medicament for treating an infection caused by a coronavirus, preferably a beta coronavirus, most preferred a SARS-CoV-2.
  • a coronavirus preferably a beta coronavirus, most preferred a SARS-CoV-2.
  • Another aspect of the invention is a method of reducing a coronavirus, preferably a beta coronavirus, most preferred a SARS-CoV-2 virus viral load in a subject in need thereof, comprising administering a polypeptide or pharmaceutical composition as described herein.
  • Another aspect of the invention is the use of a polypeptide or pharmaceutical composition as described herein in the manufacture of a medicament for reducing a coronavirus, preferably a beta coronavirus, most preferred a SARS-CoV-2 virus viral load.
  • Another aspect of the invention is a polypeptide or pharmaceutical composition as described herein for use in reducing a coronavirus, preferably a beta coronavirus, most preferred a SARS-CoV-2 virus viral load.
  • the present invention furthermore relates to a polypeptide for use in the treatment or prevention of an infection by a coronavirus, preferably a beta coronavirus, most preferably a SARS-CoV-2 virus, characterized in that said polypeptide is a mimetic polypeptide of the HR1 region of the S2 subunit of the Spike protein of the said coronavirus, wherein the polypeptide comprises three alpha helices forming an antiparallel trimer of helices, wherein helix one and helix two are linked via a first linker and helix two and helix three are linked via a second linker and wherein helix two is inverted.
  • a coronavirus preferably a beta coronavirus, most preferably a SARS-CoV-2 virus
  • the inventors detected significant antibody (IgG and IgA) responses in sera from COVID-19 patients against the HR1 -mimetic proteins as further detailed in Example 6.
  • the HR1 mimetic proteins can reproduce immunogenic epitopes of relevance in SARS-CoV-2 infection
  • the reactivity of sera obtained three months after the onset of COVID- 19 when all patients had developed a sustained SARS-CoV-2-specific response against RBD (Fig. 9) and nucleocapsid protein was tested.
  • Significant IgG responses in the COVID-19 patients’ sera against the three protein variants L3A, L3B and L3C compared to the controls were detected indicating the presence of immune responses against HR1 .
  • the present invention therefore relates to the use of the polypeptides as described herein for the manufacture of a vaccine suitable for treating or preventing an infection with a coronavirus, preferably a beta coronavirus, most preferably a SARS-CoV-2 virus.
  • a coronavirus preferably a beta coronavirus, most preferably a SARS-CoV-2 virus.
  • the present invention also relates to the polypeptides as described herein or a composition comprising thereof for use as a vaccine for treating or preventing an infection with a coronavirus, preferably a beta coronavirus, most preferably a SARS-CoV-2 virus.
  • a coronavirus preferably a beta coronavirus
  • the inventors have shown the capacity of the polypeptides of present invention to detect the presence of antibodies in sera from COVID-19 patients by ELISA at a similar level to antibodies against the Receptor Binding Domain, which is so far considered the most immunogenic region. Strikingly, the IgA response detected was even higher to the polypeptides of present invention than the response to the RBD.
  • the polypeptides of present invention can therefore furthermore be used for the detection of antibodies against SARS-CoV-2 in patient sera of patients that have had an immune response against the virus as a result of a current or past infection.
  • the present invention therefore also relates to an in vitro method of detecting the presence of antibodies directed against the HR1 region of the S2 subunit of the Spike protein of a coronavirus, preferably a beta coronavirus, most preferably a SARS-CoV-2 virus in a test sample, wherein the method comprises contacting a test sample with the polypeptides as described herein and detecting the presence of a signal, wherein the presence of the signal is indicative of the presence of said antibodies in the test sample.
  • the antibodies are selected from IgG, IgA and/or IgM.
  • Table 2 Amino acid changes of the three a-helices, a-helix 1 , 2 and 3 as present in L3A, L3B and L3C compared to the three a-helices of wildtype HR1 (SEQ ID NOs.1 -3):
  • Table 3 Amino acid sequences of each of the three a-helices, a-helix 1 , 2 and 3 of the three CoVS-HR1 variants L3A, L3B and L3C; the positions in which amino acid changes were made are shown in bold:
  • the DNA encoding the protein sequences were synthesized and cloned into pET303 expression vectors by Thermo Fisher Scientific (Waltham, Massachusetts, MA, USA). The sequences included a N-terminal methionine and a C-terminal histidine tag with sequence GGGGSHHHHHH. The protein sequences are detailed in Table 4. E. co//bacteria (BL21 (DE3)) were transformed with the plasmids and cultured at 37°C in presence of 30 mg-mL-1 Ampicillin. Protein expression was induced with 0.5 mM IPTG and the cells were cultured overnight at 37°C.
  • lysis buffer 50 mM Tris/HCI, 500 mM NaCI, 1 mM EDTA, 1 mM p-mercaptoethanol
  • the cells were then lysed with three 30 s ultrasonication cycles on ice and the soluble and insoluble fractions were separated by 30 min ultracentrifugation at 4°C at 30000 rpm.
  • the proteins were purified from the supernatant fraction by nickel-affinity chromatography.
  • a second purification step was carried out by ion exchange chromatography on a HiT rap SP Sepharose XL column (Amersham GE Healthcare).
  • Synthetic peptides derived from the S2 HR2 sequence (SEQ ID NO. 35) were acquired from Genecust (Luxembourg), with a purity >95% ; this peptide corresponds to the first 39 residues of the HR2 region of the Spike (residues 1164-1202) to which 4 additional residues with SGGY (SEQ ID NO. 36) sequence were added. Peptides were C-terminally tagged with a SGGY sequence to confer UV absorption at 280 nm.
  • Protein and peptide concentrations were measured by UV absorption measurements at 280 nm with extinction coefficients calculated according to their respective amino acid sequences with the ExPasy ProtParam server (https://web.expasy.org/protparam/).
  • CD measurements were performed with a Jasco J-715 spectropolarimeter (Tokyo, Japan) equipped with a temperature-controlled cell holder. Measurements of the far-UV CD spectra (260-200 nm) were made with a 1 mm path length quartz cuvette. Spectra were recorded at a scan rate of 100 nm/min, 1 nm step resolution, 1 s response and 1 nm bandwidth. The resulting spectrum was usually the average of 5 scans. Each spectrum was corrected by baseline subtraction using the blank spectrum obtained with the buffer and finally the CD signal was normalized to molar ellipticity ([0], in deg dmol-Tcm2). Thermal unfolding was monitored by measuring the CD signal at 222 nm as a function of temperature using a scan rate of 1 °C min-1.
  • the apparent hydrodynamic radii of the proteins were measured using dynamic light scattering (DLS) in a DynaPro MS-X DLS instrument (Wyatt, Santa Barbara, CA).
  • Dynamics v6 software (Wyatt Technology Corporation, Santa Barbara, CA) was used in data collection and processing. Sets of DLS data were measured at 25 °C with an average number of 50 acquisitions and an acquisition time of 10 s.
  • Static scattering intensities were measured in a DynaPro MS-X DLS instrument (Wyatt, Santa Barbara, CA) or a Malvern pV instrument (Malvern Panalytical, Malvern, UK) at 25 °C, in 50 mM sodium phosphate buffer pH 7.4, at different concentrations of protein in a range of 0.2-4.5 mg mL-1.
  • the intensities were analyzed using the Debye plot as represented by equation 1 ,
  • Kc/ 1/ M w + 2 A 2 C , (1) valid for particles significantly smaller than the wavelength of the incident radiation, where the K is an optical constant of the instrument, c is the particle mass concentration, R90 is the Rayleigh ratio of scattered to incident light intensity, Mw is the weight-averaged molar mass, A2 is the 2nd virial coefficient that is representative of inter-particle interaction strength. Mw can be determined from the intercept of the plot.
  • ITC measurements were carried out in a Microcal VP-ITC calorimeter (Malvern Panalytical, Malvern, UK). The proteins were titrated with 25 injections of 5 pL peptide solution at 480 s intervals. Protein concentration in the cell was around 10 pM, while the peptide concentration in the syringe was typically 200-300 pM. The experiments were carried out in 50 mM phosphate buffer (pH 7.4) at 25 °C. The experimental thermograms were baseline corrected and the peaks were integrated to determine the heats produced by each ligand injection. Residual heats due to unspecific binding or ligand dilution were estimated from the final peaks of the titrations.
  • CoVS-HR1 proteins ability to bind soluble trimeric Spike (S) proteins was determined by ELISA. Briefly, 96-well ELISA plates (Maxisorp, Nunc) were coated at 4 °C overnight with recombinant trimeric SARS-CoV-2 Spike protein (Abyntek Biopharma- ProSci Inc., Poway, CA, Catalog Number 10-075, Spike sequence Gln14 - Gln1208, Protein Accession Number: QHD43416.1 ) in 0.1 M bicarbonate buffer (pH 9.6).
  • CoVS-HR1 molecules L3A, L3B and L3C were added at different concentrations and incubated for 2 h at room temperature.
  • protein constructs derived from HIV-1 gp41 sequences which are HR1 -mimics of HIV gp41 disclosed in [9] and [29] were also tested.
  • CoVS-HR1 molecules were mixed with HR2 V39E peptide at 1 :2 molar ratio.
  • Vero 76 cells were plated on a 96 well plate at 12500 cells/well. 50 pL of serial 4-fold dilutions of CoVS-HR1 proteins (2-fold concentrated) were incubated with the cells for 30 min. Cells were then infected by adding 50 pL WT SARS-CoV-2 strain (UK D614G genotype or BA.1 Omicron genotype) at Multiplicities Of Infection (MOI) of 80. After 2 days, cells are fixed with methanol for 20 min, washed with PBS and stained with anti-Nucleocapside Antibody (Genetex GTX135357) at 1/200 dilution in permwash (B&D) for 45 min at room temperature.
  • WT SARS-CoV-2 strain UK D614G genotype or BA.1 Omicron genotype
  • MOI Multiplicities Of Infection
  • CoVS-HR1 antigenicity against sera of infected patients was determined by ELISA. Sera samples were collected 3 months after recovery from COVID-19 infection. As control, sera from healthy donors were also assayed. All patients and healthy donors gave their written informed consent (COVID-HUS ethics committee approved, reference CE: 2020-34).
  • 96-well ELISA plates (Maxisorp, Nunc) were coated at 4 °C overnight with CoVS-HR1 molecules in 0.1 M bicarbonate buffer (pH 9.6). After blocking with 5% non-fat powdered milk in PBS for 1 h at 25 °C, 1/1000 diluted sera (100 pL diluted in 1 % BSA 0.05% Tween solution) were added and incubated for 30 min at room temperature.
  • the plate was then washed five times and CoVS-HR1 binding to IgG or IgA was detected by incubating for 1 hour at room temperature with 100 pL goat anti-human IgG or anti-human IgA antibody conjugated to horseradish peroxidase (HRP) (Abeam) at 1/5000 dilution, respectively.
  • HRP horseradish peroxidase
  • IgG or IgA binding was then revealed with tetramethylbenzidine (TMB) substrate buffer, the reaction was stopped with 1 M H2SO4 and optical density was read at 450 nm with a Molecular Device Plate Reader equipped with SoftMax Pro 6 program. Background binding was measured in plates without CoVS-HR1 proteins and subtracted from the data.
  • His-tagged RBD was used to detect the anti- RBD antibodies present in the patients’ sera. Data was analyzed using Origin software (OriginLab, Northampton, MA).
  • the CoVS-HR1 protein was co-crystalized in complex with the V39E HR2 peptide.
  • a concentrated protein-peptide mixture (about 9 mg/mL) was prepared in 10 mM Tris/HCI buffer at a 1 :2 protein peptide molar ratio. Screening of crystallization conditions was carried out using the sitting drop vapor-diffusion method with the crystal screening kit “Structure Screen 1 and 2 Eco Screen” from Molecular Dimensions (Suffolk, UK). Droplets consisting in mixing 2 pL complex solution with 2 pL reservoir solution were equilibrated at 298 K against 200 pL reservoir solution. Best crystals were obtained in 0.1 M sodium HEPES pH 7.5, 20% w/v PEG 4000, 10% isopropanol.
  • the L3A, L3B and L3C CoVS-HR1 proteins were produced recombinantly by overexpression in E. coli with high yields in the soluble fraction and could be easily purified by two-step standard chromatographic methods. All the proteins were highly soluble (>10 mg mL-1) in standard buffers. The proteins are highly a-helical according to their CD spectra (Fig. 3), in good agreement with the design model (Fig. 1 ).
  • L3B and L3C show much sharper denaturation peaks than L3A, with higher unfolding enthalpies, indicating a more stable and cooperative structure. This indicates that the choice of mutations was more effective in stabilizing the coiled-coil structure in these variants.
  • L3C is more thermostable than L3B by about 13°C, as a result of higher a-helical propensity of its sequence produced by the substitution of glycine residues.
  • Table 6 Thermodynamic parameters of binding of CoVS-HR1 proteins and HR2 V39E peptide measured by ITC.
  • Example 4 Binding assays of the three variants to recombinant trimeric Spike by ELISA
  • Example 5 Capacity of the CoVS-HR1 proteins to inhibit infection by SARS-CoV-2
  • Example 6 Reactivity of sera obtained three months after the onset of COVID-19
  • Example 7 High resolution structure of the CoVS-HR1 -L3B protein in complex with the HR2 region.
  • Example 8 Live virus neutralization. The ability of the CoVS-HR1 proteins L3B and L3C to neutralize various coronavirus was tested in a live virus neutralization assay against MERS-CoV/EMC, SARS-CoV HKU-39849 and SARS-CoV-2 BavPat1/2020.
  • samples were serially diluted, and pre-incubated with virus (1 h), then mixture was added to cells and incubated (20h for CoV-1 and 2, 40h MERS).
  • Cells were formalin-fixed, permeabilization and incubation with an antibody against the nucleocapsid protein, followed by a secondary IgG peroxidase conjugate and TrueBlue substrate. Nucleopcapsid positivity was the readout via ELISPOT.
  • IC50 Neutralization titers
  • L3B and L3C neutralizes both SARS-CoV-1 in live virus neutralization assay.
  • L3C shows also a neutralization activity on SARS-CoV-2.
  • SARS-CoV- 1 SARS_CoV-2 Wuhan, SARS-CoV-2 Delta (IN2), SARS-CoV-2 Omicron BA4, CoV 229E and CoV NL63.
  • samples were serially diluted, and pre-incubated with luciferase expressing virus (1 h), then mixture was added to 293T ACE2 cells and incubated for 48h. Cells were formalin-fixed and lysed. Readout was luciferase activity as measure of infection via a luminometer.
  • IC50 Neutralization titers
  • L3C neutralized SARS-CoV-2 VOCs Wuhan, Delta and BA4 as well SARS-CoV-1.
  • L3B neutralizes SARS-CoV-1 and SARS-CoV-2 Wuhan.

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Abstract

The present invention relates to novel mimetic polypeptides of the HR1 region of the S2 subunit of the Spike protein of coronaviruses that are capable of inhibiting host cell infection by a coronavirus. The invention furthermore relates to the use of such polypeptides for the prevention or treatment of an infection caused by coronaviruses as well as the detection of the infection.

Description

NOVEL MIMETIC POLYPEPTIDES OF THE HR1 REGION OF THE S2 SUBUNIT OF CORONAVIRUSES
Technical field
The invention relates to novel mimetic polypeptides of the HR1 region of the S2 subunit of the Spike protein of coronaviruses that are capable of inhibiting host cell infection by a coronavirus. The invention furthermore relates to the use of such polypeptides for the prevention or treatment of an infection caused by coronaviruses.
Background of the invention
Since SARS-CoV-2 emerged in late 2019, a huge amount of research has dramatically increased the understanding of the molecular basis of the disease and has provided novel strategies to fight it. Vaccines based in immunization with the Spike (S) protein, either using mRNA or viral vectors, have been very successful to decrease viral transmission and severity of the disease. However, the immunity of vaccinated people appears to decline after few months from vaccination and new variants of the virus that may escape the protection of current vaccines are continuously emerging. Moreover, no effective antivirals have been approved to treat the infection. All these problems make it necessary to continue the development of new treatments, as well as new immunization strategies.
As in other coronaviruses, SARS-CoV-2 Spike (S) protein decorates the virus surface and promotes its entry into the host cells. Like other Class-I fusion proteins, S protein is a trimer of heterodimers composed of S1 and S2 subunits. The S1 subunit consists of the N-terminal domain (NTD), the receptor-binding domain (RBD) and two C-terminal domains (CTD). Three S1 subunits cover the S2 trimer and maintain it in its prefusion conformation. S2 contains a fusion peptide (FP) and two heptad-repeat regions (HR1 and HR2) that are essential to promote membrane fusion. The S2 prefusion structure is organized around a coiled-coil trimer formed by its central helix (CH) and the connector domain (CD), located between the HR1 and HR2 regions. The protein is embedded in the viral membrane by a transmembrane (TM) segment that is followed by an internal short C-terminal tail (CT).
Cell infection by SARS-CoV-2 starts with S1 binding to the angiotensin-converting enzyme 2 receptor (ACE2) using the receptor-binding domain (RBD). Then, proteolysis of the S2 subunit at the S2’ site mediated by host proteases (TMPRSS2 in plasma membrane or Cathepsins in the endosomes) triggers a conformational transition in which HR1 becomes extended to continue the CH trimeric coiled-coil resulting in insertion of the FP into the cell membrane [1]. Then, S1 becomes shed and S2 folds on itself as a trimer of hairpins that for a 6-helix bundle structure (6HB), in which three HR2 pack externally against the grooves of a central trimeric HR1 helical bundle [2], This process brings into close proximity the viral and cell membranes promoting their fusion and subsequent insertion of viral content onto the cell.
Because of their importance in viral fusion, HR1 and HR2 are potential targets for coronavirus treatment [2,3]. HR2 has a particularly high sequence conservation in SARS-CoV-2 [4], as well as between different coronaviruses [3].
S2-mediated fusion of SARS-CoV-2, as well as other coronaviruses, is inhibited by peptides derived from HR2 [2, 3, 5, 6]. HR1 -based peptides are much less active inhibitors [5, 6] but stabilized trimeric helical bundles of HIV-1 gp41 HR1 have shown inhibitory activity of HIV-1 [7-9], as well as MERS-CoV, HCoV-OC43 and SARS-CoV-2 [10]. This evidence indicates that both HR1 and HR2 are exposed during coronavirus fusion and susceptible to inhibition.
Moreover, vaccination with stabilized HIV-1 gp41 HR1 trimers can elicit neutralizing antibodies against HIV-1 [11] and HIV-1 infected patients elicit neutralizing antibody B cell responses that target HR1 epitopes [12, 13]. It is conceivable that similar antibody responses may be elicited during coronavirus infection.
During SARS-CoV-2 infection, antibody responses are mostly directed against the nucleocapsid protein and the spike protein and, within this, against the S1 subunit [14], Most potently neutralizing antibody (Ab) responses in COVID-19 convalescent patients are directed to the RBD, but mutations in emerging SARS-CoV-2 variants tend to reduce their neutralization activity [15]. Neutralizing responses targeting S2 are scarcer but interesting [16], because, since S2 is highly conserved, they may be less sensitive to mutations than more variable regions such as RBD or NTD. Moreover, S2-targetting neutralizing antibody responses that cross-react with other related coronaviruses have been reported, even in uninfected individuals
[17], This type of memory B cell immunity may confer durable broad coronavirus protection
[18].
Epitope mapping has so far detected a few immunogenic epitopes in S2 [19] mostly located at sequence 765-835, comprising the fusion peptide and the S2 cleavage site, and residues 1140-1 160 at the S2 stem region immediately upstream of HR2. Both epitopes have been described as neutralizing [20] and immunodominant in COVID-19 patients [21], Neutralizing monoclonal antibodies (mAbs) targeting the 1140-1160 linear helical epitope has been recently described [22-24], There is however a lack of highly immunogenic epitopes within HR1 and HR2. A possible reason is that HR1 and HR2 are highly protected from the immune system due to their importance in the conformational changes driving membrane fusion. It is also possible that conformational variability of these regions could make sensitive epitopes to be only transiently exposed. Nevertheless, immune responses to conformational epitopes may have gone unnoticed in epitope mapping studies using linear peptides.
Given the current situation of the pandemic caused by SARS-CoV-2 and the fact that the virus will continue spreading and evolving in the coming years taken together with the current lack of specific and efficient treatment options, there is a great need for the development of post infection treatments, measures preventing the infection as well as diagnostics for reliable and sensitive infection detection. Since coronaviruses constantly evolve further, such treatment/preventive measures shall ideally target structures that are conserved among various coronavirus species ensuring that efficacy against more than just one species is provided.
Summary of the Invention
The present invention therefore relates to a polypeptide capable of inhibiting host cell infection by a coronavirus, preferably a beta coronavirus, most preferably a SARS-CoV-2 virus, wherein said polypeptide comprises three a-helices and wherein
(i) a-helix 1 comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO.1 ,
(ii) a-helix 2 comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO.2, and
(iii) a-helix 3 comprises an amino acid sequence that is at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO.3.
In one embodiment the said three a-helices form an antiparallel trimer of helices and, preferably, said polypeptide is a mimetic polypeptide of the heptad-repeat region 1 (HR1 ) of the S2 subunit of the SARS-CoV-2 Spike protein.
In a further embodiment of the polypeptide of the present invention at least one amino acid in each a-helix at sequence positions “e” and/or “g” in the heptad repeats (as shown in Figure 1) is substituted by a lysine (K), arginine (R), aspartic acid (D) or glutamic acid (E), preferably lysine (K) or glutamic acid (E). In a most preferred aspect, at least two of the amino acids are substituted in different a-helices at “e” and “g” positions close in space, so that their side chains can form mutually favorable charge-charge interactions or hydrogen bonds.
In a further embodiment of the polypeptide of present invention
(i) a-helix 1 comprises at least one of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.1 : N1 D, Q13E, V38K, A45K, and/or V63K, or a combination thereof,
(ii) a-helix 2 comprises at least one of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.2: V1 1 K, S13E, A29R, A31 E, V36E, Q38E, S50E, I56E and/or A63R, or a combination thereof,
(iii) a-helix 3 comprises at least one of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.3: 118E, A29K, A43K, V50E, G58R, L68E, D72Q and/or E75Q, or a combination thereof.
In a further embodiment of the polypeptide of present invention
(i) a-helix 1 comprises at least one of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.1 : N1 D, Q13K, A31 E, V38E, A45D, A59R and/or V63E, or a combination thereof,
(ii) a-helix 2 comprises at least one of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.2: L6D, V11 E, S13K, A31 K, V36E, Q38K, A43E, A45R, S50E, D51 K, A63E and/or N68R, or a combination thereof,
(iii) a-helix 2 comprises at least one of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.3; A1 1 E, A29K, Q36K, A43K, L68K or a combination thereof.
In yet a further embodiment of present invention at least one glycine residue (G) has been replaced in at least one of the three a-helices, preferably in two, most preferably in all three a- helices by a polar amino acid, preferably a lysine (K), arginine (R), threonine (T), or serine (S), most preferred lysine (K). In a preferred aspect the at least one glycine residue (G) is located in the middle of the a-helix.
In a further embodiment of the polypeptide of present invention
(i) a-helix 1 further comprises at least one of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.1 : G19K, G33K, G58K, or a combination thereof, (ii) a-helix 2 further comprises at least one of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.2: G16K, G41T, G55K, or a combination thereof,
(iii) a-helix 3 further comprises at least one of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.3; G19K, G33K, G58S, or a combination thereof.
In one preferred embodiment of the polypeptide of present invention
(i) the a-helix 1 consists of the amino acid sequence of SEQ ID NO.4, and
(ii) the a-helix 2 consists of the amino acid sequence of SEQ ID NO.5, and
(iii) the a-helix 3 consists of the amino acid sequence of SEQ ID NO.6.
In another preferred embodiment of the polypeptide of present invention
(i) the a-helix 1 consists of the amino acid sequence of SEQ ID NO.7, and
(ii) the a-helix 2 consists of the amino acid sequence of SEQ ID NO.8, and
(iii) the a-helix 3 consists of the amino acid sequence of SEQ ID NO.9.
In yet another preferred embodiment of the polypeptide of present invention
(i) the a-helix 1 consists of the amino acid sequence of SEQ ID NO.10, and
(ii) the a-helix 2 consists of the amino acid sequence of SEQ ID NO.11 , and
(iii) the a-helix 3 consists of the amino acid sequence of SEQ ID NO.12.
In a further embodiment of the polypeptide of the present invention at least one amino acid in each a-helix at sequence positions “a” and/or “d” in the heptad repeats (as shown in Figure 1) making internal contacts with opposite a-helices is substituted by a non-polar amino acid, preferably a leucine (L), isoleucine (I), valine (V), phenylalanine (F) or alanine (A), mostly preferred leucine (L) or isoleucine (I).
In a further embodiment of the polypeptide, a-helix 1 and a-helix 2 are linked via a first linker and a-helix 2 and a-helix 3 are linked via a second linker, and wherein the first and/or second linker is between 3 to 6 amino acids long, preferably 4 or 5 amino acids long, most preferred 4 amino acids long.
In a preferred embodiment of the polypeptide, the amino acids of the linker are selected from glycine (G), alanine (A), serine (S), aspartic acid (D), asparagine (N), lysine (K), arginine (R), proline (P) and glutamic acid (E), or a combination thereof. In a most preferred embodiment, the amino acid composition of the linker connecting a-helix 1 and a-helix 2 is selected from the sequences GEPA (SEQ ID NO. 16) or GAPA (SEQ ID NO. 17), or GEPC (SEQ ID NO. 18), and the linker connecting a-helix 2 and a-helix 3 is selected from the sequences SGSG (SEQ ID NO. 19), SGDG (SEQ ID NO. 20), KGSG (SEQ ID NO. 21) or KGDG (SEQ ID NO. 22).
In yet a further embodiment of present invention at least one pair, preferably two pairs, of residues have been replaced by a cysteine (C) in the polypeptide so as to form at least one disulfide bond, preferably two disulfide bonds.
In an embodiment of the polypeptide,
(i) a-helix 1 further comprises at least one of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.1 : L3C,
(ii) a-helix 2 further comprises at least one of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.2: Y70C,
(iii) a-helix 3 further comprises at least one of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.3: L71C .
In one embodiment, the first linker having the sequence GEPA (SEQ ID NO. 16) or GAPA (SEQ ID NO. 17) comprises the following amino acid substitutions A4C.
In an embodiment of the polypeptide of the present invention,
(i) the a-helix 1 consists of the amino acid sequence of SEQ ID NO.23, and
(ii) the a-helix 2 consists of the amino acid sequence of SEQ ID NO.24, and
(iii)the a-helix 3 consists of the amino acid sequence of SEQ ID NO.25.
In an embodiment of the polypeptide of the present invention,
(i) the a-helix 1 consists of the amino acid sequence of SEQ ID NO.27, and
(ii) the a-helix 2 consists of the amino acid sequence of SEQ ID NO.28, and
(iii) the a-helix 3 consists of the amino acid sequence of SEQ ID NO.29.
In an embodiment of the polypeptide of the present invention,
(i) the a-helix 1 consists of the amino acid sequence of SEQ ID NO.31 , and
(ii) the a-helix 2 consists of the amino acid sequence of SEQ ID NO.32, and
(iii) the a-helix 3 consists of the amino acid sequence of SEQ ID NO.33. In one embodiment the polypeptide of present invention is characterized in that it comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO.13.
In a preferred embodiment the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO. 26, SEQ ID NO. 30 and SEQ ID NO. 34, preferably wherein the polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO. 26, SEQ ID NO. 30 and SEQ ID NO. 34.
In a preferred embodiment the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15, preferably wherein the polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15.
In an embodiment, the polypeptide of the present invention has a neutralization titer against at least one, at least 2 or at least 3 coronavirus selected from the group consisting of MERS-CoV, SARS-CoV-1 , SARS-CoV-2 of less than 100 pM, preferably of less than 50pM, more preferably of less than 25 pM.
In an embodiment, the polypeptide of the present invention has a neutralization titer against at least one, at least 2, at least 3 or at least 4 or at least 5 SARS-CoV-2 variants of concern (VOCs) of less than 100 pM, preferably of less than 50pM, more preferably of less than 25 pM. SARS-CoV-2 VOCs may be selected from the group consisting of Alpha or Alpha-like, Beta or Beta-like, Gamma or Gamma-like, Delta or Delta-like and Omicron or Omicron-like, most preferably selected from the group consisting of Alpha, Beta, Gamma, Delta and Omicron
The present invention furthermore relates to a pharmaceutical composition comprising the polypeptides of any one of the preceding claims further comprising pharmaceutically acceptable excipients.
The present invention furthermore relates to a polypeptide for use in the treatment or prevention of an infection by a coronavirus, preferably a beta coronavirus, most preferably a SARS-CoV-2 virus, characterized in that said polypeptide is a mimetic polypeptide of the HR1 region of the S2 subunit of the Spike protein of the said coronavirus, wherein the polypeptide comprises three alpha helices forming an antiparallel trimer of helices, wherein helix one and helix two are linked via a first linker and helix two and helix three are linked via a second linker and wherein helix two is inverted.
In one embodiment the invention relates to the polypeptide or the pharmaceutical composition as described herein for use in the treatment or prevention of an infection caused by a coronavirus, preferably a beta coronavirus, most preferably a SARS-CoV-2 virus.
The present invention furthermore relates to the use of the polypeptides as described herein for the in vitro diagnosis of an infection with a coronavirus, preferably a beta coronavirus, most preferably a SARS-CoV-2 virus.
The present invention furthermore relates to the use of the polypeptides as described herein for the neutralization of an infection with a coronavirus, preferably a beta coronavirus, most preferably a SARS-CoV-2 virus.
The present invention also relates to a vaccine comprising the polypeptides of any one of the preceding claims for treating or preventing an infection with a coronavirus, preferably a beta coronavirus, most preferably a SARS-CoV-2 virus. In a preferred embodiment the polypeptides of present invention can be combined with other immunogens.
The present invention further relates to an in vitro method of detecting the presence of antibodies directed against the HR1 region of the S2 subunit of the Spike protein of a coronavirus, preferably a beta coronavirus, most preferably a SARS-CoV-2 virus in a test sample, wherein the method comprises contacting a test sample with the polypeptides as described herein and detecting the presence of a signal, wherein the presence of the signal is indicative of the presence of said antibodies in the test sample. In a preferred embodiment the antibodies are selected from IgG, IgA and/or IgM.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 : Design of HR1 mimetic proteins. A) Ribbon representation of the X-ray crystal structure of the six-helix bundle formed by three HR1 regions (light gray) and three HR2 regions (dark grey) of SARS-CoV-2 S2 protein (PDB id. 6LXT [2]). B) Ribbon representation showing the chain topology of the CoVS-HR1 mimetic proteins. The two parallel helices are in light gray and the loops and the antiparallel helix in dark grey. C) and D) Schematic representation of the sequence topology of the CoVS-HR1 proteins indicating the engineered amino acids in bold for L3A (C) and L3B (D) variants. The dashed lines indicate salt bridges or hydrogen bonds between the engineered side chains. The glycine residues engineered in the L3C variant are underlined in D. Amino acids in core positions “a” and “d” in the heptad repeats of the coiled-coil structure are indicated on top of the first helical sequence. The “stutter” regions correspond to discontinuities in the canonical heptad repeats.
Figure 2: Helix-wheel representation of the trimeric coiled-coil structure of the CoVS-HR1 L3B protein. The “a b c d e f g” positions of the heptad repeats in each helix wheel of the coiled-coil are indicated with letters. The coiled-coil has been divided in the three regions separated by the stutters for the sake of clarity. The dashed lines indicate the engineered interactions by amino acid substitutions, as indicated in Figure 1 D.
Figure 3: Far-UV CD spectra of the CoVS-HR1 proteins L3A, L3B and L3C (Fig.2 A, B, C) in free form and in presence of V39E peptide at different molar ratio. Spectra were recorded at 25°C in 50 mM sodium phosphate buffer pH 7.4 at a protein concentration of 30 pM.
Figure 4: Molecular size of the CoVS-HR1 proteins measured by light scattering. A) Debye plot corresponding to the light scattered as a function of the protein mass concentration. The intercept corresponds to inverse of the weight averaged molar mass, Mw of the particles. Theoretical masses of the monomeric proteins L3A, L3B and L3C are 26.4, 26.5 and 27.1 kDa respectively. B) Apparent hydrodynamic radii of the proteins measured by dynamic light scattering. The hydrodynamic radius estimated with the program HYDROPRO using the design model is 3.2 nm.
Figure 5: Thermal denaturation of the CoVS-HR1 proteins measured by differential scanning calorimetry (DSC). Experiments were carried out at pH 7.4 in 50 mM sodium phosphate buffer. A) Comparison of the DSC thermograms of the three protein variants. B) Effect of the addition of the V39E peptide at different molar ratios.
Figure 6: Isothermal titration calorimetry (ITC) analysis of the binding of V39E peptide to the CoVS-HR1 proteins. The upper panels show the experimental ITC thermograms. The lower panels show the normalized binding isotherms fitted using a binding model of n identical and independent sites.
Figure 7: Binding of the CoVS-HR1 proteins to recombinant trimeric Spike. A) ELISA experiments with immobilized recombinant trimeric Spike with different concentrations of covS- HR1 L3A, L3B and L3C proteins. CovNHR and covNHR-N-ddS correspond to HR1 mimetics of HIV-1 gp41. B) The same experiments as in panel A but in competition with V39 peptide, added at 2:1 peptide:protein ratio.
Figure 8: Inhibition by the CoVS-HR1 proteins of SARS-CoV2-infection on Vero 76 cells. Cells were infected by primary viruses (B1 D614G genotype, panel A, and Omicron BA.1 , panel B) in the presence of CoVS-HR1 proteins at different concentrations. The percentage of inhibition was calculated by reduction in the percentage of infected cells treated with the inhibitory proteins compared to untreated control cells. Data correspond to mean ± standard deviations of 4 independent experiments. Continuous lines correspond to nonlinear regression curves using a sigmoidal Hill’s function.
Figure 9: Antibody binding present in infected patients’ sera 3 months after SARS-CoV-2 infection towards CoVS-HR1 molecules or RBD. A) IgG binding towards RBD, L3A, L3B and L3C. B) IgA binding towards RBD and L3C. The antibody binding in sera from healthy donors for each antigen are labeled with C.
Figure 10: Crystallographic structure of CoVS-HR1 -L3B in complex with the V39E peptide (dark ribbons) superimposed to the crystallographic structure of the 6HB post-fusion structure of S2 (Protein Data Bank entry: 6LXT) (grey ribbons). The RMSD between the two structures is 0.634 A. Two HR2 regions of 6LXT have been deleted for the sake of clarity.
Fig. 1 1 : Ribbon representation of the model structure of CoVS-HR1 -L3C-dSS. The locations of the disulfide bonds C3-147 and C77-C225 are indicated with the arrows.
Fig. 12: Inhibition by the CoVS-HR1 L3C and L3C-dSS proteins of WT SARS-CoV2-infection on Vero 76 cells. Cells were infected by primary viruses (B1 D614G genotype) in the presence of CoVS-HR1 proteins at different concentrations.
Detailed description
The present invention may be understood more readily by reference to the following detailed description of the preferred embodiments of the invention, and to the examples included therein.
As described in more detail above there is a great need for the development of novel post infection treatments, measures preventing the infection itself, as well as diagnostics for infection detection. Since coronaviruses constantly evolve further, such treatments/preventive measures shall ideally target structures that are conserved among various coronavirus species ensuring that efficacy against more than just one species is provided. POLYPEPTIDES OF PRESENT INVENTION
The inventors have herein shown the successful design and the production of several mimetic proteins of the HR 1 region of the Spike protein of a coronavirus, specifically SARS-CoV-2. The inventors could show that such mimetic proteins, collectively named CoVS-HR1 , spontaneously acquire the predicted a-helical structure, as depicted in Fig.1 and Fig. 10 and further described in Example 1 and Example 7. In addition, these polypeptides showed a very high stability and structural cooperativity as further detailed in Example 3 and shown in Fig. 5.
The polypeptides bind HR2-derived synthetic peptides with very high affinity (Fig. 6) and can also bind to trimeric recombinant S protein through its HR2 region (Fig. 7). All three polypeptides of present invention showed strong binding to the Spike and the level of detected binding at sub nM Spike concentration runs in the order L3C > L3B > L3A but rapidly saturates at higher concentration.
Furthermore, the inventors could surprisingly show that the protein variants with higher stability of the trimeric helical bundle sequence displayed stronger inhibitory activity against SARS- CoV-2 in vitro (Fig.7 and Example 5).
As shown in Example 1 , the polypeptide was modeled by using the X-ray crystal structure of the six-helical bundle formed by HR1 and HR2 in the S2 post-fusion structure. To model the target structure of an antiparallel trimer of helices of HR1 , the HR2 chains were deleted from the model and one of the HR1 helices, helix 2, was upturned, its sequence reversed and then aligned to the original one ensuring the correct core coiled-coil packing. Due to the antiparallel orientation of the reversed helix, the side chain CA-CB bonds have different spatial orientation compared to the native ones and this may perturb interhelical side chain packing in the coiled- coil structure. To compensate this, the alignment was made trying to superimpose the CB atoms of the core residue side chains. Then, side chain clashes were removed by energy minimization.
The present invention therefore relates to a polypeptide capable of inhibiting host cell infection by a coronavirus, preferably a beta coronavirus, most preferably a SARS-CoV-2 virus, wherein said polypeptide comprises three a-helices and wherein
(i) a-helix 1 comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO.1 , (ii) a-helix 2 comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO.2, and
(iii) a-helix 3 comprises an amino acid sequence that is at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO.3.
SEQ ID NO. 1 refers to the wildtype amino acid sequence of a-helix 1 of HR1 of the S2 subunit of the SARS-CoV-2 Spike protein.
SEQ ID NO. 2 refers to the reversed wildtype amino acid sequence of a-helix 2 of HR1 of the S2 subunit of the SARS-CoV-2 Spike protein.
SEQ ID NO. 3 refers to the wildtype amino acid sequence of a-helix 3 of HR1 of the S2 subunit of the SARS-CoV-2 Spike protein.
All sequences are listed in the example section herein below in Table 1 .
In one embodiment the said three a-helices form an antiparallel trimer of helices and said polypeptide is a mimetic polypeptide of the heptad-repeat region 1 (HR1 ) of the S2 subunit of the SARS-CoV-2 Spike protein.
An heptad repeat is a polypeptide sequence where a pattern of seven amino acid residues is repeated throughout the sequence. The positions of the heptad repeat are typically designated “a b c d e f g”, where “a” and “d” are typically non-polar residues, most frequently lie, Leu or Vai, and “e” and “g” are frequently polar or charged residues, most frequently Lys or Glu.
To enhance the stability of the antiparallel trimeric bundle and reduce exposed hydrophobicity, several surface-exposed residues at “e” and “g” positions of the coiled-coil heptad repeats (see Fig. 2) were replaced by charged or polar amino acids to engineer proper salt bridges and hydrogen bonds between the antiparallel helix and the other two helices. Additional mutations at solvent exposed positions were made to increase net positive change. No mutations were carried out to modify the hydrophobic groove between the two parallel HR1 helices to preserve the HR2 binding potential. Two variants named L3A and L3B were engineered using different sets of amino acid substitutions shown in bold in Figs 1 C and 1 D as also further summarized in table 2 in Example 1 .
In one embodiment of the polypeptide of the present invention at least one amino acid in each a-helix at sequence positions “e” and/or “g” in the heptad repeats is substituted by a lysine (K), arginine (R), aspartic acid (D) or glutamic acid (E), preferably lysine (K) or glutamic acid (E). In a most preferred aspect, at least two of amino acids are substituted in different a-helices at “e” and “g” positions close in space, so that their side chains can form mutually favorable charge-charge interactions or hydrogen bonds.
Additional stabilization of the coiled-coil trimeric bundle structure may be achieved by replacing buried polar amino acids at core positions “a” or “d” in the heptad repeats, as previously demonstrated by the inventors in a recent publication related to a highly similar construct based of HIV-1 gp41 HR1 [25].
Therefore, in a further embodiment of the polypeptide of the present invention at least one amino acid in each a-helix at sequence positions “a” and/or “d” in the heptad repeats (as shown in Figure 1 ) making internal contacts with opposite a-helices is substituted by a non-polar amino acid, preferably a leucine (L), isoleucine (I), valine (V), phenylalanine (F) or alanine (A), mostly preferred leucine (L) or isoleucine (I).
In yet a further embodiment of the polypeptide of present invention
(i) a-helix 1 comprises at least one, at least two, at least three, or at least four of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.1 : N1 D, Q13E, V38K, A45K, and/or V63K, or a combination thereof,
(ii) a-helix 2 comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.2: V11 K, S13E, A29R, A31 E, V36E, Q38E, S50E, I56E and/or A63R, or a combination thereof,
(iii) a-helix 2 comprises at least one, at least two, at least three, at least four, at least five, at least six, or at least seven, of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.3: 118E, A29K, A43K, V50E, G58R, L68E, D72Q and/or E75Q, or a combination thereof.
In a further embodiment of the polypeptide of present invention
(i) a-helix 1 comprises at least one, at least two, at least three, at least four, at least five or at least six of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.1 : N1 D, Q13K, A31 E, V38E, A45D, A59R and/or V63E, or a combination thereof,
(ii) a-helix 2 comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or at least eleven of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.2: L6D, V1 1 E, S13K, A31 K, V36E, Q38K, A43E, A45R, S50E, D51 K, A63E and/or N68R, or a combination thereof,
(iii) a-helix 3 comprises at least one, at least two, at least three, or at least four of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.3: A1 1 E, A29K, Q36K, A43K, L68K, or a combination thereof.
In a preferred embodiment of the polypeptide of present invention
(i) the a-helix 1 consists of the amino acid sequence of SEQ ID NO.4, and
(ii) the a-helix 2 consists of the amino acid sequence of SEQ ID NO.5, and
(iii) the a-helix 3 consists of the amino acid sequence of SEQ ID NO.6.
In another preferred embodiment of the polypeptide of present invention
(i) the a-helix 1 consists of the amino acid sequence of SEQ ID NO.7, and
(ii) the a-helix 2 consists of the amino acid sequence of SEQ ID NO.8, and
(iii) the a-helix 3 consists of the amino acid sequence of SEQ ID NO.9.
In a third variant, named L3C, glycine residues in the middle of the a-helical regions were mutated to amino acids comprising polar side chains to increase a-helix propensity. The glycine residues in a-helices 1 to 3 can be replaced by polar amino acids, such as lysine (K), arginine (R), threonine (T) or serine (S). These amino acids are solvent-exposed in the model and, opposite to glycine, stabilize the a-helix conformation.
Therefore, in a further embodiment of present invention at least one glycine residue (G) has been replaced in at least one of the three a-helices, preferably in two, most preferably in all three a-helices by a polar amino acid, preferably a lysine (K), arginine (R), threonine (T) or serine (S), most preferred lysine (K). In a preferred aspect the at least one glycine residue (G) is located in the middle of the a-helix. In a most preferred embodiment, all glycine residues that are located in the middle of each of the a-helices are exchanged by polar amino acids, such as lysine (K), arginine (R), threonine (T) or serine (S). As used herein the middle of the a-helix refers to amino acids 16 to 58 of each helix.
In a further embodiment of the polypeptide of present invention
(i) a-helix 1 further comprises at least one, or at least two of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.1 : G19K, G33K, G58K, or a combination thereof, (ii) a-helix 2 further comprises at least one, or at least two of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.2: G16K, G41T, G55K, or a combination thereof,
(iii) a-helix 3 further comprises at least one, or at least two of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.3; G19K, G33K, G58S, or a combination thereof.
In yet another preferred embodiment of the polypeptide of present invention
(i) the a-helix 1 consists of the amino acid sequence of SEQ ID NO.10, and
(ii) the a-helix 2 consists of the amino acid sequence of SEQ ID NO.11 , and
(iii) the a-helix 3 consists of the amino acid sequence of SEQ ID NO.12.
Four-residue loops were manually built to connect each pair of helix termini to create a helix- loop-helix-loop-helix topology (Fig 1 B). It has been found that these linkers are important for keeping the topology of the polypeptide and can contain 3 to 6 amino acids, more preferred is a length of 4 to 5 amino acids, most preferred 4 amino acids.
Therefore, in a further embodiment of the polypeptide, a-helix 1 and a-helix 2 are linked via a first linker and a-helix 2 and a-helix 3 are linked via a second linker. Preferably, the first linker links the C-terminal end of a-helix 1 to the N-terminal end of the a-helix 2 and the second linker links the C-terminal end of a-helix 2 to the N-terminal end of the a-helix 3. In a preferred embodiment the first and/or second linker is between 3 to 6 amino acids long, preferably 4 or 5 amino acids long, most preferred 4 amino acids long to ensure correct folding and stability of the polypeptide. In a preferred embodiment of the polypeptide the amino acids of the linker are selected from glycine (G), alanine (A), serine (S), aspartic acid (D), asparagine (N), lysine (K), arginine (R), proline (P) or glutamic acid (E). These amino acids can be combined in any order.
In a most preferred embodiment of present invention both, the first and the second linkers are 4 amino acids long. The most preferred sequences of the linkers of present invention are shown here below:
Figure imgf000016_0001
Figure imgf000017_0001
The full amino acid sequences of the three variants L3A, L3B and L3C containing all mutations as described herein are shown in Table 4 of Example 1. As can be seen in Table 4 the sequences of the three polypeptides are highly similar, all three showing binding as well as efficacy. The amino acid sequences of L3A, L3B and L3C are furthermore depicted in the sequence listing as SEQ ID NO.13 (L3C), SEQ ID NO.14 (L3B) and SEQ ID NO.15 (L3A).
In one embodiment the polypeptide of present invention is characterized in that it comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO.13.
In a preferred embodiment the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO.13, SEQ ID NO.14 or SEQ ID NO.15, preferably wherein the polypeptide consists of the amino acid sequence of SEQ ID NO.13, SEQ ID NO.14 or SEQ ID NO.15.
PHARMACEUTICAL COMPOSITIONS
The present invention also relates to a pharmaceutical composition comprising the polypeptides as described herein further comprising pharmaceutically acceptable excipients.
The polypeptides of the present invention can be used alone or in combination, or in combination with other agents having coronavirus inhibitory activity. For example, these agents may be agents that have been reported to have inhibitory activity against coronaviruses or have therapeutic effects against coronavirus diseases, such as coronavirus pneumonia, such as Favipiravir, Nelfinavir, Arbidol, Lopinavir, Ritonavir, Chloroquine Phosphate, Darunavir or Remdesivir, etc.
The composition may include a suitable carrier, such as a pharmaceutically acceptable carrier. Such a composition can be used for external use, for example, as an external preparation, a smear preparation for external use, such as a gel for external use or an infiltration preparation for external use. Such compositions can be coated on articles that need to inhibit viruses, such as, but not limited to, masks, paper towels, gloves, clothing, such as protective clothing, and the like. Alternatively, the composition can be added as an active ingredient to hand sanitizers, such as hand sanitizer, shower gel and the like. Such compositions can be used to inhibit coronavirus in vitro to prevent and/or reduce viral infections. Such compositions can be used to prevent or treat coronavirus infections or diseases caused by coronaviruses in subjects.
The pharmaceutical compositions of present invention can be formulated in any known dosage form, such as tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, suppositories, or freeze-dried powder injections.
The polypeptides of present invention can also be formulated using a nucleic acid vector encoding the polypeptides of the present invention. For example, to protect against coronavirus disease, an immune response may result from expression of the polypeptides in a host following administration of a nucleic acid vector encoding the immunogen to the host.
The pharmaceutical compositions can be administered to a subject by various well known administration methods, such as for example by injection, including subcutaneous, intravenous, intramuscular and intraperitoneal injection, by intracavitary administration, such as for example transrectal, vaginal and sublingual, respiratory administration, such as for example through the nasal cavity, mucosal administration, or topical administration.
USES AND TREATMENT
The present in invention also relates to a polypeptide or pharmaceutical composition as described herein for use in the treatment of an infection caused by a coronavirus, preferably a beta coronavirus, most preferred a SARS-CoV-2 virus.
The capacity of the polypeptides of present invention to inhibit infection by SARS-CoV-2 was tested in an in vitro inhibition assay using Vero 76 cells infected with SARS-CoV-2 viruses (see Example 5). It could surprisingly be shown that in presence of the three polypeptides tested the level of infection decreases in a dose-dependent manner (Fig. 8). The inhibitory activity runs in the order L3C > L3B > L3A, consistently with the order in structural stability of the proteins, as well as with their capacity to interact with HR2. It could thus be shown that by interacting with the HR2 region in the Spike protein of the S2 subunit, the HR1 mimetic proteins can block the conformational transition of S2 that promotes membrane fusion and infection.
In yet a further embodiment the use of the polypeptides as described herein for the inhibition of infection with a coronavirus, preferably a beta coronavirus, most preferred a SARS-CoV-2 virus, is envisaged. In an embodiment, the coronavirus is a beta coronavirus selected from the group consisting of SARS-CoV-1 , MERS-CoV and SARS-CoV-2, preferably SARS-CoV-2.
In the preferred embodiment wherein the coronavirus is a SARS-CoV-2, the SARS-CoV-2 is preferably a SARS-CoV-2 variant of concern (VOC), more preferably a SARS-CoV-2 VOC selected from the group consisting of Alpha or Alpha-like, Beta or Beta-like, Gamma or Gamma-like, Delta or Delta-like and Omicron or Omicron-like, most preferably selected from the group consisting of Alpha, Beta, Gamma, Delta and Omicron.
Unless it was specified otherwise SARS-CoV-2 VOCs are as defined by World Health Organization (https://www.who.int/en/activities/tracking-SARS-CoV-2-variants/ as well as https://www.pango. network/ and https://cov-lineages.org/ for further details on their lineages).
The present in invention also relates to a method of treatment of an infection caused by a coronavirus, preferably a beta coronavirus, most preferably a SARS-CoV-2 virus, in a patient in need thereof, comprising administering a polypeptide or pharmaceutical composition as described herein.
The present invention also relates to the use of a polypeptide or pharmaceutical composition as described herein in the manufacture of a medicament for treating an infection caused by a coronavirus, preferably a beta coronavirus, most preferred a SARS-CoV-2.
Another aspect of the invention is a method of reducing a coronavirus, preferably a beta coronavirus, most preferred a SARS-CoV-2 virus viral load in a subject in need thereof, comprising administering a polypeptide or pharmaceutical composition as described herein.
Another aspect of the invention is the use of a polypeptide or pharmaceutical composition as described herein in the manufacture of a medicament for reducing a coronavirus, preferably a beta coronavirus, most preferred a SARS-CoV-2 virus viral load.
Another aspect of the invention is a polypeptide or pharmaceutical composition as described herein for use in reducing a coronavirus, preferably a beta coronavirus, most preferred a SARS-CoV-2 virus viral load.
The present invention furthermore relates to a polypeptide for use in the treatment or prevention of an infection by a coronavirus, preferably a beta coronavirus, most preferably a SARS-CoV-2 virus, characterized in that said polypeptide is a mimetic polypeptide of the HR1 region of the S2 subunit of the Spike protein of the said coronavirus, wherein the polypeptide comprises three alpha helices forming an antiparallel trimer of helices, wherein helix one and helix two are linked via a first linker and helix two and helix three are linked via a second linker and wherein helix two is inverted. Strikingly, the inventors detected significant antibody (IgG and IgA) responses in sera from COVID-19 patients against the HR1 -mimetic proteins as further detailed in Example 6. To investigate if the HR1 mimetic proteins can reproduce immunogenic epitopes of relevance in SARS-CoV-2 infection, the reactivity of sera obtained three months after the onset of COVID- 19 when all patients had developed a sustained SARS-CoV-2-specific response against RBD (Fig. 9) and nucleocapsid protein was tested. Significant IgG responses in the COVID-19 patients’ sera against the three protein variants L3A, L3B and L3C compared to the controls were detected indicating the presence of immune responses against HR1 . These antibody responses were much weaker in control sera of healthy donors. Moreover, the mean IgG responses clearly correlate with the relative stability of the proteins, with the most stable protein L3C showing the highest antibody binding. Although the mean IgG response against L3C was lower than against RBD, a significant percentage of patients displayed high anti-L3C antibodies with similar level to against RBD. Interestingly, the level of IgG response against anti-L3C did not correlate with that of RBD, suggesting a distinct antigen specific B cell maturation process for L3C and RBD. Moreover, the IgA responses in the sera against L3C were significantly higher than against RDB. These results clearly show that the HR1 mimetic proteins are highly antigenic and therefore imitate relevant immunogenic HR1 epitopes in S2 that elicit antibody responses during the course of the disease. Moreover, the correlation between antibody response and stability of the three variants strongly suggests that these epitopes are of conformational nature.
Again, the level of antibodies binding correlated with the stability of the trimeric HR1 -mimetic structure, suggesting that the detected epitopes are conformational. These results reveal that a stabilized S2 HR1 trimeric helical bundle structure 1 ) can display strong inhibitory activity against SARS-CoV-2 and could therefore act as a new potential therapeutic agent, and 2) is antigenic and may consequently be an alternative and innovative immunogen for the development of novel robust vaccines against SARS-CoV-2. Moreover, antibody binding to the S2 subunit has been shown to neutralize SARS-CoV-2 infection [20-24],
In a further aspect the present invention therefore relates to the use of the polypeptides as described herein for the manufacture of a vaccine suitable for treating or preventing an infection with a coronavirus, preferably a beta coronavirus, most preferably a SARS-CoV-2 virus.
The present invention also relates to the polypeptides as described herein or a composition comprising thereof for use as a vaccine for treating or preventing an infection with a coronavirus, preferably a beta coronavirus, most preferably a SARS-CoV-2 virus. The inventors have shown the capacity of the polypeptides of present invention to detect the presence of antibodies in sera from COVID-19 patients by ELISA at a similar level to antibodies against the Receptor Binding Domain, which is so far considered the most immunogenic region. Strikingly, the IgA response detected was even higher to the polypeptides of present invention than the response to the RBD. The polypeptides of present invention can therefore furthermore be used for the detection of antibodies against SARS-CoV-2 in patient sera of patients that have had an immune response against the virus as a result of a current or past infection.
The present invention therefore also relates to an in vitro method of detecting the presence of antibodies directed against the HR1 region of the S2 subunit of the Spike protein of a coronavirus, preferably a beta coronavirus, most preferably a SARS-CoV-2 virus in a test sample, wherein the method comprises contacting a test sample with the polypeptides as described herein and detecting the presence of a signal, wherein the presence of the signal is indicative of the presence of said antibodies in the test sample. In a preferred embodiment the antibodies are selected from IgG, IgA and/or IgM.
Also provided is a kit for detecting the presence of antibodies directed against the HR1 region of the S2 subunit of the Spike protein of a coronavirus, preferably a beta coronavirus, most preferably a SARS-CoV-2 virus comprising the polypeptides as described herein.
EXAMPLES
Example 1 : Protein design and engineering
Computer modeling was carried out using SwissPDBviewer [26] and YASARA structure [27], As template, we used the published X-ray crystal structure of the six-helical bundle formed by HR1 and HR2 in the S2 post-fusion structure (Fig.1 A; PDB id. 6LXT [2]). To model an antiparallel trimer of helices, the HR2 chains were deleted from the model and one of the HR1 helices was upturned and aligned to the original one. To preserve the correct core coiled-coil packing, the amino acid sequence of the upturned helix was also reversed. Due to the antiparallel orientation of the reversed helix, the side chain CA-CB bonds have different spatial orientation compared the native ones and this may perturb interhelical side chain packing in the coiled-coil structure. To compensate this, the alignment was made trying to superimpose the CB atoms of the core residue side chains. Then, side chain clashes were removed by energy minimization. Four-residue loops were manually built to connect each pair of helix termini to create a helix-loop-helix-loop-helix topology (Fig .1 B). The amino acid composition of the loops was selected using RossetaDesign web server http://rosettadesign.med.unc.edu [28]. To enhance the stability of the antiparallel trimeric bundle and reduce exposed hydrophobicity, several surface-exposed residues at e and g positions of the coiled-coil heptad repeats were replaced by charged or polar amino acids to engineer proper salt bridges and hydrogen bonds between the antiparallel helix and the other two helices. Additional mutations at solvent exposed positions were made to increase net positive change. No mutations were carried out to modify the hydrophobic groove between the two parallel HR1 helices to preserve the HR2 binding potential. Two variants code named L3A and L3B were engineered using different sets of amino acid substitutions (Figs.l C and 1 D). In a third variant code named L3C, glycine residues in the middle of a-helical regions were mutated to polar side chains to increase a-helix propensity. Finally, all the models were subjected to energy minimization. The sequences of the three wildtype a-helices of HR1 , a-helix 1 , 2 and 3, their mutations as well the full-length sequences of the three variants L3A, L3B and L3C are shown in Tables 1 -4 here below. Table 1 : Amino acid sequences of the three wildtype a-helices, a-helix 1 , 2 and 3 of HR1 :
Figure imgf000022_0001
Table 2: Amino acid changes of the three a-helices, a-helix 1 , 2 and 3 as present in L3A, L3B and L3C compared to the three a-helices of wildtype HR1 (SEQ ID NOs.1 -3):
Figure imgf000022_0002
Figure imgf000023_0001
Table 3: Amino acid sequences of each of the three a-helices, a-helix 1 , 2 and 3 of the three CoVS-HR1 variants L3A, L3B and L3C; the positions in which amino acid changes were made are shown in bold:
Figure imgf000023_0002
Figure imgf000024_0001
Table 4: Amino acid sequences of the CoVS-HR1 variants L3C (SEQ ID NO.13), L3B (SEQ
ID NO.14) and L3A (SEQ ID NO.15); the amino acid changes made are shown in bold.
Figure imgf000024_0002
Variants of CoVS-HR1 variants in which a pair of disulfide bonds were introduced to conformationally stabilize the protein by tethering each chain end with its closest loop were also designed. The sequences of the a-helix 1 , 2 and 3 as well as the full-length sequences of the three variants L3A-dSS, L3B-dSS and L3C-dSS are shown in Tables 5-6 here below. Table 5: Amino acid sequences of each of the three a-helices, a-helix 1 , 2 and 3 of the three CoVS-HR1 variants L3A-dSS, L3B-dSS and L3C-dSS; the cysteine residues establishing the disulfide bonds are shown in bold and underlined:
Figure imgf000024_0003
Figure imgf000025_0001
Table 6: Amino acid sequences of the CoVS-HR1 variants L3C-dSS (SEQ ID NO.26), L3B (SEQ ID NO.30) and L3A (SEQ ID NO.34); the cysteine residues establishing the disulfide bonds are shown in bold and underlined:
Figure imgf000025_0002
Example 2: Materials and methods
Protein production and HR2 peptides
The DNA encoding the protein sequences were synthesized and cloned into pET303 expression vectors by Thermo Fisher Scientific (Waltham, Massachusetts, MA, USA). The sequences included a N-terminal methionine and a C-terminal histidine tag with sequence GGGGSHHHHHH. The protein sequences are detailed in Table 4. E. co//bacteria (BL21 (DE3)) were transformed with the plasmids and cultured at 37°C in presence of 30 mg-mL-1 Ampicillin. Protein expression was induced with 0.5 mM IPTG and the cells were cultured overnight at 37°C. Cells were collected by centrifugation and resuspended in lysis buffer (50 mM Tris/HCI, 500 mM NaCI, 1 mM EDTA, 1 mM p-mercaptoethanol) containing a cocktail of protease inhibitors (Sigma-Aldrich). The cells were then lysed with three 30 s ultrasonication cycles on ice and the soluble and insoluble fractions were separated by 30 min ultracentrifugation at 4°C at 30000 rpm. The proteins were purified from the supernatant fraction by nickel-affinity chromatography. A second purification step was carried out by ion exchange chromatography on a HiT rap SP Sepharose XL column (Amersham GE Healthcare). Protein purity was assessed by SDS-PAGE and the identity of each protein was confirmed by mass spectrometry. Synthetic peptides derived from the S2 HR2 sequence (SEQ ID NO. 35) were acquired from Genecust (Luxembourg), with a purity >95% ; this peptide corresponds to the first 39 residues of the HR2 region of the Spike (residues 1164-1202) to which 4 additional residues with SGGY (SEQ ID NO. 36) sequence were added. Peptides were C-terminally tagged with a SGGY sequence to confer UV absorption at 280 nm. Protein and peptide concentrations were measured by UV absorption measurements at 280 nm with extinction coefficients calculated according to their respective amino acid sequences with the ExPasy ProtParam server (https://web.expasy.org/protparam/).
Circular dichroism (CD)
CD measurements were performed with a Jasco J-715 spectropolarimeter (Tokyo, Japan) equipped with a temperature-controlled cell holder. Measurements of the far-UV CD spectra (260-200 nm) were made with a 1 mm path length quartz cuvette. Spectra were recorded at a scan rate of 100 nm/min, 1 nm step resolution, 1 s response and 1 nm bandwidth. The resulting spectrum was usually the average of 5 scans. Each spectrum was corrected by baseline subtraction using the blank spectrum obtained with the buffer and finally the CD signal was normalized to molar ellipticity ([0], in deg dmol-Tcm2). Thermal unfolding was monitored by measuring the CD signal at 222 nm as a function of temperature using a scan rate of 1 °C min-1.
Molecular size characterization
The apparent hydrodynamic radii of the proteins were measured using dynamic light scattering (DLS) in a DynaPro MS-X DLS instrument (Wyatt, Santa Barbara, CA). Dynamics v6 software (Wyatt Technology Corporation, Santa Barbara, CA) was used in data collection and processing. Sets of DLS data were measured at 25 °C with an average number of 50 acquisitions and an acquisition time of 10 s. Static scattering intensities were measured in a DynaPro MS-X DLS instrument (Wyatt, Santa Barbara, CA) or a Malvern pV instrument (Malvern Panalytical, Malvern, UK) at 25 °C, in 50 mM sodium phosphate buffer pH 7.4, at different concentrations of protein in a range of 0.2-4.5 mg mL-1. The intensities were analyzed using the Debye plot as represented by equation 1 ,
Kc/ = 1/ Mw + 2 A2C , (1) valid for particles significantly smaller than the wavelength of the incident radiation, where the K is an optical constant of the instrument, c is the particle mass concentration, R90 is the Rayleigh ratio of scattered to incident light intensity, Mw is the weight-averaged molar mass, A2 is the 2nd virial coefficient that is representative of inter-particle interaction strength. Mw can be determined from the intercept of the plot.
Differential scanning calorimetry
DSC experiments were carried out in a MicroCai PEAQ-DSC microcalorimeter equipped with autosampler (Malvern Panalytical, Malvern, UK). Scans were run from 5 to 130°C at a scan rate of 120 °C h-1. The experiments were carried out in 50 mM sodium phosphate buffer pH 7.4 and 50 mM glycine/HCI buffer pH 2.5. Protein concentration was typically 30 pM. Instrumental baselines were recorded before each experiment with both cells filled with buffer and subtracted from the experimental thermograms of the protein samples. Consecutive reheating runs were carried out to determine the reversibility of the thermal denaturation. The partial molar heat capacity (Cp) was calculated from the experimental DSC thermograms using Origin software (OriginLab, Northampton, MA).
Isothermal titration calorimetry
ITC measurements were carried out in a Microcal VP-ITC calorimeter (Malvern Panalytical, Malvern, UK). The proteins were titrated with 25 injections of 5 pL peptide solution at 480 s intervals. Protein concentration in the cell was around 10 pM, while the peptide concentration in the syringe was typically 200-300 pM. The experiments were carried out in 50 mM phosphate buffer (pH 7.4) at 25 °C. The experimental thermograms were baseline corrected and the peaks were integrated to determine the heats produced by each ligand injection. Residual heats due to unspecific binding or ligand dilution were estimated from the final peaks of the titrations. Each heat was normalized per mole of injected ligand. The resulting binding isotherms were fitted using a binding model of independent and equivalent sites, allowing the determination of the binding constant, Kb, the binding enthalpy, AHb, and the binding stoichiometry, n.
S protein binding assays
CoVS-HR1 proteins ability to bind soluble trimeric Spike (S) proteins was determined by ELISA. Briefly, 96-well ELISA plates (Maxisorp, Nunc) were coated at 4 °C overnight with recombinant trimeric SARS-CoV-2 Spike protein (Abyntek Biopharma- ProSci Inc., Poway, CA, Catalog Number 10-075, Spike sequence Gln14 - Gln1208, Protein Accession Number: QHD43416.1 ) in 0.1 M bicarbonate buffer (pH 9.6). After saturation with 2% BSA, 0.05% Tween in PBS for 1 h at 25 °C, 0.3-8.0 nM of CoVS-HR1 molecules L3A, L3B and L3C (100 pL diluted in 1 % BSA 0.05% Tween solution) were added at different concentrations and incubated for 2 h at room temperature. As negative controls, protein constructs derived from HIV-1 gp41 sequences which are HR1 -mimics of HIV gp41 disclosed in [9] and [29] were also tested. In some competition experiments, CoVS-HR1 molecules were mixed with HR2 V39E peptide at 1 :2 molar ratio. The plate was then washed five times and CoVS-HR1 binding was detected with 100 pL anti-6X Histag antibody conjugated to horseradish peroxidase (HRP) (Abeam) at 1/10000 dilution incubated for 1 hour at room temperature. Antibody binding was then revealed with tetramethylbenzidine (TMB) substrate buffer, the reaction was stopped with 1 M H2SO4 and optical density was read at 450 nm with a Molecular Device Plate Reader equipped with SoftMax Pro 6 program. Background binding was measured in plates without Spike protein and subtracted from the data. The percentage of binding was calculated using the readings with wells coated with His-tagged Spike incubated with PBS buffer instead of CoVS-HR1 molecules as control for 100% binding.
Virus inhibition assays
One day prior infection, Vero 76 cells were plated on a 96 well plate at 12500 cells/well. 50 pL of serial 4-fold dilutions of CoVS-HR1 proteins (2-fold concentrated) were incubated with the cells for 30 min. Cells were then infected by adding 50 pL WT SARS-CoV-2 strain (UK D614G genotype or BA.1 Omicron genotype) at Multiplicities Of Infection (MOI) of 80. After 2 days, cells are fixed with methanol for 20 min, washed with PBS and stained with anti-Nucleocapside Antibody (Genetex GTX135357) at 1/200 dilution in permwash (B&D) for 45 min at room temperature. Ab is revealed by incubation with a donkey anti-Rabbit monoclonal Ab (Alexa 647; A31573, Invitrogen) diluted at 1/200 in PBS 5% FCS for 45 min at room temperature. In parallel, total cells were detected by Sytox green (S7020, Invitrogen) staining. Total cells (Sytox green positive) and infected cells (nucleocapside positive) were counted using SpectraMax MiniMax Imaging Cytometer (Molecular Devices LLC). The percentage of infected cells was recorded. The 50% inhibitory concentration (IC50) was defined as the protein concentration leading to a 50 % reduction in the percentage of infected cells.
Detection of antibody responses in patients’ sera
CoVS-HR1 antigenicity against sera of infected patients was determined by ELISA. Sera samples were collected 3 months after recovery from COVID-19 infection. As control, sera from healthy donors were also assayed. All patients and healthy donors gave their written informed consent (COVID-HUS ethics committee approved, reference CE: 2020-34). 96-well ELISA plates (Maxisorp, Nunc) were coated at 4 °C overnight with CoVS-HR1 molecules in 0.1 M bicarbonate buffer (pH 9.6). After blocking with 5% non-fat powdered milk in PBS for 1 h at 25 °C, 1/1000 diluted sera (100 pL diluted in 1 % BSA 0.05% Tween solution) were added and incubated for 30 min at room temperature. The plate was then washed five times and CoVS-HR1 binding to IgG or IgA was detected by incubating for 1 hour at room temperature with 100 pL goat anti-human IgG or anti-human IgA antibody conjugated to horseradish peroxidase (HRP) (Abeam) at 1/5000 dilution, respectively. IgG or IgA binding was then revealed with tetramethylbenzidine (TMB) substrate buffer, the reaction was stopped with 1 M H2SO4 and optical density was read at 450 nm with a Molecular Device Plate Reader equipped with SoftMax Pro 6 program. Background binding was measured in plates without CoVS-HR1 proteins and subtracted from the data. In parallel, His-tagged RBD was used to detect the anti- RBD antibodies present in the patients’ sera. Data was analyzed using Origin software (OriginLab, Northampton, MA).
X-ray crystallography
The CoVS-HR1 protein was co-crystalized in complex with the V39E HR2 peptide. For crystallization, a concentrated protein-peptide mixture (about 9 mg/mL) was prepared in 10 mM Tris/HCI buffer at a 1 :2 protein peptide molar ratio. Screening of crystallization conditions was carried out using the sitting drop vapor-diffusion method with the crystal screening kit “Structure Screen 1 and 2 Eco Screen” from Molecular Dimensions (Suffolk, UK). Droplets consisting in mixing 2 pL complex solution with 2 pL reservoir solution were equilibrated at 298 K against 200 pL reservoir solution. Best crystals were obtained in 0.1 M sodium HEPES pH 7.5, 20% w/v PEG 4000, 10% isopropanol.
For X-ray drifraction, crystals were flash-cooled in liquid nitrogen. Data sets were collected at 100 K at the XALOC at the ALBA synchrotron (Barcelona, Spain). Diffraction data were indexed and integrated with the AutoPROC toolbox. Data scaling was performed using the program Aimless from the CCP4 suite. Solution and refinement of the structures were performed using the PHENIX suite. Molecular-replacement phasing using PHASER was performed with the coordinates of the crystallographic structure of the S2 post-fusion core (PDB entry 6LXT). Manual model building was performed using COOT. Refinement was performed using phenix. refine in PHENIX. Quality of the structure was checked using MOLPROBITY and PDB REDO.
Example 3: Biophysical characterization of single-chain HR1 mimetic proteins
The L3A, L3B and L3C CoVS-HR1 proteins were produced recombinantly by overexpression in E. coli with high yields in the soluble fraction and could be easily purified by two-step standard chromatographic methods. All the proteins were highly soluble (>10 mg mL-1) in standard buffers. The proteins are highly a-helical according to their CD spectra (Fig. 3), in good agreement with the design model (Fig. 1 ).
Molecular size in solution measured by light scattering was consistent with mainly monomeric proteins (Fig. 4), although L3A and L3B showed a slight tendency to self-association. L3C was however fully monomeric at all concentrations tested.
The three proteins are highly stable against thermal denaturation, showing melting temperatures above 80°C (Fig. 5A). L3B and L3C show much sharper denaturation peaks than L3A, with higher unfolding enthalpies, indicating a more stable and cooperative structure. This indicates that the choice of mutations was more effective in stabilizing the coiled-coil structure in these variants. Among them, L3C is more thermostable than L3B by about 13°C, as a result of higher a-helical propensity of its sequence produced by the substitution of glycine residues.
Binding of an HR2-derived peptide V39E was probed by CD, DSC and ITC. Addition of peptide to the proteins increased slightly the a-helix structure as observed in the CD spectra (Fig. 3). In presence of the peptide, the denaturation peaks in the DSC thermograms showed an increase of their area, as a result of peptide dissociation at high temperature (Fig. 5B). Titration of the proteins with the peptides by ITC showed a 1 :1 binding stoichiometry and considerably negative binding enthalpy (Table 6). Analysis of the binding isotherms yields dissociation constants in the low nM range (Table 6). These results demonstrate that the three proteins can interact with HR2-derived peptides with high affinity. Interestingly, despite the fact that the binding affinities are similar for the three protein variants, the binding enthalpy decreases in the order L3A > L3B > L3C proteins and therefore there is a strong enthalpy-entropy compensation. This suggests HR1 -HR2 complex formation involves considerable conformational ordering and tightening. This conformational tightening upon binding decreases with the increase in stability of the variant. Accordingly, HR2 binding to L3C involves the lowest entropy costs as a result of a highest intrinsic stability of the HR1 groove.
Table 6: Thermodynamic parameters of binding of CoVS-HR1 proteins and HR2 V39E peptide measured by ITC.
Figure imgf000031_0001
Regarding the doubly disulfide bound variants, the protein L3C-dSS is highly soluble, shows a molecular size consistent with a monomer (hydrodynamic radius, Rh = 3.2 nm) and has a 72% a-helix structure according to the CD spectrum at pH 7.4. It is highly thermostable, with a melting temperature 1 1.5°C higher than L3C (114.5°C vs 103°C). L3C-dSS binds the HR2 peptide with a Kd of approximately 100 nM at 35°C, comparable to L3C.
Example 4: Binding assays of the three variants to recombinant trimeric Spike by ELISA
To investigate if the proteins can interact with the HR2 target region in a context more alike to the native Spike, the binding of the three variants to recombinant trimeric Spike by ELISA was tested (Fig. 7). The three proteins showed strong binding to the Spike whereas two small chimeric proteins that mimics gp41 HR1 [29] did not show any significant binding. The level of detected binding at sub nM Spike concentration runs in the order L3C > L3B > L3A but rapidly saturates at higher concentration. Competition experiments with V39E peptide demonstrate that the binding is specific to the HR2 region. This result is consistent with the observed binding affinity of the proteins for the V39E HR2 peptide and supports again a correlation between conformational stability and affinity.
Example 5: Capacity of the CoVS-HR1 proteins to inhibit infection by SARS-CoV-2
The capacity of the CoVS-HR1 proteins to inhibit infection by SARS-CoV-2 was tested in an in vitro inhibition assay using Vero 76 cells infected with SARS-CoV-2 viruses (UK D614G genotype and BA.1 Omicron genotype). In presence of the CoVS-HR1 proteins the level of infection decreases in a dose-dependent manner (Fig. 8). Once again, the inhibitory activity runs in the order L3C > L3B > L3A, consistently with the order in structural stability of the proteins, as well as with their capacity to interact with HR2. Collectively, these results suggest that by interacting with the HR2 region in the Spike, the HR1 mimetic proteins can block the conformational transition of S2 that promotes membrane fusion and infection. Inhibition assays comparing L3C-dSS with L3C were carried out in Vero 76 cells using WT B1 SARS-CoV-2 (Figure 12). The two proteins inhibit infection with similar IC50, in good consistency with their similar affinity for HR2.
Example 6: Reactivity of sera obtained three months after the onset of COVID-19
To investigate if the HR1 mimetic proteins can reproduce immunogenic epitopes of relevance in SARS-CoV-2 infection, we tested the reactivity of sera obtained three months after the onset of COVID-19. At that time, patients developed a sustained SARS-CoV-2-specific response against RBD (Fig. 9) and nucleocapside protein. The patient’s sera also displayed significant neutralizing activity. We detected significant Ab responses in the COVID-19 patients’ sera against the three protein variants compared to the controls, indicating the presence of immune responses against HR1. These antibody responses were much weaker in control sera of healthy donors. Moreover, the mean Ab responses clearly correlate with the relative stability of the proteins, with the most stable protein L3C showing the highest Ab binding. Although the mean Ab response against L3C was lower than against RBD, a significant percentage of patients displayed high anti-L3C Abs with similar level against RBD. These results clearly indicate that the HR1 mimetic proteins are antigenic and therefore imitate relevant immunogenic HR1 epitopes in S2 that elicit Ab responses during the course of the disease. Moreover, the correlation between Ab response and stability of the three variants strongly suggest that these epitopes are of conformational nature.
Example 7: High resolution structure of the CoVS-HR1 -L3B protein in complex with the HR2 region.
To investigate the structure of the proteins and the details of the interaction interface between the CoVS-HR1 proteins and their HR2 target we produced crystals of the L3B protein in complex with the V39E peptide. X-ray diffraction datasets were collected at 2.0 A resolution allowing structure determination (Fig. 10). The structure of the L3B protein is highly similar to the designed model. The V39E peptide also adopts a a-helical conformation flanked by extended regions in the complex and interactions at the protein-peptide interface formed by a- helix 1 and a-helix 3 in L3B and the HR2 peptide are virtually identical to those observed between HR1 and HR2 in the post-fusion S2 structure. These data confirm that the CoVS-HR1 proteins mimic accurately a fully stable HR2 structure with a strong capacity to bind HR2 in a functional-like manner.
Example 8: Live virus neutralization. The ability of the CoVS-HR1 proteins L3B and L3C to neutralize various coronavirus was tested in a live virus neutralization assay against MERS-CoV/EMC, SARS-CoV HKU-39849 and SARS-CoV-2 BavPat1/2020.
The following cell lines were used Vero, ATCC CCL-81 and Vero C1008 (Vero 76, clone E6, Vero E6), ATCC CRL-158.
Briefly, samples were serially diluted, and pre-incubated with virus (1 h), then mixture was added to cells and incubated (20h for CoV-1 and 2, 40h MERS). Cells were formalin-fixed, permeabilization and incubation with an antibody against the nucleocapsid protein, followed by a secondary IgG peroxidase conjugate and TrueBlue substrate. Nucleopcapsid positivity was the readout via ELISPOT.
Neutralization titers (IC50) were determined via the method disclosed in Zielinska et aL, 2005 [30]. The start concentration tested was 50pM. The mean spot counts above and below the 50% reduction point were used to calculate the 50% inhibitory concentration.
Results are summarized in the table below.
Figure imgf000033_0001
*”>” indicates that IC50 could not be determined at highest tested concentration.
L3B and L3C neutralizes both SARS-CoV-1 in live virus neutralization assay. L3C shows also a neutralization activity on SARS-CoV-2.
Example 9: Pseudovirion neutralization
An assay of pseudovirion neutralization was carried out with the following viruses: SARS-CoV- 1 , SARS_CoV-2 Wuhan, SARS-CoV-2 Delta (IN2), SARS-CoV-2 Omicron BA4, CoV 229E and CoV NL63.
Briefly, samples were serially diluted, and pre-incubated with luciferase expressing virus (1 h), then mixture was added to 293T ACE2 cells and incubated for 48h. Cells were formalin-fixed and lysed. Readout was luciferase activity as measure of infection via a luminometer.
The start concentration tested was 50pM. Neutralization titers (IC50) were determined as the protein concentration at which infectivity was inhibited by 50%, respectively, using a four Parameter Logistic Regression (4PL) curve fit, including transformation for variance stabilization post-fit scaling was applied to achieve 0 and 100% at fitted plateaus, the 50% reduction point of the curve is used for the 50% inhibitory concentration.
Results are summarized in the table below.
Figure imgf000034_0001
*”>” indicates that IC50 could not be determined at highest tested concentration.
L3C neutralized SARS-CoV-2 VOCs Wuhan, Delta and BA4 as well SARS-CoV-1. L3B neutralizes SARS-CoV-1 and SARS-CoV-2 Wuhan.
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Claims

1. A polypeptide capable of inhibiting host cell infection by a coronavirus, preferably a beta coronavirus, most preferably a SARS-CoV-2 virus, wherein said polypeptide comprises three a-helices and wherein
(i) a-helix 1 comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO.1 ,
(ii) a-helix 2 comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO.2, and
(iii) a-helix 3 comprises an amino acid sequence that is at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO.3.
2. The polypeptide of claim 1 , wherein the said three a-helices form an antiparallel trimer of helices and, preferably, wherein said polypeptide is a mimetic polypeptide of the heptad-repeat region 1 (HR1 ) of the S2 subunit of the SARS-CoV-2 Spike protein.
3. The polypeptide of claims 1 or 2, wherein at least one amino acid in each a-helix at sequence positions “e” and/or “g” in the heptad repeats is substituted by a lysine (K), arginine (R), aspartic acid (D) or glutamic acid (E), preferably lysine (K) or glutamic acid (E).
4. The polypeptide of any one of claims 1 to 3, wherein
(i) a-helix 1 comprises at least one of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.1 : N1 D, Q13E, V38K, A45K, and/or V63K, or a combination thereof,
(ii) a-helix 2 comprises at least one of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.2: V1 1 K, S13E, A29R, A31 E, V36E, Q38E, S50E, I56E and/or A63R, or a combination thereof,
(iii) a-helix 3 comprises at least one of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.3: 118E, A29K, A43K, V50E, G58R, L68E, D72Q and/or E75Q, or a combination thereof.
5. The polypeptide of any one of claims 1 to 3, wherein (i) a-helix 1 comprises at least one of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.1 : N1 D, Q13K, A31 E, V38E, A45D, A59R and/or V63E, or a combination thereof,
(ii) a-helix 2 comprises at least one of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.2: L6D, V11 E, S13K, A31 K, V36E, Q38K, A43E, A45R, S50E, D51 K, A63E and/or N68R, or a combination thereof,
(iii) a-helix 3 comprises at least one of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.3; A11 E, A29K, Q36K, A43K, L68K or a combination thereof. The polypeptide of any one of claims 1 to 5, wherein
(i) a-helix 1 further comprises at least one of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.1 : G19K, G33K, G58K, or a combination thereof,
(ii) a-helix 2 further comprises at least one of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.2: G16K, G41T, G55K, or a combination thereof,
(iii) a-helix 3 further comprises at least one of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.3; G19K, G33K, G58S, or a combination thereof. The polypeptide of any one of claims 1 to 6, wherein
(i) a-helix 1 further comprises at least one of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.1 : L3C,
(ii) a-helix 2 further comprises at least one of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.2: Y70C,
(iii) a-helix 3 further comprises at least one of the following amino acid substitutions based on the amino acid sequence of SEQ ID NO.3: L71C The polypeptide of any one of claims 1 to 7, wherein
(i) the a-helix 1 consists of the amino acid sequence of SEQ ID NO.4, and the a-helix 2 consists of the amino acid sequence of SEQ ID NO.5, and the a-helix 3 consists of the amino acid sequence of SEQ ID NO.6; or
(ii) the a-helix 1 consists of the amino acid sequence of SEQ ID NO.7, and the a-helix 2 consists of the amino acid sequence of SEQ ID NO.8, and the a-helix 3 consists of the amino acid sequence of SEQ ID NO.9; or (iii) the a-helix 1 consists of the amino acid sequence of SEQ ID NO.10, and the a-helix 2 consists of the amino acid sequence of SEQ ID NO.1 1 , and the a-helix 3 consists of the amino acid sequence of SEQ ID NO.12; or
(iv) the a-helix 1 consists of the amino acid sequence of SEQ ID NO.23, and the a-helix 2 consists of the amino acid sequence of SEQ ID NO.24, and the a-helix 3 consists of the amino acid sequence of SEQ ID NO.25; or
(v) the a-helix 1 consists of the amino acid sequence of SEQ ID NO.27, and the a-helix 2 consists of the amino acid sequence of SEQ ID NO.28, and the a-helix 3 consists of the amino acid sequence of SEQ ID NO.29; or
(vi) the a-helix 1 consists of the amino acid sequence of SEQ ID NO.31 , and the a-helix 2 consists of the amino acid sequence of SEQ ID NO.32, and the a-helix 3 consists of the amino acid sequence of SEQ ID NO.33. The polypeptide of any one of claims 1 to 7, wherein a-helix 1 and a-helix 2 are linked via a first linker and a-helix 2 and a-helix 3 are linked via a second linker and wherein the first and/or second linker is between 3 to 6 amino acids, preferably 4 or 5 amino acids, most preferred 4 amino acids long; and preferably, wherein the amino acids of the linker are selected from glycine (G), alanine (A), serine (S), aspartic acid (D), asparagine (N), lysine (K), arginine (R), proline (P) or glutamic acid (E), or a combination thereof. The polypeptide of any one of claims 1 to 8, characterized in that it comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO.13. The polypeptide of any one of claims 1 to 8, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO. 26, SEQ ID NO. 30 and SEQ ID NO. 34, preferably wherein the polypeptide consists of the amino acid sequence selected from the group consisting of SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15 SEQ ID NO. 26, SEQ ID NO. 30 and SEQ ID NO. 34. A pharmaceutical composition comprising the polypeptides of any one of the preceding claims further comprising pharmaceutically acceptable excipients. A polypeptide for use in the treatment or prevention of an infection by a coronavirus, preferably a beta coronavirus, most preferably a SARS-CoV-2 virus, characterized in that said polypeptide is a mimetic polypeptide of the HR1 region of the S2 subunit of the Spike protein of the said coronavirus, wherein the polypeptide comprises three alpha helices forming an antiparallel trimer of helices, wherein helix one and helix two are linked via a first linker and helix two and helix three are linked via a second linker and wherein helix two is inverted. The polypeptide or the pharmaceutical composition according to any one of the preceding claims for use in the treatment or prevention of an infection caused by a coronavirus, preferably a beta coronavirus, most preferably a SARS-CoV-2 virus. Use of the polypeptides of any one of claims 1 to 11 for the neutralization of an infection with a coronavirus, preferably a beta coronavirus, most preferably a SARS-CoV-2 virus. A vaccine comprising the polypeptides of any one of claims 1 to 11 for treating or preventing an infection with a coronavirus, preferably a beta coronavirus, most preferably a SARS-CoV-2 virus. An in vitro method of detecting the presence of antibodies directed against the HR1 region of the S2 subunit of the Spike protein of a coronavirus in a test sample, wherein the method comprises contacting a test sample with the polypeptides of any one of the claims 1 to 1 1 and detecting the presence of a signal, wherein the presence of the signal is indicative of the presence of said antibodies in the test sample. A kit for detecting the presence of antibodies directed against the HR1 region of the S2 subunit of the Spike protein of a coronavirus, preferably a beta coronavirus, most preferably a SARS-CoV-2 virus comprising the polypeptides of any one of the claims 1 to 11.
PCT/EP2023/055655 2022-03-08 2023-03-07 Novel mimetic polypeptides of the hr1 region of the s2 subunit of coronaviruses WO2023170016A1 (en)

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