WO2022043449A1 - Vaccins à base de protéine antigénique fusionnée à un échafaudage nanostructurant - Google Patents

Vaccins à base de protéine antigénique fusionnée à un échafaudage nanostructurant Download PDF

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WO2022043449A1
WO2022043449A1 PCT/EP2021/073632 EP2021073632W WO2022043449A1 WO 2022043449 A1 WO2022043449 A1 WO 2022043449A1 EP 2021073632 W EP2021073632 W EP 2021073632W WO 2022043449 A1 WO2022043449 A1 WO 2022043449A1
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Roman Jerala
Tina FINK
Vida FORSTNERIC
Špela MALENŠEK
Jana AUPIC
Duško LAINŠCEK
Mojca Bencina
Iva Hafner Bratkovic
Mateja MANCEK
Peter PECAN
Petra DEKLEVA
Tjaša PLAPER
Sara OREHEK
Žiga STRMŠEK
Esih HANA
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Kemijski Institut
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    • 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
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6075—Viral proteins
    • 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
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A61K2039/627—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier characterised by the linker
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/735—Fusion polypeptide containing domain for protein-protein interaction containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)
    • 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
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • Vaccines based on an antigen protein fused to a nanostructuring scaffold FIELD OF THE INVENTION Genetically fused antigenic protein domain with a scaffolding polypeptide that self assembles into soluble oligomeric nanoparticles that comprise at least six copies of the antigen protein domain.
  • DNA plasmid encoding protein antigen domains of the viral Spike protein of SARS-CoV-2 genetically fused to the scaffolding polypeptide is used as a vaccine against Covid-19.
  • BACKGROUND OF THE INVENTION Covid-19 is a pandemic viral disease caused by infection with SARS-CoV-2 that affected millions of people across the world and claimed hundreds of thousands lives(1–3).
  • DNA plasmid delivery including low immunogenicity, cost-effective production, stability at ambient temperature without the need for a cold chain make it a suitable vaccine platform although no DNA plasmid vaccines has been approved(4, 5).
  • Antibodies should preferentially be focused to the domains and epitopes that can prevent viral recognition of the receptor, viral fusion with cell membrane or hinder viral replication in other ways.
  • the most suitable targets seem to be protein domains through which virus attaches to the cellular receptors such as the receptor binding domain (RBD) of the trimeric spike protein of the SARS-CoV-2, whose tertiary structure in the complex with ACE2 receptor has been determined(6, 7).
  • RBD receptor binding domain
  • Masking binding epitope by the antibodies prevents viral binding to the receptor and infection of target cells.
  • RBD has been used in several vaccine candidates against SARS-CoV-2, similar as before for the related pathogenic coronaviruses (8, 9) and monoclonal antibodies against the RBD have been effective in therapy(10, 11).
  • RBD Spike protein has other domains that are involved in the fusion with human cells, such as the HRC domain that is not involved in recognition of the receptor protein but plays a direct role in fusion. This domain could be very interesting as a vaccine as this segment is highly conserved among coronaviruses in contrast to the RBD, therefore vaccine based on HRC could be used against multiple coronavirus types, such as for example SARS-CoV-2, SARS-CoV, MERS-CoV and other emerging viruses of the same type.
  • HRC also called HR2 domain
  • HRC also called HR2 domain
  • bacterialy produced denatured HRC peptides have been used that generated relatively weak immune response in comparison to the RBD(12, 13).
  • the ability of viral proteins or its domains to induce formation of antibodies depends on the structure and size of antigen.
  • Viral surface proteins are typically presented to the host the form or nanoparticles tens of nm in diameter that present tens of copies of viral proteins. Affinity maturation, class switching and memory cell formation take place in germinal centers inside lymph node follicles(14), therefore transport of protein antigens to lymph nodes is preferable in comparison to the insoluble aggregates at the site of injection.
  • antibodies that do not prevent viral entry may even facilitate viral entry into the cell through an antibody dependent enhancement mechanism (ADE), most likely mediated by the Fc domain of antibodies and Fc ⁇ receptors, as shown before for SARS CoV and MERS CoV, as a highly undesirable property of a vaccine(29–31). Therefore focusing immune response to the receptor binding domain (RBD) of the Spike protein may increase the probability of inducing neutralizing antibodies. It has been shown that RBD as an immunogen indeed induces formation of neutralizing antibodies(24, 26) Presentation of the target antigen domain in the multimeric form may augment cellular response and formation of antibodies. This could be accomplished by chemical conjugation of a viral protein domain to the nanoparticle or by a genetic fusion to the scaffold-forming polypeptide.
  • ADE antibody dependent enhancement mechanism
  • immunogenic domains to the virus-like capsule are structural envelope proteins of bacterial, plant or animal viruses of such as Q ⁇ , HPV, JCV, HBcAg, cowpea chlorotic mottle virus capsids(32) and many others, nonviral protein compartments such as ferritin, lumazine synthase and encapsulin(17, 33–36) and de novo designed protein or DNA cages(37–43).
  • structural envelope proteins of bacterial, plant or animal viruses of such as Q ⁇ , HPV, JCV, HBcAg, cowpea chlorotic mottle virus capsids(32) and many others
  • nonviral protein compartments such as ferritin, lumazine synthase and encapsulin(17, 33–36) and de novo designed protein or DNA cages(37–43).
  • An important issue of this strategy is that antibodies or T cell response may also be targeted against the scaffolding protein, which could impair the efficiency of the subsequent immunizations with the same vaccine type or it
  • the size of the scaffold that forms nanoparticles is typically on the order of 100-150 amino acid residues (e.g. for ferritin, Q ⁇ , lumazine synthase).
  • fusion of peptide tags that induce formation of large amyloid fibrils comprising peptide antigens has also been used for the generation of vaccines (46, 47).
  • the amyloid or helical assembly-promoting short peptide tag induced formation of long insoluble fibers.
  • the addition of peptide antigens decorated those fibers with T or B-cell targeting epitopes (47–49).
  • Proteins domains could however only be incorporated into the amyloid fibers by a combination of amyloidogenic peptides and folded protein domain with attached longer domain that by itself was not prone to aggregation but was incorporated into the amyloid fibers(50).
  • the antigens must reach lymph nodes where maturation of the adaptive immune response takes place (32).
  • Virus like particles with sizes below 500 nm can traffic through the lymphatic system and can be taken up by antigen presenting cells such as dendritic cells, therefore large insoluble aggregates are not optimal as they remain in the tissue and only degradation products can reach lymph nodes where the maturation of antibodies and class switching takes place in cooperation between follicular dendritic cells B- and T-lymphocytes.
  • Foldon is a peptide from a T4 bacteriophage fibritin that has a strong propensity for trimerization (51, 52). Fusion of a 27-aminoacid residue foldon peptide to a protein domain can direct the trimerization of an antigen, which increases its size and presents it to the B –cell receptors in form of a trimer that may increase the response. Indeed fusion of an RDB domain to a foldon peptide induced formation of trimers of RBD and has been used as a vaccine against the SARS-CoV-2(24). In this structural arrangement, the size of a protein particle is about 60 kDa and the complex has on one side exposed a trimeric foldon that is strongly immuniogenic (52).
  • a short peptide comprising 24 amino acid residues from the viral beta-annulus protein from tomato bushy stunt virus has been shown to spontaneously form nanoparticles measuring approximately 40 nm in diameter(53). This peptide has been used to attach different cargo molecules, from DNA to peptide domains (54, 55)(56). Beta-annulus peptide, however, has not been used for fusion with large folded protein domains or for vaccination. Vaccines comprising fusion of protein antigen and scaffolding protein have been in most cases used as isolated proteins injected into the patient but have not been used to encode nucleic acids to enable production of the immunogen in the body.
  • the present invention provides a subunit vaccine that is composed by genetic fusion of a selected protein antigen with a small scaffolding domain that induces formation of soluble particles of oligomers of the protein antigen in six or more copies per particle.
  • a subunit vaccine that is composed by genetic fusion of a selected protein antigen with a small scaffolding domain that induces formation of soluble particles of oligomers of the protein antigen in six or more copies per particle.
  • the scaffolding peptide is a short preferably less than 30 amino acid long hypoimmunogenic peptide while the precise stoichiometry or structure of the oligomer does not need to be defined.
  • scaffolding domain is composed of the following functional segments: 1. Core oligomerizing domain that induces formation of oligomers, 2.
  • Solubility enhancing and repulsion domain that maintains the solubility of the nanoparticle in a micellar arrangement.
  • oligomerization domain that maintains the defined oligomers of the protein antigen, such as e.g. in a trimeric cluster. Between those domains short linker peptides comprising preferentially from 0 to 6 amino acids, preferentially selected from glycine or serine residues.
  • Core oligomerizing domains are selected from hydrophobic amino acid residues, beta strand forming peptides or multiple copies of short beta sheet forming peptides connected by linkers of short peptide comprising at least two phenylalanine residues that have propensity for oligomerization.
  • Example of the multiple beta sheet forming peptides are segments from the beta-annulus peptide (53) or combination of dry zipper peptides (57).
  • Solubility enhancing and repulsion domain is composed of 1-4 charged amino acid residues such as Glu, Asp or Lys, Arg of the same type so that they induce repulsion between polypeptide chains and lead to the formation of micelles.
  • defined oligomerization domains are composed of peptides that form defined oligomers such as preferentially dimers, trimers, tetramers that are required to form defined protein antigen epitopes such as in viral proteins.
  • oligomerizing domains can be selected from examples in the literature such as the trimerizing foldon peptide or defined parallel homodimeric, homotrimeric or homotetrameric coiled coil peptides (58, 59).
  • Polypeptide sequence in this invention comprise the selected polypeptide antigen genetically fused via a flexible polypeptide linker to the self-assembling polypeptide scaffold that causes oligomerization of the polypeptide antigen on the self-assembled nanoparticle, where the number of polypeptide antigen domains in each self-assembled particle is equal or more than six and the self-assembling polypeptide scaffold and where the scaffolding peptide comprises the following functional domains: core oligomerizing domain based on packing based on hydrophobic amino acids, beta sheet forming peptide segments or clusters of aromatic residues comprising at least two phenylalanine residues; charged solubility and repulsion enhancing residues comprising 1-6 residues of the same charge and optionally a defined oligomerizing domains to present
  • a particularly preferred embodiment of the invention concerns a polypeptide, wherein the polypeptide antigen is fused to a beta-annulus peptide via a linker, that may comprise 0 to 10 amino acid residues, preferentially selected from glycine and serine.
  • invention provides an implementation of vaccine that comprises nucleotide sequence that codes for the protein antigen that self assembled due to the fused scaffolding domain and is able to generate strong response and neutralization of viral infection with a scaffold domain to enable formation of particles comprising six or more copies of the protein antigen domain par particle. Small monomeric protein domains typically exhibit weak response of the humoral immune system, which is strongly improved by the scaffolding segment.
  • the invention combines vaccination compositions where two or more immunizations are performed using the same target protein domain in each vaccine composition that is in each vaccination combined with a different scaffold domain to minimize the formation of immune response against the scaffold.
  • Weak immune response against the scaffold is beneficial and is minimized by the small length of the scaffolding amino acid sequence and hypoimmunogenic amino acid composition.
  • This disclosure provides vaccine compositions against infection with a virus SARS- CoV-2, where the target viral domain comprises RBD or HRC domain from the viral spike protein while the scaffold polypeptide domains are composed of the foldon peptide to which two domains of the RBD or HRC are fused to the N- and C-terminus or beta- annulus peptide or an icosahedral cage-forming lumazine synthase.
  • This invention provides increased immune response in comparison to isolated domains RBD or trimerized RBD or bacterialy produced denatured HRC because the protein antigen domains are presented in multiple compies on protein particles selfassembled by the scaffolding domain genetically fused to the scaffolding polypeptide domain.
  • Figure 1 The concept of designed soluble oligomeric protein vaccines. Selected protein antigen is genetically fused to the scaffolding domain that contains the core oligomerizing domain, charged solubility and repulsion domain and optionally defined oligomerizing domain to present protein antigen as monomer or a defined oligomer at the surface of a soluble oligomer.
  • Figure 2 Molecular models of differently scaffolded RBD domain with number of RBD domains per particle larger or equal to six (in brackets).
  • FIG. 5 Scheme of immunization and sample collection from mice.
  • RBD vaccines were administered into BALB/c mice in the form of combination plasmid DNA, complexed with transfection reagent at defined time intervals. Blood was drawn before each boost immunization to determine specific antibody titers. At the termination mice spleens were harvested fur further experiments.
  • Figure 6 Titer of total IgG antibodies against the RBD for different scaffolded RBDs and scaffold alone. Mice were immunized with different combination of RBD plasmid DNA, complexed with jetPEI-in vivo transfection reagent.
  • RBD specific antibodies rose from prime to first and second boost. Higher end point titer against RBD was observed in scaffold RBD vaccines compared to RBD alone.
  • Figure 7 Titer of total IgG antibodies reacting with a SARS-CoV-2 Spike protein for different scaffolded RBDs and against immunization by the scaffold. Mice were immunized with different combination of RBD plasmid DNA, complexed with jetPEI-in vivo transfection reagent. SARS-CoV-2 Spike protein specific antibodies rose from prime to first and second boost. Higher end point titer against SARS-CoV-2 Spike protein was observed in scaffold RBD vaccines compared to RBD alone.
  • Figure 8 Formation of different classes of antibodies against RBD for different scaffolded RBDs and immunization by the scaffold. Mice were immunized with different combination of RBD plasmid DNA, complexed with jetPEI-in vivo transfection reagent. Titers of IgA, IgM, IgG1, IgG2b and IgG3 against RBD protein were higher in scaffold RBD DNA vaccines compared to RBD DNA vaccine alone.
  • Figure 9 Weak formation of antibodies against the scaffolding domain of the RBD- scaffold constructs. Mice were immunized with different combination of RBD plasmid DNA, complexed with jetPEI-in vivo transfection reagent.
  • FIG. 10 Cytotoxic T cell killing from immunized mice against cells expressing viral S protein. Mice were immunized with different combination of RBD plasmid DNA, complexed with jetPEI-in vivo transfection reagent and at the end of the experiment spleen was harvested. The cytotoxic effect of isolated CD8+ T cells was higher in RBD- scaffold DNA vaccines compared to RBD DNA vaccines alone.
  • Figure 11 Scheme of neutralization assay based on Spike-ACE2 interaction.
  • Virus entry depends on the interaction of virus S (Spike) protein and host receptor ACE2.
  • Spike virus S
  • ACE2 virus S
  • Unbound Spike proteins are washed away before detecting ACE2-bound Spike proteins with Streptactin-HRP.
  • Figure 12 Neutralization assay based on Spike-ACE2 interaction. Sera of mice immunized with DNA vaccines comprising scaffolded RBD are capable of inhibition of Spike-ACE2 interaction. Constructs encoding scaffolded RBD are more potent than RBD in inducing neutralizing antibodies.
  • Figure 13 Neutralization of Spike pseudoviral entry into cells by generated antibodies from RBD-scaffold DNA immunized mice.
  • Sera of mice immunized with DNA vaccines comprising scaffolded RBD are capable of neutralization of Spike pseudovirus entry into cells.
  • Figure 14 Titer of total IgG antibodies reacting with a SARS-CoV-2 Spike protein for different scaffolded HRCs. Mice were immunized with different combination of HRC plasmid DNA. Higher end point titer against SARS-CoV-2 Spike protein after prime immunization was observed in scaffold HRC vaccines compared to HRC alone.
  • Figure 15 Western blot of recombinant proteins RBD and RBD-bann isolated from mammalian cell Expi293F supernatants, used in mice immunization with protein based vaccine.
  • FIG 16 Titer of total IgG antibodies against the RBD (A) and SARS-CoV-2 Spike protein (B) for protein based vaccine, composing RBD or RBD-bann protein, coupled with squalene based adjuvant.
  • RBD or SARS-CoV-2 Spike specific antibodies rose from prime to first and second boost.
  • Higher end point titer against RBD or SARS-CoV2 Spike was observed in scaffold RBD-bann protein vaccine compared to RBD alone.
  • Figure 17 Neutralization of Spike pseudoviral entry into cells by generated antibodies from RBD or RBD-bann protein based vaccine immunized mice. Neutralization titer in mice sera was determined using a pseudovirus system.
  • mice immunized with isolated proteins were diluted 50-fold, and Spike-pseudotyped virus infection of ACE2 and TMPRSS2-transfected HEK293 cells was followed by luminescence.Sera of mice immunized with protein vaccines comprising scaffolded RBD are capable of neutralization of Spike pseudovirus entry into cells.
  • Figure 18 Trafficking of labeled isolated proteins RBD or RBD-bann into the popliteal lymph nodes (PLN) after 24 or 72 hours. Alexa Fluor 647 labeled RBD proteins were injected into the foot pad (RBD-left foot, RBD-bann; right foot) of three animals.
  • the fluorescence signal of AF-647 was determined in the popliteal lymph node, depicting the presence of labeled RBD or RBD-bann.
  • RBD-bann proteins remain present within PLN for a longer period of time suggesting different status of trafficking compared to RBD protein.
  • the term “vaccine” refers to the preparation introduced into the body of human or animal that stimulates active acquired immunity to a particular infectious disease.
  • the term vaccine as used herein contains protein components that are based on the sequence of selected viral proteins.
  • the term “scaffold” as used herein relates to the polypeptide that triggers formation of the self-assembling oligomeric structure, composed of three or more copies of the scaffolding polypeptide and oligomerizes protein antigen to which it is genetically fused to in order to present multiple copies of the protein antigen per each particle.
  • the term “Spike” as used herein relates to the Spike glycoprotein of SARS-Cov-2 (UniProt: PODTC2) which attaches the virion to the cell membrane through interaction with the host receptor and mediates fusion of the virion with cellular membranes.
  • the term “RBD” as used herein relates to the receptor binding domain of Spike glycoprotein from SARS-Cov-2 encompassing amino acids 330 – 521.
  • HRC alpha helical domain of Spike glycoprotein from SARS-Cov-2 encompassing amino acids 1128 – 1210.
  • the term “ferritin” as used herein relates to Helicobacter pylori-bullfrog hybrid ferritin encompassing residues 2-9 of bullfrog ferritin lower subunit (UniProt: P07797 with mutation N8Q) and residues 3-167 of H. pylori nonheme ferritine (UniProt Q9ZLI1 with mutation I7E).
  • foldon as used herein relates to the C-terminal domain fibritin of bacteriophage T4 which in its native state forms a trimeric ⁇ -hairpin propeller and can be used as an artificial trimerization domain.
  • lumazine synthase as used herein relates to the cage-forming protein lumazine synthase of bacteria Aquifex aeolicus (UniProt: O66529).
  • B-annulus (beta-annulus, ⁇ -annulus) as used herein relates to the 24- residue beta-annulus peptide originating from the tomato bushy stunt virus, which leads to icosahedral assembly.
  • the term “pseudotyped virus SARS-CoV-2” as used herein relates to VSV-G virus, where surface G protein was replaced with SARS-CoV-2 Spike protein.
  • the term “antibody” as used herein relates to a protective protein produced by B lymphocytes in response to the presence of a foreign substance, called an antigen.
  • the term “type of antibody” as used herein relates to a protein (or protein complex) that includes one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • the term "antigen” refers to a compound such as a polypeptide, polypeptide complex, glycoprotein, nucleic acid or the like, which elicits an immune response.
  • Organism as used herein relates to any living organism.
  • the term “immune system”, as used herein, relates to an organ system in higher developed organisms composed out of specific cell subtypes, which act in order to eliminate foreign molecules from the organism.
  • the term “immune response” as used herein relates to ability of an immune system of any living organism to respond to administered antigen to formulate appropriate antibodies by antibody-producing cells, e.g. plasma cells in order to achieve a specific protection against any foreign pathogens.
  • the term “immunization” as used herein relates to administration of prepared vaccine into living organism or its body via oral or different parenteral inoculation routes, e.g. intranasal, intramuscular, subcutaneous, intradermal. Vaccine is delivered via simple needle injection, inhalation or by the use of different traumatic or non-traumatic devices after vaccine administration.
  • the first dosage of the administered vaccine into the organism is referred as prime and any following administrations of vaccines are known as boost.
  • the term “mice” as used herein relates to BALB/c OlaHsd mice, an albino laboratory-bred strain or any mice laboratory strain that is commonly used in immunological studies and immunization protocols, composed of immunization, appropriate blood draw and final termination of the study.
  • End point titer refers to ELISA-based or any other experimental determination and calculation of specific antibodies towards different antigens. EPT is depicted as the final serum dilution or titer that present absorbance value above calculated cutoff value.
  • genetic fusion refers to the polypeptide or nucleic acid that codes for the polypeptide in a single chain that comprises polypeptide of two or more constituents that are consecutive or between them are short linker polypeptides that prevent steric overlap, typically comprising 1-10 small polar amino acid residues, typically glycine or serine or similar amino acid residues.
  • cell refers to a eukaryotic or prokaryotic cell, a cellular or multicellular organism (cell line) cultured as a single cell entity that has been used as a recipient of nucleic acids and includes the daughter cells of the original cell that has been genetically modified by the inclusion of nucleic acids.
  • the term refers primarily to cells of higher developed eukaryotic organisms, preferably vertebrates, preferably mammals. This invention relies also on non-vertebrates cells, preferably plant cells.
  • the term cells also refer to human or animal primary cells or cell lines. Naturally, the descendants of one cell are not necessarily completely identical to the parents in morphological form and its DNA complement, due to the consequences of natural, random or planned mutations.
  • a “genetically modified host cell” (also “recombinant host cell”) is a host cell into which the nucleic acid has been introduced.
  • the eukaryotic genetically modified host cell is formed in such a way that a suitable nucleic acid or recombinant nucleic acid is introduced into the appropriate eukaryotic host cell.
  • the invention hereafter includes host cells and organisms that contain a nucleic acid according to the invention (transient or stable) bearing the operon record according to the invention.
  • Suitable host cells are known in the field and include eukaryotic cells. It is known that proteins can be expressed in cells of the following organisms: human, rodent, cattle, pork, poultry, rabbits and the like.
  • Host cells may include cultured cell lines of primary or immortalized cell lines.
  • T cell relates to lymphocytes, theT-subset (e.g. CD4+ and CD8+ T cells) of white blood cells, a specific mononuclear immune cell population that interacts in the adaptive immune system by recognizing antigen peptides bound to major histocompatibility complex (MHC) molecules with a T cell receptor. Recognition of antigen peptides via a TCR activates signaling pathways, which result in cytokine signaling and a cytotoxic effect.
  • MHC major histocompatibility complex
  • cytotoxic as used herein relates to cell destruction or cell killing by specific cytotoxic T cells, e.g. CD8 positive T cell subset via MHCI, bound to specific antigen.
  • Antigen specific CD8+ T cell recognition of specific antigen bound to MHCI results in cell death activation and subsequent antigen-infected cell destruction.
  • the term “specific lysis” as used herein relates to calculated percentage of antigen- infected cell death via antigen specific CD8 cytotoxic T cells. Percentage of specific lysis is calculated based on bioluminescence values of measured cells. Bioluminescence values are presented as Average Radiance (p/s/cm 2 /sr).
  • B cell as used herein, relates to lymphocytes, the B-subset of white blood cells, a specific mononuclear immune cell population that interacts in the adaptive immune system by recognizing antigen peptides bound to major histocompatibility complex molecules with a B cell receptor.
  • nucleic acids refers to a polymeric form of nucleotides (ribonucleotides or deoxyribonucleotides) of any length and is not limited to single, double or higher chains of DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers with a phosphorothioate polymer backbone made from purine and pyrimidine bases or other natural, chemical or biochemically modified, synthetic or derived nucleotide bases.
  • protein refers to the polymeric form of amino acids of any length, which expresses any function, for instance localizing to a specific location, localizing to specific DNA sequence, facilitating and triggering chemical reactions, transcription regulation, structural function, biological recognition.
  • recombinant means that a particular nucleic acid (DNA or RNA) is a product of various combinations of cloning, restriction and / or ligation or chemical synthesis leading to a construct having structurally coding or non-coding sequences different from endogenous nucleic acids in a natural host system.
  • trafficking means transportation of the material from the site of the injection to first regional lymphnode, for instance transportation of the injected protein from the footpad to popliteal lymphnode.
  • variant refers to a protein characterized by its amino acid sequence or encoding nucleotide sequence which differs from the amino acid sequence or nucleotide sequence of the corresponding wild-type (wt) protein. Such variants necessarily have less than 100% sequence identity or similarity with the wt protein.
  • the protein variant will have an amino acid sequence from about 75% to less than 100% amino acid sequence identity or similarity with the amino acid sequence of wt protein, more preferably from about 80% to less than 100%, more preferably from about 85% to less than 100%, more preferably from about 90% to less than 100%, and most preferably from about 95% to less than 100%.
  • Identity or similarity with respect to this sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical (i.e. same residue) with the wt protein residues, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.
  • Variants as defined herein are provided by insertions, susbtitutions and/or deletions in the corresponding wt amino acid or nucleotide sequence. “Conservative substitutions”, i.e. substitutions that change a given amino acid to a different amino acid with similar biochemical properties (e.g. charge, hydrophobicity and size) are in particular preferred. Variants, also called “functional variants”, show preferably the same ar a comparable biological activity as the wt protein does. Thus, in a preferred embodiment “variant” refers to a protein which differes from the corresponding wt protein by 1-5, preferably 1-3 und in particular 1 conservative substitution.
  • the insertion of the vectors into the host cells is carried out by conventional methods known from the field of science, and the methods relate to transformation or transfection and include: chemically induced insertion, electroporation, micro-injection, DNA lipofection, cellular sonication, gene bombardment, viral DNA input, as well as other methods.
  • the entry of DNA may be of transient or stable.
  • Transient refers to the insertion of a DNA with a vector that does not incorporate the DNA of the invention into the cell genome.
  • a stable insertion is achieved by incorporating DNA of the invention into the host genome.
  • the insertion of the DNA of the invention in particular for the preparation of a host organism having stably incorporated a nucleic acid, e.g.
  • a DNA, of the invention can be screened by the presence of markers.
  • the DNA sequence for markers refers to resistance to antibiotics or chemicals and may be included on a DNA vector of the invention or on a separate vector.
  • the invention relates to the preparation of vaccines that comprise oligomeric, limited to more than three copies of the polypeptide domain from the antigen protein, such as for example but not limited to viral protein, fused to the scaffolding protein that defines the oligomeric state and the number of copies of viral protein domain in each vaccine particle whereas the scaffold protein comprises less than 30 amino acid residues.
  • the scaffolding domain is composed of the fibritin trimerization foldon peptide, whereas the protein antigen domain is genetically fused both to the amino- and carboxyl- terminal end of the foldon peptide in the arrangement antigen-foldon-antigen.
  • Antigen is fused directly to the foldon of a flexible linker peptide comprising typically 0-20 amino acid residues is inserted, where the linker preferably contains glycine and serine or threonine amino acid residues or other small hydrophilic amino acid residues.
  • each particle forms a trimer comprising 6 copies of the antigen domain.
  • the scaffolding domain is composed of the beta-annulus peptide containing 24 amino acid residues that has a propensity to self-assemble into several nanometer large particles comprising 60 or more copies of the antigen per particle.
  • the scaffolding domain is fused to the protein antigen directly or via a flexible linker peptide comprising 0-20 amino acid residues, where the linker preferably contains glycine and serine or threonine amino acid residues or other small hydrophilic amino acid residues.
  • the scaffolding domain may also be composed of the lumazine synthase from Aequifex aeolicus that has a propensity to self-assemble into several nanometer large particles comprising 60 copies of the antigen per particle in an icosahedral geometry.
  • the scaffolding domain is fused to the protein antigen directly or via a flexible linker peptide comprising 0-20 amino acid residues, where the linker preferably contains glycine and serine or threonine amino acid residues or other small hydrophilic amino acid residues.
  • the next embodiment of the invention provides a vaccine in the form of nucleic acids coding for a protein antigen-scaffold polypeptide formed through the genetic fusion of the antigen polypeptide with the scaffold polypeptide that forms nanoparticles comprising more than three copies of the polypeptide domain.
  • nanostructured vaccine triggers stronger immune response than a monomeric antigenic polypeptide and induces weak response to the scaffold polypeptide that is fused to the viral polypeptide in order to assemble them in a nanoparticle comprising more than three copies of the viral polypeptide.
  • the embodiment of the vaccine can select different antigens and is represented by examples of the polypeptide domains of the Spike protein of SARS-CoV-2 protein, specifically the RBD domain (residues 330-521) or an HRC segment of the S2 domain (residues 1128-1210).
  • RBD domain residues 330-521
  • HRC segment of the S2 domain residues 1128-12
  • Example 1 Design of the HRC and RBD protein genetically fused to the scaffolding self assembling polypeptides
  • RBD domain of the Spike protein of SARS-CoV-2 encompassing residues 330 to 521 or an HRC segment of the S2 domain (residues 1128-1210) were selected as antigens for immunization.
  • Polypeptide scaffolds were designed in order to induce formation of oligomers of RBD or HRC aimed to present those domains at the surface of the generated polypeptide nanoparticles. While the foldon peptide is often used to generate protein trimers, in this type of fusion the selected antigen is clustered in a trimeric form on one side of the complex, while the trimeric foldon is exposed at the other side of the trimeric nanoparticle.
  • the beta-annulus induces the self-association of the antigen domains, where its structural model has been approximated based on the icosahedral symmetry of the parent viral scaffold. According to this model, 60 copies of the antigen would be presented in the assembly. However based on the particle size the isolated protein produced in mammalian cells the assembly may be even larger, which is beneficial for the presentation to cells of the immune system and formation of antibodies. Beta-annulus peptide therefore presents approx. 16% of the residues and since it is engaged in the assembly, this domain is buried and weakly accessible to the antibodies.
  • the beta-annulus peptide does not contain aromatic residues, with the exception of a single histidine residue, which makes it hypoimmunogenic, therefore resulting in the production of a low amount of antibodies against the scaffold.
  • Two additional scaffolded variants of the RBD and HRC were produced, one with a fusion of Bullfrog (Rana catesbeiana) and Helicobacter pylori ferritin that forms a 24-meric particle(60) and the second with Aequifex aeolicus lumazine synthase that forms an icosahedral assembly comprising 60 subunits.
  • the double N- and C-terminal fusion to the foldon could be designed for other protein antigens against viruses or other targets that present six copies of the domain in a single particle.
  • Genetic fusion with the beta-annulus peptide could also be used to design vaccines for other antigens. While smaller peptides have been fused before to the beta-annulus peptide and produced in bacteria(55, 61, 62), here we present an invention of genetic fusion of the beta-annulus peptide to the antigen for the production of vaccine and expression in mammalian cells, where beta-annulus peptide fusions haven’t been produced before.
  • Example 2 Design of a sequence of scaffold for soluble protein antigen oligomers for vaccine
  • the soluble protein oligomers are composed of the core oligomerizing domain, charged solubility and repulsion residues and optionally defined oligomerizing domain ( Figure 1).
  • the core oligomerizing domain can be composed of a cluster of hydrophobic amino acid residues that form a core resembling molten globule with not necessarily defined tertiary structure. These residues are preferentially aliphatic amino acid residues such as Leu, Ile, Val, Ala that are hypoimmunogenic.
  • the second type of core oligomeric cluster relies on beta sheet forming segments that are composed of short, preferentially 5- 20 amino acid segments and preferentially from 1- 5, preferentially to 3 beta sheet forming segments.
  • the beta- annulus peptide comprises 3 beta sheet forming segments linked by flexible linkers to enable unhindered packing.
  • the third example of core oligomerizing domain are clusters of aromatic amino acids, preferentially at least two Phe residues that have been shown to form oligomers (64, 65).
  • Scaffolding peptides can be attached to the protein antigen either at the amino- or carboxyl-terminal end, but preferentially at the carboxyterminal, so that the oligomerizing peptide is formed last and the ribosomal synthesis is not hindered by the oligomerizing domain.
  • coli cells plasmid DNA isolation, polymerase chain reaction (PCR), reverse transcription - PCR, PCR linking, nucleic acid concentration determination, DNA agarose gel electrophoresis, isolation of fragments of DNA from agarose gels, chemical synthesis of DNA, DNA restriction with restriction enzymes, cutting of plasmid vectors, ligation of DNA fragments, purification of plasmid DNA in large quantities.
  • PCR polymerase chain reaction
  • PCR reverse transcription - PCR
  • PCR linking nucleic acid concentration determination
  • DNA agarose gel electrophoresis isolation of fragments of DNA from agarose gels
  • chemical synthesis of DNA DNA restriction with restriction enzymes
  • cutting of plasmid vectors ligation of DNA fragments
  • purification of plasmid DNA in large quantities The exact course of experimental techniques and methods are well known to experts in the field and are described in the manuals of molecular biology. All the work was performed using sterile techniques, which are also well known to the experts in the field. All plasm
  • Plasmids for transfection into cell lines have been isolated using a DNA isolation kit that removes endotoxins. Results: All plasmid constructs were inserted into a pcDNA3 backbone downstream of a CMV promotor. A CD45 signal sequence was added N-terminally for the secretion of protein antigens from mammalian cells and a hexa-histidine tag was added to constructs for affinity isolation. Non-tagged constructs were prepared in parallel for immunization purposes.
  • the RBD construct and fusion constructs were prepared using a synthesized gene fragment encoding the RBD domain (aa 330-521) or RBD domain fused to peptides (beta annulus, foldon) or polypeptides (ferritin, lumazine synthase). Constructs are listed in table 1 and nucleotide sequences in sequence listing.
  • Table 1 Constructs encoding RBD and HRC-based nanoparticle vaccine variants Example 4. Characterization of RBD and HRC-based nanoparticles produced by human cells (western blot, isolation, SEC-MALS, GF) Mammalian cell line HEK293 and Expi293 were transfected with plasmids for vaccination coding fusion proteins of the RBD to the scaffolding domains in order to produce in cells the protein immunogen. Supernatants of HEK293 or Expi293 cells were harvested 3-5 days post transfection and analyzed with ELISA and western blot using anti-RBD antibodies to determine the expression and size of proteins ( Figure 3). Produced proteins were purified and characterized.
  • Expi293F cells were cultivated in Expi293TM Expression Medium at 37°C and 8% CO2 on an orbital shaking platform with shaking speed based on shaking diameter and flasks’ size.
  • Figure 4B shows the soluble protein oligomer formation in the range of 500 nm, as determined by DLS. Example 5.
  • mice Immune response in mice to immunization by the scaffolded RBD by DNA vaccine
  • BALB/c OlaHsd mice Male Envigo, Italy
  • All animal studies were approved by appropriate government institutions taking into account all the ethical considerations regarding animal studies.
  • Laboratory animals were housed in IVC cages (Techniplast), fed standard chow (Mucedola) and tap water was provided ad libitum. Mice were maintained in 12-12 hours dark-light cycle. All animals, used in the study, were healthy; accompanied with health certificate from the animal vendor.
  • Immunization ( Figure 5) was carried out under general inhalation 1,8% MAK isoflurane anesthesia (Harvard Apparatus). Animals were vaccinated with plasmid DNA (RBD, RBD- scaffold, scaffold vectors, empty pcDNA3 vector), coupled with jetPEI-in vivo transfection reagent (Polyplus Transfection) with the N/P ratio 12. Each animal received a total of 20 ⁇ g of designated plasmid DNA, complexed with transfection reagent with intramuscular injection. Vaccine (prime) was administered using 30G needle into m.tibialis anterior after appropriate area preparation. First boost was administered 2 weeks later and same was done for the second boost.
  • dilution row (1:100, 1:300, 1:900, 1:2700, 1:8100, 1:24300, 1:72900, 1:218700).
  • ELISA diluent was used for mouse sera. Mouse sera were incubated at 4°C overnight. Next day, plates were washed and afterwards goat anti mouse IgG (H+L)-HRP antibodies (Jackson ImmunoResearch), diluted 1:3000 in ELISA diluent solution. Plates were incubated 1h at RT. Next plates were washed.
  • FIG. 6 shows end point titer against RBD antibodies two weeks after prime or boost immunization, determined in mice sera from RBD DNA, RBD-scaffold, scaffold or empty vector immunized animals. EPT after prime (left panel) is enhanced in animals, vaccinated in RBD-scaffold DNA vaccines (all different scaffolds) compared to RBD DNA immunized animals. The highest titer is seen in B-annulus RBD DNA immunized animals; approximately 2log10 higher. No antibodies against RBD were seen in only scaffold immunized animals.
  • EPT after 1st boost are higher in all animals, still no antibodies are formed in animals, immunized only with scaffold vectors. Titer in animals, immunized with RBD-scaffold DNA increased. The same was observed after the 2 nd boost (right panel), where some antibodies were also formed in animals, immunized with ferritin scaffold or B-annulus. Further, some increase in EPT was observed in all animals, immunized with RBD-scaffold DNA vaccines. Clearly, all constructs, encoding for RBD- scaffold vaccines were more immunogenic as RBD DNA alone, suggesting higher immune protection against SARS-CoV-2.
  • Figure 7 shows end point titer against Spike SARS-CoV-2 antibodies two weeks after prime or boost immunization, determined in mice sera from RBD DNA, RBD- scaffold, scaffold or empty vector immunized animals.
  • EPT after prime is enhanced in animals, vaccinated in RBD-scaffold DNA vaccines (all different scaffolds) compared to RBD DNA immunized animals. No antibodies against Spike SARS-CoV-2 were seen in only scaffold immunized animals.
  • EPT after 1 st boost are higher in all animals, still no antibodies are formed in animals, immunized only with scaffold vectors. Titer in animals, immunized with RBD-scaffold DNA increased.
  • mice spleens from RBD DNA or RBD-scaffold DNA immunized animals were harvested at the end of the immunization protocol.
  • Single cell suspension from spleens were obtained using tissue dissociator gentleMACSTM Dissociator, gentleMACS C tubes and MACS buffer according to the manufacturer’s instruction (Miltenyi Biotec).
  • CD8+ T cell from spleen cell suspension were isolated using CD8a+ T Cell Isolation Kit according to the manufacturer’s instruction (Miltenyi Biotec).
  • NIH-3T3 cells were isolated based on negative selection using LS columns, obtaining up to 108 of maximal number of labelled cells.
  • RBD specific cytotoxicity mouse NIH-3T3 cells NIH-3T3 cells were seeded into 24-well plates (1*105/well); next day cells were transfected with pCG1-hACE2 and pCMV-TMPRSS2 plasmids. The next day, cells were infected with Spike pseudovirus. Next day isolated CD8a+T cells (1*10 5 /well) were added in RPMI1640 cell medium.
  • Bioluminescence was determined using IVISIII (Perkin Elmer) after the addition of D- luciferin (500 ⁇ g/ml), showing the state of RBD-specific cytotoxicity of CD8+T cells, isolated from RBD DNA or RBD-scaffold DNA vaccinated animals. Bioluminscence values are presented as Average Radiance (p/s/cm 2 /sr), which were determined using Living Image® software.
  • Figure 10 shows improved specific lysis of NIH-3T3 infected cells, when CD8+ isolated cells from RBD-scaffold immunized mice were added compared to lysis of NIH-3T3 cells treated with CD8+ T cells from alone RBD DNA vaccinated animals. Mean % of all specific lysis off RBD-scaffold DNA vaccinated animals isolated CD8 cells exhibited more than 80% RBD-specific cytotoxic activity, compared to only RBD, where approximately 40% of specific lysis was observed. Each dot in graphs presents spleen cells from designated animal that was immunized with appropriate RBD DNA or RBD- scaffold DNA vaccine.
  • Example 7 Neutralization of ACE2 spike interaction by generated antibodies SARS-CoV-2 entry into host cells depends on the interaction between virus Spike protein and human receptor ACE2 that is present on the surface of human cells. Thus in vitro assay using isolated proteins can be used to follow the capacity of antibodies to block Spike interaction with ACE2.
  • ACE2-Fc Genescript
  • PBS phosphate buffer saline
  • Serum dilutions were prepared in 1% BSA/PBS-T (1% bovine serum albumin in phosphate buffer saline with Tween 20) and incubated with Spike protein at final concentration of 0.1 ⁇ g/ml for 1h at 37°C. After blocking in 1% BSA/PBS-T for 1 h at 37°C plates were washed in PBS-T and serum- Spike samples were added to wells and incubated for 2h at room temperature. After wash the plate was incubated with streptactin-HRP (1:10000) in 1% BSA/ PBS-T for 1h at room temperature. After the final wash, TMB substrate was added and the reaction was stopped with addition of acid solution (3M H3PO4).
  • BSA/PBS-T 1% bovine serum albumin in phosphate buffer saline with Tween 20
  • mice immunized with scaffolded RBD DNA vaccines develop potent neutralizing antibodies when compared to RBD vaccine as demonstrated with the loss of Spike protein binding to ACE2, depicted in Figure 12.
  • Example 8 Neutralization of viral entry into cells by generated antibodies Pseudovirus system based on vesicular stomatitis virus (Berger Rentsch and Zimmer, Plos One, 2011) was used to determine virus neutralization capacity of immunized mice sera. Plasmids and VSV ⁇ G*/G virus were provided by Stefan Pölhmann. Pseudovirus preparation was described previously (Hoffmann et al, Cell, 2020).
  • HEK293T cells were seeded into 6-well plates (9*105/well) a day before transfection with pCG1- Spike/PEI mixture. The next day, cells were infected with VSV ⁇ G*/G virus in serum-free medium for 1h, after which medium was removed and cells were washed with PBS before complete medium supplemented with anti-VSV-G antibody (8G5F11, Kerafast) was added to the cells. After 18h cell supernatant was centrifuged and cleared pseudovirus supernatant was aliquoted and stored at -80°C until neutralization experiments.
  • HEK293 or Vero E6 were seeded (2.5*10 4 /well) a day before transfection with plasmids encoding ACE2, TMPRSS2 and Renilla luciferase. Immunized mice sera were preincubated with spike pseudovirus for 30min before addition to the cells. The next day, medium was removed and cells were lysed in Passive lysis buffer. Luciferin substrate was used to detect Firefly luciferase activity as a measure of pseudovirus infection and coelenterazine H to follow Renilla luciferase activity for determination of transfection efficiency and normalization.
  • mice immunized with scaffolded RBD DNA vaccines neutralize pseudotype virus infection of HEK293 cells that were transfected with plasmids encoding ACE2 and TMPRSS2.
  • Mice sera from B-annulus-RBD DNA immunized mice exhibited the best neutralization effect compared to others.
  • Example 9 Immune response in mice to immunization by the scaffolded HRC by DNA vaccine To test the immunogenicity of HRC or HRC-scaffold DNA vaccines female 8-10 weeks BALB/c OlaHsd mice (Harlan Envigo, Italy) were used for immunization protocols. All animal studies were approved by appropriate government institutions taking into account all the ethical considerations regarding animal studies.
  • Mouse sera were prepared by centrifugation of blood samples 3000RPM/ 20min at 4°C. In mouse sera specific anti-Spike SARS CoV-2 total IgG were determined by ELISA test in order to test the immunogenicity of designed HRC DNA vaccines. ELISA tests were performed to determine End point titer as stated in the Example 5. Graphs present values of calculated EPT; each dot represent single animal. Titers below 1:100 are considered as negative value and are not presented in the graphs, but are taken into account when mean is calculated. Results: Figure 14 shows end point titer against Spike SARS CoV-2 antibodies two weeks after prime immunization, determined in mice sera from HRC DNA, HRC-scaffold or empty vector immunized animals.
  • Example 5 in mouse sera specific anti-RBD and anti-Spike SARS CoV-2 total IgG were determined by ELISA test in order to test the immunogenicity of designed RBD protein vaccines. ELISA tests were performed to determine End point titer as stated in Example 5, only that the next dilution row was used (1:10 4 , 1:10 5 , 1:10 6 , 1:10 7 , 1:10 8 , 1:10 9 , 1:10 10 , 1:10 11 ). Graphs present values of calculated EPT; each dot represent single animal. Titers below 1:100 are considered as negative value and are not presented in the graphs, but are taken into account when mean is calculated.
  • Results Figure 15 shows picure of performed western blot analysis of isolated recombinant protein RBD or RBD-bann, isolated from mammalian supernatant, that were used in protein based vaccine immunization experiments. End point titer against RBD (A) and Spike SARS CoV-2 antibodies (B) two weeks after prime and boost immunization, determined in mice sera from RBD protein or RBD-bann protein, coupled with or without adjuvant Addavax immunized animals are depicted in Figure 16. Clearly, all protein vaccines, coupled with adjuvant were more immunogenic compared to naked protein injection.
  • RBD-bann protein vaccine elicit higher immune response; RBD-bann with adjuvant is more immunogenic as RBD, coupled with adjuvant alone, suggesting higher immune protection against SARS-CoV-2.
  • sera of mice immunized with protein based vaccine in combination with adjuvant neutralize pseudotype virus infection of HEK293 cells that were transfected with plasmids encoding ACE2 and TMPRSS2.
  • Mice sera from RBD-bann protein, coupled with adjuvant Addavax immunized mice exhibited the best neutralization effect compared to other tested mice sera.
  • the RBD and RBD-ban proteins were labeled with the fluorescent dye Alexa Fluor647 (AF647), using AlexaFluor 647 conjugation kit.
  • Alexa Fluor647 Alexa Fluor 647 conjugation kit.
  • the modifier reagent 100 ⁇ L was added to a solution of protein (1 mL, 1 mg/mL PBS). Afterward, the solution was mixed with a lyophilized AlexaFluor 647 conjugation mix. After 10 min of incubation at 25 °C, the reaction was terminated with the addition of the quencher reagent (100 ⁇ L).
  • the labeled protein was desalted using a PD-10 column and was concentrated (300 ⁇ L, approx.2 mg/mL PBS). A total of 50 ⁇ g of AF-647 labeled RBD or RBD-bann protein was injected into the mouse foot pad (RBDAF647 into the left and RBD-bann-AF647 into the right foot). Total three animals were used for the experiment. Protein trafficking based on fluorescence measurement was observed using the in vivo imaging system IVIS® Lumina Series III. The fluorescence signal that depicted the presence of the AF647-labeled protein was determined. Fluorescence was measured using AlexaFluor 647 Probe settings. The mice were exposed for 0.5–5 s using bining factor 8, f number 2.
  • FIG. 18 shows trafficking analysis of AF647 labelled RBD or RBD-bann protein.
  • RBD protein was transported to popliteal lymph node faster compared to injected labelled RBD-bann protein.
  • the fluorescence values of area surrounding lymph nodes are higher at 24 hours in left foot, where labelled RBD protein was injected, compared to right foot that received labelled RBD-bann protein, suggesting faster trafficking of monomeric RBD protein.
  • Higher fluorescence values at 72 hours for RBD-bann injected feet depict that RBD-bann protein retained in lymph nodes for longer period of time, compared to RBD protein, indicating higher immunogenicity due to different trafficking and prolonged mantainace within the lymph node.

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Abstract

Le SARS-CoV-2 est la source d'une maladie virale pandémique affectant des millions de personnes dans le monde et est responsable de centaines de milliers de morts depuis son apparition fin 2019. Un vaccin efficace devrait déclencher la formation d'une réponse immunitaire humorale et cellulaire protectrice contre les composants viraux qui soit empêchera l'entrée du virus dans des cellules, soit tuera les cellules infectées par le virus. La présente invention concerne d'une manière générale la mise en œuvre de vaccins composés de séquences nucléotidiques codant pour des domaines de protéines antigéniques génétiquement fusionnés avec des polypeptides d'échafaudage qui s'auto-assemblent en nanoparticules oligomères solubles. Les nanoparticules comprennent au moins six copies du domaine de protéine antigénique comprenant (mais sans s'y limiter) un domaine de liaison au récepteur (RBD) ou des domaines HRC ou une protéine de spicule du SARS-Cov-2 extracellulaire complète et sont bénéfiques par rapport à des protéines monomères, du fait d'une avidité accrue. La présente divulgation concerne une amélioration supplémentaire pour des vaccins efficaces sur la base de différentes combinaisons du domaine de protéine cible avec différents échafaudages conduisant à une réponse amplifiée contre l'antigène et non contre les domaines d'échafaudage du vaccin.
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CN114702578A (zh) * 2022-06-06 2022-07-05 百斯医学诊断科技(北京)有限公司 新型冠状病毒Omicron突变株特异性抗体及其应用
CN114702578B (zh) * 2022-06-06 2022-09-27 百斯医学诊断科技(北京)有限公司 新型冠状病毒Omicron突变株特异性抗体及其应用

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