mRNA VACCINE FORMULATIONS AND METHODS OF USING THE SAME

FEDERAL FUNDING STATEMENT

[0001] This invention was made with government support under RAPID grant (2029579) awarded by the NSF and the National Institutes of Health under award number P20GM130448. The government has certain rights in the invention.

SEQUENCE LISTING

[0002] This application contains a sequence listing in paper format and in computer readable format, the teachings and content of which are hereby incorporated by reference.

FIELD OF THE DISCLOSURE

[0003] This disclosure relates to mRNA compositions, more particularly to mRNA vaccine formulations.

BACKGROUND

[0004] The coronavirus disease 2019 (COVID-19), caused by the novel pathogenic SARS-coronavirus 2 (SARS-CoV-2) that emerged in December 2019 in Wuhan China, is rapidly spreading globally. SARS-CoV-2 is a novel virus that belongs to the genus betacoronavirus and it is related to Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), Middle East Respiratory Syndrome coronavirus (MERS-CoV) and some bat and pangolin coronaviruses. Similar to SARS-CoV-2 coronaviruses, SARS-CoV-2 utilizes the spike (S) protein to infect host cells by binding to human angiotensinconverting enzyme 2 (ACE2), but with much higher affinity. The S protein, which exists as a trimer displayed on the virus surface, contains ACE2 receptor binding domain [RBD]; this knowledge is relevant to vaccine development, since antibodies capable of blocking this interaction can neutralize the virus and stop the infection of cells. Coronavirus virions exhibit the S glycoprotein that binds to host-cell receptors and mediates cell entry via fusion of the host cell and viral membranes. S proteins are trimeric class I fusion glycoproteins that are expressed as a single polypeptide that is subsequently cleaved into S1 and S2 subunits by cellular proteases. The S1 subunit contains the receptor-binding domain (RBD), which, in the case of SARS-CoV-2, recognizes the angiotensin-converting enzyme 2 (ACE2) receptor on the host-cell surface. The S2 subunit mediates membrane fusion and contains an additional protease cleavage site, referred to as S2', that is adjacent to a hydrophobic fusion peptide. Binding of the RBD to ACE2 triggers S1 dissociation, allowing for a large rearrangement of S2 as it transitions from a metastable prefusion conformation to a highly stable postfusion conformation. During this rearrangement, the fusion peptide is inserted into the host-cell membrane after cleavage at S2', and two heptad repeats in each protomer associate to form a six-helix bundle that brings together the N- and C-termini of the S2 subunits as well as the viral and host-cell membranes. Attachment and entry - both mediated by the S protein - are essential for the viral life cycle, making the S protein a primary target of neutralizing antibodies and a critical vaccine antigen- Stabilization of the prefusion S protein can be accomplished through a series of mutations that may increase the yield in recombinant expression systems and improve the induction of neutralizing antibodies.

[0005] Lipid nanoparticle (LNP) mRNA vaccine has been revolutionary for preventing severe COVID19 disease and may extend to treating other infectious diseases and cancer. Despite rapid clinical advancement, a major limitation is LNP-RNA temperature instability, requiring storage at or below -20-0 °C for the RNA to retain activity. This cold chain requirement greatly limits efficacy and widespread deployment and is considered a contributing factor to the emergence of SARS-CoV-2 variants due to under vaccination.

[0006] In addition to LNP, a variety of other nanoparticle systems have been evaluated for delivery of mRNA and therapeutic RNA. Zinc is best perhaps best known for its role in stabilizing RNA and protein interactions in cells and tissues, and as such, the nanoscale biomolecular interactions of zinc oxide (ZnO) nanoparticle has been of considerable interest. Various forms of protamine have been considered cell penetrating peptide and studied for delivery of siRNA and mRNA. [0007] Consequently, there is an urgent need to develop safe and effective vaccines to protect the human population. What is needed are compositions that are effective at reducing the incidence the severity and/or the transmissibility of the pathogenic SARS-CoV-2. What is further needed are are improved mRNA-based SARS-CoV-2 vaccines that do not require extremely low temperature storage and logistically complex cold chain distribution, factors which have limited their usage in remote areas and low resource countries.

SUMMARY OF THE DISCLOSURE

[0008] The present disclosure addresses key unmet needs in the art, providing stabilized RNA and/or mRNA vaccines for SARS-CoV-2 that can be distributed and stored at higher temperatures for longer periods of time than those currently available.

[0009] In one aspect of this disclosure, RNA temperature-stabilization is achieved by binding RNA to zinc nanoparticle coated with protamine (ZNP). As shown herein, ZNP- RNA was characterized by transmission electron microscopy, UV, dynamic light scatter and zeta potential analysis, with RNA payloads ranging from 20.1 -to-124 micrograms RNA per milligram ZNP. Zinc oxide nanoparticle binding to RNA was confirmed by gel shift and best protected RNA from hydrolysis at physiological temperature (37 °C) in comparison to other inorganic nanoparticles commonly used for RNA delivery. RNA structural stabilization was shown by circular dichroism (CD) spectroscopy, dependent on nanoparticle: RNA stoichiometry. Whereas protamine ratio (1 :1 , 1 :10, 1 :100) did not impact in vitro translation efficiency, protamine coating enhanced expression in cell free lysates and cellular fluorescence after delivery of ZNP-mRNA encoding green fluorescent protein (GFP). Temperature-stabilization was shown by differential scanning calorimetry (DSC) in three independent experiments, the RNA melting temperature increasing from 63.9-64.7 to 70.1 -71.6 °C in the presence of nanoparticle. ZNP-RNA suspensions in phosphate buffered saline (PBS) could therefore be stored at 30, 40 and 50 °C for up to two weeks retaining intact RNA as shown by RNA agarose gel electrophoresis (RAGE). ZNP- mRNA (mCherry) incubated at these temperatures also retained expression activity in cell free isolates. ZNP delivery into HEK293 cells was shown for spike mRNA. RNA could also be loaded onto ZNP with cationic antiviral peptide LL37 albeit with less efficiency than protamine which is about 97% efficient and the RNA eluted in Tris-EDTA-urea-heparin buffer. These data suggest ZnO-protamine- RNA (ZNP-RNA) may be useful as an alternative to LNP to increase temperature resistance and enhance pharmaceutical performance of mRNA and potentially other therapeutic RNAs.

[0010] In one aspect, this disclosure relates to a composition comprising a nucleic acid encoding a SARS-CoV-2 S protein; and a pharmaceutically acceptable carrier comprising a zinc oxide nanoparticle. In some forms, the composition further includes a further ingredient selected from the group consisting of a solvent, a dispersion media, a coating, a stabilizing agent, a diluent, a preservative, an antimicrobial agent, an antifungal agent, an isotonic agent, and an adsorption delaying agent. In some forms, the protein has at least 90% sequence homology with SEQ ID NO. 1. In some forms, the pharmaceutically-acceptable carrier comprises a stabilizing agent and/or a preservative and/or an antimicrobial agent. In some forms, the nucleic acid is at a concentration greater than 100 micrograms per milligram weight of the nanoparticle. In some forms, the zinc oxide nanoparticle comprises a cell penetrating peptide. In some forms, the zinc oxide nanoparticle comprises protamine. In some forms, the nucleic acid further comprises a sequence encoding a CD40L. In some forms, the nucleic acid encodes a tetrameric CD40L trimer. In some forms, the SARS-CoV-2 S protein and the CD40L are encoded by a polycistronic mRNA. In some forms, the nucleic acid encodes a SARS-CoV-2 S protein / CD40L fusion protein. In some forms, the vector includes an inserted sequence having at least 90% sequence homology with SEQ ID NO. 9 or 10. In some forms, the nucleic acid is present at a concentration of greater than 100 pg/mg of nanoparticle.

[0011] In another aspect of the disclosure, methods for making and using the composition described herein are provided. Such uses include reducing the incidence, severity, transmissibility of SARS-CoV-2 infection when administered to a subject in need thereof. Further information regarding these aspects and others are provided below. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0013] Fig. 1 is an illustration of a preferred amino acid sequence of the SpikeFTm-2A- _CD5 SPD-CD40L polypeptide;

[0014] Fig. 2 is an illustration of a preferred amino acid sequence of the Spike-SPD- CD40L polypeptide;

[0015] Fig. 3 is photograph illustrating protein expression by the four plasmid constructs provided above in transfected HEK 293 cells probed with anti-COVID-19 spike protein specific neutralizing mAbs;

[0016] Figure 4 is an illustration of the in vivo distribution. Two mice were administered a 2 mg/kg single intravenous dose in 200 mL PBS of either ZnO-NP or cy5.5-ZnO-PEG NP and imaged directly in the bio-imager or sacrificed at 5 hours the brain, heart, lungs, liver, spleen and kidneys were removed and imaged in the bioimager (A). The corresponding mice were sacrificed after 3 days for histopathological analysis (B). At 5 hours, the tissues were removed and weighed, homogenized in PBS buffer and their relative fluorescence and zinc content per milligram tissue determined by fluorescence spectroscopy and ICP/MS analysis (C). Also at 5 hours blood was collected and blood cell counts were determined and compared to nude mouse reference standards (D).

[0017] Figure 5: Uptake of cy5.5-labeled ZnO NP into Caco-2 3-D organoids (A), cy5.5-ASO delivery into human A375 melanoma cells by ZnO NP or Co3O4 NP relative to cy5.5-ASO control (B) with improved uptake by ZnO, Co3O4 or NiO NP confirmed by flow cytometry (C), finally splicing correction of the aberrant RBD transcript by nanoparticle delivery of ASO targeting that cryptic site (D).

[0018] Figure 6: (A) Various nanoscale physiometacomposite materials were fabricated as described in the materials and methods and their nanoscale confirmed by TEM and NTA analysis (see supplemental data). The exact compositions of each type are provided in the summary table including all the oxide combinations, ZnS, FeZnS, MnZnS and MnZnS and others (B). The biocompatibility of the different compositions after 48 hour treatment of continuous exposure in serum containing media to NIH3T3 cells is also shown (C).

[0019] Figure 7: (A) PMC materials were spiked into PBS, serum, tumor or liver homogenates and their fluorescence versus concentration curves obtained, (B) Bioluminescence assays were conducted in the presence of FeZnS or MnZnS with/without Luciferase enzyme and substrate and the optimal excitation, emission and intensity 3-D plots obtained, (C) similarly these materials or MnZnSe were spiked into tissue slurries and homogenates and the 3-D fluorescence patterns obtained, (D and E) finally ex vivo slices of mouse brain, liver and lung were injected with MnZnS or MnZnSe and imaged directly in the bio-imager.

[0020] Figure 8: ZnO NP, CoZnO or NiZnO increase cy5.5-poly l:C labeling of 3-D tumor spheroid as shown by bio-imager relative to RNA alone or untreated controls (A), NiZnO or higher order PMC containing iron and manganese inhibited 3-D tumor spheroid growth and ablated these structures (B), and (C) ZnO or CoZnO PMC inhibited B16F10 cell invasion in the scratch assay, the PMC treated material still having the gap filled in by untreated or poly l:C negative controls.

[0021] Figure 9: (A) High throughput proteomic analysis of B16F10/BALB-C tumor, (b) RBD protein interference or (C) RBD or BCL-xL targeted ASO in B16F10, A375 or 132N1 , ERK/AKT RT-PCR after ZnO or Ni/ZnO (D) or treating drug-resistant canine mucosal melanoma cells with LL37 peptide or RBD ASO and aptamer complexes (F)

[0022] Figure 10: 20 ug/ml inhibition of PMC inhibition of B-Gal biochemical activity by various PMC including MnZnS and FeZnS relative to ZnO NP or silver nanoparticle control.

[0023] Figure 11 illustrates A) Differential scanning calorimetry (DSC) analysis of poly l:C melting temperature increases from 63.9-64.1 degrees Celsius to greater than 70 deg C in the presence of ZnO NP. B) Stabilizing effect of ZnO NP on RNA structure is shown by circular dichroism (CD) spectroscopy at increasing stoichiometry of ZnO NP:poly l:C RNA accentuates the pattern indicating stability-enhancement

[0024] Figure 12 illustrates temperature-stability (physico-chemical stability) imparted to TY-RNA, when bound to ZnO NP via protamine, dried to a powder and resuspended in sterile PBS can be stored at elevated temperatures for prolonged periods of time (2 weeks). Luciferase mRNA formulations also show expression in vitro (data not shown).

[0025] Figure 13 illustrates protamine increases TY-RNA payload onto ZnO NP more than LL-37 antiviral peptide.

[0026] Figure 14 illustrates spike protein expression by mRNA. Expression and validity of mRNA-encoded SARS-CoV-2 spike protein was evaluated by immunocytometric analysis of HEK-293A transfected cells probed with anti-spike mAbs (R2F4 and CR3022). HEK293A cells transfected with the plasmid constructs used to generate mRNA served as positive controls, whereas non-transfected cells served as negative controls.

[0027] Figures 15A-E illustrate ZNP-RNA characterization by transmission electron microscopy (Fig. 15A), UV spectroscopy (Fig. 15B), nanoparticle size analysis (Fig. 15C), zeta potential (Fig. 15D) and payload (Fig 15E).

[0028] Figures 16A-D illustrate binding, stability and expression of ZNP-RNA and ZNP-mRNA as shown by RNA agarose gel electrophoresis (RAGE) (Fig. 16A), in vitro translation (Fig. 16B), cell free mRNA expression in lysates (Fig. 16C) and GFP mRNA cell delivery (Fig. 16D)

[0029] Figures 17A-B illustrate RNA Temperature-stabilization with Fig. 17A providing results from RAGE analysis of heat treated RNA , and Fig. 17B providing an expression analysis from heat treated ZNP-mRNA.

[0030] Figures 18A-F illustrate different RNA biological activities with Fig. 18A illustrating loading efficiency and expression of SARS-CoV-2 spike mRNA< Figs. 18B-F illustrating protein expression by immunostaining of HEK293A cells transfected with SARS-CoV-2 spike mRNA formulated in/as: (Fig. 18B) Zinc oxide nanoparticle-mRNA); (Fig. 18C) Protamine coated Zinc oxide nanoparticle (ZNP- mRNA) (Fig. 18D) Double protamine coated - one over the ZNP and other over mRNA (ZNP-protamine-mRNA- protamine) (Fig. 18E) Invitrogen™ Lipofectamine™ MessengerMAX™ transfection reagent (positive control); and (Fig. 18F) Mock-transfected cells which served as a negative control. The cells were probed with a recombinant humanized anti-SARS-CoV- 2 spike-specific neutralizing monoclonal antibody. The secondary antibody was antihuman IgG-Alkaline Phosphatase, and Fast Red was the substrate. Arrows show the cells expressing the spike protein.

[0031] Fig. 19 is a photograph illustrating that RNA elution from the particle is protamine-dependent using protamine high (1 :1 ), medium (1 :10), and low (1 :100);

[0032] Fig. 20 is a photograph illustrating the impact of buffer components on RNA elution from the particles;

[0033] Fig. 21 is an illustration of the sequence alignment of low molecular weight protamine, LL37, and penetratin peptide;

[0034] Fig. 22 is a graph illustrating the evidence for LL37 functioning as an RNA- binding peptide (RBP);

[0035] Fig. 23 is a photograph and a graph illustrating quantitative RNA agarose gel electrophoresis (RAGE) with from 0.093 to 3 micrograms RNA loaded per lane, stained, and analyzed as described in materials and methods.

DETAILED DESCRIPTION

[0036] The following detailed description and examples set forth preferred materials and procedures used in accordance with the present disclosure. It is to be understood, however, that this description and these examples are provided by way of illustration only, and nothing therein shall be deemed to be a limitation upon the overall scope of the present disclosure.

[0037] “Sequence Identity” as it is known in the art refers to a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, namely a reference sequence and a given sequence to be compared with the reference sequence. Sequence identity is determined by comparing the given sequence to the reference sequence after the sequences have been optimally aligned to produce the highest degree of sequence similarity, as determined by the match between strings of such sequences. Upon such alignment, sequence identity is ascertained on a position- by-position basis, e.g., the sequences are “identical” at a particular position if at that position, the nucleotides or amino acid residues are identical. The total number of such position identities is then divided by the total number of nucleotides or residues in the reference sequence to give % sequence identity. Sequence identity can be readily calculated by known methods, including but not limited to, those described in Computational Molecular Biology, Lesk, A. N., ed., Oxford University Press, New York (1988), Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H. G., eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinge, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York (1991 ); and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988), the teachings of which are incorporated herein by reference. Preferred methods to determine the sequence identity are designed to give the largest match between the sequences tested. Methods to determine sequence identity are codified in publicly available computer programs which determine sequence identity between given sequences. Examples of such programs include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research, 12(1 ):387 (1984)), BLASTP, BLASTN and FASTA (Altschul, S. F. et al., J. Molec. Biol., 215:403-410 (1990). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCVI NLM NIH Bethesda, MD 20894, Altschul, S. F. et al., J. Molec. Biol., 215:403-410 (1990), the teachings of which are incorporated herein by reference). These programs optimally align sequences using default gap weights in order to produce the highest level of sequence identity between the given and reference sequences. As an illustration, by a polynucleotide having a nucleotide sequence having at least, for example, 85%, preferably 90%, even more preferably 95% “sequence identity” to a reference nucleotide sequence, it is intended that the nucleotide sequence of the given polynucleotide is identical to the reference sequence except that the given polynucleotide sequence may include up to 15, preferably up to 10, even more preferably up to 5 point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, in a polynucleotide having a nucleotide sequence having at least 85%, preferably 90%, even more preferably 95% identity relative to the reference nucleotide sequence, up to 15%, preferably 10%, even more preferably 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 15%, preferably 10%, even more preferably 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5’ or 3’ terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. Analogously, by a polypeptide having a given amino acid sequence having at least, for example, 85%, preferably 90%, even more preferably 95% sequence identity to a reference amino acid sequence, it is intended that the given amino acid sequence of the polypeptide is identical to the reference sequence except that the given polypeptide sequence may include up to 15, preferably up to 10, even more preferably up to 5 amino acid alterations per each 100 amino acids of the reference amino acid sequence. In other words, to obtain a given polypeptide sequence having at least 85%, preferably 90%, even more preferably 95% sequence identity with a reference amino acid sequence, up to 15%, preferably up to 10%, even more preferably up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 15%, preferably up to 10%, even more preferably up to 5% of the total number of amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or the carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in the one or more contiguous groups within the reference sequence. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. However, conservative substitutions are not included as a match when determining sequence identity. [0038] “Sequence homology”, as used herein, refers to a method of determining the relatedness of two sequences. To determine sequence homology, two or more sequences are optimally aligned, and gaps are introduced if necessary. However, in contrast to “sequence identity”, conservative amino acid substitutions are counted as a match when determining sequence homology. In other words, to obtain a polypeptide or polynucleotide having 95% sequence homology with a reference sequence, 85%, preferably 90%, even more preferably 95% of the amino acid residues or nucleotides in the reference sequence must match or comprise a conservative substitution with another amino acid or nucleotide, or a number of amino acids or nucleotides up to 15%, preferably up to 10%, even more preferably up to 5% of the total amino acid residues or nucleotides, not including conservative substitutions, in the reference sequence may be inserted into the reference sequence. Preferably the homologous sequence comprises at least a stretch of 50, even more preferably 100, even more preferably 250, even more preferably 500 nucleotides.

[0039] A “conservative substitution” refers to the substitution of an amino acid residue or nucleotide with another amino acid residue or nucleotide having similar characteristics or properties including size, hydrophobicity, etc., such that the overall functionality does not change significantly.

[0040] It will be found that the immunogenic compositions comprising any of the disclosed vaccine candidates as provided herewith are very effective in reducing the severity of or incidence of clinical signs associated with coronavirus infections including COVID-19 up to and including the prevention of such signs. Further, such immunogenic compositions reduce the transmissibility of COVID-19.

[0041] The immunogenic compositions described herein can further include one or more other immunomodulatory agents such as, e. g., interleukins, interferons, or other cytokines. The immunogenic compositions can also include Gentamicin and Merthiolate. In another preferred embodiment, the present disclosure contemplates vaccine compositions comprising from about 1 ug/ml to about 60 pg/ml of antibiotics, and more preferably less than about 30 pg/ml of antibiotics. [0042] The terms “immunogenic protein”, “immunogenic polypeptide” or “immunogenic amino acid sequence” as used herein refer to any amino acid sequence which elicits an immune response in a host against a pathogen comprising said immunogenic protein, immunogenic polypeptide or immunogenic amino acid sequence. An “immunogenic protein”, “immunogenic polypeptide” or “immunogenic amino acid sequence” as used herein, includes the full-length sequence of any proteins, analogs thereof, or immunogenic fragments thereof. By “immunogenic fragment” is meant a fragment of a protein which includes one or more epitopes and thus elicits the immunological response against the relevant pathogen. Such fragments can be identified using any number of epitope mapping techniques, well known in the art. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, New Jersey. For example, linear epitopes may be determined by e.g., concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports. Such techniques are known in the art and described in, e.g., U.S. Patent No. 4,708,871 ; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81 :3998-4002; Geysen et al. (1986) Molec. Immunol. 23:709-715. Similarly, conformational epitopes are readily identified by determining spatial conformation of amino acids such as by, e.g., x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, supra. Synthetic antigens are also included within the definition, for example, polyepitopes, flanking epitopes, and other recombinant or synthetically derived antigens. See, e.g., Bergmann et al. (1993) Eur. J. Immunol. 23:2777-2781 ; Bergmann et al. (1996), J. Immunol. 157:3242-3249; Suhrbier, A. (1997), Immunol, and Cell Biol. 75:402-408; Gardner et al., (1998) 12th World AIDS Conference, Geneva, Switzerland, June 28-July 3, 1998. It is understood that immunogenic proteins of the present disclosure include the S protein sequence, the F-Tm sequence, the SPD sequence, and the CD40L sequence.

[0043] In the present description, the terms polypeptide, peptide and protein are interchangeable. [0044] In the present description, COVID-19 (the disease) and SARS-CoV2 (the causative agent) are used interchangeably.

[0045] Additionally, any composition or vaccine candidate of the disclosure can include one or more pharmaceutical-acceptable or veterinary-acceptable carriers. As used herein, “a pharmaceutical-acceptable carrier” or “veterinary-acceptable carrier” includes any and all solvents, dispersion media, coatings, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like. In some forms, the pharmaceutical or veterinary acceptable carrier is selected from the group consisting of a solvent, a dispersion media, a coating, a stabilizing agent, a preservative, an antimicrobial agent, an antifungal agent, an isotonic agent, an adsorption delaying agent, and any combination thereof.

[0046] It is understood that the immunogenic compositions described herein can be administered to any animal susceptible to coronavirus infection including humans, dogs, cats, ferrets, bats, cattle, camels, hamsters, horses, chimps, gorillas, anteaters, dolphins, alligators, and sheep. Further, administration of any of the immunogenic compositions described herein will reduce the incidence of, severity of, and transmission of SARS-CoV2.

[0047] An “immunogenic or immunological composition” refers to a composition of matter that (i) comprises at least one antigen which elicits an immunological response in the host of a cellular and/ or antibody-mediated immune response to the composition or vaccine of interest, or (ii) comprises a nucleic acid such as a plasmid DNA or messenger RNA encoding such an antigen. Usually, an “immunological response” includes but is not limited to one or more of the following effects: the production or activation of antibodies, B cells, helper T cells, suppressor T cells, and/or cytotoxic T cells and/or yS T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest. Preferably, the host will display either a therapeutic or protective immunological response such that resistance to new infection will be enhanced and/or the clinical severity of the disease reduced. Such protection will be demonstrated by either a reduction in the severity or prevalence of, up to and including a lack of symptoms normally displayed by an infected host, a quicker recovery time and/or a lowered viral titer in the infected host.

[0048] A “reduction” in terms of incidence of symptoms, severity of symptoms, or transmissibility of infection is understood to encompass a comparison to a subject or group of subjects that has not received an administration of a composition of the present disclosure. Preferably, the reduction is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or even 100% in comparison to an animal that has not received an administration or dose of a composition described herein. It is understood that this percentage of reduction can be in terms of the number of symptoms, incidence of symptoms, severity of symptoms, and transmissibility of infection. Further, this can apply to individual animals or groups of animals wherein the reduction is in terms of a percentage of animals infected or showing symptoms vs uninfected or not showing symptoms.

[0049] “Adjuvants” as used herein, can include aluminum hydroxide and aluminum phosphate, saponins e.g., Quil A, QS-21 (Cambridge Biotech Inc., Cambridge MA), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, AL), water-in-oil emulsion, oil- in-water emulsion, water-in-oil-in-water emulsion. The emulsion can be based in particular on light liquid paraffin oil (European Pharmacopea type); isoprenoid oil such as squalane or squalene oil resulting from the oligomerization of alkenes, in particular of isobutene or decene; esters of acids or of alcohols containing a linear alkyl group, more particularly plant oils, ethyl oleate, propylene glycol di-(caprylateZcaprate), glyceryl tri- (caprylate/caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearic acid esters. The oil is used in combination with emulsifiers to form the emulsion. The emulsifiers are preferably nonionic surfactants, in particular esters of sorbitan, of mannide (e.g. anhydromannitol oleate), of glycol, of polyglycerol, of propylene glycol and of oleic, isostearic, ricinoleic or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene-polyoxyethylene copolymer blocks, in particular the Pluronic products, especially L121 . See Hunter et al., The Theory and Practical Application of Adjuvants (Ed. Stewart-Tull, D. E. S.). JohnWiley and Sons, NY, pp51 -94 (1995) and Todd et al., Vaccine 15:564-570 (1997). For example, it is possible to use the SPT emulsion described on page 147 of “Vaccine Design, The Subunit and Adjuvant Approach” edited by M. Powell and M. Newman, Plenum Press, 1995, and the emulsion MF59 described on page 183 of this same book.

[0050] A further instance of an adjuvant is a compound chosen from the polymers of acrylic or methacrylic acid and the copolymers of maleic anhydride and alkenyl derivative. Advantageous adjuvant compounds are the polymers of acrylic or methacrylic acid which are cross-linked, especially with polyalkenyl ethers of sugars or polyalcohols. These compounds are known by the term carbomer (Phameuropa Vol. 8, No. 2, June 1996). Persons skilled in the art can also refer to U. S. Patent No. 2,909,462 which describes such acrylic polymers cross-linked with a polyhydroxylated compound having at least 3 hydroxyl groups, preferably not more than 8, the hydrogen atoms of at least three hydroxyls being replaced by unsaturated aliphatic radicals having at least 2 carbon atoms. The preferred radicals are those containing from 2 to 4 carbon atoms, e.g. vinyls, allyls and other ethylenically unsaturated groups. The unsaturated radicals may themselves contain other substituents, such as methyl. The products sold under the name Carbopol; (BF Goodrich, Ohio, USA) are particularly appropriate. They are cross-linked with an allyl sucrose or with allyl pentaerythritol. Among then, there may be mentioned Carbopol 974P, 934P and 971 P. Among the copolymers of maleic anhydride and alkenyl derivative, the copolymers EMA (Monsanto) which are copolymers of maleic anhydride and ethylene. The dissolution of these polymers in water leads to an acid solution that will be neutralized, preferably to physiological pH, in order to give the adjuvant solution into which the immunogenic, immunological or vaccine composition itself will be incorporated.

[0051] Further suitable adjuvants include, but are not limited to, the RIBI adjuvant system (Ribi Inc.), Block co-polymer (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), monophosphoryl lipid A, Avridine lipid-amine adjuvant, heat-labile enterotoxin from E. coli (recombinant or otherwise), cholera toxin, IMS 1314 or muramyl dipeptide among many others.

[0052] As used herein, “a pharmaceutical-acceptable carrier” or “veterinary- acceptable carrier” includes any and all solvents, dispersion media, coatings, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like.

OVERVIEW

[0053] With the COVID-19 pandemic and deaths from metastatic cancer at an all- time high, the search for a chemotherapeutic nanoparticle that can deliver targeted therapies to affected tissues is imperative. SARS-COV-2 infects cells in the lung, but the virus is also detected and causes pathology in the liver, kidney, spleen and brain. Zinc oxide (ZnO) nanoparticle (NP) is now considered a chemotherapeutic nanoparticle and its antimicrobial, antiviral and anticancer activity has now been well-characterized. There are surprisingly few in vivo studies of the distribution and in vivo tolerance of ZnO NP which we here describe by cross-comparing the bioimaging, fluorescence and ICP/MS signals in these tissues of primary concern for SARS-COV-2 and cancer metastasis. In cells and tissues, zinc is perhaps best known for the role it plays in stabilizing protein and RNA structure and interaction. Yet despite the recent clinical approval of antisense oligomer (ASO) and aptamer oligonucleotides and the well accepted role of RAS and this pathway in a variety of metastatic cancers, ZnO NP delivery of RAS binding protein (RBD) targeted ASO or aptamer has only recently been described by the inventors’ group. Although ZnO does emit a weak fluorescent signal its emission range is not conducive to ex vivo/in vivo imaging, but the inventors have recently demonstrated for the first time the unique optical properties of the zinc-based physiometacomposites (PMC), red-shifted and increased in fluorescence intensity when doped with cobalt (Co) or Nickel (Ni) or when synthesized as the sulfide [S] or selenide [Se] derivatives when doped with manganese (Mn) or iron (Fe) as more novel second generation PMC nanomaterials. Importantly these two latter materials show enhanced inhibition of the β-Gal enzyme associated with antimicrobial activity exhibit viral inhibition of porcine reproductive respiratory virus (PRRS) a safer surrogate animal model for SARS-CoV, and enhanced anticancer activity in combination with RAS- targeted ASO or aptamer or that targeting a putative regulatory region in SARS-CoV-2.

[0054] The inventors’ research group has studied the interaction mechanisms of zinc oxide nanoparticle (ZnO NP) to RNA extensively and patented 2-dimensional fluorescence difference spectroscopy as a quality control assay for RNA and protein bound ZnO NP. One area of particular interest among the inventors’ group has been the anticancer/antitumor properties of this material with more recent results as a RAS- targeted nanomedicine. We began initially to examine what was known about the immunogenicity of ZnO NP. ZnO NP being capable of activating cellular and molecular immunity we next examined the biological activity of the ZnO-poly l:C complex. Having broken the DNA vaccine cold chain with a US patent in 2013. Knowing that RNA vaccines are under even greater such restrictions, we have begun to examine the effect of zinc-based nanoparticles on RNA stability, expression and activity. For example, we developed a scalable method to synthesize zinc-based physiometacomposite (PMC) nanoparticles finding that these have improved biocompatibility, fluorescence and bioluminescence characteristics. As an initial coronavirus model, porcine reproductive respiratory virus (PRRSV) was chosen, the nanoparticles screened, with MnZnS demonstrating significant inhibition (Fig. 5). As model RNA molecules we have used poly l:C or torula yeast RNA (TY-RNA) and examined the effect initially of ZnO NP on RNA structure and temperature stability by differential scanning calorimetry (DSC) and by circular dichroism spectroscopy (CD) (Fig. 6). We next examined the temperaturestability imparted by binding protamine to ZnO NP prior to RNA attachment (Fig. 7) and its increase in particle-associated RNA relative to antiviral LL37 peptide (Fig. 8).

[0055] Certain embodiments of the present disclosure relate to compositions, including without limitation immunogenic compositions and/or vaccine compositions, comprising nucleic acids encoding a full-length or partial-length S protein of SARS-CoV- 2 and a pharmaceutically or veterinary acceptable carrier comprising a Zinc oxide (ZnO) nanoparticle (NP) or physiometacomposite (PMC).

[0056] Turning first to the nucleic acids used in these compositions, these are generally characterized in that they are selected from one or more of: a) a nucleotide sequence encoding a specific fragment of the sequence of SEQ ID No. 1 or one of its fragments; b) a nucleotide sequence homologous to a nucleotide sequence such as defined in a); c) a nucleotide sequence complementary to a nucleotide sequence such as defined in a) or b), and a nucleotide sequence of their corresponding RNA; d) a nucleotide sequence capable of hybridizing under stringent conditions with a sequence such as defined in a), b) or c); e) a nucleotide sequence comprising a sequence such as defined in a), b), c) or d); and f) a nucleotide sequence modified by a nucleotide sequence such as defined in a), b), c), d) or e).

[0057] Nucleotide, polynucleotide or nucleic acid sequence will be understood according to the present disclosure as meaning both a double-stranded or singlestranded DNA in the monomeric and dimeric (so-called in tandem) forms and the transcription products of said DNAs. Nucleic acids of this disclosure can include, without limitation, RNAs such as messenger RNAs (mRNAs), DNAs such as plasmid DNAs (pDNAs), RNA/DNA chimeras and like nucleic acid forms known in the art. These nucleic acids optionally include backbone modifications (e.g., phosphorothioate or morpholino linkages) and/or nucleobase modifications.

[0058] It must be understood that the present disclosure does not relate to the genomic nucleotide sequences taken in their natural environment, that is to say in the natural state. It concerns sequences which it has been possible to isolate, purify or partially purify, starting from separation methods such as, for example, ion-exchange chromatography, by exclusion based on molecular size, or by affinity, or alternatively fractionation techniques based on solubility in different solvents, or starting from methods of genetic engineering such as amplification, cloning and subcloning, it being possible for the sequences of the disclosure to be carried by vectors. All genes and vectors in the present application were generated using synthetic biology.

[0059] Complementary nucleotide sequence of a sequence of the disclosure is understood as meaning any DNA whose nucleotides are complementary to those of the sequence of the disclosure, and whose orientation is reversed (antiparallel sequence).

[0060] Hybridization under conditions of stringency with a nucleotide sequence according to the disclosure is understood as meaning a hybridization under conditions of temperature and ionic strength chosen in such a way that they allow the maintenance of the hybridization between two fragments of complementary DNA.

[0061] Among said nucleotide sequences according to the disclosure, are those corresponding to S sequences, and coding for polypeptides, such as, for example, SEQ ID No. 1. The nucleotide sequence fragments according to the disclosure can be obtained, for example, by specific amplification, such as PCR, or after digestion with appropriate restriction enzymes of nucleotide sequences according to the disclosure, these methods in particular being described in the work of Sambrook et al., 1989. Said representative fragments can likewise be obtained by chemical synthesis when their size is not very large and according to methods well known to persons skilled in the art.

[0062] Modified nucleotide sequence will be understood as meaning any nucleotide sequence obtained by mutagenesis according to techniques well known to the person skilled in the art, and containing modifications with respect to the normal sequences according to the disclosure, for example mutations in the regulatory and/or promoter sequences of polypeptide expression, especially leading to a modification of the rate of expression of said polypeptide or to a modulation of the replicative cycle.

[0063] Modified nucleotide sequence will likewise be understood as meaning any nucleotide sequence coding for a modified polypeptide such as defined below.

[0064] The present disclosure relates to nucleotide sequences of SARS-CoV-2 according to the disclosure, characterized in that they are selected from the sequences encoding SEQ ID No. 1 or sequences having at least 80, 85, 88, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.6, 99.7, 99.8, 99.9, or even 100% sequence identity or homology to SEQ ID NO. 1 , or one of their fragments.

[0065] The disclosure likewise relates to nucleotide sequences characterized in that they comprise a nucleotide sequence selected from: a) a nucleotide sequence encoding SEQ ID No. 1 , or one of their fragments; b) a nucleotide sequence of a specific fragment of a sequence such as defined in a); c) a homologous nucleotide sequence having at least 80, 85, 88, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.6, 99.7, 99.8, 99.9, or even 100%% identity with a sequence such as defined in a) or b); d) a complementary nucleotide sequence or sequence of RNA corresponding to a sequence such as defined in a), b) or c); and e) a nucleotide sequence modified by a sequence such as defined in a), b), c) or d).

[0066] As far as homology with the nucleotide sequences encoding SEQ ID No. 1 or one of their fragments is concerned, the homologous, especially specific, sequences having a percentage identity with SEQ ID No.1 , or one of their fragments of at least 80, 85, 88, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.6, 99.7, 99.8, 99.9, or even 100% are preferred. Said specific homologous sequences can comprise, for example, the sequences corresponding to S protein sequences of SARS-CoV-2. In the same manner, these specific homologous sequences can correspond to variations linked to mutations within strains of SARS-CoV-2 and especially correspond to truncations, substitutions, deletions and/or additions of at least one nucleotide.

[0067] Compositions according to this aspect of the disclosure generally comprise a nucleotide sequence that is (a) extracted or derived from the genome of SARS-CoV-2, and/or (b) encodes a SARS-CoV-2 gene product, for instance the portion encoding the SARS-CoV-2 S protein of SEQ ID NO. 1. In some forms, the nucleotide sequence encodes SARS-CoV-2 S protein that has at least 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.6, 99.7, 99.8, 99.9, or even 100% sequence homology with SEQ ID NO. 1 and optionally include one or more of a signal sequence, receptor binding domains, a linker, a fusion protein transmembrane, a 2A cleavable peptide, a signal sequence, a surfactant protein tetramerization domain, and CD40 ligand (CD40L). In some forms, the signal sequence is a F signal protein, preferably SEQ ID No. 2. In some forms, the linker is SEQ ID NO. 3. In some forms, a tag such as a His tag or FLAG tag is included, but not required. In some forms, there are no tags. In some forms, the fusion protein transmembrane is SEQ ID NO. 4. In some forms, the 2A cleavable peptide is SEQ ID NO. 5. In some forms, the signal sequence is a CD5 signal sequence, preferably SEQ ID NO. 6. In some forms, the surfactant protein tetramerization domain is SEQ ID NO. 7. In some forms, the CD40L is SEQ ID NO. 8.

[0068] Any SARS-CoV-2 S protein would be effective as the source of the SARS- CoV-2 S encoding sequence and/or polypeptide as used herein. In some forms, the nucleic acid encodes a polypeptide having at least 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.6, 99.7, 99.8, 99.9, or even 100% sequence homology or sequence identity with SEQ ID NO. 1. A suitable SARS-CoV-2 S protein is that of SEQ ID NO. 1 , but it will be understood by those of skill in the art that this sequence could vary by as much as 10-20% in sequence homology and still retain the antigenic characteristics that render it useful in immunogenic compositions. The antigenic characteristics of an immunological composition can be, for example, estimated by challenge experiments. Moreover, the antigenic characteristic of a modified antigen is still retained, when the modified antigen confers at least 70%, preferably 80%, more preferably 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% of the protective immunity as compared to the SARS-CoV-2 S protein of SEQ ID NO. 1. In some forms, immunogenic portions of a SARS-CoV-2 S protein are used as the antigenic component in the composition. The term “immunogenic portion” as used herein refers to truncated and/or substituted forms, or fragments of SARS-CoV-2 S protein and/or polynucleotide, respectively. Preferably, such truncated and/or substituted forms, or fragments will comprise at least 6 contiguous amino acids from the full-length S polypeptide. More preferably, the truncated or substituted forms, or fragments will have at least 10, more preferably at least 15, and still more preferably at least 19 contiguous amino acids from the full-length S polypeptide. It is further understood that such sequences may be a part of larger fragments or truncated forms. Preferably, such truncated or substituted forms, or fragments will comprise at least 18 contiguous nucleotides from the full-length S nucleotide sequence, e.g. of SEQ ID NO. 1. More preferably, the truncated or substituted forms, or fragments will have at least 30, more preferably at least 45, and still more preferably at least 57 contiguous nucleotides the full-length S nucleotide sequence.

[0069] Turning next to zinc oxide nanoparticles, a variety of ZnO NPs are suitable for use in compositions according to this aspect of the disclosure, including without limitation NPs derived from zinc oxide nanopowder compositions characterized by average particle sizes <100 nm or by specific surface areas of 15-25 m ² /g sold by, e.g., Sigma-Aldrich (St. Louis, MO, USA). ZnO NPs be modified to incorporate one or more functional agents that impart cell- or tissue specificity, emit a detectable signal, etc. Specificity may be imparted, for instance, by incorporation of moieties such as PMCs that are selectively bound or taken up by certain cell or tissue types, while detectable signals can be emitted by fluorophores or bioloumenescent PMCs as described in greater detail below. The ZnO NPs, in certain embodiments, further comprise a nucleic acid binding peptide such as a protamine such as recombinant human PRM1 or PRM2, or a pharmaceutically acceptable salt or other derivative thereof. Without wishing to be bound by any theory, it is believed that the inclusion of protamine in compositions of the present disclosure promotes condensation and stabilization of nucleic acids within the compositions.

[0070] In a further aspect of the present disclosure, an immunogenic composition effective for lessening the severity and/or reducing the incidence of clinical symptoms associated with SARS-CoV-2 infection, and/or reducing the transmissibility of SARS- CoV-2, comprising a nucleic acid encoding a SARS-CoV-2 S protein is provided. Preferably, the SARS-CoV-2 S protein encoded by the nucleic acid is selected from the group consisting of: 1 ) a polypeptide comprising the sequence of SEQ ID NO: 1 ; 2) any polypeptide that is at least 90% homologous to the polypeptide of 1 ); 3) any immunogenic portion of the polypeptides of 1 ) and/or 2); or 4) the immunogenic portion of 3), comprising at least 10 contiguous amino acids included in the sequence of SEQ ID NO: 1.

[0071] In preferred forms, these immunogenic portions will have the immunogenic characteristics of SARS-CoV-2 S protein that has at least 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.6, 99.7, 99.8, 99.9, or even 100% sequence homology with SEQ ID NO. 1.

[0072] Those of skill in the art will understand that compositions of the present disclosure may incorporate known injectable, physiologically acceptable, sterile solutions. For preparing a ready-to-use solution for parenteral injection or infusion, aqueous isotonic solutions, such as e.g. saline or corresponding plasma protein solutions are readily available. In addition, the immunogenic and vaccine compositions of the present disclosure can include diluents, isotonic agents, stabilizers, or adjuvants. Diluents can include water, saline, dextrose, ethanol, glycerol, and the like. Isotonic agents can include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Stabilizers include albumin and alkali salts of ethylendiamintetracetic acid, among others. Suitable adjuvants, are those described above.

[0073] According to a further aspect, the immunogenic composition of the present disclosure further comprises a pharmaceutical acceptable salt, preferably a phosphate salt in physiologically acceptable concentrations. Preferably, the pH of said immunogenic composition is adjusted to a physiological pH, meaning between about 6.5 and 7.5.

[0074] The immunogenic compositions described herein can further include one or more other immunomodulatory agents such as, e. g., interleukins, interferons, or other cytokines. The immunogenic compositions can also include Gentamicin and Merthiolate. In another preferred embodiment, the present disclosure contemplates vaccine compositions comprising from about 1 ug/ml to about 60 pg/ml of antibiotics, and more preferably less than about 30 pg/ml of antibiotics.

[0075] It will be found that the immunogenic compositions comprising nucleic acids encoding recombinant SARS-CoV-2 S protein as provided herewith are very effective in reducing the severity of or incidence of clinical signs associated with SARS-CoV-2 infections up to and including the prevention of such signs. Additionally, the immunogenic compositions will be effective at reducing the transmissibility of SARS- CoV-2.

[0076] Another aspect of the present disclosure relates to a kit. Generally, the kit includes a container comprising at least one dose of a vaccine composition protein as provided herewith, wherein one dose comprises a pharmaceutically effective quantity of an immunogenic composition comprising a nucleic acid encoding SARS-CoV-2 S protein. Said container can comprise from 1 to 250 doses of the immunogenic composition. In some preferred forms, the container contains 1 , 10, 25, 50, 100, 150, 200, or 250 doses of the immunogenic composition of nucleic acid encoding SARS- CoV-2 S protein. It may be advantageous, in the case of containers comprising more than one dose of the immunogenic composition, for such compositions to further comprise an anti-microbiological active agent. Those agents are for example, antibiotics including Gentamicin and Merthiolate and the like. Thus, one aspect of the present disclosure relates to a container that comprises from 1 to 250 doses of the immunogenic composition of a nucleic acid encoding SARS-CoV-2 S protein, wherein one dose comprises a pharmaceutically effective quantity of the immunogenic composition, and an antimicrobial selected from Gentamicin and/or Merthiolate, preferably from about 1 pg/ml to about 60 pg/ml of antibiotics, and more preferably less than about 30 pg/ml. In preferred forms, the kit also includes an instruction manual, including the information for the intramuscular application of at least one dose of the immunogenic composition of SARS-CoV-2 S protein into animals, to lessen the incidence and/or severity of clinical symptoms associated with SARS-CoV-2 infection. Moreover, according to a further aspect, said instruction manual comprises the information of a second or further administration(s) of at least one dose of the immunogenic composition of SARS-CoV-2 S, wherein the second administration or any further administration is at least 14 days beyond the initial or any former administration. In some preferred forms, said instruction manual also includes the information, to administer an immune stimulant. Preferably, said immune stimulant shall be given at least twice. Preferably, at least 3, more preferably at least 5, and even more preferably at least 7 days are between the first and the second or any further administration of the immune stimulant. Preferably, the immune stimulant is given at least 10 days, preferably 15, even more preferably 20, and still even more preferably at least 22 days beyond the initial administration of the immunogenic composition of SARS-CoV-2 S protein. It is understood that any immune stimulant known to a person skilled in the art can also be used. “Immune stimulant” as used herein, means any agent or composition that can trigger a general immune response, preferably without initiating or increasing a specific immune response, for example the immune response against a specific pathogen. It is further instructed to administer the immune stimulant in a suitable dose. The kit may also comprise a second container, including at least one dose of the immune stimulant.

[0077] A further aspect of the present disclosure relates to the kit as described above, comprising the immunogenic composition of SARS-CoV-2 S as provided herewith and the instruction manual, wherein the instruction manual further includes the information to administer the SARS-CoV-2 S immunogenic composition together, or around the same time as, with an immunogenic composition that comprises an additional antigen effective for reducing the severity of or incidence of clinical signs related to another mammalian pathogen. Preferably, the manual contains the information of when the SARS-CoV-2 S containing composition and the immunogenic composition that comprises an additional antigen are administered. [0078] A further aspect, relates to the use of any of the compositions provided herewith as a medicament, including as a veterinary medicament, even more preferably as a vaccine. Moreover, the present disclosure also relates to the use of any of the compositions described herein, for the preparation of a medicament for lessening the severity of clinical symptoms associated with SARS-CoV-2 infection. Preferably, the medicament is for the prevention of a SARS-CoV-2 infection in an animal susceptible to infection with SARS-CoV-2.

[0079] A further aspect relates to a method for (1 ) the prevention of an infection, or re-infection with SARS-CoV-2 or (2) the reduction in incidence or severity of or elimination of clinical symptoms caused by SARS-CoV-2 in a subject, comprising administering any of the immunogenic compositions provided herewith to a subject in need thereof. It is understood that the reduction is in comparison to a subject that has not received an administration of a composition of the present disclosure. Preferably, the reduction is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or even 100% in comparison to an animal that has not received an administration or dose of a composition described herein. It is understood that this percentage of reduction can be in terms of the number of symptoms, incidence of symptoms, or severity of symptoms. Further, this can apply to individual subjects, animals, or groups of subjects or animals. Preferably, one dose or two doses of the immunogenic composition is/are administered, wherein one dose preferably comprises at least about 2 pg SARS-CoV-2 S protein. A further aspect relates to the method of treatment as described above, wherein a second application of the immunogenic composition is administered. Preferably, the second administration is done with the same immunogenic composition, preferably having the same amount of SARS-CoV-2 S protein. Preferably, the second administration is done at least 14 days beyond the initial administration, even more preferably at least 4 weeks beyond the initial administration. In preferred forms, the method is effective after just a single dose of the immunogenic composition and does not require a second or subsequent administration in order to confer the protective benefits upon the subject.

[0080] According to a further aspect, the present disclosure provides a multivalent combination vaccine which includes an immunological agent effective for reducing the incidence of or lessening the severity of SARS-CoV-2 infection, and at least one immunological active component against another disease-causing organism in mammals.

[0081] In particular the immunological agent effective for reducing the incidence of or lessening the severity of SARS-CoV-2 infection is a SARS-CoV-2 antigen. Preferably, said SARS-CoV-2 antigen is a SARS-CoV-2 S protein as provided herewith, or any immunogenic composition as described above, that comprises SARS-CoV-2 S protein, such as a SARS-CoV-2 S protein that has at least 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.6, 99.7, 99.8, 99.9, or even 100% sequence homology with SEQ ID NO. 1.

[0082] An “immunological active component” as used herein means a component that induces or stimulates the immune response in an animal to which said component is administered. Said immune response may be directed to said component or to a microorganism comprising said component. According to a further embodiment, the immunological active component is an attenuated microorganism, including modified live virus (MLV), a killed-microorganism or at least an immunological active part of a microorganism.

[0083] “Immunological active part of a microorganism” as used herein means a protein-, sugar-, and or glycoprotein containing fraction of a microorganism that comprises at least one antigen that induces or stimulates the immune response in an animal to which said component is administered. According to a preferred embodiment, said immune response is directed to said immunological active part of a microorganism or to a microorganism comprising said immunological active part.

[0084] Also included within the scope of the present disclosure are biologically functional plasmids, viral vectors and the like that contain the new recombinant nucleic acid molecules described herein, suitable host cells transfected by the vectors comprising the molecular DNA clones and the immunogenic polypeptide expression products. Some particularly preferred immunogenic protein expression products will have the amino acid sequence set forth in SEQ ID NO: 1. The biologically active variants thereof are further encompassed by the disclosure. One of ordinary skill in the art would know how to modify, substitute, delete, etc., amino acid(s) from the polypeptide sequence and produce biologically active variants that retain the same, or substantially the same, activity as the parent sequence without undue effort.

[0085] To produce the immunogenic nucleic acid products of this disclosure, the process may include in vitro transcription utilizing a DNA substrate such as a plasmid DNA. Alternatively, or additionally, the process may include chemical synthesis of oligonucleotides using, e.g., phosphoram idite or other functionalized monomers.

[0086] Still further, the present disclosure relates to vaccines or immunogenic compositions comprising a pharmaceutically acceptable vehicle and a nucleic acid encoding a single polypeptide, wherein the single polypeptide consists of SEQ ID No. 1.

[0087] Additionally, the present disclosure relates to methods of immunizing a mammal against SARS-CoV-2 comprising administering to a mammal an effective amount of a vaccine or immunogenic composition described above.

[0088] The present disclosure likewise encompasses the polypeptides encoded by a nucleotide sequence according to the disclosure, preferably a polypeptide whose sequence is represented by a fragment, especially a specific fragment, these six amino acid sequences corresponding to the polypeptides which can be encoded according to SEQ ID No. 1.

[0089] The disclosure also relates to the polypeptides, characterized in that they comprise a polypeptide selected from: a) a specific fragment of at least 5 amino acids of a polypeptide of an amino acid sequence according to the disclosure; b) a polypeptide homologous to a polypeptide such as defined in a); c) a specific biologically active fragment of a polypeptide such as defined in a) or b); and d) a polypeptide modified by a polypeptide such as defined in a), b) or c).

[0090] Among the polypeptides according to the disclosure, the polypeptide of amino acid sequence SEQ ID No. 1 , is also preferred, these polypeptides being especially capable of specifically recognizing the antibodies produced during infection by SARS- CoV-2. These polypeptides thus have epitopes specific for the SARS-CoV-2 and can thus be used in particular in the diagnostic field or as immunogenic agent to confer protection in an animal against infection by SARS-CoV-2. [0091] In the present description, the terms polypeptide, peptide and protein are interchangeable.

[0092] It must be understood that the disclosure does not relate to the polypeptides in natural form, that is to say that they are not taken in their natural environment but that they can be isolated or obtained by purification from natural sources, or else obtained by genetic recombination, or alternatively by chemical synthesis and that they can thus contain unnatural amino acids, as will be described below.

[0093] Polypeptide fragment according to the disclosure is understood as designating a polypeptide containing at least 5 consecutive amino acids, preferably 10 consecutive amino acids or 15 consecutive amino acids.

[0094] In the present disclosure, specific polypeptide fragment is understood as designating the consecutive polypeptide fragment encoded by a specific fragment nucleotide sequence according to the disclosure.

[0095] Homologous polypeptide will be understood as designating the polypeptides having, with respect to the natural polypeptide, certain modifications such as, in particular, a deletion, addition or substitution of at least one amino acid, a truncation, a prolongation, a chimeric fusion, and/or a mutation. Among the homologous polypeptides, those are preferred whose amino acid sequence has at least 80, 85, 88, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.6, 99.7, 99.8, 99.9, or even 100%, homology with the sequences of amino acids of polypeptides according to the disclosure.

[0096] Specific homologous polypeptide will be understood as designating the homologous polypeptides such as defined above and having a specific fragment of polypeptide according to the disclosure.

[0097] In the case of a substitution, one or more consecutive or nonconsecutive amino acids are replaced by “equivalent” amino acids. The expression “equivalent” amino acid is directed here at designating any amino acid capable of being substituted by one of the amino acids of the base structure without, however, essentially modifying the biological activities of the corresponding peptides and such that they will be defined by the following. These equivalent amino acids can be determined either by depending on their structural homology with the amino acids which they substitute, or on results of comparative tests of biological activity between the different polypeptides, which are capable of being carried out. By way of example, the possibilities of substitutions capable of being carried out without resulting in an extensive modification of the biological activity of the corresponding modified polypeptides will be mentioned, the replacement, for example, of leucine by valine or isoleucine, of aspartic acid by glutamic acid, of glutamine by asparagine, of arginine by lysine etc., the reverse substitutions naturally being envisageable under the same conditions.

[0098] The specific homologous polypeptides likewise correspond to polypeptides encoded by the specific homologous nucleotide sequences such as defined above and thus comprise in the present definition the polypeptides which are mutated or correspond to variants which can exist in SARS-CoV-2, and which especially correspond to truncations, substitutions, deletions and/or additions of at least one amino acid residue.

[0099] Specific biologically active fragment of a polypeptide according to the disclosure will be understood in particular as designating a specific polypeptide fragment, such as defined above, having at least one of the characteristics of polypeptides according to the disclosure, especially in that it is: capable of inducing an immunogenic reaction directed against a SARS-CoV-2; and/or capable of being recognized by a specific antibody of a polypeptide according to the disclosure; and/or capable of linking to a polypeptide or to a nucleotide sequence of SARS-CoV-2; and/or capable of exerting a physiological activity, even partial, such as, for example, a dissemination or structural (capsid) activity; and/or capable of modulating, of inducing or of inhibiting the expression of SARS-CoV-2 gene or one of its variants, and/or capable of modulating the replication cycle of SARS-CoV-2 in the cell and/or the host organism.

[0100] The polypeptide fragments according to the disclosure can correspond to isolated or purified fragments naturally present in a SARS-CoV-2 or correspond to fragments which can be obtained by cleavage of said polypeptide by a proteolytic enzyme, such as trypsin or chymotrypsin or collagenase, or by a chemical reagent, such as cyanogen bromide (CNBr) or alternatively by placing said polypeptide in a very acidic environment, for example at pH 2.5. Such polypeptide fragments can likewise just as easily be prepared by chemical synthesis, from hosts transformed by an expression vector according to the disclosure containing a nucleic acid allowing the expression of said fragments, placed under the control of appropriate regulation and/or expression elements.

[0101] “Modified polypeptide” of a polypeptide according to the disclosure is understood as designating a polypeptide obtained by genetic recombination or by chemical synthesis as will be described below, having at least one modification with respect to the normal sequence. These modifications will especially be able to bear on amino acids at the origin of a specificity, of pathogenicity and/or of virulence, or at the origin of the structural conformation, and of the capacity of membrane insertion of the polypeptide according to the disclosure. It will thus be possible to create polypeptides of equivalent, increased or decreased activity, and of equivalent, narrower, or wider specificity. Among the modified polypeptides, it is necessary to mention the polypeptides in which up to 5 amino acids can be modified, truncated at the N- or C- terminal end, or even deleted or added.

[0102] As is indicated, the modifications of the polypeptide will especially have as objective: to render it capable of modulating, of inhibiting or of inducing the expression of at least one SARS-CoV-2 gene and/or capable of modulating the replication cycle of SARS-CoV-2 in the cell and/or the host organism, of allowing its incorporation into vaccine compositions, and/or of modifying its bioavailability as a compound for therapeutic use.

[0103] The methods allowing said modulations on eukaryotic or prokaryotic cells to be demonstrated are well known to the person skilled in the art. It is likewise well understood that it will be possible to use the nucleotide sequences coding for said modified polypeptides for said modulations, for example through vectors according to the disclosure and described below, in order, for example, to prevent or to treat the pathologies linked to the infection. [0104] The preceding modified polypeptides can be obtained by using combinatorial chemistry, in which it is possible to systematically vary parts of the polypeptide before testing them on models, cell cultures or microorganisms for example, to select the compounds which are most active or have the properties sought.

[0105] Chemical synthesis likewise has the advantage of being able to use unnatural amino acids, or nonpeptide bonds. Thus, in order to improve the duration of life of the polypeptides according to the disclosure, it may be of interest to use unnatural amino acids, for example in D form, or else amino acid analogs, especially sulfur-containing forms, for example.

[0106] Finally; it will be possible to integrate the structure of the polypeptides according to the disclosure, its specific or modified homologous forms, into chemical structures of polypeptide type or others. Thus, it may be of interest to provide at the island C-terminal ends compounds not recognized by the proteases.

[0107] The nucleotide sequences coding for a polypeptide according to the disclosure are likewise part of the disclosure.

[0108] The disclosure likewise relates to nucleotide sequences utilizable as a primer or probe, characterized in that said sequences are selected from the nucleotide sequences according to the disclosure.

[0109] The disclosure additionally relates to the use of a nucleotide sequence according to the disclosure as a primer or probe for the detection and/or the amplification of nucleic acid sequences.

[0110] The nucleotide sequences according to the disclosure can thus be used to amplify nucleotide sequences, especially by the PCR technique (polymerase chain reaction) (Erlich, 1989; Innis et al., 1990; Rolfs et al., 1991 ; and White et al., 1997). These oligodeoxyribonucleotide or oligoribonucleotide primers advantageously have a length of at least 8 nucleotides, preferably of at least 12 nucleotides, and even more preferentially at least 20 nucleotides. Other amplification techniques of the target nucleic acid can be advantageously employed as alternatives to PCR. [0111] The nucleotide sequences of the disclosure, in particular the primers according to the disclosure, can likewise be employed in other procedures of amplification of a target nucleic acid, such as: the TAS technique (Transcription-based Amplification System), described by Kwoh et al. in 1989; the 3SR technique (Self- Sustained Sequence Replication), described by Guatelli et al. in 1990; the NASBA technique (Nucleic Acid Sequence Based Amplification), described by Kievitis et al. in 1991 ; the SDA technique (Strand Displacement Amplification) (Walker et al., 1992); the TMA technique (Transcription Mediated Amplification).

[0112] The polynucleotides of the disclosure can also be employed in techniques of amplification or of modification of the nucleic acid serving as a probe, such as: the LCR technique (Ligase Chain Reaction), described by Landegren et al. in 1988 and improved by Barany et al. in 1991 , which employs a thermostable ligase; the RCR technique (Repair Chain Reaction), described by Segev in 1992; the CPR technique (Cycling Probe Reaction), described by Duck et al. in 1990; the amplification technique with Q- beta replicase, described by Miele et al. in 1983 and especially improved by Chu et al. in 1986, Lizardi et al. in 1988, then by Burg et al. as well as by Stone et al. in 1996.

[0113] In the case where the target polynucleotide to be detected is possibly an RNA, for example an mRNA, it will be possible to use, prior to the employment of an amplification reaction with the aid of at least one primer according to the disclosure or to the employment of a detection procedure with the aid of at least one probe of the disclosure, an enzyme of reverse transcriptase type in order to obtain a cDNA from the RNA contained in the biological sample. The cDNA obtained will thus serve as a target for the primer(s) or the probe(s) employed in the amplification or detection procedure according to the disclosure.

[0114] The detection probe will be chosen in such a manner that it hybridizes with the target sequence or the amplicon generated from the target sequence. By way of sequence, such a probe will advantageously have a sequence of at least 12 nucleotides, in particular of at least 20 nucleotides, and preferably of at least 100 nucleotides. [0115] The disclosure also comprises the nucleotide sequences utilizable as a probe or primer according to the disclosure, characterized in that they are labeled with a radioactive compound or with a nonradioactive compound. The unlabeled nucleotide sequences can be used directly as probes or primers, although the sequences are generally labeled with a radioactive element ( ³² P, ³⁵ S, ³ H, ¹²⁵ l) or with a nonradioactive molecule (biotin, acetylaminofluorene, digoxigenin, 5-bromodeoxyuridine, fluorescein) to obtain probes which are utilizable for numerous applications. Examples of nonradioactive labeling of nucleotide sequences are described, for example, in French Patent No. 78.10975 or by Urdea et al. or by Sanchez-Pescador et al. in 1988. In the latter case, it will also be possible to use one of the labeling methods described in patents FR-2 422 956 and FR-2 518 755.

[0116] The hybridization technique can be carried out in various manners (Matthews et al., 1988). The most general method consists in immobilizing the nucleic acid extract of cells on a support (such as nitrocellulose, nylon, polystyrene) and in incubating, under well-defined conditions, the immobilized target nucleic acid with the probe. After hybridization, the excess of probe is eliminated and the hybrid molecules formed are detected by the appropriate method (measurement of the radioactivity, of the fluorescence or of the enzymatic activity linked to the probe).

[0117] The disclosure likewise comprises the nucleotide sequences according to the disclosure, characterized in that they are immobilized on a support, covalently or noncovalently.

[0118] According to another advantageous mode of employing nucleotide sequences according to the disclosure, the latter can be used immobilized on a support and can thus serve to capture, by specific hybridization, the target nucleic acid obtained from the biological sample to be tested. If necessary, the solid support is separated from the sample and the hybridization complex formed between said capture probe and the target nucleic acid is then detected with the aid of a second probe, a so-called detection probe, labeled with an easily detectable element. [0119] Another subject of the present disclosure is a vector for the cloning and/or expression of a sequence, characterized in that it contains a nucleotide sequence according to the disclosure.

[0120] The vectors according to the disclosure, characterized in that they contain the elements allowing the expression in a determined host cell, are likewise part of the disclosure. The vector must then contain a promoter, signals of initiation and termination of translation, as well as appropriate regions of regulation of transcription. It must be able to be maintained stably in the host cell and can optionally have particular signals specifying the secretion of the translated protein. These different elements are chosen as a function of the host cell used. To this end, the nucleotide sequences according to the disclosure can be inserted into autonomous replication vectors within the chosen host, or integrated vectors of the chosen host. Such vectors will be prepared according to the methods currently used by the person skilled in the art, and it will be possible to introduce the clones resulting therefrom into an appropriate host by standard methods, such as, for example, lipofection, electroporation and thermal shock. The vectors according to the disclosure are, for example, vectors of plasmid or viral origin.

[0121] It is today possible to produce recombinant polypeptides in relatively large quantity by genetic engineering using the cells transformed by expression vectors according to the disclosure or using transgenic animals according to the disclosure. The procedures for preparation of a polypeptide of the disclosure in recombinant form, characterized in that they employ a vector and/or a cell transformed by a vector according to the disclosure and/or a transgenic animal comprising one of said transformed cells according to the disclosure, are themselves comprised in the present disclosure. Among said procedures for preparation of a polypeptide of the disclosure in recombinant form, the preparation procedures employing a vector, and/or a cell transformed by said vector and/or a transgenic animal comprising one of said transformed cells, containing a nucleotide sequence according to the disclosure coding for a polypeptide of SARS-CoV-2, are preferred. The recombinant polypeptides obtained as indicated above can just as well be present in glycosylated form as in nonglycosylated form and can or cannot have the natural tertiary structure. [0122] More particularly, the disclosure relates to a procedure for preparation of a polypeptide of the disclosure comprising the following steps: a) culture of transformed cells under conditions allowing the expression of a recombinant polypeptide by a nucleotide sequence according to the disclosure; b) if need be, recovery of said recombinant polypeptide.

[0123] When the procedure for preparation of a polypeptide of the disclosure employs a transgenic animal according to the disclosure, the recombinant polypeptide is then extracted from said animal.

[0124] The disclosure also relates to a polypeptide which is capable of being obtained by a procedure of the disclosure such as described previously.

[0125] The disclosure also comprises a procedure for preparation of a synthetic polypeptide, characterized in that it uses a sequence of amino acids of polypeptides according to the disclosure. The disclosure likewise relates to a synthetic polypeptide obtained by a procedure according to the disclosure.

[0126] The polypeptides according to the disclosure can likewise be prepared by techniques which are conventional in the field of the synthesis of peptides. This synthesis can be carried out in homogeneous solution or in solid phase. For example, reference can be made to the technique of synthesis in homogeneous solution described by Houben-Weyl in 1974. This method of synthesis consists in successively condensing, two by two, the successive amino acids in the order required, or in condensing amino acids and fragments formed previously and already containing several amino acids in the appropriate order, or alternatively several fragments previously prepared in this way, it being understood that it will be necessary to protect beforehand all the reactive functions carried by these amino acids or fragments, with the exception of amine functions of one and carboxyls of the other or vice-versa, which must normally be involved in the formation of peptide bonds, especially after activation of the carboxyl function, according to the methods well known in the synthesis of peptides. According to another preferred technique of the disclosure, recourse will be made to the technique described by Merrifield. To make a peptide chain according to the Merrifield procedure, recourse is made to a very porous polymeric resin, on which is immobilized the first C-terminal amino acid of the chain. This amino acid is immobilized on a resin through its carboxyl group and its amine function is protected. The amino acids which are going to form the peptide chain are thus immobilized, one after the other, on the amino group, which is deprotected beforehand each time, of the portion of the peptide chain already formed, and which is attached to the resin. When the whole of the desired peptide chain has been formed, the protective groups of the different amino acids forming the peptide chain are eliminated and the peptide is detached from the resin with the aid of an acid.

[0127] The disclosure additionally relates to hybrid polypeptides having at least one polypeptide according to the disclosure, and a sequence of a polypeptide capable of inducing an immune response in man or animals.

[0128] Advantageously, the antigenic determinant is such that it is capable of inducing a humoral and/or cellular response. It will be possible for such a determinant to comprise a polypeptide according to the disclosure in glycosylated form used with a view to obtaining immunogenic compositions capable of inducing the synthesis of antibodies directed against multiple epitopes. Said polypeptides or their glycosylated fragments are likewise part of the disclosure. These hybrid molecules can be formed, in part, of a polypeptide carrier molecule or of fragments thereof according to the disclosure, associated with a possibly immunogenic part, in particular an epitope of the diphtheria toxin, the tetanus toxin, a surface antigen of the hepatitis B virus (patent FR 79 21811 ), the VP1 antigen of the poliomyelitis virus or any other viral or bacterial toxin or antigen. The procedures for synthesis of hybrid molecules encompass the methods used in genetic engineering for constructing hybrid nucleotide sequences coding for the polypeptide sequences sought. It will be possible, for example, to refer advantageously to the technique for obtainment of genes coding for fusion proteins described by Minton in 1984. Said hybrid nucleotide sequences coding for a hybrid polypeptide as well as the hybrid polypeptides according to the disclosure characterized in that they are recombinant polypeptides obtained by the expression of said hybrid nucleotide sequences are likewise part of the disclosure. [0129] The disclosure likewise comprises the vectors characterized in that they contain one of said hybrid nucleotide sequences. The host cells transformed by said vectors, the transgenic animals comprising one of said transformed cells as well as the procedures for preparation of recombinant polypeptides using said vectors, said transformed cells and/or said transgenic animals are, of course, likewise part of the disclosure.

[0130] Further, the disclosure relates to the polypeptides according to the disclosure, labeled with the aid of an adequate label such as of the enzymatic, fluorescent or radioactive type. Such methods comprise, for example, the following steps: 1 ) deposition of determined quantities of a polypeptide composition according to the disclosure in the wells of a microtiter plate; 2) introduction into said wells of increasing dilutions of serum, or of a biological sample other than that defined previously, having to be analyzed,; 3) incubation of the microplate; and 4) introduction into the wells of the microtiter plate of labeled antibodies directed against mammal immunoglobulins, the labeling of these antibodies having been carried out with the aid of an enzyme selected from those which are capable of hydrolyzing a substrate by modifying the absorption of the radiation of the latter, at least at a determined wavelength, for example at 550 nm, detection, by comparison with a control test, of the quantity of hydrolyzed substrate.

[0131] The polypeptides according to the disclosure allow monoclonal or polyclonal antibodies to be prepared which are characterized in that they specifically recognize the polypeptides according to the disclosure. It will advantageously be possible to prepare the monoclonal antibodies from hybridomas according to the technique described by Kohler and Milstein in 1975. It will be possible to prepare the polyclonal antibodies, for example, by immunization of an animal, in particular a mouse, with a polypeptide or a DNA, according to the disclosure, associated with an adjuvant of the immune response, and then purification of the specific antibodies contained in the serum of the immunized animals on an affinity column on which the polypeptide which has served as an antigen has previously been immobilized. The polyclonal antibodies according to the disclosure can also be prepared by purification, on an affinity column on which a polypeptide according to the disclosure has previously been immobilized, of the antibodies contained in the serum of animals infected by a SARS-CoV-2. [0132] The disclosure likewise relates to a pharmaceutical composition comprising a compound selected from the following compounds: a) a nucleotide sequence according to the disclosure; b) a polypeptide according to the disclosure; c) a vector, a viral particle or a cell transformed according to the disclosure; d) an antibody according to the disclosure; and e) a compound capable of being selected by a selection method according to the disclosure; possibly in combination with a pharmaceutically acceptable carrier and, if need be, with one or more adjuvants of the appropriate immunity.

[0133] The disclosure further relates to a nanomaterial that is capable of operating as a pharmaceutically acceptable carrier and/or as a polypeptide/nucleic acid binding, stabilization, and delivery enhancing agent.

[0134] The disclosure also relates to an immunogenic and/or vaccine composition, characterized in that it comprises a compound selected from the following compounds: a) a nucleotide sequence according to the disclosure; b) a polypeptide according to the disclosure; c) a vector or a viral particle according to the disclosure; and d) a cell according to the disclosure.

[0135] In one embodiment, the vaccine composition according to the disclosure is characterized in that it comprises a mixture of at least two of said compounds a), b), c) and d) above and in that one of the two said compounds is related to the SARS-CoV-2.

[0136] In another embodiment of the disclosure, the vaccine composition is characterized in that it comprises at least one compound a), b), c), or d) above which is related to SARS-CoV-2. In still another embodiment, the vaccine composition is characterized in that it comprises at least one compound a), b), c), or d) above which is related to SARS-CoV-2 S protein.

[0137] A compound related to the SARS-CoV-2 is understood here as respectively designating a compound obtained from the genomic sequence of the SARS-CoV-2 and/or S protein of SARS-CoV-2.

[0138] The disclosure is additionally aimed at an immunogenic and/or vaccine composition, characterized in that it comprises at least one of the following compounds: 1 ) a nucleotide sequence encoding SEQ ID No. 1 or one of its fragments or homologues; 2) a polypeptide of sequence SEQ ID No. 1, SEQ ID No. 9, or SEQ ID No. 10 or one of their fragments, or a modification thereof; 3) a vector or a viral particle comprising a nucleotide sequence encoding SEQ ID No. 1 or one of its fragments or homologues; 4) a transformed cell capable of expressing a polypeptide of sequence SEQ ID No. 1 , or one of its fragments, or a modification thereof; or 5) a mixture of at least two of said compounds.

[0139] The disclosure also comprises an immunogenic and/or vaccine composition according to the disclosure, characterized in that it comprises said mixture of at least two of said compounds as a combination product for simultaneous, separate or protracted use for the prevention or the treatment of infection by a SARS-CoV-2.

[0140] The disclosure is likewise directed at a pharmaceutical composition according to the disclosure, for the prevention or the treatment of an infection by a SARS-CoV-2.

[0141] It is understood that “prevention” as used in the present disclosure, includes the complete prevention of infection by a SARS-CoV-2, but also encompasses a reduction in the severity of or incidence of clinical signs associated with or caused by SARS-CoV-2 infection. Such prevention is also referred to herein as a protective effect.

[0142] The disclosure likewise concerns the use of a composition according to the disclosure, for the preparation of a medicament intended for the prevention or the treatment of infection by a SARS-CoV-2.

[0143] Under another aspect, the disclosure relates to a vector, a viral particle or a cell according to the disclosure, for the treatment and/or the prevention of a disease by gene therapy.

[0144] Finally, the disclosure comprises the use of a vector, of a viral particle or of a cell according to the disclosure for the preparation of a medicament intended for the treatment and/or the prevention of a disease by gene therapy.

[0145] The polypeptides of the disclosure entering into the immunogenic or vaccine compositions according to the disclosure can be selected by techniques known to the person skilled in the art such as, for example, depending on the capacity of said polypeptides to stimulate the T cells, which is translated, for example, by their proliferation or the secretion of interleukins, and which leads to the production of antibodies directed against said polypeptides.

[0146] The pharmaceutical compositions according to the disclosure will contain an effective quantity of the compounds of the disclosure, that is to say in sufficient quantity of said compound(s) allowing the desired effect to be obtained, such as, for example, the modulation of the cellular replication of SARS-CoV-2. The person skilled in the art will know how to determine this quantity, as a function, for example, of the age and of the weight of the individual to be treated, of the state of advancement of the pathology, of the possible secondary effects and by means of a test of evaluation of the effects obtained on a population range, these tests being known in these fields of application.

[0147] According to the disclosure, said vaccine combinations will preferably be combined with a pharmaceutically or veterinary acceptable carrier and, if need be, with one or more adjuvants of the appropriate immunity.

[0148] Today, various types of vaccines are available for protecting animals or man against infectious diseases: attenuated living microorganisms (M. bovis--BCG for tuberculosis), inactivated microorganisms (influenza virus), a cellular extracts (Bordetella pertussis for whooping cough), recombinant proteins (surface antigen of the hepatitis B virus), polysaccharides (pneumococcal). Vaccines prepared from synthetic peptides or genetically modified microorganisms expressing heterologous antigens are in the course of experimentation. More recently still, recombined plasmid DNAs carrying genes coding for protective antigens have been proposed as an alternative vaccine strategy. This type of vaccination is carried out with a particular plasmid originating from a plasmid of E. coli which does not replicate in vivo and which codes uniquely for the vaccinating protein. Animals have been immunized by simply injecting the naked plasmid DNA into the muscle. This technique leads to the expression of the vaccine protein in situ and to an immune response of cellular type (CTL) and of humoral type (antibody). This double induction of the immune response is one of the principal advantages of the vaccination technique with naked DNA.

[0149] The constitutive nucleotide sequence of the vaccine composition according to the disclosure can be injected into the host after having been coupled to compounds which favor the penetration of this polynucleotide into the interior of the cell or its transport to the cell nucleus. The resultant conjugates can be encapsulated in polymeric microparticles, as described in the international application No. WO 94/27238 (Medisorb Technologies International).

[0150] According to another embodiment of the vaccine composition according to the disclosure, the nucleotide sequence, preferably a DNA, is complexed with DEAE- dextran (Pagano et al., 1967) or with nuclear proteins (Kaneda et al., 1989), with lipids (Feigner et al., 1987) or encapsulated in liposomes (Fraley et al., 1980) or else introduced in the form of a gel facilitating its transfection into the cells (Midoux et al., 1993, Pastore et al., 1994). The polynucleotide or the vector according to the disclosure can also be in suspension in a buffer solution or be combined with liposomes.

[0151] Advantageously, such a vaccine will be prepared according to the technique described by Tacson et al. or Huygen et al. in 1996 or alternatively according to the technique described by Davis et al. in the international application No. WO 95/11307.

[0152] Such a vaccine can likewise be prepared in the form of a composition containing a vector according to the disclosure, placed under the control of regulation elements allowing its expression in man or animal. It will be possible, for example, to use, by way of in vivo expression vector of the polypeptide antigen of interest, the plasmid pcDNA3 or the plasmid pcDNA1/neo, both marketed by Invitrogen (R&D Systems, Abingdon, United Kingdom). It is also possible to use the plasmid V1 Jns.tPA, described by Shiver et al. in 1995. Such a vaccine will advantageously comprise, apart from the recombinant vector, a saline solution, for example a sodium chloride solution.

[0153] As far as the vaccine formulations are concerned, these can comprise adjuvants of the appropriate immunity which are known to the person skilled in the art, such as, for example, those described above.

[0154] These compounds can be administered by the systemic route, in particular by the intravenous route, by the intramuscular, intradermal or subcutaneous route, or by the oral route. In a more preferred manner, the vaccine composition comprising polypeptides according to the disclosure will be administered by the intramuscular route, through the food or by nebulization several times, staggered over time. [0155] Their administration modes, dosages and optimum pharmaceutical forms can be determined according to the criteria generally taken into account in the establishment of a treatment adapted to an animal such as, for example, the age or the weight, the seriousness of its general condition, the tolerance to the treatment and the secondary effects noted. Preferably, the vaccine of the present disclosure is administered in an amount that is protective or provides a protective effect against SARS-CoV-2 infection.

[0156] For example, in the case of a vaccine according to the present disclosure comprising a polypeptide encoded by a nucleotide sequence of the genome of SARS- CoV-2, or a homologue or fragment thereof, the polypeptide will be administered one time or several times, spread out over time, directly or by means of a transformed cell capable of expressing the polypeptide, in an amount of about 0.1 to 10 pg per kilogram weight of the animal, preferably about 0.2 to about 5 pg/kg, more preferably about 0.5 to about 2 pg/kg for a dose.

[0157] The present disclosure likewise relates to the use of nucleotide sequences of SARS-CoV-2 according to the disclosure for the construction of autoreplicative retroviral vectors and the therapeutic applications of these, especially in the field of gene therapy in vivo.

[0158] The feasibility of gene therapy applied to man no longer needs to be demonstrated and this relates to numerous therapeutic applications like genetic diseases, infectious diseases and cancers. Numerous documents of the prior art describe the means of employing gene therapy, especially through viral vectors. Generally speaking, the vectors are obtained by deletion of at least some of the viral genes which are replaced by the genes of therapeutic interest. Such vectors can be propagated in a complementation cell line which supplies in trans the deleted viral functions in order to generate a defective viral vector particle for replication but capable of infecting a host cell. To date, the retroviral vectors are amongst the most widely used and their mode of infection is widely described in the literature accessible to the person skilled in the art.

[0159] The principle of gene therapy is to deliver a functional gene, called a gene of interest, of which the RNA or the corresponding protein will produce the desired biochemical effect in the targeted cells or tissues. On the one hand, the insertion of genes allows the prolonged expression of complex and unstable molecules such as RNAs or proteins which can be extremely difficult or even impossible to obtain or to administer directly. On the other hand, the controlled insertion of the desired gene into the interior of targeted specific cells allows the expression product to be regulated in defined tissues. For this, it is necessary to be able to insert the desired therapeutic gene into the interior of chosen cells and thus to have available a method of insertion capable of specifically targeting the cells or the tissues chosen. Some preferred genes of interest for the present disclosure are those that encode the S protein.

[0160] Among the methods of insertion of genes, such as, for example, microinjection, especially the injection of naked plasmid DNA, electroporation, homologous recombination, the use of viral particles, such as retroviruses, is widespread. However, applied in vivo, the gene transfer systems of recombinant retroviral type at the same time have a weak infectious power (insufficient concentration of viral particles) and a lack of specificity with regard to chosen target cells.

[0161] The production of cell-specific viral vectors, having a tissue-specific tropism, and whose gene of interest can be translated adequately by the target cells, is realizable, for example, by fusing a specific ligand of the target host cells to the N- terminal part of a surface protein of the envelope of SARS-CoV-2. It is possible to mention, for example, the construction of retroviral particles having the CD4 molecule on the surface of the envelope so as to target the human cells infected by the HIV virus, viral particles having a peptide hormone fused to an envelope protein to specifically infect the cells expressing the corresponding receptor or else alternatively viral particles having a fused polypeptide capable of immobilizing on the receptor of the epidermal growth factor (EGF). In another approach, single-chain fragments of antibodies directed against surface antigens of the target cells are inserted by fusion with the N-terminal part of the envelope protein.

[0162] For the purposes of the present disclosure, a gene of interest in use in the disclosure can be obtained from a eukaryotic or prokaryotic organism or from a virus by any conventional technique. It is, preferably, capable of producing an expression product having a therapeutic effect and it can be a product homologous to the cell host or, alternatively, heterologous. In the scope of the present disclosure, a gene of interest can code for an (1 ) intracellular or (2) membrane product present on the surface of the host cell or (3) secreted outside the host cell. It can therefore comprise appropriate additional elements such as, for example, a sequence coding for a secretion signal. These signals are known to the person skilled in the art.

[0163] In accordance with the aims pursued by the present disclosure, a gene of interest can code for a protein corresponding to all or part of a native protein as found in nature. It can likewise be a chimeric protein, for example arising from the fusion of polypeptides of various origins or from a mutant having improved and/or modified biological properties. Such a mutant can be obtained, by conventional biological techniques, by substitution, deletion and/or addition of one or more amino acid residues.

[0164] The disclosure thus relates to the vectors characterized in that they comprise a nucleotide sequence of SARS-CoV-2 according to the disclosure, and in that they additionally comprise a gene of interest (“GOI”).

[0165] The present disclosure likewise relates to viral particles generated from said vector according to the disclosure. It additionally relates to methods for the preparation of viral particles according to the disclosure, characterized in that they employ a vector according to the disclosure, including viral pseudoparticles (VLP, virus-like particles).

[0166] The disclosure likewise relates to animal cells transfected by a vector according to the disclosure. Likewise comprised in the disclosure are animal cells, especially mammalian, infected by a viral particle according to the disclosure.

[0167] Additional genetically engineered vaccines, which are desirable in the present disclosure, are produced by techniques known in the art. Such techniques involve, but are not limited to, further manipulation of recombinant DNA, modification of or substitutions to the amino acid sequences of the recombinant proteins and the like. Genetically engineered vaccines based on recombinant DNA technology are made, for instance, by identifying alternative portions of the viral gene encoding proteins responsible for inducing a stronger immune or protective response in animals (e.g., proteins derived from the S). Such identified genes or immuno-dominant fragments can be cloned into standard protein expression vectors, such as the baculovirus vector, and used to infect appropriate host cells. The host cells are cultured, thus expressing the desired vaccine proteins, which can be purified to the desired extent and formulated into a suitable vaccine product.

[0168] If the clones retain any undesirable natural abilities of causing disease, it is also possible to pinpoint the nucleotide sequences in the viral genome responsible for any residual virulence, and genetically engineer the virus avirulent through, for example, site-directed mutagenesis. Site-directed mutagenesis is able to add, delete or change one or more nucleotides. An oligonucleotide is synthesized containing the desired mutation and annealed to a portion of single stranded viral DNA. The hybrid molecule, which results from that procedure, is employed to transform bacteria. Then doublestranded DNA, which is isolated containing the appropriate mutation, is used to produce full-length DNA by ligation to a restriction fragment of the latter that is subsequently transfected into a suitable cell culture. Ligation of the genome into the suitable vector for transfer may be accomplished through any standard technique known to those of ordinary skill in the art. Transfection of the vector into host cells for the production of viral progeny may be done using any of the conventional methods such as calciumphosphate or DEAE-dextran mediated transfection, electroporation, protoplast fusion and other well-known techniques. The cloned virus then exhibits the desired mutation. Alternatively, two oligonucleotides can be synthesized which contain the appropriate mutation. These may be annealed to form double-stranded DNA that can be inserted in the viral DNA to produce full-length DNA.

[0169] Genetically engineered proteins, useful in vaccines, for instance, may be expressed in insect cells, yeast cells or mammalian cells. The genetically engineered proteins, which may be purified or isolated by conventional methods, can be directly inoculated into animals to confer protection against viral infection caused by SARS- CoV-2. An insect cell line (like HI-FIVE) can be transformed with a transfer vector containing nucleic acid molecules obtained from the virus or copied from the viral genome which encodes one or more of the immuno-dominant proteins of the virus. The transfer vector includes, for example, linearized baculovirus DNA and a plasmid containing the desired polynucleotides. The host cell line may be co-transfected with the linearized baculovirus DNA and a plasmid in order to make a recombinant baculovirus.

[0170] An immunologically effective amount of one of the vaccines or immunogenic compositions of the present disclosure is administered to an animal in need of protection against viral infection, pneumonia, fever, cough, and loss of taste and/or smell. The immunologically effective amount or the immunogenic amount that inoculates the animal can be easily determined or readily titrated by routine testing. An effective amount is one in which a sufficient immunological response to the vaccine is attained to protect the animal exposed to SARS-CoV-2. Preferably, the animal receiving a dose of a vaccine or immunogenic composition according to this disclosure is protected to an extent in which one to all of the adverse physiological symptoms or effects of the viral disease are significantly reduced, ameliorated or totally prevented.

[0171] The vaccine can be administered in a single dose or in repeated doses with single doses being preferred. Single dose vaccines provide protection after a single dose without the need for any booster or subsequent dosages. Protection can include the complete prevention of clinical signs of infection, or a lessening of the severity, duration, or likelihood of the manifestation of one or more clinical signs of infection.

[0172] Desirably, the vaccine is administered to a human or animal not yet exposed to the SARS-CoV-2 virus. When administered as a liquid, the present vaccine may be prepared in the form of an aqueous solution, syrup, an elixir, a tincture and the like. Such formulations are known in the art and are typically prepared by dissolution of the antigen and other typical additives in the appropriate carrier or solvent systems. Suitable carriers or solvents include, but are not limited to, water, saline, ethanol, ethylene glycol, glycerol, etc. Typical additives are, for example, certified dyes, flavors, sweeteners and antimicrobial preservatives such as thimerosal (sodium ethylmercurithiosalicylate). Such solutions may be stabilized, for example, by addition of partially hydrolyzed gelatin, sorbitol or cell culture medium, and may be buffered by conventional methods using reagents known in the art, such as sodium hydrogen phosphate, sodium dihydrogen phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate, a mixture thereof, and the like. [0173] Liquid formulations also may include suspensions and emulsions that contain suspending or emulsifying agents in combination with other standard co-formulants. These types of liquid formulations may be prepared by conventional methods. Suspensions, for example, may be prepared using a colloid mill. Emulsions, for example, may be prepared using a homogenizer. Fig. 12 illustrates the temperature stability for ZnO-Protamine-TYRNA that had been stored as a suspension in PBS in a closed eppendorf tube for 2 weeks at the specified temperatures at a high (H) concentration of protamine coating.

[0174] Parenteral formulations, designed for injection into body fluid systems, require proper isotonicity and pH buffering to the corresponding levels of body fluids. Isotonicity can be appropriately adjusted with sodium chloride and other salts as needed. Suitable solvents, such as ethanol or propylene glycol, can be used to increase the solubility of the ingredients in the formulation and the stability of the liquid preparation. Further additives that can be employed in the present vaccine include, but are not limited to, dextrose, conventional antioxidants and conventional chelating agents such as ethylenediamine tetraacetic acid (EDTA). Parenteral dosage forms must also be sterilized prior to use.

[0175] It should be appreciated that all scientific and technological terms used herein have the same meaning as commonly understood by those of ordinary skill in the art.

[0176] Another aspect of the present disclosure is the preparation of the combination vaccine(s) or immunogenic compositions. Such combinations can be between the different vaccine components described herein. For example, a vaccine of the present disclosure can include both protein portions and DNA portions of SARS-CoV-2, as described herein, which are administered concurrently or separately. Additionally, the combinations can be between the SARS-CoV-2 vaccine components described herein and antigens of other disease-causing organisms, such as those described above.

[0177] According to a further aspect, the vaccine or immunogenic composition is first dehydrated. If the composition is first lyophilized or dehydrated by other methods, then, prior to vaccination, said composition is rehydrated in aqueous (e.g. saline, PBS (phosphate buffered saline)) or non-aqueous solutions (e.g. oil emulsion (mineral oil, or vegetable/metabolizable oil based/single or double emulsion based), aluminum-based, carbomer based adjuvant).

[0178] Compositions according to the disclosure may be applied orally, though compositions of similar type are most frequently delivered parenterally, particularly intravenously, intramuscularly, transdermally, intradermally, intratracheally, intravaginally or intranasally. In an animal body, it can prove advantageous to apply the pharmaceutical compositions as described above via an intravenous injection or by direct injection into target tissues. For systemic application, the intravenous, intravascular, intramuscular, intranasal, intraarterial, intraperitoneal, oral, or intrathecal routes are preferred. A more local application can be effected subcutaneously, intradermally, intracutaneously, intracardially, intralobally, intramedullarly, intrapulmonarily or directly in or near the tissue to be treated (connective-, bone-, muscle-, nerve-, epithelial tissue). Depending on the desired duration and effectiveness of the treatment, the compositions according to the disclosure may be administered once or several times, also intermittently, for instance on a daily basis for several days, weeks or months, and in different dosages.

[0179] In a further aspect of the disclosure, a method for protecting humans from SARS-CoV-2 infection transmitted by animals is provided. In general, the method comprises administering at least one dose of a composition comprising the S protein from SARS-CoV-2 to a susceptible animal. Preferred susceptible animals are provided above. Such administration prevents or reduces the transmission from an infected animal to humans. In preferred forms, the transmission to humans will be reduced by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or even 100%. Such a method removes a potential host or reservoir for SARS-CoV-2 and reduces the overall circulation of SARS- CoV-2. Preferred compositions are selected from those disclosed herein.

[0180] In a further aspect of the disclosure, a method of inducing an immunological response against SARS-CoV-2 is provided. In general, the method comprises the steps of administering a composition of the present disclosure, to an animal in need of prevention or treatment of SARS-CoV-2 associated disease. In some forms, the S protein has at least 90% sequence homology with SEQ ID NO. 1. In some forms, the pharmaceutical-acceptable carrier comprises a stabilizing agent and/or a preservative and/or an antimicrobial agent. In some forms, the protein is present in the final composition in an amount from 0.2 to about 400 pg/ml. In some forms, the composition further comprises an immune stimulant. In some forms, the composition further comprises at least one immunological active component against another diseasecausing organism. In some forms, the animal in need thereof is selected from the group consisting of humans, dogs, cats, ferrets, bats, cattle, camels, hamsters, horses, chimps, gorillas, anteaters, dolphins, alligators, and sheep. In some forms, the composition is administered a first time and a second time. In some forms, the second time is at least 10 days after the first time.

[0181] Immunogenic compositions of the present disclosure exhibit improved stability relative to currently available mRNA vaccines, advantageously permitting distribution without the need for an unbroken cold-chain, permitting higher temperature storage of vaccine compositions once distributed, and potentially extending the useful shelf life of multi-dosage vaccine and immunogenic composition forms both before and after they are opened. Likely the most relevant data to this application are the in vivo distribution (Fig. 4), the zinc-based compositions studied so far and biocompatibility (Fig. 6 panel b, c). Figures 1 , 12 and 13 relate to the temperature-stability conferred to RNA by binding to ZnO or ZnO-protamine.

[0182] In addition to the significant temperature-stabilization advantages to the ZnO- protamine-RNA formulations, there are additional advantages that will have an impact on the activity of RNA vaccines. First, protamine in addition to being a condensing agent for nucleic acids is considered a cell penetrating peptide (CPP) itself. Accordingly, when it complexes to RNA it can form RNA-protein nanoparticles that have some ability to carry nucleic acids into cells even in the absence of a core metal nanoparticle. Thus in addition to a stabilizing element, protamine increases binding to the nanoparticle (Fig. 13) but also importantly likely influences intracellular delivery of the nucleic acid to some extent. Second, the LL37 peptide, while less effective at loading RNA onto zinc oxide (Fig. 13), there is some evidence to suggest this peptide’s antiviral activity and/or more recently role in COVID-19 immunology. Thus, the peptide component can contribute to the activity of the formulation, more so than just stabilizing the nucleic acid, increasing the RNA particle association, or enhancing delivery, it can actually contribute to the immunology of the vaccine and/or its antiviral activity. Lastly, overall from a nanomaterial perspective, the data illustrates that zinc oxide nanoparticles (ZnO NP) interacts with RNA, protects RNA from hydrolysis and nuclease-RNase mediated degradation, and also contributes to potentiating the immune response of RNA or peptide.

[0183] In one aspect, the disclosure provides a composition comprising a nucleic acid encoding a SARS-CoV-2 S protein and a pharmaceutically acceptable carrier comprising a zinc oxide nanoparticle. In some forms the pharmaceutically acceptable carrier further comprises a solvent, a dispersion media, a coating, a stabilizing agent, a diluent, a preservative, an antimicrobial agent, an antifungal agent, an isotonic agent, an adsorption delaying agent, and any combination thereof. In some forms, the protein has at least 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or even 100% sequence homology with SEQ ID NO. 1. In some forms, the pharmaceutical-acceptable carrier comprises a stabilizing agent and/or a preservative and/or an antimicrobial agent. In some forms, the nucleic acid is present at a concentration greater than 100 micrograms per milligram weight of nanoparticle. In some forms, the zinc nanoparticle comprises or further comprises a cell penetrating peptide. In some forms, the zinc nanoparticle comprises or further comprises protamine. In some forms, the nucleic acid further comprises a sequence encoding a CD40L. In some forms, the nucleic acid encodes a tetrameric CD40L trimer. In some forms, the SARS-CoV-2 S protein and/or the CD40L are encoded by a polycistronic mRNA. In some forms, the nucleic acid encodes a SARS- CoV-2 S protein / CD40L fusion protein. In some forms, the vector includes an inserted sequence having at least 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or even 100%% sequence homology with SEQ ID NO. 9 or 10. In some forms, the nucleic acid is present at a concentration of greater than 100 pg/mg of nanoparticle. In some forms, the nucleic acid is RNA or mRNA. In some forms, the nucleic acid is a component in a lipid nanoparticle immunogenic composition. In some forms, the lipid nanoparticle immunogenic composition is an mRNA immunogenic composition.

[0184] In another aspect of the disclosure, a method of temperature stabilizing a lipid nanoparticle RNA or mRNA immunogenic composition is provided. In general, the method comprises the step of binding the RNA or mRNA to a zinc nanoparticle. In some forms, the method further comprises the step of coating the zinc nanoparticle with protamine. In some forms, the RNA or mRNA encodes a SARS-CoV-2 S protein. In some forms, the SARS-CoV-2 S protein has at least 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or even 100%% sequence homology with SEQ ID NO. 1. In some forms, the immunogenic composition further comprises a pharmaceutical-acceptable carrier. In some forms, the pharmaceutical-acceptable carrier comprises a stabilizing agent and/or a preservative and/or an antimicrobial agent. In some forms, the RNA or mRNA is present in said immunogenic composition at a concentration greater than 100 micrograms per milligram weight of nanoparticle. In some forms, the zinc nanoparticle comprises a cell penetrating peptide. In some forms, the zinc nanoparticle comprises protamine. In some forms, the RNA or mRNA further comprises a sequence encoding a CD40L. In some forms, the RNA or mRNA further comprises a nucleic acid encoding a tetrameric CD40L trimer. In some forms, the SARS-CoV-2 S protein is encoded by a polycistronic mRNA. In some forms, the CD40L protein is encoded by a polycistronic mRNA. In some forms, the RNA or mRNA includes a nucleic acid encoding a SARS- CoV-2 S protein / CD40L fusion protein. In some forms, the RNA or mRNA includes an inserted sequence having at least 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or even 100%% sequence homology with SEQ ID NO. 9 or 10. In some forms, the RNA or mRNA is present at a concentration of greater than 100 pg/mg of nanoparticle. In some forms, the temperature stabilization provides an increase in intact RNA or mRNA after at least two weeks of storage at a temperature above freezing when compared to RNA or mRNA that is not bound to a zinc nanoparticle. In some forms, the temperature stabilization provides an increase in expression activity of the RNA or mRNA after at least two weeks of storage at a temperature above freezing when compared to RNA or mRNA that is not bound to a zinc nanoparticle.

[0185] In another aspect, a method of increasing the amount of intact RNA or mRNA during storage at a temperature above freezing is provided. Generally, this method comprises the step of binding the RNA or mRNA to a zinc nanoparticle, wherein the increase of the amount of intact RNA or mRNA is in comparison to RNA or mRNA that is not bound to a zinc nanoparticle. In some forms, the method further comprises the step of coating the zinc nanoparticle with protamine. In some forms, the RNA or mRNA encodes a SARS-CoV-2 S protein. In some forms, the SARS-CoV-2 S protein has at least 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or even 100%% sequence homology with SEQ ID NO. 1. In some forms, the immunogenic composition further comprises a pharmaceutical-acceptable carrier. In some forms, the pharmaceutical-acceptable carrier comprises a stabilizing agent and/or a preservative and/or an antimicrobial agent. In some forms, the RNA or mRNA is present in said immunogenic composition at a concentration greater than 100 micrograms per milligram weight of nanoparticle. In some forms, the zinc nanoparticle comprises a cell penetrating peptide. In some forms, the zinc nanoparticle comprises protamine. In some forms, the RNA or mRNA further comprises a sequence encoding a CD40L. In some forms, the nucleic acid encodes a tetrameric CD40L trimer. In some forms, the SARS-CoV-2 S protein is encoded by a polycistronic mRNA. In some forms, the CD40L protein is encoded by a polycistronic mRNA. In some forms, the RNA or mRNA includes a nucleic acid encoding a SARS- CoV-2 S protein / CD40L fusion protein. In some forms, the RNA or mRNA includes an inserted sequence having at least 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or even 100%% sequence homology with SEQ ID NO. 9 or 10. In some forms, the RNA or mRNA is present at a concentration of greater than 100 pg/mg of nanoparticle. In some forms, the storage is for a period of at least 2 weeks.

[0186] In another aspect, the disclosure provides a method of increasing the expression activity of RNA or mRNA during storage at a temperature above freezing. In general, the method comprises the step of binding the RNA or mRNA to a zinc nanoparticle, wherein the increase in expression activity of the RNA or mRNA is in comparison to RNA or mRNA that is not bound to a zinc nanoparticle. In some forms, the method further comprises the step of coating the zinc nanoparticle with protamine. In some forms, the RNA or mRNA encodes a SARS-CoV-2 S protein. In some forms, the SARS-CoV-2 S protein has at least 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or even 100%% sequence homology with SEQ ID NO. 1. In some forms, the immunogenic composition further comprises a pharmaceutical-acceptable carrier. In some forms, the pharmaceutical-acceptable carrier comprises a stabilizing agent and/or a preservative and/or an antimicrobial agent. In some forms, the RNA or mRNA is present in said immunogenic composition at a concentration greater than 100 micrograms per milligram weight of nanoparticle. In some forms, the zinc nanoparticle comprises a cell penetrating peptide. In some forms, the zinc nanoparticle comprises protamine. In some forms, the RNA or mRNA further comprises a sequence encoding a CD40L. In some forms, the RNA or mRNA further comprises a nucleic acid encoding a tetrameric CD40L trimer. In some forms, the SARS-CoV-2 S protein is encoded by a polycistronic mRNA. In some forms, the CD40L protein is encoded by a polycistronic mRNA. In some forms, the RNA or mRNA includes a nucleic acid encoding a SARS- CoV-2 S protein / CD40L fusion protein. In some forms, the RNA or mRNA includes an inserted sequence having at least 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or even 100%% sequence homology with SEQ ID NO. 9 or 10. In some forms, the RNA or mRNA is present at a concentration of greater than 100 pg/mg of nanoparticle.

[0187] In summary, over and above its ability to provide temperature-stabilization to RNA, the ZnO-protamine-RNA likely has several other characteristics which will make it a successful RNA and/or mRNA vaccine formulation including high RNA loading, delivery, and immunological activity.

EXAMPLES

[0188] The principles of this disclosure are further illustrated by the following nonlimiting examples:

Example 1 : ZnO Nanoparticles

I. IN VIVO DISTRIBUTION AND TOLERANCE OF ZNO NP:

[0189] SARS-COV-2 infects lung and is detected in kidney, liver and brain and metastasis of drug-resistant cancer to these organs can cause significant suffering and lead to rapid physiological decline in humans, companion animals and many other organisms. Antimicrobial, antiviral and anticancer activity has been previously reported for zinc oxide nanoparticle (ZnO NP), but its in vivo characteristics have been insufficiently described. To address this 2 mg/kg ZnO or cy5.5-labeled ZnO via standard click chemistry was administered into the tail vein of nude mice, imaged in the bio- imager and 5 hours later distribution into these key physiological organs was quantified by fluorescence and by ICP/MS (Fig. 4).

[0190] Significantly, Fig. 4 shows ZnO NP or its cy5.5 fluorophore conjugate tissue distribution to liver, kidney, spleen, lungs and brain with no overt toxicity in these organs and blood counts in the normal range relative to untreated or reference mice. Importantly ZnO tissue uptake was in the following order; liver>spleen>kidney>lung>heart>brain. At 5 hours or 3 days after intravenous administration no tissue damage or change in blood cell count was observed relative to control PBS injected mice or reference standards.

II. TISSUE UPTAKE AND CELL DELIVERY OF ZNO NP

[0191] Robust uptake of the cy5.5-ZnO NP could be seen into 3-D cultures by confocal fluorescence microscopy. This experiment was conducted in triplicate for CACO2 gut organoids growing on matrigel and the nanoparticle very clearly distributed readily into the tissue. Antisense oligomer (ASO) have been clinically approved to treat rare disease and we designed an ASO to target the cryptic splice site associated with melanoma drug resistance. ZnO NP as well as cobalt oxide (CO3O4) NP increased cy5.5-ASO uptake into melanoma cells, indeed the Co-NP appeared to provide for functional nuclear delivery which was confirmed in our functional assay (data not shown). ASO delivery by ZnO, Co or NiO NP was confirmed by flow cytometry and the molecular mechanism was as expected correction of the aberrant spliced Ras binding domain (RBD) as shown by RT-PCR (Fig. 5).

[0192] As can be seen in Fig. 6, cy5.5-ZnO is readily taken into 3-D organoid tissues (2a) and increases the uptake and intracellular distribution of cy5.5-ASO into melanoma cells (2b) as shown by confocal fluorescence microscopy with apparent nuclear ASO location for CO3O4 NP. Increased ASO uptake stimulated by the NP delivery was confirmed by flow cytometry, where the ZnO, CO3O4 or NiO NP complexed to cy5.5- ASO had a 3-log order shift in cellular fluorescence compared to cy5.5-ASO oligo only control (2c). The functional effect of NP delivery of ASO was shown by RT-PCR with the corrected transcript appearing when treated with NP-ASO complex versus untreated controls (2D). With the increased cell and tissue uptake of ZnO, cell association and nuclear delivery of Ni and Co materials these data suggested the possibility of synthesizing composites combining these biometals as described next.

III. Zn-BASED PHYSIOMETACOMPOSITES (PMC).

[0193] Metamaterials or nanoparticle composites have unique physico-chemical properties but their delivery, antiviral and anticancer activity has never been explored. An initial library containing nickel (Ni) and cobalt (Co) in the context of precious metals was synthesized by the Mirkin group. Recently we described the synthesis of cobaltzinc oxide (CoZnO) and nickel-zinc oxide (NiZnO) and here the zinc-based series was expanded to include manganese (Mn) and iron (Fe), sulfides [S] and selenides [Se] in addition to oxides [0], Nanoscale and nanorod shape of these materials was confirmed by transmission electron microscopy (TEM) and nanoparticle tracking analysis (data not shown). The biocompatibility of these PMC nanomaterials was tested at 10, 20 and 25 ug/ml with highly sensitive non-transformed NIH3T3 cells (Fig. 6).

[0194] Importantly, Fig. 6 shows the outstanding biocompatibility of the Zn-based PMC nanoscale materials, particularly the MnZnSe, MnZnS or alternatively FeZnS. Very little cytotoxicity is seen for MnZnS and FeZnS or MnZnSe after 48 hour continuous exposure to untransformed highly sensitive NIH3T3 cells at 10, 20 or 25 microgr/ml concentration (2c). The MgZnO material was relatively toxic at the two higher doses, either 20 or 25 microgr/ml whereas the FeZnS and NiZnO were intermediate and better tolerated.

IV. PHYSIOMETACOMPOSITE (PMC) FLUORESCENCE AND BIOLUMINESCENCE.

[0195] Early highly fluorescent quantum dot materials were zinc sulfide (ZnS) based, but doped with toxic non-physiological metals such as lead or cadmium limiting their biological utility. Instead here we developed alternative physiometacomposite (PMC) compositions of ZnS or zinc selenide (ZnSe) doped with iron (Fe) or manganese (Mn) at a similar percentage (1 , 3 or 5%) as they were for Co and Ni. The fluorescent and bioluminescence of these PMC derivatives was outstanding and tested in serum containing media, liver and tumor homogenates and slurries of the other key organs (kidney, spleen, lung and brain) (Fig. 7).

[0196] Importantly Fig. 7 shows the unquenchable fluorescent yield of the PMCs in serum, liver and tumor homogenate (7a). FeZnS and MnZnS also potentiate bioluminescence causing a red-shifted signal at 10 ⁴ -10 ⁴ relative light units above background (7b). The PMC show some tissue-specificity for example MnZnSe fluorescence shifts in the kidney slurry and the MnZnS fluorescence is enhanced on the liver homogenate (7c). FeZnS also red-shifts in the kidney homogenate to emission at 725 nm. Dose-dependent signal intensity is also enhanced in ex vivo brain, liver (7d) and lung tissue (7e), especially for MnZnSe and MnZnS. These data build on our previous patent of 2-dimensional fluorescence difference spectroscopy (2-D FDS), taking it into the 3 ^rd dimension for fluorescence-based bioimaging.

V. 3-D TUMOR SPHEROID IMAGING AND INHIBITION

[0197] Previously poly l:C RNA had been combined with a iron-zinc oxide composite shown to potentiate its anticancer activity. Here we cy5.5-labeled poly l:C and showed that the PMC were able to label 3-D tumor spheroids imaged in the bio-imager far- red/NIR range. Intravital staining of the spheroids clearly showed an increase in the kill zone when treated with 20 microgr/ml PMC nanoparticle consistent with antitumor activity indeed the dense inner layer of cells which appears dark on light microscope examination breaks apart after nanoparticle treatment. Both ZnO and CoZnO PMC were able to inhibit drug-resistant melanoma (B16F10) cell invasion in the classic scratch assay (Fig. 8).

[0198] As shown in Fig. 8, PMC are able to load and label 3-D spheroids with cy5.5- poly l:C (8a). 3-D tumor spheroids treated with PMC break apart and many of the cells within the dense interior dye as shown by green/red intravtial staining (8b). The PMCs or ZnO NP control also inhibit cancer cell invasion in the scratch assay (9c). These data suggest the anticancer or antitumor activity of ZnO NP and second-generation PMC materials which was tested next.

VII. ANTICANCER ACTIVITY OF PMCs. [0199] Anticancer activity of the PMC derivations and their ASO and aptamer complexes was tested next. First melanoma tumor at the time of metastasis was harvested and subjected to high throughput proteomics analysis confirming the import of proteins in the RAS/ERK/AKT pathways and secondarily BCL apoptosis pathways. To address this melanoma or model glioblastoma line (132N1 ) were treated with NP delivered RBD decoy protein interference or ASO targeting RBD or BCL-xL demonstrating outstanding inhibition (Fig. 9).

[0200] As shown in Fig. 9, proteomics analysis revealed multiple proteins in the RAS/ERK/AKT associated with metastatic melanoma (9a, c). This could be targeted wither by PMC delivery of RBD decoy as a protein interference approach for CoZnO or CoFeZnO ternary or quaternary composites (9b), or by ASO targeting RBD or BCL-xL. Dogs are considered an excellent comparative oncology model for melanoma especially for rarer drug resistant forms, the canine mucosal melanoma are activated in the ERK/AKT pathway and are considered an excellent preclinical model for nanomedicine testing. These cells could be greatly inhibited by NiZnO even at early timepoints, but ironically in concert with RBD targeted ASO or aptamer no enhanced anticancer activity was seen, except for ZnO or MnZnS (9e).

VII. ANTIVIRAL ACTIVITY OF PMCs

[0201] We next tested the enzyme inhibition and the design of ASO targeting putative conserved regulatory sites in SARS-COV-2 (Fig. 10).

[0202] As shown in Fig. 10, Antibacterial activity of ZnO NP has been correlated to b-Gal enzyme inhibition, and here we show that FeZnS and particularly MnZnS PMC give a 3-log order enzyme inhibition, a safer surrogate model to COVID19 that infects the lungs of pigs but not humans.

Results and Discussion

Conclusions

[0203] In conclusion these data support the further preclinical evaluation of the Zn- based physiometacomposite (PMC) nanomaterials for imaging and treatment of cancer and infectious disease. Especially for certain rarer drug-resistant forms of cancer such as mucosal melanoma and other ERK/AKT-activated metastatic forms where canine represents an excellent comparative oncology model for pre-clinical testing of protein and RNA-based nanomedicines. Here, Zn-based conjugates, either ZnO NP or the cy5.5 derivative are shown to distribute into liver, kidney, lung, spleen and brain. 3-D culture experiments suggest these materials are well taken up by tissues and are able to deliver ASO into cells as shown by confocal microscopy and flow cytometry. At least at the 20 mg/kg dosage after a single intravenous administration no overt toxicity was seen in the two animals treated and assessed after 5 hours or 3 days, and in cell culture at least for 48 hours of continuous exposure normal cell controls such as the NIH3T3 standard cells tested displayed very little cytotoxicity up to 25 microgr/ml, and in some cases notably MnZnS and MnZnSe cells could be treated for much longer up to 3 or even 4 days with 98-99% viability (data not shown). The MnZnS and MnZnSe in particular gave enhanced fluorescence and could be used to image these tissues based on their outstanding red-shifted fluorescence yield when exposed to ex vivo tissues, or their homogenates or slurries, notably including tumor, lung and brain slices or tissues of significance for both COVID and cancer metastasis. PMC RBD or RBD targeted ASO or aptamer showed dose-dependent cancer cell inhibition and could be used to inhibit drug-resistant rarer forms of melanoma such as canine mucosal melanoma (M5) line used here. Finally 3-log order enzyme inhibition was shown for MnZnS as well as its significant antiviral activity (2-log order) and given its activity with ASO we designed ASO sequences to target regulatory regions within NSARS-COV-2. These data overall support the evaluation of PMC conjugates of Ras-targeted ASO, aptamer and sequences targeting NSARS-COV-2 as a new anticancer/antiviral approach.

[0204] Zn-based physiometacomposite nanoparticles are biocompatible and indigenously fluorescent. Importantly PMC have antiviral activity and can achieve significant RNA payloads and impart structural retention and temperature stabilization to RNA. PMC materials are thus of interest for pre-clinical applications of RNA vaccines and RNA-based therapeutics.

Materials and methods.

Nanomaterials and Reagents: [0205] All nanoparticles used were obtained pure from Sigma-Aldrich or PlasmaChem GmbH (Berlin, Germany), except for MnZnSe was provided by Dr. Emily McLaurin formerly in the Department of Chemistry Kansas State University, MnZnS and FeZnS or NiZnO were provided by Dr. Garry Glaspell US Army Corps of Engineers, Cobalt Zinc Oxide (CoZnO) cobalt ferrite PMC were synthesized by Dr. KC Ghosh’s laboratory (Missouri State University). Poly inosinic:poly cytidylic acid [poly(l:C)] was obtained from Sigma-Aldrich (Cat# P958250MG'). Cy5.5-labelled SSO (sequence: 3- CCUCUUACCUCAGUUACA-5) was obtained from Trilink Biotechnologies. Clinical- grade LL-37 peptide was obtained from our collaborator Dr. Cheng Kao Indiana University. NIH3T3 and A375 cells for cytotoxicity studies were obtained from ATCC. All NP and RNA were precipitated from 70% alcohol/H2O washed once with 100% alcohol, air dried in the biosafety cabinet prior to RNA and protein complexation, cell or animal administration. Copper (Cu) was purchased from PlasmaChem 10-100 nm in size.. Manganese Zinc Sulfide (MnZnS (1 , 3, 5%)), 50/50% Nickel Zinc Oxide (NiZnO), 10/90% NiZnO and Iron Zinc Sulfide (FeZnS) were all synthesized by Dr. G Glaspell’s laboratory (Virginia Commonwealth University). Cobalt Oxide (CO3O4), Nickel Oxide (NiO), 2/98% Scalable MnZnS and FeZnS , pure powders were physically mixed, heated to a flux, allowed to cool in an oxygen purged atmosphere and jet ball milled to nanoscale confirmed by transmission electron microscopy and nanoparticle tracking analysis [see supplemental data]. The NPs were washed with double-distilled water, 70% ethanol/water, ethanol, and were stored dry prior to use. Costar (Coming, NY, USA) 96-well black, clear bottom assay plates were used for the assays. Luciferase enzyme (Photinus pyralis, >10x10 ¹ ° (units/mg protein) was obtained from Sigma Aldrich and diluted it to a 0.2% solution [1 :500 dilution with PBS buffer], PBS buffer at 10X concentration was diluted to a 10% solution with de-ionized water [ddH20], Luciferase enzyme substrate buffer (ATP, Mg) was diluted to a 1 :1 vol/vol ratio with PBS buffer. Obtained β-Galactosidase ( β-Gal) from Aspergillus oryzae was obtained from Sigma Aldrich (>8.0 units/mg solid, Louis, Ml, USA) and was diluted to a 1 mg/kg solution in spectral grade H2O. B-Gal substrate, Resorufin [3-D-galactopyranoside was purchased in a 10 g vial from Marker Gene Technologies (Eugene, OR, USA) and was diluted down into ten 10 mg/kg aliquots in spectral grade H2O and re-suspended into a 1 mg/kg solution for experimentation. Fluorescence emission, excitation and intensities were determined by Spectramax Paradigm.

Animal Model Imaging:

[0206] Animal procedures followed approved IACUC 4064.1. Female 6 week old BALB/C Nu/Nu mice were obtained from Charles River and allowed to acclimate for several weeks prior to the experiment. These were anesthetized using oxygen/isoflurane prior to administration with treatment and bioimaging. Animals were intravenously administered with 100 μl of with PBS or ZnO NP or ZnO NP-Cy5.5 at the dose rate of 2 mg/kg body weight. Imaging was done using a Pearl® Trilogy Small Animal Imaging System (LI-COR Biosciences, USA) immediately before and after PBS administration, 5 days after ZnO NP administration and at regular time points until 6 h after ZnO NP-Cy5.5 administration. After the defined time points, animals were euthanized under anesthesia. Nanoparticle analysis was provided by the Nanotechnology Innovation Center Kansas State.

[0207] ICP - MS: For this procedure, tissues were collected from mice treated with, (1 ) PBS alone (n=1 ), (2) ZnO NP (n=2), and (3) ZnO NP-Cy5.5 (n=2). The concentration of zinc (Zn) in the mouse tissues was determined using ICP-MS analysis following the standard protocol. In brief, tissues were digested using 2 ml of 70% nitric acid (HNOs) for liver and 1 ml for brain, heart, lungs, spleen and kidneys. The digestion was performed in SC154 HotBlock® (Environmental Express, USA) at 90°C overnight. Following overnight digestion, all the tissue digests were diluted by addition of 9 ml deionized water. The diluted digests were further diluted by combining 1 ml of the digest with 4 ml of 2% HNO3 and filtered using 0.2 pm filter. Zinc concentration was measured on a PerkinElmer NexION® 350D ICP-MS.

[0208] Tissues were collected from mice treated with, (1 ) PBS alone (n=1 ), (2) ZnO NP (n=2), and (3) ZnO-Cy5.5 NP (n=2). Fluorescence was measured in the following tissues:

(1 ) PBS treated: Liver and Kidney (2) ZnO NP treated: a. Mouse 1 : Liver and kidneys b. Mouse 2: brain, heart, lungs, spleen and kidneys

(3) ZnO NP-Cy5.5 treated: brain, heart, lungs, spleen and kidneys from both mice From the tissues collected, a portion was cut off, weighed and homogenized using SONICS VCX Vibra 130 Tissue Sonicator (PRO Scientific Inc.) at an amplitude of 50 at a pulse rate of 10s(on) and 5s (off) for 20 minutes. From the homogenate, 200 μl was transferred to a 96-well plate and fluorescence was measured using SpectraMax® i3x multimode microplate reader (Molecular Devices, California, USA). Excitation and emission wavelengths used were 660 nm and 695 nm respectively. PBS was used as the blank. The experiment was done in triplicates. Histopathology and hematology analyses were performed at the Veterinary Medical Diagnostic Laboratory, KSU. The remainder of the collected tissues were fixed in 10% neutral buffered formalin. Sections of fixed tissue were routinely processed on a Sakura Tissue-TEK VIP 6 Processor prior to paraffin embedding. Slides were cut at and routinely stained with hematoxilin and eosin on a Leica Autostainer XL ST5010. Representative images at 10x magnification were captured on an Olympus LC20 camera mounted on an Olympus BX53F2 light microscope with CellSens (Olympus Corporation).

Cellular Delivery and Nanomaterial Binding Assays:

[0209] 3D Spheroid Culture of Caco2 cells: Human Caco2 cells (ATCC®, passage 30) were seeded onto a 35mm sterile glass-bottomed cell culture dish (FluoroDish™- World Precision Instruments) to form 3D spheroids in a thin layer of 10% Matrigel (Corning® Matrigel® Basement Membrane Matrix, LDEV-free). Culture medium was comprised of 1X Minimum Essential Media (MEM, L-glutamine free), 10% Fetal bovine serum, 1 % L-glutamine, and 1 % Pen/Strep. Caco2 spheroids were in culture for approximately 24-hours prior to imaging. Delivery of ZnO-PEG-Cy5.5 (ZnOCy5.5) nanoparticles (NPs) to human Caco2 spheroids: Immediately prior to delivery, ZnOCy5.5 NPs were diluted to a stock concentration in Ham’s F12 medium (160 pg/mL) sonicated for 60 seconds at room temperature (Fisher Scientific 60 Sonic Dismembrator Model F60 Cell Disrupter). Caco2 Matrigel-embedded spheroids were exposed to 20 pg/mL of ZnOCy5.5 NPs in culture media overnight in a 5% CO2 humidified incubator at 37 °C. Post-treatment (16-hours), the Caco2 cells were evaluated for NP uptake by confocal microscopy using a FluoView FV1000 Inverted Confocal Microscope. Images were acquired with a 60X oil objective and the following laser settings (CHS1 : Cy5.5, 795v, 1x Gain, 7% offset, laser 635 (1 %), TD1 :235v, 1x Gain, 0% offset, laser 488 (11 %). Images were imported into SlideBook Version 5.0 (SB 5.0.0.14 5/13/2010) for presentation.

[0210] Delivery: B16F10 cells were incubated with cy5.5-ASO control or complexes with cy5.5-ZnO-NP cy5.5-ASO:Co3O4 NP incubated for 24 h, rinsed with PBS and imaged by confocal microscopy.

[0211] Similarly, NIH3T3 cells were treated with Cas9-GFP fusion protein or the MgO-NP control or MgO-Cas9 NP and imaged by fluorescence microscopy. B16F10 melanoma cells were incubated with cy5.5-ASO control or the complexes with ZnO, CO3O4 or NiO NP and 24 h later, rinsed with PBS, trypsinized and analyzed for cellular fluorescence by flow cytometry (K-State VDL core lab). B16F10 melanoma spheroids were allowed to establish for several days and treated with cy5.5-poly l:C viral mimetic RNA, the ZnO NP or cobalt (Co/ZnO) or nickel (Ni/ZnO) composites and imaged by fluorescence microscopy. Engineered human melanoma cells we previously reported under the control of an ASO inducible luciferase expression and delivery in the 5 replicate wells quantified by relative luminescence per well.

[0212] Mouse tumor was isolated at the time of metastasis, 20 mg samples lysed, the proteins extracted (2-3 tumors were pooled) and standardized to A280 (Molecular Devices Spectramax i3x, Sunnyvale, CA, USA). Slides were incubated with Cy3- Streptavidin (Sigma Aldrich, St. Louis, MO, USA), dried by centrifugation and stored under dark conditions and imaged using Molecular Devices Genepix 4000B (Sunnyvale, CA, USA). Nanobio interaction was confirmed by CD, FT-IR and zeta potential and payload estimated as previously described. The molecular dynamics simulation was performed with the peptide in the water far above the surface and observed the adsorption process (2a). Fluorescent microscopy was conducted on an Olympus IX73 within poly-D-lysine coated 8 chamber slide, cells inoculum density (5x10 ⁴ ) after o/n adherence exposed to 20 ug/ml NP with maximal payload of Cas9-GFP (Applied Biological Materials Inc. Richmond, BC, Canada) or cy5.5-SSO versus cy5.5-SSO control (200 nM) assayed in the Texas Red/Rhodamine filter/channel. 3-D spheroids were formed within Insphero Corp plates, 48 h later the media was changed with NP as above containing Rhoda-poly l:C or stained with Invitrogen live/dead stain and imaged in the bio-imager (Licor Pearl Trilogy) or on the fluorescence microscope respectively.

Biolummescent/fluorescent Readings:

[0213] Bio-luminescence and fluorescent readings were taken on PerkinElmer (Caliper) LifeSciences IVIS Lumina II imager. Fluorescent readings were set to sixty second exposure, medium binning, 1 F/stop, dsRed emission filter, and intensity viewed through Rainbow setting. 200 μl of NP (FeZnS and 3% MnZnS) ± luciferase were added to an all-black FB brand 9-well microtiter plate in PBS solution (1 :1 vol/vol). Doseresponse assay utilized NPs that elicited high relative light units (RLUs) in earlier experiments for further analysis of the effects of increasing NP dose when incubated with β-Gal. CO3O4, ZnO, NiO, MgO, 10% NiZnO, 50% NiZnO, 95% CoZnO, and 98% CoZnO nanomaterials were incubated with β-Gal in a 2:1 enzyme:substrate ratio (200 and 100 pg/ml respectively at a NP concentration of 1 , 2, 5, 10, 50, 100, 200 and 400 pg/ml dose. Time course measurements looking at enzyme: nanoparticle interaction were taken at 0, 10, 30, and 60 minutes. Dose-dependent assay utilized 1 mg of each NP was weighed out on a XS204 Mettler Toledo (Columbus, OH, USA) analytical balance, placed into Eppendorf tubes, and made into a 1 mg/ml suspension with PBS buffer stock solution. Each well had a total volume of 200 pl. Bio-fluorescence Detection was performed by the SpectraMax i3x by Molecular Devices (San Jose, CA, USA) at 1 , 10, 30, and 60 minutes.

Ex-Vivo Bio-imaging:

[0214] Mouse specimens were provided by Comparative Medicine Group. Lung sections were removed and were evenly divided into 2 sections. Ex-vivo imaging was performed in the Pearl® Trilogy Bioimaging system. 1 mg/ml of MnZnSe was diluted to a 1 :3 with HPLC water and injected into individual sample and then imaged under white light, 700, and 800 nm filter, with 85 pm resolution and “0” focus. Increasing volumes (pl) were injected (1 -20) with increasing fluorescent output. Tissue slurry/homogenate preparation consisted of heart, liver, kidney, brain, spleen, and lung from three different mice. Tissues were weighed on the XS204 Mettler Toledo (Columbus, OH, USA) analytical balance. Sectioned samples 100 mg per ml were then placed in sterile 10% PBS buffer, and homogenized via Vibra-Cell Processor VCX 130 (Newton, CT, USA) for 2 minutes, with 10 second pulses and 5 seconds rest. Slurry composition contained stromal tissue homogenized in with the sample (liver and kidney); homogenate composition had stroma removed via centrifugation and removal of supernatant to a separate tube (lung, heart, small intestine, liver, kidney and spleen).

2-D FDS Characterization:

[0215] To obtain excitation, emission, and intensity data Molecular Devices Spectramax i3x spectrophotometer was used. The microplate is scanned without the lid using the Molecular Devices Spectramax i3x spectrophotometer utilizing the Spectral Optimization Wizard that is included in the Softmax Pro 6.4.2 accompaniment software (Sunnyvale, CA, USA). The device was set to read the fluorescence endpoints of unknown wavelengths. The photomultiplier (PMT) gain was set to high, flashes per read was six, and wavelength increment was 5 nm. Before the first read, the microplate is shaken at medium intensity in a linear mode. The microplate was read from the top at a height of 1 mm. The range of excitation and emission wavelengths was set to 250- 830 nm and 270-850 nm, respectively.

[0216] PMC’s (1 mg/ml) were spiked into tissue slurry/homogenate and then placed into BRAND® 96-well black bottomed plates (CAT# 781668). 200 μl of PMC/tissue slurry/homogenate was assayed via SpectraMax i3x by Molecular Devices (San Jose, CA, USA) for 2-Dimensional Fluorescence Difference Spectroscopy (2D-FDS). For NP- Luc 2-D FDS bioluminescent readings, an all-black, FB brand 96-well titer plate was loaded with 200 μl of NP (FeZnS and MnZnS) in a 1 :1 vol/vol solution. For the enzyme:NP conjugate, Luc was added (10 pl) with 200 μl of NP and spun down at 140 RPM for one minute and re-suspended and added to the microtiter plate. The wavelength settings were set to unknown, spin before read, and optimization settings were set at excitation (250-830 nm) and emission (270-850 nm).

MTT Assay [0217] For cytotoxicity (MTT) assay, NIH3T3 fibroblast cells were seeded on a 96- well plate with 5,000 cells/well and allowed to grow for 24 h in DMEM with 10% FBS and 1 % penicillin/streptomycin. After 24 h, the medium was replaced with PMCs (5% NiZnO, MnZnS, 5% MgZnO, FeZnS and MnZnSe) dissolved in DMEM at 10, 20 and 25 pg/ml. Each treatment was tested on four wells. Four wells containing DMEM alone served as the blank and four wells with untreated cells served as the control. The cells were monitored daily for visible cytotoxicity using a light microscope. After 24/48/72/96 h with the treatment, the treatment was removed. All the wells were rinsed once with PBS, followed by the addition of 110 pL of 1 :20 mix of MTT: indicator-free DMEM. After incubating the plate for 5 h at 37°C, 85 pL of the mixture was removed from each well and 75 pL of dimethyl sulfoxide was added to solubilize the crystals. The plate was then kept at 37°C in an orbital shaker with 175 revolutions/m inute. After 15 minutes on the orbital shaker, the plate was then read on a Synergy H1 Hybrid Multi-Mode Microplate Reader for absorbance at a wavelength of 562 nm.

Virus mitigation assay.

[0218] A North American genotype-2 porcine reproductive and respiratory syndrome virus infectious clone containing green fluorescence protein (PRRSV-GFP) was utilized for in vitro anti-viral activity experiments. PRRSV-GFP was propagated and titrated on MARC-145 cells derived from African green monkey kidney cells. Assays to determine the antiviral activity of MnZnS nanoparticles (NP) were similar to previous work investigating the in vitro efficacy of fatty acid and formaldehyde based additives on reducing the titer of African swine fever virus (Niederwerder et al. 2020). Dilutions of MnZnS NP (100 pg/ml, 50 pg/ml, 20 pg/ml, 10 pg/ml) were prepared in Minimum Essential Medium (Corning® Eagle's MEM; Fisher Scientific) supplemented with fetal bovine serum, antibiotics, and anti-mycotics. Each dilution of MnZnS NP was mixed with an equal volume of PRRSV-GFP (titer 10 ⁶ 50% tissue culture infectious dose per ml, TCIDso/ml) for testing anti-viral activity. Positive controls included PRRSV-GFP mixed with an equal volume of MEM. Cytotoxicity controls included MnZnS NP mixed with an equal volume of MEM. Ten-fold serial dilutions of each NP/virus mixture or control were prepared in triplicate and added to confluent monolayers of MARC-145 cells in a 96-well tissue culture plate. Cells were incubated at 37°C in 5% CO2 for 3 days prior to examination of cells for fluorescence under an inverted microscope. PRRSV titers (TCIDso/ml) were calculated using the method of Spearman and Karber (Finney 1964).

Example 2 Spike Protein Expression

Materials and Methods

[0219] mRNAs were synthesized in vitro using mMESSAGE mMACHINE™ T7 ULTRA Transcription Kit (AM1345, Thermofisher). Briefly, pcDNA3.1 construct encoding spike protein was linearized using Xbal restriction enzyme and used as template to synthesize mRNA. After synthesis, ~100 nt long poly(A) tail was added. mRNA recovery was performed by LiCI precipitation method. Lipofectamine MessengerMAX (LMRNA001 ) was used to transfect mRNA into HEK 293A cells. Briefly, 3pL of transfection reagent and 700ng of mRNA was used and 36 hrs. post-transfection, the cells were fixed and probed with anti-spike CR3022 or R2F4 SARS-CoV-2 neutralizing mAbs to evaluate the protein expression. Figure 1 provides the amino acid sequence of the SpikeFTm-2A-cD5SPD-CD40L polypeptide. The SARS-CoV-2 spike has a Fusion [F] protein leader sequence at the N-terminus, a flexible linker, and the transmembrane domain of an F protein at the C terminus. A 2A autocleavable sequence was added to allow co-expression of human CD40L using human surfactant protein D [SPD] tetramerization motif. This cassette is designed to allow optimal expression of Spike antigen on the surface of transfected cells and concurrent secretion of the CD40L cytokine. Figure 2 provides an amino acid sequence of the Spike-SPD-CD40L polypeptide, which has a CD5 secretory signal followed by the SARS-CoV-2 spike, a flexible linker, SPD, and the CD40L functional domain. This cassette is designed to allow secretion of the spike-CD40L chimeric protein. In these figures, the F Signal is a Fusion [F] protein signal sequence; Spike is a SARS-CoV-2 S protein; Flexible linker is a (G4S)3 linker sequence that allows the spike protein to fold; F Tm is a Fusion [F] protein transmembrane domain for expression of the spike on the surface for optimal recognition by B cells; 2A peptide is a 2A autocleavable peptide to allow multicistronic expression of the surface displayed spike antigen and secreted tetrameric CD40L molecular adjuvant; CD5 signal sequence is a CD5 secretory signal sequence; SPD is a Surfactant protein D tetramerization domain; and CD40L is a molecular adjuvant/B cell agonist.

[0220] The above polypeptides were used to generate codon-optimized synthetic genes (outsourced from GenScript) and cloned into pCDNA3.1 + vector to generate the recombinant plasmid constructs below:

1 . pCDNA3 _FLp Spike _FTM -2A- _CD5 SPD- _human CD40L [designated plasmid TX001 : pTX001 ]

2. pCDNA3 _CD5 Spike_SPD- _human CD40L [designated plasmid pTX002],

[0221] The pCDNA3.1 vector has a T7 promoter that allows mRNA synthesis using the constructs above and a commercial mRNA miniprep synthesis kit. The above mentioned pTX001 [and pTX003 which has hamster CD40L instead of human] constructs have been used to generate miniprep mRNA [~ 40μg] for preliminary studies and showed that the generated mRNA is expressing spike antigen as judged by immunocytometric analyses using SARS-CoV-2 neutralizing mAbs (Fig. 14).

Example 3 Spike Protein Expression

Materials and Methods

[0222] Formulation preparation: The samples were made by weighing out 1.5mg of Zinc oxide nanoparticles (ZnO) (Sigma Alrich 100 nm) or (PlasmaChem 14 nm) and 1.5mg of protamine (Pr). ZnO was first washed with 1ml of 70% isopropyl, microcentrifuged at 5000 rpm for 5 minutes, and the supernatant was aspirated. The protamine was prepared by dissolving it in 1ml of milli-q water (Prot-hi). 3 different dilutions of protamine were created by taking 100μI of Protamine solution and adding to another tube and adding 900pl of milli-q water to make a dilution of 1 :10 (Prot-med). Another dilution of 1 :100 was made from taking 100μI from 1 :10 and adding 900pl of milli-q water (Prot-Lo) Once all 3 dilutions of Pr were created 33pl were added to the ZnO and mixed. These were microcentrifuged at 5000 rpm for 5 minutes and supernatant was aspirated. The RNA solution was made from 3 mg to 1 ml of milli-q water. 50 to 100μI RNA (3 mg/ml) was added to the ZnO and Pr sample, mixed, and washed with 1 ml of 100% cold alcohol, ethanol or isopropanol. The sample was microcentrifuged at 5000 rpm at 5 minutes and supernatant was aspirated. The samples were left to dry overnight in their certain temperatures of either 20, 30, 40, or 50 °C. To re-suspend the sample, 1 ml of PBS was added to the samples. 100μI was added into another tube and 1 ml of cold alcohol was added for reprecipitation, then microcentrifuged at 5000 rpm for 5 minutes and supernatant was aspirated. Samples were air dried briefly prior to RNA elutuion and RAGE analysis.

[0223] RNA elution: An initial elution buffer combined saturated heparin and poly- acrylic acid with 10X PBS and 50X TAE and 10% SDS at 1 :1 :1 :1 :1 vol: vol. In subsequent experiments we noted elution could also be accomplished with a second lot of saturated Heparin buffered again by TAE/PBS with adjustment to 0.1 % SDS. Saturated urea in the presence of TAE provided a high intensity RNA band from a formulation prepared as above eluted and analyzed by RAGE analysis. Final elution buffer consisted of a 1 :1 ratio of 1X TAE and 1X PBS and was saturated with Urea.

[0224] Stability analyses: Dry powder or PBS re-suspended formulations were maintained in stability chambers (30, 40 and 50 °C) for one to two days, one to two weeks or the formulations or the RNA stock solution stored in the refrigerator for the 4°C samples. In the hydrolysis experiment, 1 mg/ml nanoparticle was exposed to 1 mg/ml RNA overnight in a hot plate set to 37 °C, the samples removed and analyzed by RAGE.

[0225] RAGE analysis: To each 100 ul scale dried precipitate aliquot is added 30pl elution buffer and incubated at 37 C for 20 minutes. The agar gel was made by weighing out 2% gel 2g to 100ml of 1X TAE that was made by 2ml of 50X TAE and 98ml distilled water. The solution was initially heated up in the microwave for 10 seconds and subsequently heated in the microwave in 5 second intervals until the solution had become clear. The agarose gel was then cooled off and added to the gel platform with the comb. This was left for 20 minutes to mold into the gel. The samples were prepared for the gel by mixing 30pl of the samples with 1 μl bullseyes staining and 10pl saturated sucrose. A total of 30pl was added into each well. The gel was run at manual 100V for an hour and imaged in the gel imager.

[0226] In vitro translation: CleanCap FLuc mRNA was obtained from Trilink Biotechnologies. Nuclease-free Rabbit reticulocyte lysate was obtained from Promega. In a typical translation reaction to 1.5 uL of mRNA was added one microliter each of Cys, Leu and Met amino acids in a tube containing biocompatible concentration of NP (20 microgram/mL). After which 35 microliters of RRL was added and the tubes were incubated for 30 minutes at 30 deg C to allow for translation of the Luc mRNA to protein. Afterwards 100 uL of Luciferin/Luciferase reagent was added and the bioluminescence of the samples were determined at 562 nm Biotek Hybrid Synergy.

[0227] Payload efficiency: Tubes were run in triplicate containing RNA control, RNA with protamine, or RNA with LL37. 1 mg of 100 nm ZnO NP was washed with alcohol, to which was added 300 uL of peptide (Prot 1 :10) or LL-37 (1 :5), spun down and the RNA added (5 uls of 3 mg/ml), spun-down (in the absence of alcohol), and 5 uL aliquots removed and quantified by nano-drop.

[0228] Characterization: Particle size and zeta potential analysis were conducted as previously described (Crommelin, D.J.A et al; J. Pharm Sci 2021 ; March (110(3)); DeLong, R.K, et al; Biomaterials 2009; 30(32):6451 -6459; U.S. Patent No. 8,349,364). CD analysis was conducted as previously described (Choi, YS. et al., Biomaterials Feb. 2010, 31 (6): 1429-1443). DSC experiments were conducted in the Vaccine Characterization and Stabilization Center at the University of Kansas in the laboratory of Dr. Russ Middaugh.

[0229] Delivery: NIH 3T3 cells were seeded in 24-well plates at a density of 30,000 cells per square centimeter. 7.4 mg of protamine sulfate were dissolved in 5 milliliters of deionized water autoclaved water. The solution was sonicated for 1 min, and the solution was divided into 1.5 mg of protamine. Each vial was centrifuged, and the supernatant was discarded. The precipitate was re-suspended in 50 mcl of cold 75 % ethanol and centrifuged. The alcohol was removed. 50 mcl of DI autoclaved water was added to all vials, and 7 mcl of 1.5 mg/mL protamine solution was added. Each vial was vortexed and centrifuged at 5000 rpm for 5 min. After the removal of the supernatant, 50 mcl of DD water was added to all vials. Then, green fluorescent protein was added to one vial and green wfluorescent protein mRNA was added to the other two vials. 7 mcl of protamine solution was added to one of the vials containing green fluorescent protein mRNA. 100 mcl of cold 75% ethanol were added to all vials to further sterilize the samples; ethanol was removed by centrifugation. Each formulation was suspended in 2 ml of DMEM, all samples were dispersed readily into solution. Then, cells were treated with 20 mcg per ml Zinc Oxide formulation 12 hrs. following seeding. After 24 hrs of incubation, the treatment was removed and replaced with 10% FBS in DMEM. Cells were allowed to incubate another 24 hrs and the mean cell fluorescence per well analyzed on a Molecular Devices SpectraMax Paradigm instrument. 4 wheels were used per condition and the experiments were repeated twice both showing increased mRNA transfection relative to the GFP protein control. The plots shown in Fig. 18 represents the average of all 4 wells for one independent experiment.

[0230] mCherry mRNA cell lysate expression. 10pg/ml ZnO solution was prepared using RNase free water. Protamine solution was also prepared using RNase free water and the solute to solvent ratio was 1 :100. 10OpI of 3T3 cell suspension (sonicated) was added in 4 wells of a 96 well plate with a clear bottom. 3pl of RNase free water was added in the first well with 3T3, 2pl of RNase free water and 1 μl of mCherry RNA was added in the 2nd well. In the third well, 1 μl of mCherry RNA, 1 μl RNase free water and 1 μl of ZnO solution was added. In the fourth well, 1 μl of Pr solution, 1 μl of ZnO, and 1 μl of mCherry RNA was added. The same procedure was repeated with 10OpI of Raw cell suspension (sonicated) in 4 wells of a 96 well plate, followed by RNase free water in the first well, mCherry RNA and RNase free water in the second well, RNase free water, mCherry RNA and ZnO in the third well, and Pr solution, ZnO solution and mCherry RNA in the fourth well. Then fluorometric readings were taken on a spectroscope with the settings of fluorescence spectrum, fixed excitation at 550 nm and 587 nm and emission at 570-650 and 607-700 nm in 10 nm steps while the temperature was 37 degrees Celsius. Three readings were taken at the time periods of 0, 3 hrs, and 6 hrs.

[0231] Spike mRNA synthesis. mMESSAGE mMACHINE™ T7 ULTRA Transcription Kit (AM1345) was used to perform mRNA synthesis. In brief, a pcDNA3.1 construct encoding spike protein was linearized with Xbal restriction enzyme and used as a template DNA for mRNA synthesis. A 100 nt poly(A) tail was added after mRNA synthesis. To recover mRNA, the LiCI precipitation method was used. The recovered mRNA was quantified and stored at -80C for further use. [0232] Spike mRNA transfection. HEK293A cell monolayers were used to transfect mRNA on a 12-well plate format. The spike mRNA, zinc oxide nanoparticle, with or without protamine formulation was mixed with HEK293A cell culture media and overlaid on cells in their respective wells. The positive control was transfected with 3uL of lipofectamine MessengerMAX (LMRNA001 ) transfection reagent and 700ng of spike mRNA. Mock-transfected cells served as a negative control. To assess spike protein expression, the cells were fixed 24 hours after transfection and probed with a recombinant humanized anti-SARS-CoV-2 spike-specific neutralizing monoclonal antibody.

[0233] RESULTS AND DISCUSSION

[0234] Characterization: ZNP-RNA was characterized by transmission electron microscopy, UV spectroscopy (Fig. 15B), nanoparticle size analysis (Fig. 15C), zeta potential (Fig. 15D) and payload (Fig. 15E). Fig. 15A shows the lighter biomolecular RNA-protamine complexes associated at the nanoparticles surface. The loss of RNA from the supernatant analysis also confirming RNA association to the protamine coated zinc nanoparticle (ZNP) is shown in Fig. 15B. The nanoparticle size shifts as a consequence of protamine coating and RNA loading as shown in Fig. 15C. Zeta potential measures the effective surface charge of nanoparticle. To examine this we incubated ZnO NP with low, medium or high concentrations of protamine and measured the nanoparticle size and zeta potential before and after protamine coating and complexation to torula yeast RNA (TY-RNA). AAs expected size increases were observed for ZJNP and ZNP-RNA (Fig. 15C) a cationic surface charge shift and allowed to bind RNA shifting back in the anionic direction (Fig. 15D). Finally the range of RNA payloads with and without protamine coating was compared for several different singlestranded RNA types ranging from as low as only a few milligrams without protamine to more than one hundred for protamine coated TY-RNA, based on RNA loss from supernatant UV analysis (Fig. 15E).

[0235] RNA stability and expression activity: Binding of RNA to zinc or protamine coated zinc nanoparticle (ZNP) was shown by gel shift and circular dichroism (CD) spectroscopy, in vitro translation, mRNA expression and cell delivery was studied next (Figs. 16A-D). Binding, stability and expression of ZNP-RNA and ZNP-mRNA shown by RNA agarose gel electrophoresis (RAGE) is shown in Fig. 16A, circular dichroism is shown in Fig. 11 B, in vitro translation is shown in Fig. 16B, cell free mRNA expression in lysates is shown in Fig. 16C and GFP mRNA cell delivery is shown in Fig. 16E.

[0236] As shown in Figs. 16A-D, RNA stability after incubating torula yeast RNA (TY- RNA) with the nanoparticles in water at 37 °C followed by RNA agarose gel electrophoresis (RAGE) analysis was in the following order: ZnO > Fe3O4 > Ag > MSN ~ Cu > CNT ZnO NP (ZNP) and to a lesser extent Fe3O4, caused a slight gel shift consistent with their RNA interaction, but importantly retained RNA band staining intensity. In these cases, the band is tighter in comparison to the control RNA incubated over-night in water at 4 °C indicating some hydrolysis occurs in the control RNA but is protected in the ZnO NP and Fe3O4 samples. A demonstrable lack of RNA stain intensity or a smear pattern consistent with RNA degradation was seen with MSN, copper (Cu) NP and CNT, and silver (Ag) was intermediate. The CD data shown in Fig. 11 B shows the typical two peak CD pattern for poly I and poly C which is well-known to form an A-form RNA double helix. This is stabilized with peak enhancement when ZNP is complexed to it. Structural stability was a function of the stoichiometric ratio of poly l:C:ZnO-NP. As shown in Fig. 16B, although some variability was observed with two different batches of Luc mRNA used, both batches of ZNPe-mRNA formulations supported in vitro translation. Although mRNA expression was independent of protamine ratio with 1 :1 (high), 1 :10 (medium) and 1 :100 (low), all supporting translation, elution of RNA from the particles was protamine-dependent (Fig. 19). mCherry mRNA expression of the ZnO-protamine-mRNA was higher than ZnO-mRNA alone in both NIH3T3 and RAW264.7 cell extracts decreasing from three to six hours (Fig. 16C). mRNA cell delivery experiments were conducted with green fluorescent protein (GFP). mRNA was compared to GFP protein control and protamine enhancing cell fluorescence (Fig. 16D).

[0237] Temperature stabilization: RNA temperature stabilization was demonstrated next (Figs. 17A-B). [0238] Figures 17A-B. RNA Temperature-stabilization. Three independent DSC experiments (Fig. 11 A), RAGE analysis of heat treated RNA (Fig. 17B) and expression analysis from heat treated ZNP-mRNA (Fig. 17C).

[0239] As shown in Figs. 17A-B and Fig. 11 A, the temperature stabilization conferred to RNA by binding ZnO was investigated by differential scanning calorimetry (DSC). DSC can be used to estimate the melting temperature (Tm) for RNA. ZnO complexation to poly l:C increases its melting temperature from 63.9-64.7 °C for the RNA alone, approximately five to six degrees (Tm = 70.1 -71.1 oC) for the ZnO-poly l:C RNA species. To increase accelerated temperature stability similar to our prior work 14 or 100 nm ZnO nanoparticles were coated with protamine, the RNA was then allowed to bind, the preparations were then sedimented from alcohol, air dried and then re-suspended in phosphate buffered saline (PBS) and stored for two weeks at 30, 40 or 50 °C, versus material that was left out on the bench (20 °C) or in the refrigerator (4 °C) for 2 weeks. As shown in Fig. 17A, the RNA band co-migrating with control could be observed for all temperature storage conditions. Two different sizes of ZnO NP were tested, 14 nm versus 100 nm ZnO-NP and the RNA band staining intensity was slightly higher for 14 nm in comparison to 100 nm, as expected. Whereas some slight band broadening occurred more noticeably at the higher temperatures, intact RNA was clearly present after two weeks of storage even at room or above physiological temperature comigrating with control RNA. Importantly, this accelerated stability and retention of RNA integrity was here maintained by liquid formulations, even after high temperature exposure for two weeks. No lyophilization, concentration or dry powder step was required in order to demonstrate this stability. Finally expression of the heat treated mRNA samples in NIH3T3 cell extracts was protected by ZNP-mRNA (Fig. 17B).

[0240] Biological activity: The efficiency of RNA loading with protamine (ZNP) versus another cationic antiviral peptide, cathalecidin (LL-37)21 was evaluated next (Figs. 18A- F).

[0241] Figures 18A-F. RNA biological activity. Loading efficiency (Fig. 18A) and expression of SARS-CoV-2 spike mRNA: Protein expression was evaluated by immunostaining of HEK293A cells transfected with SARS-CoV-2 spike mRNA formulated in/as: (Fig. 18B) Zinc oxide nanoparticle-mRNA); (Fig. 18C) Protamine coated Zinc oxide nanoparticle (ZNP- mRNA) (Fig. 18D) Double protamine coated - one over the ZNP and other over mRNA (ZNP-protamine-mRNA-protamine) (Fig. 18E) Invitrogen™ Lipofectamine™ MessengerMAX™ transfection reagent (positive control); and Fig. 18(F) Mock-transfected cells which served as a negative control. The cells were probed with a recombinant humanized anti-SARS-CoV-2 spike-specific neutralizing monoclonal antibody. The secondary antibody was anti-human IgG-Alkaline Phosphatase, and Fast Red was the substrate. Arrows showing the cells expressing the spike protein.

[0242] Figs. 18A-F shows loading efficiency of RNA (TY-RNA) onto ZnO, ZNP or LL37 coated ZnO (ZN-LL37) (Fig. 18A) which was in the order; ZNP > ZN-LL37 > ZnO. Figures 18A-F also demonstrate spike mRNA expression in cells consistent with the data shown above. Spike mRNA expression comparable to commercial transfection reagent was observed both for protamine underlayer (Fig. 18B) or overlayer coating (Fig. 18C) demonstrating the activity of the formulation. Clearly the cells grew and divided much more in the case of ZNP treated cells comparing the cell density in each well to that of lipofectamine (Fig. 18D), transfection demonstrated even in the presence of a dense cell mat, indicating some toxicity of the lipid-based formulation.

[0243] CONCLUSIONS

[0244] The data support RNA temperature-stabilization of the ZnO-protamine-RNA (ZNP-RNA) formulation. Stability-indicating methods used here were RAGE, CD and DSC, each method independently verifying the stability conferred to RNA by interaction to ZnO or ZnO-protamine (ZNP). Importantly, structural stability is imparted to RNA by binding to ZnO nanoparticle as shown by CD which significantly increases RNA melting temperature as shown by DSC. An early report suggested RNA temperature stabilization by surface-functionalized mesoporous silica nanoparticles (MSN) allowing brief storage of RNA at 4 °C. In addition to MSN, a variety of other inorganic nanoparticle types including silver (Ag), iron oxide (Fe2O3/Fe3O4) copper and carbon nanotube (CNT) and composites have been reported for RNA delivery. Here the data clearly show binding to ZnO NP by gel shift better protects RNA from hydrolysis at physiological temperature in comparison to the other inorganic nanoparticle chemistries. Protamine coating seems to have a significant impact on the amount of RNAthat can be loaded onto the nanoparticle, likely owing to increasing the overall cationic surface charge for ionic interaction to RNA as shown by zeta potential. The presence of protamine does not detract from in vitro translation of mRNA or expression activity in cell extracts. However, the release of RNA from the particles after elution was protamine-dependent (Fig. 19). It is interesting to note that an outer layer of protamine seemed to enhance delivery of GFP mRNA into cells, however the RNA loading was more efficient for protamine than with LL37, another RNA binding peptide (RBP) (Fig. 20) known for its cell membrane and antiviral activity. Alignment of low molecular weight protamine to the FK16 fragment of LL37 that is thought to have this membrane cell surface activity has some interesting similarities to cell penetrating peptide (penetratin) (Fig. 21 ). This suggests that peptide sequences which balance RNA loading with release and intracellular delivery may be of some interest in the future. Indeed biomolecular interaction of RNA to ZNP is strong requiring multiple buffer components to elute the RNA off the particle (Fig. 22), again enforcing the stability of the RNA on particle to ZNP. Overall these data suggest ZNP provides temperature stability to RNA, and as such ZNP-mRNA represents a potential breakthrough for mRNA vaccination in situations where the samples cannot be maintained in cold storage. The application of ZNP to improve RNA payload, temperature stability and RNA delivery for other forms of therapeutic RNA is an important next step. RAGE analysis of different amounts of RNA was performed as described for the other RAGE analyses. Fig. 23 illustrates that the higher the concentration of RNA loaded into the lane, the higher the fluorescence when using RAGE analysis.

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