WO2023018384A1 - A vaccine composition against coronavirus infection - Google Patents

Abstract

The present disclosure includes a vaccine composition against infection of Coronavirus in a subject. Particularly, the vaccine composition comprises a plurality copy of a mRNA having polynucleotide sequence as setting forth in SEQ ID No. 1, a lipid nanoparticles mixture used for encapsulating the pluralities copies of the mRNA forming a colloidal dispersion of the mRNA-lipid particles thereto, one or more buffering agent for stabilizing the colloidal dispersion, wherein the lipid nanoparticles and the plurality copies of the mRNA are in a predetermined weight ratio of 12-36: 1.

Description

A VACCINE COMPOSITION AGAINST CORONAVIRUS INFECTION

Technical Field

The present disclosure relates a to vaccine composition effective in eliciting active immunity in a subject against Coronavirus infection. Particularly, the disclosed composition utilizes polynucleotides, in the form of mRNA, encoding for a specific antigenic peptide originated from a given strain or type of coronavirus for evoking the desired active immunity in the subject upon administrating the composition in a pharmaceutically effective dosage to the subject.

Background

Coronavirus disease 2019 (COVID-19), also known as the coronavirus or COVID, is a contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Of those people who develop noticeable symptoms enough to be classed as patients, most (81%) develop mild to moderate symptoms (up to mild pneumonia), while 14% develop severe symptoms (dyspnea, hypoxia, or more than 50% lung involvement on imaging), and 5% suffer critical symptoms (respiratory failure, shock, or multiorgan dysfunction). Despite of its serious clinical implication and relatively high mortality rate, there is no known medicament usable as treatment for the infection and only supportive care is generally provided to relieve the symptoms especially for those critically ill such that the patients gain enough time to develop the needed immunity themselves to fight off the infection. Therefore, the primary treatment is symptomatic. To curb the spread of the SARS-CoV-2 infection, vaccines and drugs have been developed by pharmaceutical companies and even approved by different government agencies in a speedy manner which was not seen before. For instance, there are at least 163 candidate vaccines under development based on 6 technology platforms: inactivated vaccines, protein subunit vaccines, mRNA vaccines, DNA vaccines, non-replicating/replicating viral vector vaccines, and viral-like particle vaccine, (the WHO landscape report by 19 July 2020). Among those, 23 candidates are entering clinical development (phase 1- 3) including 5 mRNA vaccines, 5 inactivated vaccines, 5 protein subunit vaccines, 4 DNA vaccines, 3 non -replicating viral vector vaccines, and 1 Viral-like particle vaccine.

To date, mRNA-based vaccines such as Modema, Pfizer-BioNTech and Johnson & Johnson’s Janssen are the few major brands being employed for various vaccination programs implemented in different countries as an effort to institute herd immunity thereto thus stopping the spread. There are so many uncertainties in terms of side effects, immunogenicity, and potential inherent in these vaccines due to the rush in developing and approving them. Further research may be needed to explore and unlock greater potential of the mRNA-based vaccine to finally produce a safer yet capable of generating better neutralizing antibodies and T-cell immune responses in the vaccinated subjects.

Summary

The present disclosure aims to provide a vaccine composition effective against Coronavirus upon administrating one or more effective dosages to a subject within a predetermined period of time.

Another object of the present disclosure is to offer a vaccine composition operable based upon a mRNA platform, which is believed to be able to induce production of relatively wide spectrum of antibodies and more target-oriented against coronavirus coming into contact with a subject being vaccinated by the disclosed composition.

Further object of the present disclosure is directed to a vaccine composition incorporated with specially prepared carrier system capable of yielding or generating an improved immunogenicity response in a subject upon administrating one or more effective dosages to the subjects.

At least one of the preceding objects is met, in whole or in part, by the present disclosure, in which one of the embodiments of the present disclosure is a vaccine composition against infection of Coronavirus comprising a plurality copies of a mRNA having polynucleotide sequence as setting forth in SEQ ID No. 1 capable of being translated into a peptide corresponding to at least a portion of a S-Spike protein of the Coronavirus; a lipid nanoparticles mixture used for encapsulating the pluralities copies of the mRNA forming a colloidal dispersion of the mRNA-lipid particles thereto; and one or more buffering agent for stabilizing the colloidal dispersion, wherein the lipid nanoparticles and the plurality copies of the mRNA are in a predetermined weight ratio of 12-36:1, wherein the translated peptide is free from di -proline (2P) or hexa-proline (6P).

In more embodiments, the pluralities copies of the mRNA is encapsulated by way of suspending the copies of the mRNA in the a buffering solution to form a suspension, mixing a lipid nanoparticles mixture in a liquid alcohol to form a lipid solution, mixing the suspension and the lipid solution at a controlled rate corresponding to a weight ratio of 1: 12-36 to form a mRNA-lipid mixture, diluting the mRNA-lipid mixture through adding in an aqueous phase carrying the mRNA, producing a colloidal dispersion of mRNA-lipid particles by flowing a buffering saline into the mRNA-lipid mixture, collecting the colloidal dispersion, diluting the liquid alcohol by flowing a second buffering saline into the collected colloidal suspension, concentrating the colloidal dispersion using tangential flow ultrafiltration, performing a diafiltration to remove the remaining liquid alcohol, performing a second diafiltration for introducing a cryoprotectant buffer into the colloidal dispersion, and adjusting the colloidal dispersion with a storage buffer.

For more embodiments, the plurality copies of mRNA are in an amount of 10-50 microgram for at least sufficiently inducing production of wide spectrum of neutralizing antibodies against and establishment of an active immunity against Coronavirus, preferably SARS-CoV-2.

For a number of embodiments, the polynucleotide sequence of the mRNA is modified by way of substituting uridine with Pseudouridine to improve the expression of the encoded peptide in the subject receiving the disclosed vaccine composition.

Still, in some embodiments, the buffering agent is any one or combination of Tris (2-Amino-2- hydroxymethyl-propane- 1,3 -diol, hydrochloric acid, sucrose, and water.

Brief Description of Drawings

Fig. 1 is a graph presenting percentage of RBD-ACE2 binding inhibition of immunized mice sera at 2 weeks after the 2nd immunization of CC-19 mRNA (S, SI and S2)/LNPl(Acuitas Therapuetic) by surrogate viral neutralization (sVNT) assays with the sera of vaccinated mice diluted at 1:7200 whereas the PBS groups (*) were diluted at 1:10, normal value <20%; S = a transmembrane domain and cytoplasmic domain deleted SASR-Cov-2 Spike protein without 2 prolines substitution;

Fig. 2 is a graph showing the neutralizing antibody response in mice two weeks after the first and second immunization with 30 microgram mRNA-S/LNPl; S = a transmembrane domain and cytoplasmic domain deleted SASR-Cov-2 Spike protein without 2 prolines substitution;

Fig. 3 is a graph showing the results of the induction of T cell response in BALB/c mice postimmunization of 30 microgram SI, S2 and S mRNA/LNPl, where T cell responses were analyzed on 14 days after the second immunization and the T cell responses were measured by IFN-y ELISpot in splenocytes stimulated for 20h with overlapping peptide pools spanning the SARS-CoV-2 SI (pooled #1-5) and S2 (pooled #6-10), with bars representing the mean ± SD of each pooled peptide (SFU=spot-forming units);

Fig. 4 are graphs (a) SI (upper) and (b) S2 (lower) showing immunogenic peptides generated and pooled in connection to T cells response in mice immunized with PBS, S (which comprises S 1 & S2 without transmembrane and cytoplasmic domains), S 1 and S2 mRNA vaccine, where IFN-y- secreting cells from each group were analyzed by EFISpot assay when stimulated by SI (upper) or S2 (lower) peptides pooled that the number of IFN-y- secreting cells were expressed as spot forming unit/million splenocytes with data being presented for individual mouse and bars indicating the mean of spot forming unit;

Fig. 5 is a graph showing kinetics of the neutralizing antibody (NAb) responses in Cynomolgus macaque immunized with 50 microgram CC-19 mRNA-S/ENPl where microneutralization assay was used to assess the NAb titers against wild-type SARS-CoV-2 virus in sera obtained at indicated timepoint and the titers were expressed as the reciprocal of the highest serum dilution at which the virus infectivity was reduced by 50% relative to the virus control with the collected data being presented as the mean ± SD for each group, dotted line representing limit of detection, squares representing the vaccinated animals, circles representing the control animals;

Fig. 6 is a graph summarizing frequency of spike- specific IFN-y positive T cells in 50 microgram CC-19 mRNA- S/ENP1 immunized macaques where T-cell responses were measured by IFN-y EFISPOT in PBMCs stimulated with overlapping peptide pools corresponding to the spike protein of SARS-CoV-2 at indicated timepoint with the dotted line being the cut-off titer, straight line being the mean value, the red squares denoting the CC-19 vaccinated animals, blue circles denoting the PBS- control animals, SFU being abbreviated for spot- forming units;

Fig. 7 is a graph showing generation of the neutralizing antibody responses in mice two weeks after the second immunization with the various doses of CC-19/ENP1, CC-19/ENP2 and mRNA-S-2P/LNP2 (30, 10 and 1 microgram), where microneutralization titers were expressed as the reciprocal of the highest serum dilution at which the virus infectivity was reduced by 50% relative to the virus control or MN50 that the data are presented for individual mouse from different experiments (* : Exp I, ** : Exp II and # : Exp III) and the horizontal lines indicate the geometric mean titers; of note mice were immunized 2 doses of each vaccine candidates in 3 weeks interval;

Fig. 8 is a graph of SARS-Cov2 wild-type live-virus micro-neutralization assay (MN-50) results in comparison between CC-19 mRNA/LNP2 vaccine/ (encoding for SARS-Cov2 transmembrane and cytoplasmic-deleted-spike protein) consists without and with 2 proline substitutions (K986P/V987P) (di-proline mutation or S-2P) at 3 different dosages in mice, each dot representing an individual mouse MN-50 titer and each solid horizontal line with the value representing the geometric mean titer of each study group; of note mice were immunized 2 doses of each vaccine candidates in 3 weeks interval;

Fig. 9 is a graph illustrating the outcome of T cell responses in B ALB/c mice immunized with the various dosages of CC-19/LNP1, CC-19/LNP2 and mRNA-S-2P/LNP2 (30, 10 and 1 microgram), where T cell responses were analyzed on 14 days after the second immunization and the T cell responses were measured by IFN-y ELISpot in splenocytes stimulated for 20 hours with overlapping peptide pools spanning the SARS-CoV-2 SI (pooled #1-5) and S2 (pooled #6-10) that the bars represent the mean ± SD of each pooled peptide from different experiments (*: Exp I, **: Exp II and # : Exp III) and the SFU being the abbreviation of spot-forming units; of note mice were immunized 2 doses of each vaccine candidates in 3 weeks interval;

Fig. 10 is a graph showing the neutralizing antibodies results of pseudovirus neutralization test (psVNT), against wild-type virus and variants of concern, in the sera of BALB/c mice two weeks after receiving the second CC-19 mRNA-S/LNP2 vaccination respectively at the dose of 1, 10 and 30 microgram;

Fig. 11 is a graph showing the results of pseudovirus neutralizing test (psVNT) against wild-type virus and different variants of Covid- 19 in subjects from a phase 1 trial being respectively vaccinated at 10, 25, and 50 microgram of CC-19 mRNA for 3 weeks interval comparing to sera panels from those vaccinated with Pfizer, AstraZeneca and Sinovac, each dot representing the reciprocal psVNT titer of each individual, bar and value representing geometric mean titer (GMT) of each group; Fig. 12 is a polynucleotide sequence of the SEQ ID No. 1 covering a 5’; UTP region, a translatable S-spike region, a 3 ’-UTP region and a 101 poly-A tail region. The translated protein is a secreted, TM and CT domain deleted, perfusion non- stabilized Spike protein which does not comprise of 2 or 6 proline substitutions.

Detailed Description

Hereinafter, the disclosure shall be described according to the preferred embodiments and by referring to the accompanying description and drawings. However, it is to be understood that referring the description to the preferred embodiments of the disclosure and to the drawings is merely to facilitate discussion of the various disclosed embodiments and it is envisioned that those skilled in the art may devise various modifications without departing from the scope of the appended claim.

Unless specified otherwise, the terms "comprising" and "comprise" as used herein, and grammatical variants thereof, are intended to represent "open" or "inclusive" language such that they include recited elements but also permit inclusion of additional, un-recited elements.

As used herein, the phrase “in embodiments” means in some embodiments but not necessarily in all embodiments.

The terms “LNP” and “lipid nanoparticles mixture” are used interchangeably throughout this specification referring to a mixture, which comprises at least one or more lipid types (i.e. ionizable lipids, cationic lipids, non-cationic lipids, polymer-conjugated lipids, helper lipids, etc., and one or more excipients including lipid-aggregation inhibitors, pharmaceutically acceptable salts, buffering agents, sucrose, etc. with each ingredient being prepared in a predetermined ratio or proportion for delivering the mRNA encoding at least a portion of a spike S, preferably free of any transmembrane and cytoplasmic domains, of SARS-CoV-2 into a subject to evoke active immunity thereof unless mentioned otherwise.

The terms “helper lipids”, as a lipid component of the lipid nanoparticles (LNP), refers to lipid molecules that are able to maintain or support the LNP structure or increase LNP stability. Some examples of the lipid molecules used as helper lipids, but not limit to, such as phosphatidylcholine (i.e. DSPC), cholesterol, PEG-lipid, etc. As used herein, the terms “approximately” or "about", in the context of concentrations of components, conditions, other measurement values, etc., means +/- 5% of the stated value, or +/- 4% of the stated value, or +/- 3% of the stated value, or +/- 2% of the stated value, or +/- 1% of the stated value, or +/- 0.5% of the stated value, or +/- 0% of the stated value.

According to one aspect of the present disclosure, a vaccine composition effective to act against infection of Coronavirus, preferably SARS-CoV-2, is describe hereinafter. Essentially, the disclosed vaccine composition comprises a plurality copies of a mRNA having polynucleotide sequence as setting forth in SEQ ID No. 1 capable of being translated into a peptide corresponding to at least a portion of a S-Spike protein of the SARS-Cov2; a lipid nanoparticles mixture used for encapsulating the pluralities copies of the mRNA forming a colloidal dispersion of the mRNA-lipid particles thereto; and one or more buffering agent for stabilizing the colloidal dispersion, wherein the lipid nanoparticles and the plurality copies of the mRNA are in a predetermined weight ratio. Further, the translated peptide is a secreted form of an extracellular domain of the SARS-Cov2 S-spike protein (without transmembrane and cytoplasmic domains) and free from di-proline (2P) or hexa-proline (6P) substitutions. Inventors of the present disclosure found that portion of or complete S-spike protein originated from SARS-CoV-2, has the capacity to evoke wide spectrum of neutralizing antibodies effective in binding onto the viruses coming into contact thus eliminating the viruses prior having it to infect the subject or host. The portion of or complete peptide of the S-spike protein applicable to induce the host immunogenic response can be encoded by the mRNA and subsequently delivered to the subject for expression thereby to eventually trigger production of the corresponding neutralizing antibodies and T-cell immune responses capable of acting against the coronavirus. For some embodiments, the portion of S-spike protein employed in the disclosed vaccine composition can be encoded by a corresponding polynucleotide sequence, in the form of mRNA, as setting forth in SEQ ID No. 1 of Fig. 12. It is important to note that portion, preferably at least 80% of the give sequence, of the referred polynucleotide sequence can be used in the production or manufacturing of the disclosed vaccine composition as well in other embodiments instead of the exact sequence being illustrated in Fig. 12. These shorter fragments or portions are likely being used for offering the like vaccine composition featuring better and safer immunogenic response elicited in connection to a specially prepared carrier system.

Pursuant to several preferred embodiments, the polynucleotide sequence of the mRNA is modified by way of substituting uridine with pseudouridine. Specifically, the nucleoside modified mRNA is generated by replacing every uridine with pseudouridine (y). The nucleoside-modified mRNA employed in the disclosed vaccine composition may offer several advantages over the unmodified counterpart. For example, the modified mRNA, may lead to higher protein expression because the modification permits stable, and even longer, translation due to extended half-life. Also, the mRNA substituted with the Pseudouridine becomes less stimulatory towards several host defense RNA sensors including protein kinase R (PKR), toll- like receptor (TLR)3, TLR7, TLR8 and retinoic acid- inducible gene I (RIG-I). The like chemical properties of the mRNA modified with Pseudouridine also partly attributes to the extended half-life and improved expression in the subject received the vaccination through the disclosed composition.

For more embodiments, the plurality copies of mRNA are in an amount of 10 to 50 microgram to at least sufficiently induce production of wide spectrum of neutralizing antibodies against and establishment of an active immunity against Coronavirus, preferably SARS-CoV-2. Considering that the immunization process on the subject utilizing the disclosed vaccine composition may require injection or administration of multiple doses spanning across a predetermined period of time, the preferred amount of the mRNA in each dose may range from 10-50 microgram. Accordingly, the subject may receive or need a total of 10-50 microgram of the mRNA throughout a complete immunization course. For an immunization course of multiple doses, the amount of the mRNA contained in the vaccine composition may be varied to finally yield the desired level of active immunity against SARS-CoV-2. Also, the amount of the mRNA incorporated into the disclosed vaccine composition can be varied depending on the carrier used for delivering the payload.

Efficient delivery of the mRNA encoding for the immunogenic peptide is the one of the key factors determining the clinical translation and protection rate of the mRNA-based vaccine. Among potential nonviral vectors, lipid nanoparticles (LNPs) are proven efficacious clinically. Particularly, LNPs efficiently encapsulate and condense the RNA deposited thereto, protect the encapsulated RNA or mRNA against degradation within the extracellular space, and localize the payload at the membrane of the target cell facilitating cellular uptake and endosomal escape into the cytosol for translation and being processed by the cellular proteasome. Such advantages allow the generated peptide epitopes to enter the endoplasmic reticulum to be loaded onto major histocompatibility complex (MHC) class I molecules. Followed by transporting the MHC molecules to the surface of the cell for presenting the antigenic epitopes to CD8 T cells along with costimulatory signals to finally yield the antigen- specific antibodies capable of acting against Coronavirus present in the body of the subject. Inventors of the present disclosure discovered the combined use of the mRNA and a specially prepared lipid nanoparticles mixture, namely LNP1 (Acuitas Therapuetics), and LNP2 (Genevant Sciences), is able to generate effective immune response in the vaccinated host compared to commercially used LNP found in one or more presently available mRNA-based vaccine. More specifically, the experiments performed by the inventors of the present disclosure unveiled that the disclosed vaccine composition utilizing the mentioned lipid nanoparticle mixture or LNP2 elicits better and more long-lasting immune response in the vaccinated host for better protection against SARS-CoV-2. The formation of the lipid- mRNA nanoparticles facilitates dispersion within the disclosed composition possibly having an interfacial layer functionable to mask or change the physiochemical properties of the encapsulated mRNA freeing the mRNA from being detected by the host defenses against insertion of foreign RNA into the body of the subject. The lipid nanoparticles mixture of the present disclosure also favor diffusion of the payload across the cellular membrane and ready for subsequent expression thereby in the cells of the subject at a site received injection of the disclosed vaccine composition. In order to successfully encapsulate the copies of mRNA to attain the advantages as described in the setting forth, the lipid nanoparticles mixture and the plurality copies of the mRNA are preferably in a predetermined weight ratio of 12-36: 1.

For many embodiments, the lipid nanoparticles (LNPs) or lipid nanoparticles mixture may comprise one or more lipid types (i.e. ionizable lipids/or amino lipids, cationic lipids, noncationic lipids, polymer-conjugated lipids, helper lipids, or the likes etc.) and one or more excipients (i.e. lipid-aggregation inhibitors, pharmaceutically acceptable salts, buffering agents, sucrose, etc.), in a predetermined ratio or proportion for delivering the mRNA encoding at least a portion of peptide or protein, preferably a portion of S spike protein. For example, the lipid components of LNP may comprise an amino lipid, and three helper lipids (namely DSPC, Cholesterol, and PEG). For the buff ers/or buffering agents, it also may be selected from one or a combination of potassium phosphate, potassium chloride, sodium phosphate, sodium chloride, sodium acetate, Tris, hydrochloric acid, water, ethanol, and antioxidants. Further, most of the buffer materials will be removed from the final mRNA-LNP vaccine, only the storage buffer which may be sucrose-Tris pH8 buffer composed in the final vaccine. In accordance with several embodiments, the plurality copies of the mRNA are encapsulated with a lipid nanoparticles mixture in a weight ratio of 1: 12-36 to form a mRNA-lipid mixture or mRNA-lipid particles for further dispersion with one or more buffering agents for particles stability by decreasing particle aggregation. A colloidal form of the mRNA-lipid mixture or mRNA-lipid particles is subsequently preserved in a storage buffer. One skilled in the field shall appreciate that fact that the mRNA can be reacted or encapsulated with the lipid nanoparticle mixture through one or more manners, processes, or steps other than the embodiments set out above. Such modification about the manners, processes, and/or steps to encapsulate the mRNA utilizing the lipid nanoparticles mixture or composition, as described, shall be deemed under the scope of the present disclosure.

The disclosed vaccine composition may be effective against several stains of the Coronavirus, but not limited to, SARS-CoV-2. In more particular, peptide encoded by the mRNA may elicit production of one or more neutralizing antibodies which can actually act against other strains of Coronavirus having antigenic surface epitopes or proteins similar or almost similar to the one encoded by the mRNA. Thus, the subject received vaccination through a single course, one or multiple doses, of the disclosed composition may equipped with active immunity towards other Coronavirus in addition to SARS-CoV-2.

To stabilize the reagents in the disclosed vaccine composition, one or more buffering agents may be used to render the reagents more resistant against pH and temperature fluctuation without substantial decomposition or degradation throughout the shelf life of the disclosed vaccine due to transportation and movement between storage facilities. The buffering agent is any one or combination of Tris (2-Amino-2-hydroxymethyl-propane-l,3-diol), hydrochloric acid, sucrose, and water.

The following example is intended to further illustrate the disclosure, without any intent for the disclosure to be limited to the specific embodiments described therein.

Example 1

A plasmid DNA template was transformed into a Stable competent cells (NEB® Stable Competent E. coli, New England BioLabs Inc.) by heat shock and incubated on LB agar plates with Kanamycin. Plates were cultured at 30 °C for approximately 24 hours. Three colonies were selected and inoculated in 50-mL animal-free LB medium with antibiotics for around 11 hours to reach OD600 between 0.6 to 1.2. Then 9 vials of glycerol stocks were prepared and stored at =£ -80 °C ± 10 °C. This cell stock was assigned as Research Cell Bank. One vial of RCB was inoculated in 2 x 100 mL animal-free medium without antibiotic and inoculated at 30 °C, 200 rpm for approximately 16 hours. Fermentation and harvesting: Cells were harvested by centrifugation. Cell paste was collected.

The cell paste was lysed and concentrated by Tangential Flow Filtration (TFF). The supernatant was loaded onto Bestarose 6FF column. After washing and eluting, fractions were pooled and further loaded onto columns; PlasmidCap Mustang and Fractogel EMD DEAE. The final eluted samples were concentrated and exchanged into Tris-buffer using ultrafiltration system. The plasmid DNA was obtained after final sterilization using 0.2 pm filtration.

The plasmid DNA template (6703 base pairs) was digested by linealization with Aflll (one cut site) by incubating the digestion reaction containing pDNA with Afin enzyme in buffer solution at 37 °C for 200-240 minutes. The complete linearization DNA was checked by agarose gel electrophoresis. Following the linearlization, DNA was purified by pheno l/ch loro form extraction followed by ethanol precipitation. The digested DNA was further mixed with phcnol/lchloroform-isoamyl alcohol at equal volume then vigorously mixed and centrifuge at maximum speed to separate the phases. The aqueous layer was subsequently removed. Additional water was added for back extraction and washing. Finally, ethanol was added to obtain purified DNA.

The purified linear DNA was used as a transcription template for transcribing into long-chain mRNA using recombinant T7 RNA-polymerase in the present of 4 nucleotide triphosphates (ATP, CTP, GTP, Nl-Methyl-Pseudo-UTP) and a derivative of the cap trinucleotide (CleanCap® AG).

At the end of the transcription reaction and the template DNA was hydrolyzed with DNase I. The reaction was treated with Proteinase K to degrade enzymes. The crude mRNA product was obtained after removal of nucleotides (NTPs), DNA fragment and enzymes by diafiltration into water. Purification was performed using reverse-phase high-pressure liquid chromatography (RP-HPLC) to further remove any remaining contaminant; residual protein, residual DNA, short RNA fragments, residual salt, endotoxin and double strand RNA (dsRNA). The purified mRNA was exchanged and diafiltrated into sodium citrate, pH 6.4. A purified ChulaCovl9 mRNA DS comprising a polynucleotide sequence as setting forth in SEQ ID No. 1 was obtained after concentration and processed by 0.2 pm filtration. Example 2

The encapsulation was performed by controlled mixing of mRNA and lipids in a solvent environment conducive to the formation of sub-100 nm particles. Particularly, the ChulaCovl9 mRNA DS stock solution is prepared by dilution of the mRNA in acetate buffer (100 mM) at 20 ±5 °C. Embodiments of the LNPs may comprise different lipid components in a predetermined ratio for the desired delivery of the mRNA encoding at least a portion of a spike S protein of SARS-CoV-2 into a subject to build a protective immunogenic response. For instance, one LNP embodiment may comprise ALC-0315 or other amino lipids, DSPC, Cholesterol, and PEG-DMA in an approximate molar ratio of 50, 10, 38.5, and 1.5.

The major goals of encapsulation process, concentration and diafiltration, and dilution and sterile filtration are to obtain mRNA encapsulation efficiency of at least 85%, mRNA integrity of 60%, mRNA concentration of 0.16-0.24 mg/mL, the particle size within 50-120 nanometers, and with purity as specified according to the World Health Organization (WHO): Annex 3 - POST-ECBS version 2021 (Evaluation of the quality, safety and efficacy of messenger RNA vaccines for the prevention of infectious diseases: regulatory considerations).

In brief, the processes included 4 Lipids (ionized lipid, PEG-lipid, DSPC, cholesterol) were weighed according to the mentioned optimized ratio above and then combined using ethanol to achieve quantitative transfer and the mixture was warmed to dissolve the lipids then allowed to cool and filtered through a 0.2 pm filter. The mRNA CC- 19 was prepared by diluted in acetate buffer (100 mM) at room temperature. The lipid and mRNA solutions are mixed at room temperature in a controlled rate maintaining a lipid to drug weight ratio of 12-36:1 depended on the optimization. Upon mixing, RNA-Lipid particles were formed as with aqueous mRNA solution it was reducing the lipid solubility of the ethanolic lipid solution. Immediately after mixing, a stream phosphate-buffer saline (PBS) is introduced, further diluting the ethanol content and also raising the solution pH. The final buffer exchange was with 10 % sucrose 5 mM Tris pH 8 as a final cryoprotectant buffer.

Example 3

Since safety and delivery efficiency of mRNA vaccine depends on formulation of lipid nanoparticle (LNP); lipid composition and ratio, CC-19 immunogens encapsulated in various LNP formulations; LNP1 (Acuitas Therapeutics) and LNP2 (Genevant Sciences) were evaluated in proof-of- concept studies in mice and non-human primates. Challenge study was performed in KI 8- hACE2 transgenic mice model after confirming the immunogenicity in proof-of-concept studies. A proof-of-concept study of ChulaCovl9 or CC-19 mRNA vaccine candidates were evaluated in mice and non-human primates to select a safe and immunogenic SARS-CoV-2 immunogen. These mRNA vaccine candidates include mRNA encoding S (SI and S2 extracellular portion of a S-Spike protein without transmembrane and cytoplasmic domains, SI or S2 which were formulated or encapsulated in lipid nanoparticle formulation- 1 (LNP1, Acuitas Therapeutics) (Table 1). An immunogen (mRNA candidate) inducing highest immune response in mice, in this case it was the mRNA-S (or later on called ChulaCovl9 or CC-19), was selected to further use in a second POC for assessment of safety and immunogenicity in non-human primates.

Table 1 CC-19 mRNA/LNPl Vaccine composition

Example 4

To evaluate the immunogenicity of CC-19 mRNA/LNPl vaccine candidates which encoding SARS CoV-2 spike (S, SI and S2) in BALB/c mice; (S is transmembrane and cytoplasmic domain deleted spike protein, and S 1 and S2 are spike protein subunit 1 and spike protein subunit 2, respectively.

4-6 weeks old female BALB/c mice (5 mice/group) were immunized intramuscularly, 2 doses in 4-week interval with either CC-19 mRNA/LNPl vaccine candidates (mRNA-S, mRNA-Sl or mRNA-S2). Control group was immunized with PBS. Mice were bled at week 0 (baseline) and week 6 for antibodies measurement using surrogate virus neutralization test (sVNT) and microneutralizing test against wild type SARS CoV-2 virus at 50% reduction (MN50). The principle of surrogate viral neutralization or sVNT analysis is to measure circulating antibodies against SARS-CoV-2 that block the interaction between the receptor binding domain (RBD) of the viral spike glycoprotein with the ACE2 cell surface receptor (Genscript, USA). Mice were sacrificed and harvested the spleens on week 8 for T-cell analysis. IFN-y ELISpot assay was performed with the peptide pools of 15-mer peptides overlapping by 10 amino acids of SARS-CoV-2 spike protein subunit 1 (SI) and 2 (S2); 5 peptide pools per protein (total 253 peptides) as stimulating antigens. Concanavalin A and culture media were used as positive and negative control, respectively. Results are expressed as spot-forming unit (SFU) per million splenocytes.

Table 2 The protocol for mice immunization with CC-19 mRNA/LNPl vaccines (N=20)

Circulating antibodies against SARS-CoV-2 were preliminary analyzed at 2 weeks after the second immunization by sVNT assay. The highest sVNT was observed in mice immunized mRNA-S while mRNA-Sl immunization showed lower activity. As expected, sVNT was not observed in mice immunized with mRNA-S2 and PBS control as shown in Fig. 1.

As the CC-19 mRNA-S immunization showed the highest sVNT activity, neutralizing antibody titers were then further analyzed by microneutralizing test against wild type SARS- CoV- 2 virus at 50% reduction (MN50). The neutralizing antibody titer could be detected at the moderate level at 2 weeks after the first immunization. The MN50 titers at this time-point were ranged from 80 - 160 (Geometric mean titer, GMT = 121). At 2 weeks after the second immunization (week 6), a marked increase of MN50 level were observed which ranged from 20,480 - 81,960 (GMT = 40,960). The neutralizing antibody was further increased when analyzed at week 8, MN50 titers were ranged from 40,960 - 163,840 (GMT = 71,316) as shown in Fig. 2. Microneutralization assay was used to assess NAb titers against wild-type SARS-CoV-2 virus in sera obtained at indicated time point. The titers were expressed as the reciprocal of the highest serum dilution at which the virus infectivity was reduced by 50% relative to the virus control or MN50. Data are presented for individual mouse in Fig. 2 with the horizontal lines indicating the geometric mean titers. Example 5

To evaluate SARS-Cov2 specific T cell responses measured by IFN-y-ELISpot assay in comparison between 3 different mRNA/LNPl immunogens: S-mRNA, Sl-mRNA and S2-mRNA tested in mice, the results showed splenocytes from mice immunized with 30 microgram mRNA-S/LNPl secreted IFN-y when stimulated with peptide pools from both S 1 and S2 regions with 1,466 and 1,078 SFU per million splenocytes; whereas mice that were immunized with either 30 microgram mRNA-S 1 or mRNA-S2 induced IFN-y secretion only after stimulated with the corresponding peptide pools from each region. This could be implied that mRNA-S was a potent immunogen and induced better breadth of epitope specific T-cell responses (Fig. 3). T cell responses in individual mouse for each peptide pool is shown in Fig. 4 that the peptide pool #3-5 and pool #9 from SI and S2 respectively were the most potent peptides pools in induction of IFN-y secretion. The analyzed regions of peptide pool #3-5 and #9 corresponded to the receptor- binding domain (RBD) in SI and the central helix in S2, respectively.

In a first proof-of-concept study, BALB/c mice were immunized via i.m. route at Day 0 and boosted on Day 28. The finding showed CC-19 mRNA-S/LNPl induced the highest titer followed by CC-19 mRNA-S 1 when screened by using surrogate viral neutralization test (sVNT). sVNT titer could not detected in CC- 19 mRNA-S2 immunized mice sera. In the CC- 19 mRNA-S group, NAb, MN50 titer at 2 weeks after the first immunization was 121 in GMT; and markedly increased to GMT=40,960 at 2 weeks after the boost. CC-19 mRNA-S/LNPl also elicited higher, and more breadth specific T cell responses measured by IFNy-ELISpot assays as compared to CC-19 mRNA- S 1 or S2/ LNP1 formulation. From these promisingly high humoral and cellular immune responses, CC-19 mRNA-S was then selected for further studies in non-human primates.

Example 6

To evaluate the safety and immunogenicity of CC-19 mRNA vaccine which encoding SARS CoV-2 spike (S) formulated with LNP1 (mRNA-S/LNPl) in cynomolgus macaques (Macaca fascicularis).

Seven animals 2.5 to 6.5-year-old female cynomolgus macaques, with body weights ranging from 2.7-4.1 kg, were used in this experiment. All monkeys had been tested negative for antibodies against SARS-CoV-2. The animals were housed in individual cages in an animal biosafety level one (ABSL-1) containment facility, maintained at constant room temperature (25°C) with a 12-h light/12-h dark photoperiod, and fed fresh fruits and vegetables every day. Before bleeding and immunization, monkeys were anesthetized with Zoletil 2-5 mg/kg and dexmedetomidine 20-50 microgram/kg. Monkeys were immunized according to protocol in Table 3. Seven animals were randomized into two groups as follows; i) 50 microgram of CC-19 mRNA-S/LNPl (N=5) and ii) PBS-control (mock) (N=2). All animals were received an intramuscular injection in the deltoid muscle (0.3 mL of the vaccine) on Days 0 and 30. Venous blood was collected on Days 0, 15, 30, 45, 60, 75, 90, 120, 150 and 180. Serum was separated for biochemical examination and stored at -20°C until performed the microneutralizing assay. EDTA blood samples were collected for hematological examination and ELISpot assay.

Table 3 Immunization protocol for cynomolgus macaques with ChulaCoV-19 or CC-19 mRNA-S/LNPl vaccine (N=7) (i.m = intramuscular injection)

Following immunization, each animal was monitored daily for clinical signs including changes in activity, rectal temperature, appetite, rash, frequency of defecation and stool consistency. At the injection site, the Draize skin irritation test was performed after vaccination following the OECD-Test guideline (TG) 404. Briefly, the signs of erythema and edema were scored (from 0 to 4 depending on the severity) at 1, 4, 24, 48 and 72 hr. If an irritation persists for longer than 3 days (72 hrs.), it should be followed every day until the irritation resolves. Hematological (hematocrit, hemoglobin level and white blood cell count) and biochemical analysis (aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin (ALB), alkaline phosphatase ( ALP) , total bilirubin ( TBIL) , direct bilirubin ( DBIL) , cholesterol (CHOL), glucose (GLU), total protein (TP), blood urea nitrogen (BUN), triglyceride (TRIG), uric acid (UA), globulin (Globu) and creatinine (CREA) were performed at Day 0 as the normal (baseline) levels and every 30 days after vaccination until the end of the experiment.

Cynomolgus macaques were immunized two times at Day 0 and Day 30 with 50 microgram CC-19 mRNA- S7 LNP1 (Groupl; n= 5) and PBS- control (Group2; n=2). Following each immunization, all animals showed normal in activity and appetite and had normal weight gain. No systemic abnormalities were observed for any group. Particularly, none of the monkeys showed skin rash at the vaccination site. Similarly, the hematological and biochemical (ALT, AST, TP, ALB, ALP, TBIL, DBIL, BUN and CRE) parameters in all macaques remained constant within the typical range throughout the experiment.

Also, animals in both vaccine group (n= 5) and the control group (n= 2) have normal physical appearances by examination. All animals in both vaccine and control groups have normal weight gain from baseline (Day 0) to the end of experiment (Day 180). Animals in both vaccine and control groups have comparable mean rectal temperature from baseline to the end of experiment (Day 180). There were no statistically significant differences in all blood chemistry parameters (AST, ALT, ALB, ALP, TBIL and DBIL) of liver function test at any timepoints both in group 1 (CC-19) and group 2 (PBS -control). All parameters were generally situated within the reference ranges.

At predetermined time-point, immune responses were measured by microneutralizing test against wild type SARS-CoV-2 virus at 50% reduction (MN50) at Faculty of Science, Mahidol University. IFN-y ELISpot assay was performed by in vitro PBMC stimulation with the peptide pools of 15-mer peptides overlapping by 10 amino acids of SARS-CoV-2 spike protein subunit 1 (SI) and 2 (S2); 5 peptide pools per protein (total 253 peptides). Concanavalin A and culture media were used as positive and negative control, respectively. Results are expressed as spot- forming unit (SFU) per million cells. There were no statistically significant differences in BUN and CREA (creatinine) at any timepoints both in group 1 (CC-19) and group 2 (PBS -control). Both parameters were generally situated within the reference ranges.

Neutralizing antibody (NAb) play an important role in the protective immune response to the SARS-CoV-2 infection. To evaluate the NAb levels and the importance of multiple immunizations, the kinetics of the NAb responses were determined by microneutralization assay (Fig. 5). All serum samples from animals before immunization (Day 0, baseline) showed no NAb. Two weeks after the first immunization (Dayl5), 4 out of 5 of vaccinated animals showed seroconversion with geometric mean titer (GMT) 28.99 and showed slightly increased of the NAb on four weeks after the first immunization (MN50 titers= 20- 320, GMT= 105. 56) . At 2 weeks after the second immunization (Day 45), the NAb titer were significantly increased and reached a peak with GMT= 5120 (MN50 titers= 2560- 10240). Kinetically, the NAb titers were declined in later time points, i.e., approximately 5-fold in 15 days later (Day 60), 24-fold in 75 days later (Day 120) and 42-fold in 135 days later (Day 180). The NAb titers (GMT of 121) were still detected up to 5 months post-last immunization in all macaques. As expected, no NAb was detected in the control group (Fig. 5).

SARS-CoV-2 spike specific T cell responses were detected by IFN-y ELISPOT assay (Fig. 6). All PBMC samples from animals before immunization (Day 0, baseline) showed no IFN- y specific T cell response. One month after the first immunization (Day30), 2 out of 5 of vaccinated animals showed positive IFN- y T cell responses of 19 SFU/ million PBMCs. Interestingly at Day 60 and Day 90, all animals showed significantly IFN- y specific T cell responses of 223 and 590 SFU/million PBMCs, respectively. The IFN- y specific T cell responses were declined on Dayl20 to 158 SFU/ million PBMCs. As expected, no T cell response was observed at any timepoint in the PBS (mock) animals (Fig. 6).

The results showed after immunization with CC-19 mRNA-S/LNPl on Day 0 and Day 30, the humoral and cell- mediated immune responses were elicited significantly. No systemic side effects were observed. The second immunization elicited significant difference of NAb titers (peak at 2 weeks after the second dose of immunization) comparing to the first immunization. The kinetics of NAb response after the immunization showed a time -dependent decline in the NAb titers. The results showed that CC-19 mRNA- S/ LNP1 vaccine can induce effective and durable humoral immunity (at least 6 months) and provide strong cell-mediated immunity against SARS-CoV-2, is safe in monkeys, and the vaccine may be a good candidate for clinical trials.

Since the SARS- CoV-2 virus is spreading around the world, development of an effective vaccine with high immunogenicity and safety is crucial for control and prevention of the global COVID-19 pandemic. Early studies in mice showed our CC-19 mRNA-based vaccine can induce a robust humoral and cell- mediated immune responses. The present disclosure further explored the immunogenicity of CC-19 mRNA vaccine in cynomolgus macaque, a non- human primate model of SARS-CoV-2 infection and pathology (1). Here, we showed that both NAb and T cell responses were significantly elevated after the second immunization.

In advance of the experiments performed, four SARS-CoV-2 vaccine candidates were evaluated the protective efficacy in macaques, including an adenovirus-vectored vaccine (ChAdOxl nCoV- 19), a DNA vaccine and two inactivated vaccines (PiCoVacc and BBIBP-CorV). For ChAdOxl nCoV-19, a single vaccination can induce low titers of NAb (5-40) and also low titers of SARS-CoV-2 spike specific T-cell response (<50 SFU/million PBMCs), however, a significantly reduced viral load in bronchoalveolar lavage fluid and respiratory tract tissue and no pneumonia was observed in vaccinated animals challenged with SARS-CoV-2 compared with control (2). The SARS- CoV-2 DNA vaccine experiment demonstrated that the vaccine-induced NAb titer is correlated with the protective efficacy, however, overall NAb titer in DNA-vaccinated macaques is less than 100 (3). For the PiCoVacc, three immunizations with high dose conferred a complete protection in macaques against SARS-CoV-2 challenge, overthought the NAb titer before challenge is less than 100 (4). Interestingly, for BBIBP- CorV, all macaques vaccinated even at low dose showed detectable high NAb titers (GMT=215) with undetectable viral load in the lung after virus challenge (5). Comparison of our NAb titers to the earlier studies reveals that our CC- 19 mRNA-based vaccine can elicit very high NAb after 2 doses injection.

In conclusion, the present disclosure demonstrates the CC-19 mRNA-S encapsulated in LNP1 (Acuitas Therapeutics) is safe, with no local or systemic adverse reactions, as assessed by hematological and biochemical analyses and can induce high NAb response in the cynomolgous macaques. The safety and immunogenicity profile of the CC-19 mRNA-S vaccine indicates the potent immunogen which could be used in human trials.

Example 7

To explore which LNP formulation when encapsulate this disclosed mRNA immunogen can induce better specific immunogenicity, we have conducted a comparison between LNP 1 and LNP2, and to explore whether prefusion stabilization of Spike protein is essential, this disclosed S-mRNA immunogen, which did not contain 2 proline (2-P) substitutions is comparable to the previous reports of S-mRNA contained 2 proline substitutions (S-2P), both mRNA immunogens were evaluated in mice.

Forty female BALB/c mice 4-6 weeks old were randomly divided into 8 groups (5 mice/group) as shown in Table 4. Briefly, mice (5 mice/group) were two times immunized intramuscularly with different immunization and bleeding schedule depended on the concentration of CC-19 mRNA- S/ LNP2, mRNA- S- 2P/ LNP2 or CC-19 mRNA- S/ LNP1 vaccines. For the high dose, Group 1 and 4, mice were received 30 microgram vaccines on week 0 and week 4 (4-week interval), whereas the medium-dose (10 microgram) and low-dose (1 microgram), Group 2, 3, 5, 6, 7 and 8, mice were vaccinated on week 0 and week 3 (3 -week interval). Serum samples were collected at indicated time points to evaluate the antibody production. Mice was sacrificed on week 8 (Group 1, 4) and week 5 (Group 2, 3, 5, 6, 7 and 8) for assessment of fFN-y T-cell response in splenocytes (ELISpot).

Table 4 Summary of arrangement for mice immunization with CC-19 mRNA-S VS mRNA-2P-S encapsulated in LNP2 (N=30)

All groups of mice immunized with CC-19 mRNA-S encapsulated either LNP1 or LNP2 and mRNA- S-2P encapsulated in LNP2 with various doses induced high NAb with a dose-dependent response even at the lowest dose (1 microgram) (Fig. 7). By using the LNP2 formulation, CC-19 mRNA-S showed comparable NAb titers with mRNA-S-2P at 30 microgram and 1 microgram doses (GMT 62,064 and 11,763) and showed a higher NAb titer at 10 microgram dose (GMT 54,047 VS 20,480). Interestingly, we observed significant difference of NAb induction between LNP1 and LNP2 formulations when encapsulated with CC-19 mRNA-S. The LNP2 formulation induced higher NAb titers than the LNP1 around 1.5 to 5.3 folds (Fig. 8).

Splenocytes from mice immunized with various doses of CC-19/LNP1, CC-19/LNP2 and mRNA-S-2P/LNP2 were analyzed as summed frequency of spike-specific IFN-y positive T cells (Fig. 8). In contrast to the NAb responses, the T cell response is not a dose level-dependence. By using a LNP2 formulation, the highest magnitude responses were observed in 10 microgram dose of both CC-19 and S-2P immunized mice. An analysis of the individual pool peptides in all vaccinated groups showed that peptide pool #3-5 and pool #9 from S 1 and S2, respectively were the most potent peptides pools in induction of IFN-y secretion (Fig. 9).

The present disclosure characterized the immunogenicity of CC-19 mRNA vaccine in BALB/c mice. Based on the above results indicating CC-19 mRNA-S encapsulated in two LNP formulations induced remarkably high NAb against SARS-CoV-2 in a dose-dependent manner. However, the higher immunogenicity responses were observed in the novel LNP2 formulation.

Therefore, the CC-19 mRNA-S/LNP2 vaccine was selected for the clinical trial candidate. The CC-19 mRNA-S/LNP2 candidate is currently manufacturing for the GMP clinical batch for Phase 1/2 clinical trials.

Example 8

The sera of BALB/c mice after the second CC-19 mRNA-S/LNP2 vaccination at dose of 1 , 10 and 30 microgram, for two weeks (from the example?) was tested the neutralizing antibodies by using pseudovirus neutralization assay (psVNT) as shown in Fig. 10.

The experimental results show that all sera of vaccinated mice in any concentration (low, moderate, high) when tested with viral candidate having spike of wild-type strain are capable of potent viral protection. The GMT is at 6,142, 26,416 and 87,096 respectively. Further, when the sera were tested with the alpha, and beta strains, the level of tested the neutralizing antibodies remain or decrease not less than 2.8 folds relatively to wild-type strain. Interestingly, when the sera were tested with the delta strain, the levels of the neutralizing antibodies are decreased to 3.7-5.9 folds. This means the delta spike is highly tolerant to the immunization. As a result, the vaccine candidate should induce the high level of the cross-neutralizing antibodies for various strain protection. Example 9

To evaluate whether this disclosed CC-19 mRNA sequence when encapsulated with a proper LNP in this case is LNP2 can protect the animals from SARS-Cov2 virus challenges, eight female K18-hACE2 mice (B6.Cg-Tg(K18-ACE2)2Prlmn/J), 5-6 weeks of age (from The Jackson Laboratory, USA) were randomly divided into 2 groups. Four mice in group 1 were infected with SARS-CoV-2 intranasally at a dose of 2x104 pfu (50 uL) (Seo SH and Jang Y, 2020). Four mice in group 2 were infected with the lower dose 2x103 pfu (50 uL). The infected mice were daily monitored for body weight change and mortality. Six -day post infection, mice were sacrificed for determining virus titers in different tissues (nasal turbinate, brain, lung, and kidney) and for histopathology. Virus titers were quantified by RT-qPCR and by determining the logio TCID50 values. The SARS-CoV-2 infection schedule and protocol are summarized in Table 5.

Table 5 SARS-CoV-2 infection protocol in K18-hACE2 transgenic mice

Seventeen female K18-hACE2 mice (B6.Cg-Tg(K18-ACE2)2Prlmn/J), 5-6 weeks of age (from The Jackson Laboratory, USA) were randomly divided into 3 groups. Group 1 and groups 2, 6 mice/group were immunized intramuscularly via quadricep muscles with 2 doses, 3 weeks apart of CC-19 mRNA/LNP2 at dose of 10 microgram and 1 microgram, respectively. In negative control, group 3 (5 mice), mice were immunized with PBS. At 2 weeks after the second immunization, mice were challenge intranasally with 2xl0⁴ (20,000) pfu (50 pL) of SARS-CoV-2. Blood was collected at week 0, week 2, week 5 and week 5 plus 6 days for antibody kinetic measurement. Six-day post challenge, mice were sacrificed for determining virus titers in different tissues (nasal turbinate, brain, lung, and kidney) and for histopathology. Virus titers were quantified by RT- qPCR and by determining the logio TCID50 values. The immunization and challenge schedule and protocol are summarized in Table 6. Table 6 Experimental design for CC-19 mRNA/LNP2 immunization in K18-hACE2 transgenic mice and the challenge study

The SARS-Cov-2 viral challenge study was performed in K18-hACE2 mice. In brief, all control mice (n=5) on Day 5 worsening symptoms, while mice received 2 doses of either 1 or 10 microgram 21 days apart (n=6 each group), were normal with no symptoms. More importantly, in comparison to the controls, the vaccinated mice showed no detectable SARS-Cov2 viremia and had a significant reduction of tissue viral loads. The GMT of MN-50 before the viral challenge was 5, 120 and 4,480 at the 10 and 1 microgram dosed mice, respectively. In addition, all vaccinated mice at the 10 microgram dose, and 5 of 6 mice at 1 microgram dose also elicited SARS-Cov-2 specific IgA antibody.

Example 10

The toxicological studies in animals of CC-19 mRNA/LNP2 vaccine wase performed at National Laboratory Animal Center, Mahidol University (NLAC-MU) during 22 November 2020 to 22 March 2021 by based on WHO Guidelines on Nonclinical Evaluation of Vaccines. WHO Technical Report Series, No.927, 2005 Annexl. Repeat-dose toxicity evaluations were performed in Wistar rats which is a well characterized model for vaccine toxicity studies including Poliovirus vaccine (Viki B, et al., 2018), Enterovirus 71 vaccine (Xiao-bing Z, et al., 2013), Rabies vaccine (Rajni G, et al., 2014) and Rotavirus vaccine (Jagdish K.Z., et al., 2014). Several parameters were monitored during repeat-dose studies generally include food and water consumption, body weight and body temperature. The blood chemistry, hematology, organ weights with macroscopic evaluation, as well as histopathology on a range of tissues including pivotal organs such as the lung, brain, liver and spleen were evaluated at the end of experiment. The two repeated-dose toxicity experiments were performed as a preliminary toxicity study of CC-19 mRNA/LNP2 was designed and conducted considerations for First-in-human trials. The toxicity experiment was performed by using the same immunization schedule with the decreased dose (from 100 microgram to 50 microgram) which derived from BNT162bl study. Finally, Wistar rats were received 2 doses, separated by 21 days of the highest dose (50 microgram) of CC-19 mRNA/LNP2 vaccine. Particularly, a total of 40 Wistar rats were injected intramuscularly (bilateral) with total 50 microgram CC- 19 mRNA/LNP2 (vaccine concentration=0.25 mg/mL), 2 times, 3 weeks interval (DI and D21). The list of animals in each group is summarized in Table 7.

Table 7 Arrangement for two repeated-dose toxicity experiments

For 3 repeated-dose toxicity experiments, a total of 100 Wistar rats were injected intramuscularly with 25 and 50 microgram CC-19 mRNA/LNP2 (CC-19 vaccine), 3 times, 3 weeks interval (DI, D21, and D42). The list of animals in each group is shown in Table 8.

Table 8 Arrangement for three repeated-dose toxicity experiments

All observations were started from Day 0 (the baseline). Animals were monitored daily for skin, fur, eye, mucous membrane, occurrence of secretion and excretion, autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), gait, posture, response of handling, clonic and tonic movement, stereotypes (e.g. excessive grooming, repetitive circling), bizarre behavior (e.g. self-mutilation, walking backwards). Weight and food consumption were measured daily on the first week after vaccination and once a week thereafter.

All animals were subjected to 15-18 h of fasting prior to blood collection. Blood was collected on three days after the first vaccination (Day3) and three days (Day24) after the second vaccination. The blood was assessed for hematological and biochemical tests. Hematological parameters were reported including hemoglobin (Hb), hematocrit (Het), leukocyte totals, neutrophil, lymphocyte, monocyte, eosinophil, basophil, platelet count. Blood biochemical parameters were measured including blood urea nitrogen (BUN), uric acid, creatinine (CRE), alkaline phosphatase (ALP), total protein (TP), albumin, direct bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and electrolytes.

Anatomorphological studies (gross necropsy) were performed immediately after the euthanasia of each animal on Day 24. All organs and sites of vaccine administration were examined macroscopically. The following organs were removed and weights recorded: adrenal glands, brain, bronchi, epididymis, large intestine, small intestine, eye, heart, kidneys, liver, lung, lymph node (mandibular), ovaries and oviduct, pancreas, spleen, testes, thymus, thyroid and parathyroid glands, urinary bladder and uterus.

The following tissue samples were processed by fixation in 4% formaldehyde and then sectioned and stained with a hematoxylin and eosin (H&E): 1) injection site, 2) immune organs: lymph node (mandibular and mesenteric), thymus, spleen, bone marrow, Peyer’s patches, bronchus, 3) pivotal organs: brain, kidneys, liver, lung and reproductive organs (epididymis, ovaries and oviducts, prostate, seminal vesicles, testes, uterus, vagina) and 4) all gross lesions. Finally, all sections will be examined by a board-certified veterinary pathologist.

The daily clinical observations of all animals both pilot toxicity (2 Doses injection) and repeatdose toxicity (3 Doses injections) showed no clinical signs cause of toxicity. In overall, the body weights of all animals were continued to gain throughout the study and the feed consumptions were normal in all animals.

In both 2 Doses and 3 Doses injections, the significant difference of hematology changes with a dose related effect included decreases (within normal range) in RBC, HCT, MCV, MCH, PLT, neutrophil and lymphocyte and increases (outside normal range) in WBC and monocytes. A decrease of RBC, HCT and its indices and increase of WBC and monocyte might be related to inflammatory responses after vaccine administrations in rats. Although, the potential clinical relevance is not known, these findings were reversible by the end of the 2-week recovery period. The significant difference of biochemistry changes with a dose related effect included increases in globulin, albumin, alanine amino transferase (ALT), aspartate amino transferase (AST) and alkaline phosphatase (ALP). Of five parameters, only the ALP is slightly increased outside the normal range and generally reversed by the end of the 2-week recovery period. This finding was corresponded with hepatic alterations (increased liver weights and the minimal microvesicular fatty change in the liver) which is possibly caused by the systemic inflammation following the vaccine administration.

For 2 Doses injection, a mild local inflammation regarding the vaccine injection was observed in some animals when compared to control group. The liver, kidney, spleen and popliteal lymph node showed a significant increase in organ weights whereas the thymus showed a significant decrease in organ weights. The histopathology demonstrated thymic atrophy, minor microvesicular fatty in liver, diffuse lymphocyte hyperplasia of popliteal lymph node and a high myeloid to erythroid precursor ratio in bone marrow which might be a consequence of inflammatory reaction from the vaccination. For 3 Doses injection, the similar findings for organ weights with a dose related effect were observed. The liver, kidney, spleen and popliteal lymph node showed a significant increase in organ weights whereas the thymus showed a significant decrease in organ weights. Interestingly, all organ weight changes mostly recovered following a 2-week recovery period.

The safety evaluation of CC-19 mRNA/LNP2 vaccine in Wistar rats reveal no special hazard for humans based on a pilot toxicity (2 Doses injection) and conventional studies of repeat-dose toxicity (3-Dose injection).

Example 11

For the thermostability test, the CC-19 can keep at 2-8 degree Celsius and room temperature (25 degree Celsius for at least 3 months and two weeks, respectively, which may be more convenient than other mRNA vaccines.

Example 12

For the clinical trial Phase 1 (n= 36, age 18-55 years) for evaluation its safety and optimal dosages, the subjects were vaccinated at 10, 25, and 50 microgram for 3 weeks interval. The result has shown that the three dosages are able to enhance T-cell immunity in all subjected, while the neutralizing antibodies are increased in different level, for examples, the 50 microgram of CC-19 mRNA is highly effective than those lower doses. In comparison with the sera panel from subjects who were vaccinated with other SARS-Cov-2vaccines (i.e. Pfizer, AstraZeneca, Sinovac), the average levels of anti-receptor-binding domain (anti-RBD), and pseudovirus neutralizing antibody (psVNT) are summarized in Table 9 and Fig.10.

Table 9 Immunogenicity Results at 1 Week after the Second Dose of the CC-19-mRNA Vaccinated Adults aged 18-55 from the Phase 1 Trial (ChulaVAcOOl) (N=36, 12 per group) in comparison to the sera from subjects vaccinated with other Covid- 19 vaccines

Example 13

A Phase 2 trial has been conducted to evaluate the safety, tolerability, and reactogenicity of the disclosed composition, CC-19 vaccine at the concentration of 50 microgram was administered intramuscularly (IM) according to subjects a repeat vaccination schedule (given 21 days apart) in healthy adults aged 18-59 years up to visit 10 (Day 50 ±3). In the same experiments, the immunogenicity was measured as neutralizing antibody titer (measured by Micro-viral neutralizing test [MN-50 or Micro VNT50]) pursuant to the repeated vaccination of the CC-19 vaccine of 50 microgram at Visit 9 (Day 29 +3).

In this Phase 2 study, the total enrollment is 150 for CC-19 vaccine and placebo in a ratio of 4:1 i.e., 120:30. The study was to evaluate safety and immunogenicity in comparison with the placebo by gauging the seroconversion rates as the primary objective. Based on the Phase 1 study, summarized in example 12, the results has suggested that CC-19 vaccine at 50 microgram dose is highly immunogenic to elicit high GMT of all binding- and neutralizing antibodies tested in this study. The CC-19 vaccine also induced strong T cell responses. The dose of 50 microgram was therefore selected for the Phase 2 study, and all participants received 2 doses of the CC-19 vaccine.

In this Phase 2 study, the safety assessment of CC-19 vaccine, given intramuscularly at 50 microgram for 2 doses 3 weeks apart, was compared with the placebo group. Any local reaction pain Dose 1: 91.7% vs 20%, Dose 2: 64% vs 66.7%, Any systemic reaction: Dose 1: 97 vs 63%, Dose2: 86% vs 56.7%, respectively. There was no fever report at Dose 1, 34.7% reported fever at Dose 2 of CC-19 vs 0% in placebo group. Chill at Dose 1: 5.8% vs 3.3%, Dose 2: 36.4% vs 3.3%; Headache at Dose 1: 19.2% vs 40%, Dose 2: 55.9% vs 13%; Fatigue at Dose 1: 41.7%/30%, Dose 2: 44.1% vs 0%; Myalgia at Dose 1: 7.5% vs 13.3%, Dose 2: 44.1% vs 0%; Arthralgia at Dose 1: 5% vs 13.3%, Dose 2: 11.9% vs 0%, respectively. Most adverse effects (AEs) are mild, few are moderate. Both vaccine-related local and systemic AEs are temporary. The mean duration of local AEs is 2.27 vs 1.17 days, and of systemic AEs is 1.68 vs 1.67 days reported in the CC-19 vs placebo arms respectively. There is no vaccine-related systemic adverse effect (SAE).

After 29 days, the study was amended to allow all subjects in the placebo arm receiving Pfizer/BNT vaccine and their immunogenicity were also studied. The safety profiles in this phase 2 were similar to those observed in earlier study (phase 1). The 2-dose vaccine regimen of CC- 19 was effective in triggering the immune response. 100% seroconversion was attained post 2 doses at Day 50 (approximately 1 month after the second dose). The exploratory comparison analysis showed CC-19 vaccine generated significantly higher SARS-CoV2-specific B-cells and T-cells than those induced by Pfizer/BNT vaccine.

CC-19 vaccine elicited both strong SARS-CoV2 specific antibody and T-cells Responses in this study. The immunogenic response observed was compared with the results of immunogenic response reported by Placebo and by Pfizer/BNT vaccine. All subjects enrolled in the placebo were given Pfizer/BNT vaccine after Day 29 (1 week after the second dose) with a similar vaccination schedule and blood samples collection timepoints. The seroconversion rate of micro VNT50 antibody of CC-19 at 50 microgram vs Placebo was 94% vs 0% at Day 29, and 100% vs 3.4% at Day 50 respectively. Also, CC-19 vaccine at 50 microgram induced 2.18- and 2.7-fold significantly higher than Pfizer/BNT against wild-type (WT) Covid- 19 viruses at Day 29 and 50, p 0.01, and p<0.001, respectively.

Further, the test using micro- VNT50 against variants of Covid- 19 were performed on Day 29 only, there are not significantly different between the 2 vaccines (CC-19 and Pfizer) against alpha, beta, and delta. More specifically, the CC-19 vaccine at 50 microgram induced 1.7- and 2.79-fold significantly higher than Pfizer/BNT did against WT at Day 29 and 50, p<0.05, p<0.001, respectively; and 1.7-fold significantly higher than Pfizer/BNT did against delta at Day 50 (p=0.01). The MicroVNT50 results against wild-type, alpha, beta, and delta variants were summarized in Table 10. The pseudovirus neutralizing antibody (psVNT50) results against WT and delta variant obtained from the CC-19 mRNA vaccine subjects in comparison to those from the placebo group and Pfizer/BNT group were summarized in Table 11 and Table 12, respectively.

Anti RBD-IgG antibody results of both CC-19 mRNA vaccine and Pfizer/BNT study groups were summarized in Table 13. The results demonstrated that CC-19 mRNA vaccine elicited anti-RBD-IgG antibody 1.84-folds higher than Pfizer/BNT vaccine at Day 50 or 4 weeks after the second dose, p <0.001.

CC-19 vaccine at 50 microgram was found being able to induce 1.84-fold significantly higher than Pfizer/BNT at Day 50. (p<0.001) in the test using anti-RBD antibody. Likewise, the CC-19 vaccine at 50 microgram and Pfizer/BNT respectively induced 95.4% vs 93.3% inhibition in the RBD-ACE2 binding Inhibition (sVNT). As to IFNv ELISpot results, CC-19 vaccine at 50 microgram induced geometric mean -lENy ELISpot (SFC/million PBMCs) 2.21 and 2.69-fold significantly higher than Pfizer/BNT against WT at Day 29 and 50, both p<0.001, respectively. The summary showed in Table 14.

Table 10 Comparison of SARS-CoV-2-specific serum neutralizing antibody as measured by Live-virus Micro VNT50- WT, Alpha, Beta and Delta at day 29 and 50 between ChulaCov-19 or CC-19 vaccine of 50 microgram and Pfizer/BNT (Note: only Day 29 results were preformed against wild-type virus and Alpha, Beta and Delta variants)

GMT: Geometric mean titer, GMTR: Geometric mean titer ratio, 95% CI: 95% confidence interval,

LL = lower limit, UL = upper limit, P-value were evaluated by Two-sample independent t-test, Ref: reference Table 11 Comparison of SARS-CoV-2-specific serum neutralizing antibody results as measured by pseudovirus neutralization test (psVNT50) against WT and Delta variant between

ChulaCovl9 (CC-19) and Placebo study groups

Table 12 Comparison of SARS-CoV-2-specific serum neutralizing antibody as measured by pseudovirus neutralization test against WT and Delta variant (psVNT50-WT and Delta) between ChulaCovl9 50 microgram and Pfizer/BNT vaccine study groups

GMT: Geometric mean titer, GMTR: Geometric mean titer ratio, 95%CI: 95% confidence interval, LL = lower limit, UL = upper limit, P-value were evaluated by Two-sample independent t-test, Ref: reference Table 13 Comparison of Anti RBD-IgG by Abbott assay (BAU/mL) results between

ChulaCovl9 vaccine and Pfizer/BNT study groups

GMT: Geometric mean titer, GMTR: Geometric mean titer ratio, 95%CI: 95% confidence interval, LL = lower limit, UL = upper limit, P-value were evaluated by Two-sample independent t-test, Ref: reference

Table 14 Comparison of IFN-y -ELISpot results between ChulaCovl9 vaccine 50 microgram and Pfizer/BNT

GMT: Geometric mean titer, GMFR: Geometric mean titer ratio, 95%CI: 95% confidence interval, LL = lower limit, UL = upper limit, P-value were evaluated by Two-sample independent t-test, Ref: reference

It is to be understood that the present disclosure may be embodied in other specific forms and is not limited to the sole embodiment described above. However, modification and equivalents of the disclosed concepts such as those which readily occur to one skilled in the art are intended to be included within the scope of the claims which are appended thereto.

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Claims

33 Claims

1. A vaccine composition against infection of Coronavirus comprising: a plurality copies of a mRNA having polynucleotide sequence as setting forth in SEQ ID No. 1 capable of being translated into a peptide corresponding to at least a portion of a S-Spike protein of the Coronavirus; a lipid nanoparticles mixture used for encapsulating the pluralities copies of the mRNA forming a colloidal dispersion of the mRNA-lipid particles thereto; and one or more excipient for stabilizing the colloidal dispersion, wherein the lipid nanoparticles and the plurality copies of the mRNA are in a predetermined weight ratio of 12-36:1, wherein the translated peptide is free from di-proline (2P) or hexa-proline (6P).

2. The vaccine composition of claim 1, wherein the lipid nanoparticles mixture comprises one or more lipid types of ionizable lipids, cationic lipids, non-cationic lipids, polymer- conjugated lipids, and helper lipids.

3. The vaccine composition of claim 1, wherein the excipient is selected from anyone or combination of lipid-aggregation inhibitors, pharmaceutically acceptable salts, buffering agents, and sucrose.

4. The vaccine composition of claim 1, wherein the plurality copies of mRNA is in an amount of 10-50 microgram.

5. The vaccine composition of claim 1, wherein the polynucleotide sequence of the mRNA is modified by way of substituting uridine with pseudouridine.