Oral vaccine for covid-19

Abstract

An oral vaccine for COVID-19 is disclosed. The oral vaccine for COVID-19 includes a delivery platform including Arthrospira platensis with a plurality of host genomes and a plurality of COVID-19 antigen delivery vectors coupled to the plurality of host genomes. Each respective host genome of the plurality of host genomes includes a nucleotide sequence identical to nucleotide sequence of SEQ ID NO. 1. Each COVID-19 antigen delivery vector of the plurality of COVID- 19 antigen delivery vectors has a nucleotide sequence identical to nucleotide sequence of SEQ ID NO. 2. Each respective COVID-19 antigen delivery vector of the plurality of COVID-19 antigen delivery vectors includes at least one antigen of COVID-19 with a weight ratio of the delivery platform to the at least one antigen of COVID-19in a range of 1: 10-3 to 1: 2.5 × 10-3 (delivery platform: at least one antigen of COVID-19).

Description

ORAL VACCINE FOR COVID-19 TECHNICAL FIELD

[0001] The present disclosure generally relates to an oral vaccine for COVID-19, and particularly, relates to a method of producing the aforementioned oral vaccine for COVID-19 using Arthrospira platensis as a delivery platform. BACKGROUND ART

[0002] COVID-19, short for "Coronavirus Disease 2019," is a highly contagious and novel respiratory illness caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2). It emerged in late 2019 in the city of Wuhan, China, and rapidly evolved into a global pandemic. COVID-19 is characterized by symptoms ranging from mild respiratory distress to severe pneumonia, and in some cases, it can lead to critical illness and death. The pandemic has had profound and far-reaching impacts on public health, economies, and daily life around the world. Response to COVID-19 has included development of vaccines, widespread testing, social distancing measures, and various public health interventions to mitigate its spread and effects.

[0003] Development and distribution of a vaccine for COVID-19 have been paramount in global fight against the pandemic. Importance of having an effective vaccine cannot be overstated, as it represents a crucial tool in controlling spread of COVID-19 virus, reducing severity of illness, and ultimately saving countless lives. COVID-19, caused by novel coronavirus SARS-CoV-2, had unleashed a global health crisis of unprecedented scale, impacting every aspect of human life. Without vaccines, world would face challenge of continued surges in cases, overburdened healthcare systems, economic devastation, and persistence of restrictive measures that strained societies. Development of COVID-19 vaccines became a symbol of hope, offering potential to return to a semblance of normalcy and bring an end to widespread suffering caused by COVID-19 virus.

[0004] Several categories of vaccines were developed for COVID-19, reflecting global scientific and pharmaceutical community's extraordinary efforts to find solutions to the pandemic. Primary categories of vaccines included mRNA vaccines, viral vector vaccines, inactivated, protein subunit vaccines, and DNA-based vaccines. Notably, mRNA vaccines represented a groundbreaking technology, using a small piece of the virus's genetic material to trigger an immune response. Viral vector vaccines used harmless viruses to deliver viralcomponents, stimulating immunity. Inactivated used deactivated virus particles or specific viral proteins to induce immune responses. These diverse approaches allowed for a broad range of vaccine options to address different needs and global distribution challenges. Accordingly, different vaccines have been developed for COVID-19. For example, Jong-Hoon Lee, et al. presented a patent on “4,4′-Diaminodiphenyl Sulfone as an Inflammasome Competitor of Oral Vaccine or Therapeutics for SARS-CoV-2 or COVID-19” (KR20200124185A). Jong-Hoon Lee prepared an inflammasome competitor dapsone as an oral vaccine for COVID-19. Zhou Yusen et al. presented a patent on “Novel coronavirus COVID-19 vaccine, preparation method and application thereof” (CN111333704B). Zhou Yusen et al. connected an antigen with immunoglobulin to prepare RBD-Fc fusion protein. These vaccines, in addition to their preventive effects against COVID-19, come with some associated risks for human.

[0005] While COVID-19 vaccines have been essential in fight against the pandemic, they have drawbacks and challenges. Some individuals experienced mild side effects, including sore arms, fatigue, and fever, after vaccination, though these were generally short-lived and less severe than the consequences of a COVID-19 infection. There were also rare instances of more serious side effects, such as blood clotting events associated with certain viral vector vaccines, which prompted temporary suspensions in some regions. Additionally, there were issues related to vaccine distribution, equity, and hesitancy, which hindered global efforts to achieve herd immunity. Addressing these challenges and monitoring vaccine safety and effectiveness became an ongoing priority in the pandemic response.

[0006] There is, therefore, a need for a non-toxic, available, easy to use, and highly effective vaccine for COVID-19. There is further a need for a simple, scalable, and cost-effective method to produce the aforementioned vaccine for COVID-19. SUMMARY OF THE DISCLOSURE

[0007] This summary is intended to provide an overview of the subject matter of this patent, and is not intended to identify essential elements or key elements of the subject matter, nor is it intended to be used to determine the scope of the claimed implementations. The proper scope of this patent may be ascertained from the claims set forth below in view of the detailed description below and the drawings.

[0008] According to one or more exemplary embodiments, the present disclosure is directed to an oral vaccine for COVID-19. In an exemplary embodiment, an exemplary oral vaccinemay include a delivery platform including Arthrospira platensis with a plurality of host genomes and a plurality of COVID-19 antigen delivery vectors coupled to an exemplary plurality of host genomes of an exemplary delivery platform. In an exemplary embodiment, each respective host genome may include a nucleotide sequence identical to nucleotide sequence of SEQ ID NO.1. In an exemplary embodiment, an exemplary COVID-19 antigen delivery vector may have a nucleotide sequence identical to nucleotide sequence of SEQ ID NO. 2. In an exemplary embodiment, an exemplary each respective COVID-19 antigen delivery vector may include at least one antigen of COVID-19 with a weight ratio of an exemplary delivery platform to an exemplary at least one antigen of COVID-19 in a range of 1: 10-3to 1: 2.5 ^ 10-3(delivery platform: at least one antigen of COVID-19).

[0010] In an exemplary embodiment, each respective COVID-19 antigen delivery vector of an exemplary plurality of COVID-19 antigen delivery vectors may include a transposon vector. In an exemplary embodiment, each respective COVID-19 antigen delivery vector of an exemplary plurality of COVID-19 antigen delivery vectors may include an exemplary at least one antigen of COVID-19 with a nucleotide ratio of each respective COVID-19 antigen delivery vector to an exemplary at least one antigen of COVID-19 in a range 50: 7 to 50; 22 (each COVID-19 antigen delivery vector: an exemplary antigen of COVID-19).

[0011] In an exemplary embodiment, an exemplary antigen of COVID-19 may have a nucleotide sequence identical to nucleotide sequence of SEQ ID NO. 3. In an exemplary embodiment, an exemplary delivery platform may be in a form of at least one of a tablet, a capsule, powder, a pearl, and combinations thereof. In an exemplary embodiment, an exemplary capsule may be made of at least one of gelatin, non-gelatin, and combinations thereof. In an exemplary embodiment, an exemplary oral vaccine may include at least 10 µg of an exemplary at least one antigen of COVID-19. In an exemplary embodiment, an exemplary oral vaccine may be an additive in food. In an exemplary embodiment, an exemplary food may include at least one of yogurt, dessert, gum, ice cream, pastilles, and combinations thereof.

[0012] According to one or more exemplary embodiments, the present disclosure is directed to a method for preparing an oral vaccine for COVID-19. In an exemplary embodiment, an exemplary method may include forming a plurality of COVID-19 antigen delivery vectors by loading a plurality of antigens of COVID-19 into a plurality of transposon vectors, forming a plurality of hybrid vectors by loading an exemplary plurality of COVID-19 antigen delivery vectors into a plurality of vectors, separating an exemplary plurality of COVID-19 antigendelivery vectors from an exemplary plurality of hybrid vectors by mixing an exemplary plurality of hybrid vectors with a separation enzyme solution, activating an exemplary separated plurality of COVID-19 antigen delivery vectors by mixing an exemplary separated plurality of COVID-19 antigen delivery vectors with an activation enzyme solution, and transferring an exemplary plurality of activated COVID-19 antigen delivery vectors into a plurality of host genomes of Arthrospira platensis by an electroporation technique.

[0013] In an exemplary embodiment, forming an exemplary plurality of hybrid vectors may further include multiplying an exemplary plurality of hybrid vectors by incubating an exemplary plurality of hybrid vectors at a temperature in a range of 4°C to16ºC for a time period in a range of 2 hours to 12 hours.

[0014] In an exemplary embodiment, forming an exemplary plurality of hybrid vectors may include loading an exemplary plurality of COVID-19 antigen delivery vectors into a plurality of plasmid vectors.

[0015] In an exemplary embodiment, forming an exemplary plurality of hybrid vectors may include loading an exemplary plurality of COVID-19 antigen delivery vectors into a plurality of pBluescript II SK(+) vectors with a nucleotide sequence identical to nucleotide sequence of SEQ ID NO.4.

[0016] In an exemplary embodiment, separating an exemplary plurality of COVID-19 antigen delivery vectors from an exemplary plurality of hybrid vectors may include mixing an exemplary plurality of hybrid vectors with an exemplary separation enzyme solution with a volume ratio of an exemplary plurality of hybrid vectors to an exemplary separation enzyme solution in a range of 20:0.4 to 20:4 (an exemplary plurality of hybrid vectors: an exemplary separation enzyme solution).

[0017] In an exemplary embodiment, mixing an exemplary plurality of hybrid vectors with an exemplary separation enzyme solution may include mixing an exemplary plurality of hybrid vectors with a solution. In an exemplary embodiment, an exemplary solution may include a separation enzyme dissolved in buffered aqueous glycerol solution with a concentration of an exemplary separation enzyme solution in a range of 10 U / µl to 50 U / µl. In an exemplary embodiment, separating an exemplary plurality of COVID-19 antigen delivery vectors from an exemplary plurality of hybrid vectors may include mixing an exemplary plurality of hybrid vectors with at least one of an EcoRI solution, a HindIII solution in a buffered aqueous glycerol solution, and combinations thereof for a time period in a range of 1 hour to16 hours.

[0018] According to one or more exemplary embodiments, the present disclosure is directed to a method of preparing an oral vaccine for COVID-19. In an exemplary embodiment, an exemplary method may include forming a plurality of COVID-19 antigen delivery vectors by loading a plurality of antigens of COVID-19 into a plurality of transposon vectors, activating an exemplary plurality of COVID-19 antigen delivery vectors by mixing an exemplary plurality of COVID-19 antigen delivery vectors with an activation enzyme solution, and transferring an exemplary plurality of activated COVID-19 antigen delivery vectors into a plurality of host genomes of Arthrospira platensis by an electroporation technique.

[0019] In an exemplary embodiment, loading an exemplary plurality of COVID-19 antigens into an exemplary plurality of transposon vectors may include isolating an exemplary plurality of COVID-19 antigens by a method including at least one of synthesizing an exemplary plurality of COVID-19 antigens, digestion ligation, and combinations thereof, forming a ligation reaction mixture by mixing an exemplary plurality of COVID-19 antigens, at least a ligase enzyme, and at least a ligase buffer solution, and an exemplary plurality of transposon vectors, and incubating an exemplary ligation reaction mixture for a time period in a range of 4 hours to 16 hours at a temperature in a range of 4°C to 37°C.

[0020] In an exemplary embodiment, activating an exemplary plurality of COVID-19 antigen delivery vectors may include mixing an exemplary plurality of COVID-19 antigen delivery vectors with an exemplary activation enzyme solution with a volume ratio of an exemplary plurality of COVID-19 antigen delivery vectors to an exemplary activation enzyme solution in a range of 8:2 to 6:8 (an exemplary plurality of COVID-19 antigen delivery vectors: an exemplary activation enzyme solution).

[0021] In an exemplary embodiment, mixing an exemplary plurality of COVID-19 antigen delivery vectors with an activation enzyme solution may include mixing an exemplary plurality of COVID-19 antigen delivery vectors with an activation enzyme dissolved water. In an exemplary embodiment, an exemplary activation enzyme solution may have a concentration in a range of 1 U / µl to 4 U / µl. In an exemplary embodiment, activating an exemplary plurality of COVID-19 antigen delivery vectors may include mixing an exemplary plurality of COVID-19 antigen delivery vectors with at least one of a DNA transposon solution, a Transposase solution in water, and combinations thereof.

[0022] In an exemplary embodiment, transferring an exemplary plurality of activated COVID- 19 antigen delivery vectors into a host genome of Arthrospira platensis may include applyinga voltage to an exemplary Arthrospira platensis in a range of 1 KV / cm to 8 KV / cm for a time period in a range of 0.5 ms to 2 ms. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The drawing figures depict one or more implementations in accord with the present teachings, by way of example only, not by way of limitation. In the figures, like reference numerals refer to the same or similar elements.

[0024] FIG. 1 illustrates a schematic view of genetic components of a COVID-19 antigen delivery vector, consistent with one or more exemplary embodiments of the present disclosure;

[0025] FIG.2A illustrates a flowchart of a method of producing an exemplary oral vaccine for COVID-19, consistent with one or more exemplary embodiments of the present disclosure;

[0026] FIG.2B illustrates a flowchart of a method of producing an exemplary oral vaccine for COVID-19 using a plasmid vector, consistent with one or more exemplary embodiments of the present disclosure;

[0027] FIG. 2C illustrates a flowchart of a method of loading a plurality of an exemplary antigens of COVID-19 into an exemplary plurality of transposon vectors, consistent with one or more exemplary embodiments of the present disclosure;

[0028] FIG. 2D illustrates a flowchart of a method for loading an exemplary plurality of COVID-19 antigen delivery vectors into a plurality of plasmid vectors, consistent with one or more exemplary embodiments of the present disclosure;

[0029] FIG. 3 illustrates a fluorescent image of protein expression of an exemplary oral vaccine, consistent with one or more exemplary embodiments of the present disclosure;

[0030] FIG.4 illustrates a fluorescent image of an exemplary modified Arthrospira platensis, consistent with one or more exemplary embodiments of the present disclosure;

[0031] FIG.5 illustrates an image of expression of an exemplary COVID 19-RBD protein in 4 replicates of transgenics and negative control by Western blot method, consistent with one or more exemplary embodiments of the present disclosure;

[0032] FIG. 6 illustrates a calibration curve of Elisa test performed for an exemplary oral vaccine, consistent with one or more exemplary embodiments of the present disclosure;

[0033] FIG.7 illustrates an image of comparison of antibody titers in blood serum of tested groups, consistent with one or more exemplary embodiments of the present disclosure; and

[0034] FIG.8 illustrates an image of comparison of mean and standard divination of COVID- 19 RBDAg titers, consistent with one or more exemplary embodiments of the present disclosure.DESCRIPTION OF EMBODIMENTS

[0036] In the following detailed description, numerous specific details are set forth by way of examples in order to provide a thorough understanding of the relevant teachings. However, it should be apparent that the present teachings may be practiced without such details. In other instances, well known methods, procedures, components, and / or circuitry have been described at a relatively high-level, without detail, in order to avoid unnecessarily obscuring aspects of the present teachings.

[0037] The novel features which are believed to be characteristic of the present disclosure, as to its structure, organization, use and method of operation, together with further objectives and advantages thereof, will be better understood from the following discussion. In the following detailed description, numerous specific details are set forth by way of examples in order to provide a thorough understanding of the relevant teachings. However, it should be apparent that the present teachings may be practiced without such details. In other instances, well known methods, procedures, components, and / or circuitry have been described at a relatively high- level, without detail, in order to avoid unnecessarily obscuring aspects of the present teachings. The following detailed description is presented to enable a person skilled in the art to make and use the methods and devices disclosed in exemplary embodiments of the present disclosure. For purposes of explanation, specific nomenclature is set forth to provide a thorough understanding of the present disclosure. However, it will be apparent to one skilled in the art that these specific details are not required to practice the disclosed exemplary embodiments. Descriptions of specific exemplary embodiments are provided only as representative examples. Various modifications to the exemplary implementations will be readily apparent to one skilled in the art, and the general principles defined herein may be applied to other implementations and applications without departing from the scope of the present disclosure. The present disclosure is not intended to be limited to the implementations shown, but is to be accorded the widest possible scope consistent with the principles and features disclosed herein.

[0038] COVID-19 is a widespread disease which may require a vaccine for preventing ongoing pandemic. In one general aspect of the present disclosure, an oral vaccine may be produced for COVID-19. In an exemplary embodiment, an exemplary oral vaccine may include a delivery platform and a plurality of COVID-19 antigen delivery vectors. In an exemplary embodiment, an exemplary delivery platform may include Arthrospira platensis. In an exemplary embodiment, an exemplary Arthrospira platensis may include a plurality of host genomes. Inan exemplary embodiment, an exemplary plurality of COVID-19 antigen delivery vectors may be coupled to an exemplary plurality of host genomes. In an exemplary embodiment, each respective host genome of an exemplary plurality of host genomes may include a nucleotide sequence identical to nucleotide sequence of SEQ ID NO.1. In an exemplary embodiment, an E value may be less than 10-15. In an exemplary embodiment, as used herein, “E value” may refer to a statistical measure associated with sequence similarity searches. In an exemplary embodiment, each respective COVID-19 antigen delivery vector may include at least one COVID-19 antigen. In an exemplary embodiment, a weight ratio of an exemplary delivery platform to an exemplary COVID-19 antigen may be in a range of 1: 10-3to 1: 2.5 ^ 10-3(delivery platform: COVID-19 antigen). In an exemplary embodiment, each respective COVID-19 antigen delivery vector of an exemplary plurality of COVID-19 antigen delivery vectors may include a nucleotide sequence identical to nucleotide sequence of SEQ ID NO.2.

[0039] In an exemplary embodiment, Arthrospira platensis may be a blue-green microalga belonging to phylum Cyanobacteria. In an exemplary embodiment, Arthrospira platensis may be selected here as an appropriate delivery platform for an exemplary oral vaccine for COVID- 19 because of plenty of advantages and effective characteristics of Arthrospira. In an exemplary embodiment, Arthrospira platensis may be renowned for remarkable nutritional content. In an exemplary embodiment, Arthrospira platensis may be a microscopic, filamentous, spiral- shaped, blue-green algae. In an exemplary embodiment, Arthrospira platensis may thrive in warm, alkaline, and brackish water bodies, particularly in subtropical and tropical regions. Arthrospira platensis may form dense colonies or mats on the water's surface. In an exemplary embodiment, Arthrospira platensis may be considered as a superfood due to a rich nutrient content. In an exemplary embodiment, Arthrospira platensis may be a complete protein source. In an exemplary embodiment, Arthrospira platensis may include all essential amino acids, B vitamins, minerals including iron and calcium, and essential fatty acids. In an exemplary embodiment, Arthrospira platensis also may be known for high concentration of antioxidants. In an exemplary embodiment, Arthrospira platensis may have gained popularity as a dietary supplement and functional food. In an exemplary embodiment, Arthrospira platensis may be believed to offer a range of health benefits, including boosting immune system, improving energy levels, aiding in detoxification, and reducing inflammation. In an exemplary embodiment, Arthrospira platensis may be generally considered safe for human consumption. In an exemplary embodiment, cultivation of an exemplary Arthrospira platensis may beenvironmentally friendly as an exemplary Arthrospira platensis may help treat wastewater, reduce carbon dioxide emissions, and minimize land and water use compared to traditional agriculture. Arthrospira platensis’s rich nutrient content and potential health benefits made it a popular choice as a dietary supplement and functional food. In an exemplary embodiment, Arthrospira platensis may function as a safe delivery platform for an exemplary oral vaccine for COVID-19 due to rich nutrient content and no side effects. In an exemplary embodiment, expressing high levels of a particular protein in Arthrospira platensis may trigger a series of cellular responses, including upregulation of other genes. In an exemplary embodiment, Arthrospira platensis’s properties of producing a specific protein in abundance may increase expression of an exemplary COVID-19 antigen while an exemplary COVID-19 antigen may be coupled to an exemplary host genome of Arthrospira platensis.

[0040] In an exemplary embodiment, each of an exemplary COVID-19 antigen delivery vector of an exemplary plurality of COVID-19 antigen delivery vectors may include one EcoRI restriction site, one Left ME sequence, one Phycocyanin operon, one Right ME sequence, and one HindIII restriction site. In an exemplary embodiment, an exemplary Phycocyanin operon may include one specific promoter of phycocyanin, one Ribosomal Binding Site (RBS) sequence, at least one COVID-19 antigen, one Green Florence Protein (GFP), and one Terminator sequence. In an exemplary embodiment, indicators may be used instead of GFP. In an exemplary embodiment, indicators may include at least one of chloramphenicol-resistance (Cm r), firefly luciferase (Luc), and combinations thereof. In an exemplary embodiment, an exemplary Phycocyanin operon may include a distance between an exemplary COVID-19 antigen and an exemplary Green Florence Protein (GFP). In an exemplary embodiment, an exemplary COVID-19 antigen may include a nucleotide sequence identical to nucleotide sequence of SEQ ID NO.3. In an exemplary embodiment, an exemplary EcoRI restriction site may include a nucleotide sequence identical to nucleotide sequence of SEQ ID NO. 5. In an exemplary embodiment, an exemplary Left ME sequence may include a nucleotide sequence identical to nucleotide sequence of SEQ ID NO.6. In an exemplary embodiment, an exemplary Right ME sequence may include a nucleotide sequence identical to nucleotide sequence of SEQ ID NO.7. In an exemplary embodiment, an exemplary HindIII restriction site may include a nucleotide sequence identical to nucleotide sequence of SEQ ID NO. 8. In an exemplary embodiment, an exemplary specific promoter of phycocyanin may include a nucleotide sequence identical to nucleotide sequence of SEQ ID NO.9. In an exemplary embodiment, anexemplary Ribosomal Binding Site (RBS) sequence may include a nucleotide sequence identical to nucleotide sequence of SEQ ID NO. 10. In an exemplary embodiment, an exemplary Green Florence Protein (GFP) may include an amino acid sequence identical to amino acid sequence of SEQ ID NO. 11. In an exemplary embodiment, an exemplary terminator sequence may include a nucleotide sequence identical to nucleotide sequence of SEQ ID NO. 12. In an exemplary embodiment, a nucleotide sequence of histidine may be attached to one end of an exemplary COVID-19 antigen sequence. In an exemplary embodiment, an exemplary histidine nucleotide sequence may be incorporated for purpose of an exemplary COVID-19 antigen purification. In an exemplary embodiment, an exemplary antigen of COVID-19 may include a COVID-19 Antigen with Receptor Binding Domain (COVID-19-AG RBD). In an exemplary embodiment, an exemplary RBD may refer to Receptor Binding Domain of a spike protein of COVID-19 virus. In an exemplary embodiment, an exemplary spike protein may be a type of glycoprotein that may be produced from surface of COVID-19 virus. In an exemplary embodiment, an exemplary RBD may be responsible for binding to human cells and initiating infection. RBD of COVID-19 virus may be often targeted in diagnostic tests and vaccines because of playing a crucial role in COVID-19 virus's ability to infect host cells.

[0041] In an exemplary embodiment, each exemplary respective COVID-19 antigen delivery vector of an exemplary plurality of COVID-19 antigen delivery vectors may include a transposon vector. As used herein, an exemplary transposon vector, or "transposon," may be a genetic tool that can move specific DNA sequences within an organism's genome. In an exemplary embodiment, each exemplary respective COVID-19 antigen delivery vector of an exemplary plurality of COVID-19 antigen delivery vectors may include an exemplary at least one antigen of COVID-19 with a nucleotide ratio of each exemplary respective COVID-19 antigen delivery vector to an exemplary at least one antigen of COVID-19 in a range 50: 7 to 50; 22 (each exemplary COVID-19 antigen delivery vector: an exemplary antigen of COVID- 19)

[0042] In an exemplary embodiment, an exemplary oral vaccine may include a form of at least one of a tablet, a capsule, powder, a pearl, and combinations thereof. In an exemplary embodiment, an exemplary capsule may include a cover for encompassing an exemplary oral vaccine. In an exemplary embodiment, an exemplary capsule may be made of at least one of gelatin, non-gelatin, and combinations thereof. In an exemplary embodiment, an exemplarytablet may be formed by pressing an exemplary oral vaccine into a tablet format. In an exemplary embodiment, each of an exemplary capsule, an exemplary powder, an exemplary pearl, and an exemplary tablet may include an exemplary delivery platform and an exemplary plurality of COVID-19 antigen delivery vectors coupled to an exemplary plurality of host genomes of an exemplary delivery platform. In an exemplary embodiment, an exemplary oral vaccine may be consumed for at least one portion for vaccination against COVID-19. In an exemplary embodiment, an exemplary at least one portion may include at least one capsule, one tablet, a predetermined amount of an exemplary powder, and one pearl of an exemplary oral vaccine. In an exemplary embodiment, an exemplary predetermined amount of an exemplary powder may include at least 10 µg of an exemplary COVID-19 antigen. In an exemplary embodiment, an exemplary at least one portion may include at least 10 µg of an exemplary COVID-19 antigen. In an exemplary embodiment, an exemplary at least one portion of an exemplary oral vaccine may offer immunity to COVID-19 for up to 3 months. In an exemplary embodiment, an exemplary oral vaccine may be an additive in food. In an exemplary embodiment, an exemplary food may include at least one of yogurt, dessert, gum, ice cream, pastilles, and combinations thereof. In an exemplary embodiment, an exemplary food with incorporation of an exemplary oral vaccine may be a therapeutic food. In an exemplary embodiment, an exemplary food with incorporation of an exemplary oral vaccine may be a prophylactic food. In an exemplary embodiment, an exemplary food with incorporation of an exemplary oral vaccine may be a preventive food.

[0043] FIG.1 illustrates a schematic view 100 of genetic components of a COVID-19 antigen delivery vector 102, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embodiment, COVID-19 antigen delivery vector 102 may include EcoRI restriction site 104, left ME sequence 106, Phycocyanin operon 108, Right ME sequence 110, and HindIII restriction site 112. In an exemplary embodiment, EcoRI restriction site 104 may include a nucleotide sequence identical to nucleotide sequence of SEQ ID NO. 5. In an exemplary embodiment, Left ME sequence 106 may include a nucleotide sequence identical to nucleotide sequence of SEQ ID NO.6. In an exemplary embodiment, Right ME sequence 110 may include a nucleotide sequence identical to nucleotide sequence of SEQ ID NO. 7. In an exemplary embodiment, HindIII restriction site 112 may include a nucleotide sequence identical to nucleotide sequence of SEQ ID NO.8.

[0044] In an exemplary embodiment, Phycocyanin operon 108 may include specific promoter of phycocyanin 114, Ribosomal Binding Site (RBS) sequence 116, at least one COVID-19 antigen 118, Green Florence Protein (GFP) 122, and terminator sequence 124. In an exemplary embodiment, Phycocyanin operon 108 may include a gene distance 120 between at least one COVID-19 antigen 118 and GFP 122. In an exemplary embodiment, Specific promoter of phycocyanin 114 may include a nucleotide sequence identical to nucleotide sequence of SEQ ID NO. 9. In an exemplary embodiment, Ribosomal Binding Site (RBS) sequence 116 may include a nucleotide sequence identical to nucleotide sequence of SEQ ID NO. 10. In an exemplary embodiment, COVID-19 antigen 118 may include a nucleotide sequence identical to nucleotide sequence of SEQ ID NO.3. In an exemplary embodiment, Green Florence Protein (GFP) 122 may include an amino acid sequence identical to amino acid sequence of SEQ ID NO. 11. In an exemplary embodiment, Terminator sequence 124 may include a nucleotide sequence identical to nucleotide sequence of SEQ ID NO.12. In an exemplary embodiment, COVID-19 antigen delivery vector 102 and an exemplary delivery platform may be used for producing an exemplary oral vaccine for COVID-19. In an exemplary embodiment, COVID- 19 antigen delivery vector 102 and an exemplary Arthrospira platensis may be used for producing an exemplary oral vaccine for COVID-19. In an exemplary embodiment, an exemplary plurality of COVID-19 antigen delivery vector 102 may be coupled to an exemplary plurality of host genome of an exemplary Arthrospira platensis. In an exemplary embodiment, methods of producing an exemplary oral vaccine for COVID-19 are illustrated in FIG.2A and FIG.2B.

[0045] FIG.2A illustrates a flowchart of a method 200 of producing an exemplary oral vaccine for COVID-19, consistent with one or more exemplary embodiments of the present disclosure. in an exemplary embodiment, method 200 may include a step 202 of forming a plurality of COVID-19 antigen delivery vectors by loading a plurality of antigens of COVID-19 into a plurality of transposon vectors, a step 204 of activating the plurality of COVID-19 antigen delivery vectors by mixing the plurality of COVID-19 antigen delivery vectors with a activation enzyme solution, and a step 206 of transferring the plurality of activated COVID-19 antigen delivery vectors into a host genome of Arthrospira platensis by electroporation.

[0046] In further detail with respect to step 202, step 202 of forming a plurality of COVID-19 antigen delivery vectors may include loading a plurality of antigens of COVID-19 into a plurality of transposon vectors. In an exemplary embodiment, an exemplary antigen ofCOVID-19 may be isolated by at least a method of synthesizing an exemplary antigen of COVID-19, digestion ligation method, and combinations thereof. In an exemplary embodiment, an exemplary antigen of COVID-19 may include a spike protein of a corona virus. In an exemplary embodiment, an exemplary antigen of COVID-19 may include a nucleotide sequence identical to nucleotide sequence of SEQ ID NO. 3. In an exemplary embodiment, loading an exemplary plurality of antigens of COVID-19 into an exemplary plurality of transposon vectors may be performed by method 230 illustrated in FIG.2C herein below.

[0047] FIG. 2C illustrates a flowchart of method 230 of loading plurality of antigens of COVID-19118 into the plurality of transposon vectors, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embodiment, method 230 may include a step 232 of isolating the plurality of COVID-19 antigens by at least a method of synthesizing an exemplary plurality of COVID-19 antigens, digestion ligation method, and combinations thereof, a step 234 of forming a first ligation reaction mixture by mixing the plurality of COVID-19 antigens, at least a first ligase enzyme, at least a first ligase buffer solution, and the plurality of transposon vectors together, and a step 236 of incubating the first ligation reaction mixture.

[0048] In further detail with respect to step 232, step 232 of isolating plurality of COVID-19 antigens 118 may include performing at least a method of synthesizing an exemplary plurality of COVID-19 antigens, digestion ligation method, and combinations thereof. In an exemplary embodiment, an exemplary plurality of COVID-19 antigens 118 may be synthesized using automated DNA synthesizers. In an exemplary embodiment, an exemplary automated DNA synthesizers may include devices that chemically create DNA fragments based on received specifications. In an exemplary embodiment, COVID-19 antigen 118 may include a nucleotide sequence identical to nucleotide sequence of SEQ ID NO.3. In an exemplary embodiment, an exemplary plurality of COVID-19 antigens 118 may be produced by an exemplary digestion ligation method. In an exemplary embodiment, an exemplary digestion ligation method may include cutting a genome of a corona virus at specific sites using enzymes. In an exemplary embodiment, an exemplary enzyme may be a restriction enzyme.

[0049] In further detail with respect to step 234, step 234 of forming a first ligation reaction mixture may include mixing plurality of COVID-19 antigens 118 with at least a first ligase enzyme, at least a first ligase buffer solution, and an exemplary plurality of transposon vectorstogether. In an exemplary embodiment, an exemplary plurality of COVID-19 antigens 118, at least a first ligase enzyme, at least a first ligase buffer solution, and an exemplary plurality of transposon vectors may be mixed together in a mixer with a stirring speed in a range of 1000 rpm to 1500 rpm. In an exemplary embodiment, an exemplary plurality of COVID-19 antigens 118, at least a first ligase enzyme, at least a first ligase buffer solution, and an exemplary plurality of transposon vectors may be mixed together for a time period in a range of 4 hours to 16 hours. In an exemplary embodiment, an exemplary first ligase enzyme may include at least one of SacI, XbaI, BamHI, EcoRI and Hind III, and combinations thereof. In an exemplary embodiment, an exemplary first ligase buffer solution may include at least one of trace acetate, potasume acetate, magnesium acetate, spirmidin, sodium dodecyl sulphate (SDS), and combinations thereof. In an exemplary embodiment, an exemplary first ligation reaction mixture may allow plurality of COVID-19 antigens 118 and an exemplary plurality of transposon vectors to be joined together; thereby, resulting in forming an exemplary plurality of COVID-19 antigen delivery vector.

[0050] In further detail with respect to step 236, step 236 of incubating the first ligation reaction mixture may include using an incubator to incubate an exemplary first ligation reaction mixture. In an exemplary embodiment, an exemplary first ligation reaction mixture may be incubated at a temperature in a range of 4°C to 37°C. In an exemplary embodiment, an exemplary first ligation reaction mixture may be incubated for a time period in a range of 4 hours to 16 hours.

[0051] In an exemplary embodiment, loading plurality of antigens of COVID-19118 into an exemplary plurality of transposon vectors may further include applying an inactivation heat to an exemplary incubated first ligation reaction mixture. In an exemplary embodiment, an exemplary inactivation heat may be used to inactivate an exemplary first ligase enzyme. In an exemplary embodiment, an exemplary incubated first ligation reaction mixture may be heated to a temperature in a range of 65ºC to 80 ºC for such inactivation purpose.

[0052] In an exemplary embodiment, an exemplary transposon vector may include a transient vector. In an exemplary embodiment, an exemplary transposon vector may include Tn5 transposons. As used herein, “Tn5 transposons” may be a type of transposable element found in bacterial genomes, particularly in Escherichia coli (E. coli). In an exemplary embodiment, exemplary Tn5 transposons may be DNA sequences that have ability to move from one location to another within a genome. In an exemplary embodiment, an exemplary transposonvector may include 1657 base pairs (bp). In an exemplary embodiment, an exemplary transposon vector may have a nucleotide sequence identical to nucleotide sequence of SEQ ID NO.13. In an exemplary embodiment, COVID-19 antigen 118 may be coupled to an exemplary transposon vector to form COVID-19 antigen delivery vector 102. In an exemplary embodiment, COVID-19 antigen delivery vector 102 may have a nucleotide sequence identical to nucleotide sequence of SEQ ID NO. 2. In an exemplary embodiment, each COVID-19 antigen delivery vector 102 of an exemplary plurality of COVID-19 antigen delivery vectors may include at least one antigen of COVID-19118. In an exemplary embodiment, a weight ratio of an exemplary delivery platform to antigen of COVID-19118 may be in a range of 1: 10-3to 1: 2.5 ^ 10-3(delivery platform: antigen of COVID-19). In an exemplary embodiment, COVID-19 antigen delivery vector 102 may include EcoRI cutting site 104, Left ME sequence 106, Phycocyanin operon 108, Right ME sequence 110, and HindIII restriction site 112. In an exemplary embodiment, Phycocyanin operon 108 may include Specific promoter of phycocyanin 114, Ribosomal Binding Site (RBS) sequence 116, at least one COVID-19 antigen 118, Green Florence Protein (GFP) 122, and Terminator sequence 124. In an exemplary embodiment, Phycocyanin operon 108 may include a gene distance 120 between at least one COVID-19 antigen 118 and GFP 122. In an exemplary embodiment, Specific promoter of phycocyanin 114 may include a nucleotide sequence identical to nucleotide sequence of SEQ ID NO. 9. In an exemplary embodiment, Ribosomal Binding Site (RBS) sequence 116 may include a nucleotide sequence identical to nucleotide sequence of SEQ ID NO. 10. In an exemplary embodiment, Green Florence Protein (GFP) 122 may include an amino acid sequence identical to amino acid sequence of SEQ ID NO.11. In an exemplary embodiment, Terminator sequence 124 may include a nucleotide sequence identical to nucleotide sequence of SEQ ID NO.12. In an exemplary embodiment, COVID-19 antigen delivery vector 102 and an exemplary delivery platform may be used for producing an exemplary oral vaccine for COVID-19. In an exemplary embodiment, COVID-19 antigen delivery vector 102 and an exemplary Arthrospira platensis may be used for producing an exemplary oral vaccine for COVID-19. In an exemplary embodiment, an exemplary plurality of COVID-19 antigen delivery vector 102 may be coupled to an exemplary plurality of host genome of an exemplary Arthrospira platensis. In an exemplary embodiment, a histidine (His) tag may be appended to antigen of COVID-19118. In an exemplary embodiment, an exemplary histidine sequence may be incorporated for purpose of purifying COVID-19 antigen 118. In an exemplary embodiment,an exemplary histidine tag may have a nucleotide sequence identical to nucleotide sequence of SEQ ID NO.14.

[0053] In an exemplary embodiment, exemplary Histidine tags, often referred to as "His tags," may be short sequences of histidine amino acids that may be added to a protein or gene product. In an exemplary embodiment, exemplary tags serve as a method for protein purification through a process called immobilized metal affinity chromatography (IMAC). In an exemplary embodiment, histidine residues may have a high affinity for metal ions including nickel or cobalt. In an exemplary embodiment, exemplary metals may be immobilized on a chromatography column. In an exemplary embodiment, when a protein with a His tag is expressed and purified, an exemplary protein may be selectively bounded to exemplary metal ions; allowing for easy and specific isolation of an exemplary protein from a complex mixture of cellular proteins. In an exemplary embodiment, antigen of COVID-19118 may be purified using IMAC.

[0054] Referring back to FIG. 2A, in further detail with respect to step 204, step 204 of activating the plurality of COVID-19 antigen delivery vectors 102 may include mixing plurality of COVID-19 antigen delivery vectors 102 with an activation enzyme solution. In an exemplary embodiment, an exemplary activation enzyme solution may activate Left ME sequence 106 and Right ME sequence 110. In an exemplary embodiment, activating plurality of COVID-19 antigen delivery vectors 102 may include mixing plurality of COVID-19 antigen delivery vectors 102 with an exemplary activation enzyme solution in a mixer for a time period in a range of 10 minutes to 60 minutes. In an exemplary embodiment, activating plurality of COVID-19 antigen delivery vectors 102 may include mixing plurality of COVID-19 antigen delivery vectors 102 with an exemplary activation enzyme solution for a time period in a range of 10 minutes to 60 minutes. In an exemplary embodiment, activating plurality of COVID-19 antigen delivery vectors 102 may include mixing plurality of COVID-19 antigen delivery vectors 102 with an exemplary activation enzyme solution at a temperature in a range of 15°C to 37°C. In an exemplary embodiment, activating plurality of COVID-19 antigen delivery vectors 102 may include mixing plurality of COVID-19 antigen delivery vectors 102 with an exemplary activation enzyme solution with a weight ratio of plurality of COVID-19 antigen delivery vectors 102 to an exemplary activation enzyme solution in a range of 8:2 to 6:8 (an exemplary plurality of COVID-19 antigen delivery vectors: an exemplary activation enzyme solution). In an exemplary embodiment, an exemplary activation enzyme solution may includeat least one of a DNA transposone solution, a Transposase solution, and combinations thereof. In an exemplary embodiment, an exemplary activation enzyme solution in water may have a concentration in a range of 1 U / µl to 4 U / µl.

[0055] In further detail with respect to step 206, step 206 of transferring an exemplary plurality of activated COVID-19 antigen delivery vectors into a host genome of Arthrospira platensis may include using an electroporation method. In an exemplary embodiment, an exemplary method may include introducing antigen delivery vector 102 into Arthrospira platensis. In an exemplary embodiment, an exemplary method may include using an electrical field to temporarily increase permeability of cell membrane of Arthrospira platensis, allowing an exemplary plurality of COVID-19 antigen delivery vectors 102 enter exemplary cells of Arthrospira platensis. In an exemplary embodiment, an electroporation reaction mixture may be prepared for an exemplary electroporation method. In an exemplary embodiment, an exemplary electroporation reaction mixture may include an Arthrospira platensis suspension mixed with a suspension of an exemplary plurality of activated COVID-19 antigen delivery vectors. In an exemplary embodiment, for preparing an exemplary Arthrospira platensis suspension, an exemplary Arthrospira platensis may be suspended in a first solution. In an exemplary embodiment, an exemplary first solution may include HEPES buffer. In an exemplary embodiment, an exemplary HEPES buffer may have a pH of 7. In an exemplary embodiment, an exemplary suspension of Arthrospira platensis in an exemplary first solution may have a weight ratio of Arthrospira platensis to exemplary first solution in a range of 0.1 mM to 1.0 mM (Arthrospira platensis: an exemplary first solution). In an exemplary embodiment, an exemplary plurality of activated COVID-19 antigen delivery vectors may be suspended in a second solution. In an exemplary embodiment, an exemplary second solution may include DOTAP Liposomal Transfection Reagent. In an exemplary embodiment, an exemplary suspension of an exemplary plurality of activated COVID-19 antigen delivery vectors in an exemplary second solution may have a weight ratio of an exemplary plurality of activated COVID-19 antigen delivery vectors to an exemplary second solution in a range of 1.0 µg / ml to 4.0 µg / ml (an exemplary plurality of activated COVID-19 antigen delivery vectors: an exemplary second solution). In an exemplary embodiment, an exemplary suspension of Arthrospira platensis and an exemplary suspension of an exemplary plurality of activated COVID-19 antigen delivery vectors may be mixed together with a volume ratio of an exemplary suspension of Arthrospira platensis to an exemplary suspension of an exemplaryplurality of activated COVID-19 antigen delivery vectors in a range of 1:100 to 1:10 (suspension of Arthrospira platensis: suspension of an exemplary plurality of activated COVID-19 antigen delivery vectors). In an exemplary embodiment, an exemplary mixture of an exemplary suspension of Arthrospira platensis and an exemplary suspension of an exemplary plurality of activated COVID-19 antigen delivery vectors may be placed in an electroporation cuvette or chamber. In an exemplary embodiment, an electrical pulse may be applied to an exemplary cuvette using an electroporation device. In an exemplary embodiment, an exemplary electrical pulse may generate an electrical field that may temporarily disrupt Arthrospira platensis’s cell membrane. In an exemplary embodiment, an exemplary electrical pulse may create pores in Arthrospira platensis’s cell membrane. In an exemplary embodiment, during brief moment when an exemplary cell membrane is permeabilized, an exemplary plurality of activated COVID-19 antigen delivery vectors may enter an exemplary Arthrospira platensis’s cell through exemplary pores. In an exemplary embodiment, once an exemplary electrical field is removed, an exemplary Arthrospira platensis’s cell membrane may reseal. In an exemplary embodiment, transferring an exemplary plurality of activated COVID-19 antigen delivery vectors into a host genome of Arthrospira platensis may include applying a voltage to an exemplary Arthrospira platensis in a range of 2 KV / cm to 7.5 KV / cm. In an exemplary embodiment, transferring an exemplary plurality of activated COVID-19 antigen delivery vectors into a host genome of Arthrospira platensis may include applying a voltage to an exemplary Arthrospira platensis for a time period in a range of 0.5 ms to 2 ms.

[0056] In an exemplary embodiment, method 200 may further include using a plasmid vector. In an exemplary embodiment, FIG.2B illustrates a flowchart of a method 210 of producing an exemplary oral vaccine for COVID-19 using a vector, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embodiment, method 210 may include two steps of 214 and 216 in addition to all steps 202-206 of method 200 described hereinabove in connection with FIG.2A. In an exemplary embodiment, method 210 may include a step 212 of forming a plurality of COVID-19 antigen delivery vectors by loading a plurality of antigens of COVID-19 into a plurality of transposon vectors, a step 214 of forming a plurality of hybrid vectors by loading the plurality of COVID-19 antigen delivery vectors into a plurality of vectors, a step 216 of separating the plurality of COVID-19 antigen delivery vectors from the plurality of hybrid vectors by mixing the plurality of hybrid vectors with a separation enzyme solution, a step 218 of activating the separated plurality of COVID-19 antigen delivery vectorsby mixing the separated plurality of COVID-19 antigen delivery vectors with an activation enzyme solution, and a step 220 of transferring the plurality of activated COVID-19 antigen delivery vectors into a plurality of host genomes of Arthrospira platensis by electroporation.

[0057] In further detail with respect to step 212, step 212 of forming a plurality of COVID-19 antigen delivery vectors may include loading a plurality of antigens of COVID-19118 into a plurality of transposon vectors. In an exemplary embodiment, antigen of COVID-19118 may be isolated by at least a method of synthesizing antigen of COVID-19118, digestion ligation, and combinations thereof. In an exemplary embodiment, antigen of COVID-19 118 may include a spike protein of a corona virus. In an exemplary embodiment, antigen of COVID-19 118 may include a nucleotide sequence identical to nucleotide sequence of SEQ ID NO.3. In an exemplary embodiment, loading an exemplary plurality of antigens of COVID-19118 into an exemplary plurality of transposon vectors may be performed by a method 230 illustrated in FIG.2C herein below.

[0058] FIG. 2C illustrates a flowchart of method 230 of loading plurality of antigens of COVID-19118 into the plurality of transposon vectors, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embodiment, method 230 may include a step 232 of isolating the plurality of COVID-19 antigens by at least a method of synthesizing antigen of COVID-19118, digestion ligation, and combinations thereof, a step 234 of forming a ligation reaction mixture by mixing the plurality of COVID-19 antigens, at least a ligase enzyme, at least a ligase buffer solution, and the transposon vector, and a step 236 of incubating the ligation reaction mixture.

[0059] In further detail with respect to step 232, step 232 of isolating plurality of COVID-19 antigens 118 may include using at least a method of synthesizing antigen of COVID-19118, digestion ligation, and combinations thereof. In an exemplary embodiment, an exemplary plurality of COVID-19 antigens 118 may be synthesized using automated DNA synthesizers. In an exemplary embodiment, an exemplary automated DNA synthesizers may be devices that chemically create DNA fragments based on received specifications. In an exemplary embodiment, COVID-19 antigen 118 may include a nucleotide sequence identical to nucleotide sequence of SEQ ID NO.3. In an exemplary embodiment, an exemplary plurality of COVID- 19 antigens 118 may be produced by an exemplary digestion ligation method. In an exemplary embodiment, an exemplary digestion ligation method may include cutting a genome of acorona virus at specific sites using enzymes. In an exemplary embodiment, an exemplary enzyme may be a restriction enzyme.

[0060] In further detail with respect to step 234, step 234 of forming a first ligation reaction mixture may include mixing plurality of COVID-19 antigens 118, at least a first ligase enzyme, at least a first ligase buffer solution, and an exemplary plurality of transposon vectors. In an exemplary embodiment, an exemplary plurality of COVID-19 antigens 118, at least a first ligase enzyme, at least a first ligase buffer solution, and an exemplary transposon vector may be mixed together in a mixer with a stirring speed in a range of 1000 rpm to 1500 rpm. In an exemplary embodiment, an exemplary plurality of COVID-19 antigens 118, at least a first ligase enzyme, at least a first ligase buffer solution, and an exemplary transposon vector may be mixed together for a time period in a range of 4 hours to 16 hours. In an exemplary embodiment, an exemplary first ligase enzyme may include at least one of SacI, XbaI, BamHI, EcoRI and Hind III, and combinations thereof. In an exemplary embodiment, an exemplary first ligase buffer solution may include at least one of trace acetate, potasume acetate, magnesium acetate, spirmidin, sodium dodecyl sulphate (SDS), and combinations thereof. In an exemplary embodiment, an exemplary first ligation reaction mixture may allow plurality of COVID-19 antigens 118 and an exemplary transposon vector be joined together forming an exemplary plurality of COVID-19 antigen delivery vector.

[0061] In further detail with respect to step 236, step 236 of incubating the first ligation reaction mixture may include using an incubator to incubate an exemplary first ligation reaction mixture. In an exemplary embodiment, an exemplary an exemplary first ligation reaction mixture may be incubated at a temperature in a range of 4°C to 37°C. In an exemplary embodiment, an exemplary an exemplary first ligation reaction mixture may be incubated for a time period in a range of 4 hours to 16 hours.

[0062] In an exemplary embodiment, loading plurality of antigens of COVID-19118 into an exemplary plurality of transposon vectors may further include inactivating an exemplary first ligase enzyme by heating an exemplary incubated first ligation reaction mixture. In an exemplary embodiment, an exemplary inactivation heat may be used to inactivate an exemplary first ligase enzyme. In an exemplary embodiment, an exemplary incubated ligation reaction mixture may be heated to a temperature in range of 65ºC to 80 ºC.

[0063] In an exemplary embodiment, an exemplary transposon vector may include a transient vector. In an exemplary embodiment, an exemplary transposon vector may include Tn5transposons. As used herein Tn5 transposons may be a type of transposable element found in bacterial genomes, particularly in Escherichia coli (E. coli). In an exemplary embodiment, exemplary Tn5 transposons may be DNA sequences that have ability to move from one location to another within a genome. In an exemplary embodiment, an exemplary transposon vector may include 1657 base pairs (bp). In an exemplary embodiment, an exemplary transposon vector may have a nucleotide sequence identical to nucleotide sequence of SEQ ID NO.13. In an exemplary embodiment, COVID-19 antigen 118 may be coupled to an exemplary transposon vector to form COVID-19 antigen delivery vector 102. In an exemplary embodiment, COVID-19 antigen delivery vector 102 may have a nucleotide sequence identical to nucleotide sequence of SEQ ID NO. 1. In an exemplary embodiment, each COVID-19 antigen delivery vector 102 of an exemplary plurality of COVID-19 antigen delivery vectors may include at least one antigen of COVID-19118. In an exemplary embodiment, a weight ratio of an exemplary delivery platform to COVID-19 antigen 118 may be in a range of 1: 10-3to 1: 2.5 ^ 10-3(delivery platform: COVID-19 antigen). In an exemplary embodiment, an exemplary COVID-19 antigen delivery vector may include EcoRI cutting site 104, Left ME sequence 106, Phycocyanin operon 108, Right ME sequence 110, and HindIII restriction site 112. In an exemplary embodiment, Phycocyanin operon 108 may include Specific promoter of phycocyanin 114, Ribosomal Binding Site (RBS) sequence 116, at least one COVID-19 antigen 118, Green Florence Protein (GFP) 122, and Terminator sequence 124. In an exemplary embodiment, Phycocyanin operon 108 may include a gene distance 120 between at least one COVID-19 antigen 118 and GFP 122. In an exemplary embodiment, Specific promoter of phycocyanin 114 may include a nucleotide sequence identical to nucleotide sequence of SEQ ID NO. 9. In an exemplary embodiment, Ribosomal Binding Site (RBS) sequence 116 may include a nucleotide sequence identical to nucleotide sequence of SEQ ID NO. 10. In an exemplary embodiment, Green Florence Protein (GFP) 122 may include an amino acid sequence identical to amino acid sequence of SEQ ID NO.11. In an exemplary embodiment, Terminator sequence 124 may include a nucleotide sequence identical to nucleotide sequence of SEQ ID NO.12. In an exemplary embodiment, COVID-19 antigen delivery vector 102 and an exemplary delivery platform may be used for producing an exemplary oral vaccine for COVID-19. In an exemplary embodiment, COVID-19 antigen delivery vector 102 and an exemplary Arthrospira platensis may be used for producing an exemplary oral vaccine for COVID-19. In an exemplary embodiment, an exemplary plurality of COVID-19 antigendelivery vector 102 may be coupled to an exemplary plurality of host genomes of an exemplary Arthrospira platensis. In an exemplary embodiment, a histidine (His) tag may be appended to antigen of COVID-19118. In an exemplary embodiment, an exemplary histidine sequence may be incorporated for purpose of purifying COVID-19 antigen 118. In an exemplary embodiment, an exemplary histidine tag may have a nucleotide sequence identical to nucleotide sequence of SEQ ID NO.14.

[0064] In an exemplary embodiment, exemplary Histidine tags, often referred to as "His tags," may be short sequences of histidine amino acids that may be added to a protein or gene product. In an exemplary embodiment, exemplary tags serve as a method for protein purification through a process called immobilized metal affinity chromatography (IMAC). In an exemplary embodiment, histidine residues may have a high affinity for metal ions including nickel or cobalt. In an exemplary embodiment, exemplary metals may be immobilized on a chromatography column. In an exemplary embodiment, when a protein with a His tag is expressed and purified, an exemplary protein may be selectively bounded to exemplary metal ions, allowing for easy and specific isolation of an exemplary protein from a complex mixture of cellular proteins. In an exemplary embodiment, antigen of COVID-19118 may be purified using IMAC.

[0065] Referring back to FIG.2B, in further detail with respect to step 214, step 214 of forming a plurality of hybrid vectors may include loading plurality of COVID-19 antigen delivery vectors 102 into a plurality of vectors. In an exemplary embodiment, loading an exemplary plurality of COVID-19 antigen delivery vectors 102 into an exemplary plurality of vectors may be performed using method 240 illustrated in FIG.2D.

[0066] FIG. 2D illustrates a flowchart of method 240 for loading an exemplary plurality of COVID-19 antigen delivery vectors102 into plurality of vectors, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embodiment, method 240 may include a step 242 of forming a second ligation reaction mixture by mixing a plurality of COVID-19 antigen delivery vectors, at least a second ligase enzyme, at least a second ligase buffer solution, and a plurality of vectors together and a step 244 of incubating the second ligation reaction mixture.

[0067] In further detail with respect to step 242, step 242 of forming an exemplary second ligation reaction mixture may include mixing plurality of COVID-19 antigen delivery vectors 102, at least an exemplary second ligase enzyme, at least an exemplary second ligase buffersolution, and an exemplary plurality of vectors in a mixer. In an exemplary embodiment, plurality of COVID-19 antigen delivery vectors 102, at least an exemplary second ligase enzyme, at least an exemplary second ligase buffer solution, and an exemplary plurality of vectors may be mixed together in an exemplary mixer with a stirring speed in a range of 1000 rpm to 1500 rpm. In an exemplary embodiment, an exemplary plurality of COVID-19 antigen delivery vectors 102, at least an exemplary second ligase enzyme, at least an exemplary ligase buffer solution, and an exemplary plurality of second vectors may be mixed together for a time period in a range of 4 hours to 16 hours. In an exemplary embodiment, an exemplary second ligase enzyme may include at least one of SacI, XbaI, BamHI, EcoRI and Hind III, and combinations thereof. In an exemplary embodiment, an exemplary second ligase buffer solution may include at least one of trace acetate, potasume acetate, magnesium acetate, spirmidin, sodium dodecyl sulphate (SDS), and combinations thereof. In an exemplary embodiment, an exemplary second ligation reaction mixture may allow plurality of COVID- 19 antigen delivery vectors 102 and an exemplary plurality of vectors be joined together. In an exemplary embodiment, an exemplary vector may include a plasmid vector. In an exemplary embodiment, forming an exemplary plurality of hybrid vectors may include loading plurality of COVID-19 antigen delivery vectors 102 into a plurality of plasmid vectors. In an exemplary embodiment, an exemplary plasmid vector for gene delivery may be a small, circular piece of DNA that may be used for at least one of duplicating COVID-19 antigen delivery vector 102, preserving COVID-19 antigen delivery vector 102 within an exemplary plasmid vector, and combinations thereof. In an exemplary embodiment, an exemplary plasmid vector may be used within a host organism. In an exemplary embodiment, an exemplary host organism may be a bacteria. In an exemplary embodiment, an exemplary bacteria may be an E. coli bacterium.

[0068] In an exemplary embodiment, an exemplary plasmid vector may include pBluescript II SK(+) vector with a nucleotide sequence identical to nucleotide sequence of SEQ ID NO.4. In an exemplary embodiment, The "SK" in an exemplary pBluescript II SK(+) vector’s name stands for "Stratagene's Klenow" and may refer to a polymerase used for creating a complementary strand of DNA during vector preparation. In an exemplary embodiment, an exemplary pBluescript II SK(+) may be a circular, double-stranded DNA plasmid. In an exemplary embodiment, an exemplary pBluescript II SK(+) may be a high-copy-number vector, meaning an exemplary pBluescript II SK(+) may replicate numerous times in a host bacterium, typically Escherichia coli (E. coli). In an exemplary embodiment, an exemplarypBluescript II SK(+)vector may include an ampicillin resistance gene (AmpR). In an exemplary embodiment, an exemplary pBluescript II SK(+) plasmid may contain multiple unique restriction enzyme recognition sites within a region called Multiple Cloning Site (MCS). In an exemplary embodiment, an exemplary MCS may be used for inserting foreign DNA fragments. In an exemplary embodiment, an exemplary foreign DNA fragment may include COVID-19 antigen delivery vector 102.

[0069] In an exemplary embodiment, use of an exemplary plasmid vector incorporating an exemplary transposon vector may serve a dual purpose in forming an exemplary oral vaccine. In an exemplary embodiment, Firstly, using an exemplary plasmid vector incorporating an exemplary transposon vector may enable multiplication of COVID-19 antigen 118 that may be included in an exemplary transposon vector. In an exemplary embodiment, COVID-19 antigen delivery vector 102 incorporating COVID-19 antigen 118 may be expressed within an exemplary host organism, allowing for production of a larger quantity of an exemplary COVID-19 antigen 118. In an exemplary embodiment, using an exemplary plasmid vector incorporating an exemplary transposon vector may serve as a means of preserving an exemplary COVID-19 antigen delivery vector 102 for an extended period of time.

[0070] In further detail with respect to step 244, step 244 of incubating an exemplary second ligation reaction mixture may include incubating an exemplary second ligation reaction mixture at a temperature in a range of 4°C to 37°C. In an exemplary embodiment, COVID-19 antigen delivery vector 102 may be replicated by a polymerase chain reaction (PCR) method. In an exemplary embodiment, an exemplary COVID-19 antigen delivery vector 102 may be replicated using at least one primer. In an exemplary embodiment, replicating an exemplary COVID-19 antigen delivery vector by an exemplary PCR method and using an exemplary at least one primer may refer to a process in which an exemplary vector may be replicated to create an exemplary COVID-19 antigen delivery vector 102. In an exemplary embodiment, an exemplary PCR method may be employed to amplify an exemplary COVID-19 antigen 118 within an exemplary vector using primers. In an exemplary embodiment, exemplary primers may include short DNA sequences with specific sequences that, when used in combination with a DNA polymerase, may attach to a target sequence in an exemplary vector and initiate replication of an exemplary vector. In an exemplary embodiment, by performing an exemplary PCR method, an exemplary COVID-19 antigen delivery vector 102 that carry an exemplary antigen of COVID-19 118 may be replicated. In an exemplary embodiment, an exemplaryprimer may include at least one of SPI-F or SPI-R, and combination thereof. In an exemplary embodiment, an exemplary primer may have a nucleotide sequence identical to nucleotide sequence of SEQ ID NO.15 and / or SEQ ID NO.16.

[0071] In an exemplary embodiment, an exemplary PCR method may include a series of temperature-dependent steps. In an exemplary embodiment, for multiplying an exemplary antigen of COVID-19 118 using an exemplary PCR method, an exemplary primer, an exemplary host organism, and an exemplary COVID-19 antigen delivery vector 102 may be mixed to form a reaction mixture. In an exemplary embodiment, an exemplary PCR method may begin with a denaturation step at a temperature in a range of 94°C to 100°C for a time period in a range of 2 minutes to 5 minutes, which may separate DNA double strands of an exemplary COVID-19 antigen delivery vector 102. In an exemplary embodiment, an exemplary denaturation step may be repeated for at least 35 cycles, causing an exemplary DNA to melt apart. In an exemplary embodiment, an exemplary reaction mixture may be heated at a temperature in a range of 50°C to 65 °C for a time period in a range of 25 seconds to 60 seconds, during which exemplary primers may bind to complementary sequences on an exemplary DNA. In an exemplary embodiment, an exemplary reaction mixture may be heated in an extension step at a temperature in a range of 70°C to 74°C for a time period in a range of 1 minute to 2 minutes, in which an exemplary DNA polymerase enzyme may synthesize new DNA strands by extending from exemplary primers. In an exemplary embodiment, a final temperature-dependent step of an exemplary PCR method may include heating an exemplary reaction mixture at a temperature in a range of 70°C to 74°C for a time period in a range of 4 minutes to 7 minutes that may ensure that any remaining single-stranded DNA is fully extended. In an exemplary embodiment, result of an exemplary PCR method may be a large quantity of an exemplary antigen of COVID-19118, which may be used for an exemplary oral vaccine.

[0072] Referring back to FIG. 2B, in further detail with respect to step 216, step 216 of separating an exemplary plurality of COVID-19 antigen delivery vectors 102 from an exemplary plurality of hybrid vectors may include mixing an exemplary plurality of hybrid vectors with a separation enzyme solution. In an exemplary embodiment, an exemplary plurality of hybrid vectors may be mixed with an exemplary separation enzyme solution in a mixer with a stirring speed in a range of 10 rpm to 50 rpm. In an exemplary embodiment, an exemplary plurality of hybrid vectors may be mixed with an exemplary separation enzymesolution for a time period in a range of 1 hour to 16 hours. In an exemplary embodiment, an exemplary plurality of hybrid vectors may be mixed with an exemplary separation enzyme solution at a temperature in a range of 30°C to 36°C. In an exemplary embodiment, an exemplary separation enzyme solution may include at least one of an EcoRI solution, a HindIII solution, and combinations thereof. In an exemplary embodiment, an exemplary EcoRI solution and an exemplary HindIII solution may include two respective restriction enzymes that may cut an exemplary plurality of COVID-19 antigen delivery vectors 102 at specific recognition sites. In an exemplary embodiment, exemplary recognition sites may be EcoRI restriction site 104 and HindIII restriction site 112. In an exemplary embodiment, an exemplary plurality of COVID-19 antigen delivery vectors 102 may be separated from an exemplary plurality of hybrid vectors at EcoRI restriction site 104 and HindIII restriction site 112. In an exemplary embodiment, a concentration of an exemplary separation enzyme solution may be in a range of 10 U / µl to 50 U / µl. In an exemplary embodiment, separating an exemplary plurality of COVID-19 antigen delivery vectors 102 from an exemplary plurality of hybrid vectors may include mixing an exemplary plurality of hybrid vectors with an exemplary separation enzyme solution with a weight ratio of an exemplary plurality of hybrid vectors to an exemplary separation enzyme solution in a range of 20:0.4 to 20:4 (an exemplary plurality of hybrid vectors: an exemplary separation enzyme solution).

[0073] In further detail with respect to step 218, step 218 of activating an exemplary plurality of COVID-19 antigen delivery vectors 102 may include mixing an exemplary plurality of COVID-19 antigen delivery vectors 102 with an activation enzyme solution. In an exemplary embodiment, an exemplary activation enzyme solution may activate Left ME sequence 106 and Right ME sequence 110. In an exemplary embodiment, activating an exemplary plurality of COVID-19 antigen delivery vectors 102 may include mixing an exemplary plurality of COVID-19 antigen delivery vectors 102 with an exemplary activation enzyme solution for a time period in a range of 30 minutes to 60 minutes. In an exemplary embodiment, activating an exemplary plurality of COVID-19 antigen delivery vectors 102 may include mixing an exemplary plurality of COVID-19 antigen delivery vectors 102 with an exemplary activation enzyme solution at a temperature in a range of 15°C to 30°C. In an exemplary embodiment, activating an exemplary plurality of COVID-19 antigen delivery vectors 102 may include mixing an exemplary plurality of COVID-19 antigen delivery vectors 102 with an exemplary activation enzyme solution with a weight ratio of an exemplary plurality of COVID-19 antigendelivery vectors 102 to an exemplary activation enzyme solution in a range of 8:2 to 6:8 (an exemplary plurality of COVID-19 antigen delivery vectors: an exemplary activation enzyme solution). In an exemplary embodiment, an exemplary activation enzyme solution may include at least one of a DNA transposone solution, a Transposase solution, and combinations thereof. In an exemplary embodiment, an exemplary activation enzyme solution in water may have a concentration in a range of 1 U / µl to 4 U / µl. In an exemplary embodiment, an exemplary activation enzyme solution may activate left ME sequence 106 and Right ME sequence 110. In an exemplary embodiment, left ME sequence 106 and Right ME sequence 110 may be restriction sites.

[0074] In further detail with respect to step 220, step 220 of transferring an exemplary plurality of activated COVID-19 antigen delivery vectors into a host genome of Arthrospira platensis may include electroporating Arthrospira platensis in the presence of activated COVID-19 antigen delivery vectors. In an exemplary embodiment, electroporating Arthrospira platensis in the presence of activated COVID-19 antigen delivery vectors may include forming a suspension of Arthrospira platensis, forming a suspension of an exemplary plurality of activated COVID-19 antigen delivery vectors, mixing an exemplary suspension of Arthrospira platensis with an exemplary suspension of an exemplary plurality of activated COVID-19 antigen delivery vectors, and electroporating an exemplary mixture of an exemplary suspension of Arthrospira platensis and an exemplary suspension of an exemplary plurality of activated COVID-19 antigen delivery vectors by applying an electric field to an exemplary mixture of an exemplary suspension of Arthrospira platensis and an exemplary suspension of an exemplary plurality of activated COVID-19 antigen delivery vectors.

[0075] In an exemplary embodiment, in step 220, an exemplary Arthrospira platensis may be suspended in a first solution. In an exemplary embodiment, an exemplary first solution may include HEPES buffer. In an exemplary embodiment, an exemplary HEPES buffer may have pH pf 7. In an exemplary embodiment, an exemplary suspension of Arthrospira platensis in an exemplary first solution may have a concentration in a range of 0.1 mM to 1.0 mM. In an exemplary embodiment, an exemplary plurality of activated COVID-19 antigen delivery vectors 102 may be suspended in a second solution. In an exemplary embodiment, an exemplary second solution may include DOTAP Liposomal Transfection Reagent. In an exemplary embodiment, an exemplary suspension of an exemplary plurality of activated COVID-19 antigen delivery vectors in an exemplary second solution may have a concentrationin a range of 1.0 µg / ml to 4.0 µg / ml. In an exemplary embodiment, an exemplary suspension of Arthrospira platensis and an exemplary suspension of an exemplary plurality of activated COVID-19 antigen delivery vectors 102 may be mixed together with a volume ratio of an exemplary suspension of Arthrospira platensis to an exemplary suspension of an exemplary plurality of activated COVID-19 antigen delivery vectors 102 in a range of 1:100 to 1:10 (suspension of Arthrospira platensis: suspension of an exemplary plurality of activated COVID-19 antigen delivery vectors). In an exemplary embodiment, an exemplary mixture of an exemplary suspension of Arthrospira platensis and an exemplary suspension of an exemplary plurality of activated COVID-19 antigen delivery vectors 102 may be placed in an electroporation cuvette or chamber. In an exemplary embodiment, an electrical pulse may be applied to an exemplary electroporation cuvette using an electroporation device. In an exemplary embodiment, an exemplary electrical pulse may generate an electrical field that may temporarily disrupt Arthrospira platensis’s cell membrane. In an exemplary embodiment, an exemplary electrical field may cause temporarily increase of permeability of Arthrospira platensis’s cell membrane, allowing for an exemplary COVID-19 antigen delivery vector 102 entering into exemplary cells of Arthrospira platensis. In an exemplary embodiment, an exemplary electrical pulse may create pores in Arthrospira platensis’s cell membrane. In an exemplary embodiment, during brief moment when Arthrospira platensis’s cell membrane is permeabilized, an exemplary COVID-19 antigen delivery vector 102 may enter an exemplary Arthrospira platensis’s cell through exemplary pores. In an exemplary embodiment, once an exemplary electrical field is removed, an exemplary Arthrospira platensis’s cell membrane may reseal. In an exemplary embodiment, transferring an exemplary plurality of activated COVID-19 antigen delivery vectors 102 into a host genome of Arthrospira platensis may include applying a voltage to an exemplary electroporation cuvette in a range of 2 KV / cm to 7.5 KV / cm. In an exemplary embodiment, transferring an exemplary plurality of activated COVID-19 antigen delivery vectors 102 into a host genome of Arthrospira platensis may include applying a voltage to an exemplary electroporation cuvette for a time period in a range of 0.5 ms to 2 ms. In an exemplary embodiment, transferring an exemplary plurality of activated COVID-19 antigen delivery vectors 102 into a host genome of Arthrospira platensis may include applying a voltage to an exemplary Arthrospira platensis in a range of 2 KV / cm to 7.5 KV / cm. In an exemplary embodiment, transferring an exemplary plurality of activated COVID-19 antigen delivery vectors into a host genome of Arthrospira platensis may includeapplying a voltage to an exemplary Arthrospira platensis for a time period in a range of 0.5 ms to 2 ms.

[0076] Example 1: Transferring an exemplary COVID-19 antigen delivery vector into a host genome

[0077] An exemplary COVID-19 antigen delivery vector was transferred into a host genome by a method similar to step 206 of method 200 or step 220 of method 210. For this objective, Arthrospira plate was harvested at logarithmic growth stage (optical density (OD) 560= 0.4). After centrifugation, Arthrospira plate cells were placed in culture medium. Exemplary Arthrospira plate cells were washed 3 times with 0.5 mM of N-2-hydroxyethylpiperazine-N´- 2-ethanesulfonic acid (HEPES) buffer solution (pH = 7) and was dissolved in an exemplary HEPES buffer solution. In the next step, 4 µl of DOTAP Liposomal Transfection Reagent was mixed with a transposon. The complex of Roch and transposon was mixed with 40 µl of ice- cold treated Arthrospira plate cells and was immediately injected into a 2 mm electroporation cuvette. Finally, an exemplary mixture was exposed to an electric pulse in a range of 2 KV / cm to 7.5 KV / cm, at 1000 Ω and 25 µF for 5.0 ms. Transgenic cells' accumulation was observed after 3 weeks of incubation at 30°C

[0078] Example 2: Replicating an exemplary COVID-19 antigen delivery vector

[0079] An exemplary COVID-19 antigen delivery vector was replicated by a method similar to step 214 of method 210. The E. coli TOP10 competent cell was prepared using optimized CaCl2method of Sambrook (according to Sambrook’s book, specifically the 1989 edition). Competent cell Preparation using CaCl2 method: The bacterium was cultured in LB Broth medium and cooled on ice for 10 minutes as it reached OD600 = 0.6-0.8. After centrifugation and removal of the supernatant, 10 ml of cold CaCl2 was added to the bacterial sediment (0.1- 0.05 M). After a 20-minute break in the cold and centrifugation, the plate was dissolved in 5 ml of cold (0.1 M) CaCl2 solution and 20% glycerol. Competent cells were stored in the freezer until use the final competent cells were kept at -80 ºC until use. In the next step, melting component was mixed with 5μl of plasmid for transformation. After mixing, one cooling step of 30 minutes on ice, one heating step of 2 minutes in water bath at 42 °C, and one cooling step of 5 minutes on ice were performed, respectively. In the next step, 600 μl of Lysogeny Broth or Luria-Bertani Broth (LB) (a liquid medium for cultivation of bacteria) was added to the sample and was incubated at 37°C while mixing within a mixer with a stirring speed of 150 rpm for 1 hour. The solution containing the transgenes was relocated to LB agar withAmpicillin with a weight ratio of 1:10, 1:100, 1:1000, and 1:10000 (the transgenes solution: LB agar). The mixtures were incubated at 37 °C for 18 hours to 24 hours. PCR method was used for amplifying an exemplary COVID-19 antigen delivery vector. The process involves a series of temperature-dependent steps. In the initial step, a temperature for denaturation at 94°C for 5 minutes was applied, which separated the DNA double strands of an exemplary COVID- 19 antigen delivery vector. In the second step, the denaturation step was repeated at 94°C for 1 minute, causing the DNA to melt apart. The annealing step follows at 58°C for 30 seconds, during which short DNA primers bind to complementary sequences on the target DNA. The extension step at 72°C for 1 minute was where a heat-resistant DNA polymerase enzyme synthesized new DNA strands by extending from the primers. It is a step in a polymerase chain reaction (PCR) process. During the extension step at 72°C for 1 minute, a heat-resistant DNA polymerase enzyme synthesizes new DNA strands by extending from the primers, a crucial phase in amplifying the target DNA.

[0080] This cycle of denaturation, annealing, and extension is repeated 35 times, doubling the amount of DNA with each cycle. Finally, a 5-minute final extension at 72°C ensures that any remaining single-stranded DNA was fully extended. The result was a large quantity of an exemplary COVID-19 antigen delivery vector, which can be used for producing an exemplary oral vaccine.

[0081] Example 3: Activating an exemplary COVID-19 antigen delivery vector

[0082] In order to activate an exemplary COVID-19 antigen delivery vector, known as the Tn5 transposon, within an in vivo environment, a transposase enzyme was employed. To achieve this, 4 µl of transposase and 2 µl of pure glycerol were added to 2 µl of transposon DNA without Mg2+. The mixture was thoroughly vortexed and allowed to incubate at room temperature (25 ºC) for 30 minutes. The resultant complex, referred to as the transposome, consisting of the active Tn5 transposon and transposase, was preserved at -20°C until it was ready for use.

[0083] Example 4: Analyzing protein expression

[0084] The bacteria were lysed using a lysis buffer solution containing 1% 2-[4-(2,4,4- trimethylpentan-2-yl)phenoxy]ethan (Triton X-100), 50 mM Tris-HCl, 150 mM NaCl, 0.25% sodium deoxycholate, 1 mM Ethylene glycol tetraacetic acid, and 1 mM NaF. To determine the protein content, a Bradford assay using a kit was conducted. The lysates were then boiled for 5 minutes, and 25 μg of the lysates were loaded onto a 12% SDS-PAGE gel. The separatedproteins were transferred onto a 0.2 μm Immune-Blot™ polyvinylidene difluoride (PVDF) membrane. To prevent non-specific binding, the membranes were blocked with a solution of 5% BSA and 0.1% Tween 20 for 1 hour. Subsequently, the membranes were incubated with HRP Anti His tag antibody as a secondary antibody at room temperature for 1 hour. Finally, the membranes were exposed to an enhanced chemiluminescence (ECL) incubator for 1-2 minutes to detect the protein bands. FIG. 3 illustrates a fluorescent image 300 of protein expression of an exemplary oral vaccine, consistent with one or more exemplary embodiments of the present disclosure. FIG.3 illustrates standard ladder band 250 bp (302), 500 bp (304), 750 bp (306), 1000 bp (308), 1500 bp (310), 2000 bp (312), 3000 bp (314), 5000 bp (316), 10000 bp (318), 1657 bp (320), and 3343 bp (322). As shown in FIG.3, standard ladder 1657 bp (320) is the transposon vector and standard ladder 3343 bp (322) is the plasmid vector.

[0085] FIG. 4 illustrates a fluorescent image 400 of an exemplary modified Arthrospira platensis, consistent with one or more exemplary embodiments of the present disclosure. As shown in FIG. 4, the GFP fluorescence is evident from genetically modified cells after exposure to blue light for 20 minutes.

[0086] Example 5: Analyzing expression of antibody COVID-19 in animals

[0087] In the animal study, each animal group included a minimum of three rats. Recommended vaccine doses for oral administration in a Arthrospira platensis -based dietary supplement are as follows:

[0088] Table 1. Recommended vaccine doses for oral administration in a Arthrospira platensis -based dietary supplement. Dose of Ag Name Code Control sample Negative control (C-) R1 (Arthrospira platensis) 30 µg 1 dose R2 80 µg 2 dose R3 150 µg 3 dose R4 0.5 µg A COVID-19 vaccine (C+) R5

[0089] as shown in Table 1, R1 is a control sample without transgenic Arthrospira platensis, R2 is a sample using 30 µg of antigen of COVID-19, R3 is a sample using 80 µg of antigen of COVID-19, R4 is a sample using 150 µg of antigen of COVID-19, and R5 is a sample that used 0.5 µg of antigen of COVID-19 via injection.

[0090] FIG.5 illustrates an image 500 of expression of an exemplary COVID 19-RBD protein in 4 replicates of transgenics and negative control by Western blot method, consistent with one or more exemplary embodiments of the present disclosure. Evaluation of Ag expression in cells cultured under different light condition by western blot (Activity of C-Phycocyanin promoter is controlled by light). CB: control- / non transgenic Spirulina under blue light condition (502) during culturing, 504 and 506 are two repeats of transgenic Spirulina including COVID-19 Ag cultured under white light condition and 508 and 510 are two repeats of transgenic Spirulina including COVID-19 Ag cultured under blue light condition.

[0091] The summary of the findings for the humoral immune response to different doses of the oral COVID-19 RBD subunit vaccine is as follows:

[0092] The negative control group (empty algae) showed a significant difference compared to all oral vaccine doses as well as the injected commercial vaccine. There was no significant difference between the 150 µg oral vaccine dose and the positive control (injected commercial vaccine). In other words, the performance of a single oral dose of the COVID-19 RBD vaccine based on whole cell microalgae Arthrospira platensis was equivalent to a commonly used subunit model vaccine. There was no significant difference between the 30 µg and 80 µg oral vaccine doses, but there was a significant difference between the 150 µg dose and the other two oral doses. The expression level with the 150 µg oral dose was significantly higher (P=0.001 & P=0.032). The differences in antibody expression between the different groups were clearly visible in FIG.3 and FIG.4. FIG. 6 illustrates a calibration curve 600 of Elisa test performed for an exemplary oral vaccine, consistent with one or more exemplary embodiments of the present disclosure. The calibration curve has a calibration equation of y=0.07171X+0.126. The coefficient of determination (R2) was 0.9687. The graph resulting from ELISA data is plotted to assess the antigen levels in algae cells based on a standard curve drawn using the formula, and the points surrounding the standard curve depict the expression levels of the antigen in various replicates.

[0093] FIG.7 illustrates an image 700 of comparison of antibody titers in the blood serum of the tested groups, consistent with one or more exemplary embodiments of the present disclosure. The calibration curve has a calibration equation of y=0.0251X+0.3173. The coefficient of determination (R2) was 0.9446. The graph resulting from ELISA data of mouse serum is plotted based on a standard curve drawn using the formula, with the points surroundingthe standard curve indicating the expression levels of antibodies in response to vaccination stimulation.

[0094] FIG. 8 illustrates an image 800 of comparison of mean and standard divination of COVID-19 RBDAg titers, consistent with one or more exemplary embodiments of the present disclosure. As shown in FIG.8, mean of COVID-19 RBDAg titer for 30 µg of an exemplary oral vaccine (802), mean of COVID-19 RBDAg titer for 80 µg of an exemplary oral vaccine (804), mean of COVID-19 RBDAg titer for 150 µg of an exemplary oral vaccine (806), control group (808), and mean of COVID-19 RBDAg titer of 0.5 µg of the injected group (810) are illustrated.

[0095] Interpretation of the evaluation factors affected by vaccination was presented in a concise manner in Table 2. The results indicated no adverse effect of oral vaccination on the evaluated factors.

[0096] Table 2. Evaluation of changes in Renal and Hepatic factors (excluding antibodies) under the influence of an exemplary oral vaccination in control and tested groups. Analyzed factors Interpretation ALT(U / L)No significant difference between the negative control and other groups. AST (U / L)No significant difference between the negative control and other groups. ALP (U / L) No significant difference between the negative control and other groups. Urea (mg / dl) No significant difference between the negative control and other groups. Cr (mg / dl) No significant difference between the negative control and other groups. T.P (g / dl) No significant difference between the negative control and the positive control groups, and the 80 and 150 µg groups. The only significant difference (P=0.049) was between the negative control and the lowest dose, which is 30 µg, with a very slight difference. Alb (mg / dl) No significant difference between the negative control and other groups. GGT (U / L) No significant difference between the negative control and other groups. Glucose (mg / dl) No significant difference between the negative control and the oral vaccine groups. The only difference (P=0.023) was between the negative control and the injected vaccine group.

[0097] Industrial Applicability

[0098] The industrial applicability of an oral vaccine utilizing Arthrospira platensis combined with COVID-19 antigens holds significant promise in the global effort to combat the COVID- 19 pandemic. Arthrospira platensis, commonly known as spirulina, is a microalgae with a well- established history of safe human consumption and remarkable nutritional benefits. Integrating this natural and versatile microorganism with COVID-19 antigens provides a novel approach to vaccine development. This innovative method offers several key advantages, including scalability, cost-effectiveness, and ease of production, making it an attractive option for large- scale manufacturing and distribution. Furthermore, an oral vaccine has the potential to enhance vaccination accessibility, as it eliminates the need for injections and may encourage higher vaccine uptake. Overall, the industrial application of this oral vaccine holds the potential to revolutionize the fight against COVID-19, offering a safe, efficient, and user-friendly immunization solution on a global scale.

[0099] While the foregoing has described what are considered to be the best mode and / or other examples, it is understood that various modifications may be made therein and that the subject matter disclosed herein may be implemented in various forms and examples, and that the teachings may be applied in numerous applications, only some of which have been described herein. It is intended by the following claims to claim any and all applications, modifications and variations that fall within the true scope of the present teachings.

[0100] Unless otherwise stated, all measurements, values, ratings, positions, magnitudes, sizes, and other specifications that are set forth in this specification, including in the claims that follow, are approximate, not exact. They are intended to have a reasonable range that is consistent with the functions to which they relate and with what is customary in the art to which they pertain.

[0101] The scope of protection is limited solely by the claims that now follow. That scope is intended and should be interpreted to be as broad as is consistent with the ordinary meaning of the language that is used in the claims when interpreted in light of this specification and the prosecution history that follows and to encompass all structural and functional equivalents. Notwithstanding, none of the claims are intended to embrace subject matter that fails to satisfy the requirement of Sections 101, 102, or 103 of the Patent Act, nor should they be interpreted in such a way. Any unintended embracement of such subject matter is hereby disclaimed.

[0102] Except as stated immediately above, nothing that has been stated or illustrated is intended or should be interpreted to cause a dedication of any component, step, feature, object, benefit, advantage, or equivalent to the public, regardless of whether it is or is not recited in the claims.

[0103] It will be understood that the terms and expressions used herein have the ordinary meaning as is accorded to such terms and expressions with respect to their corresponding respective areas of inquiry and study except where specific meanings have otherwise been set forth herein. Relational terms such as first and second and the like may be used solely to distinguish one entity or action from another without necessarily requiring or implying any actual such relationship or order between such entities or actions. The terms “comprises,” “comprising,” or any other variation thereof, are intended to cover a non- exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. An element proceeded by “a” or “an” does not, without further constraints, preclude the existence of additional identical elements in the process, method, article, or apparatus that comprises the element.

[0104] The Abstract of the Disclosure is provided to allow the reader to quickly ascertain the nature of the technical disclosure. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims. In addition, in the foregoing Detailed Description, it can be seen that various features are grouped together in various implementations. This is for purposes of streamlining the disclosure, and is not to be interpreted as reflecting an intention that the claimed implementations require more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive subject matter lies in less than all features of a single disclosed implementation. Thus, the following claims are hereby incorporated into the Detailed Description, with each claim standing on its own as a separately claimed subject matter.

[0105] While various implementations have been described, the description is intended to be exemplary, rather than limiting and it will be apparent to those of ordinary skill in the art that many more implementations and implementations are possible that are within the scope of the implementations. Although many possible combinations of features are shown in the accompanying figures and discussed in this detailed description, many other combinations of the disclosed features are possible. Any feature of any implementation may be used incombination with or substituted for any other feature or element in any other implementation unless specifically restricted. Therefore, it will be understood that any of the features shown and / or discussed in the present disclosure may be implemented together in any suitable combination. Accordingly, the implementations are not to be restricted except in light of the attached claims and their equivalents. Also, various modifications and changes may be made within the scope of the attached claims.

Claims

What is claimed is:

1. An oral vaccine for COVID-19, comprising: a delivery platform comprising Arthrospira platensis with a plurality of host genomes, each respective host genome comprising a nucleotide sequence identical to nucleotide sequence of SEQ ID NO.1; and a plurality of COVID-19 antigen delivery vectors coupled to the plurality of host genomes, each COVID-19 antigen delivery vector of the plurality of COVID-19 antigen delivery vectors has a nucleotide sequence identical to nucleotide sequence of SEQ ID NO.2, each respective COVID-19 antigen delivery vector comprising at least one antigen of COVID-19 with a weight ratio of the delivery platform to the at least one antigen of COVID-19 in a range of 1: 10-3to 1: 2.5 ^ 10-3(delivery platform: at least one antigen of COVID-19).

2. The oral vaccine of claim 1, wherein each respective COVID-19 antigen delivery vector of the plurality of COVID-19 antigen delivery vectors comprises a transposon vector.

3. The oral vaccine of claim 1, wherein each respective COVID-19 antigen delivery vector of the plurality of COVID-19 antigen delivery vectors comprises the at least one antigen of COVID-19 with a nucleotide ratio of each respective COVID-19 antigen delivery vector to the at least one antigen of COVID-19 in a range of 50: 7 to 50: 22 (each COVID-19 antigen delivery vector: the at least one antigen of COVID-19).

4. The oral vaccine of claim 1, wherein the at least one antigen of COVID-19 has a nucleotide sequence identical to nucleotide sequence of SEQ ID NO.

3.

15. The oral vaccine of claim 1, wherein the delivery platform is in a form of at least one of a tablet, a capsule, powder, a pearl, and combinations thereof, wherein the capsule is made of at least one of gelatin, non-gelatin, and combinations thereof.

6. The oral vaccine of claim 1, wherein the oral vaccine is an additive in food, the food comprising at least one of yogurt, dessert, gum, ice cream, pastilles, and combinations thereof.

7. The oral vaccine of claim 1, wherein the oral vaccine comprises at least 10 µg of the at least one antigen of COVID-19.

8. A method of preparing an oral vaccine for COVID-19, the method comprising: forming a plurality of COVID-19 antigen delivery vectors by loading a plurality of antigens of COVID-19 into a plurality of transposon vectors; forming a plurality of hybrid vectors by loading the plurality of COVID-19 antigen delivery vectors into a plurality of vectors; separating the plurality of COVID-19 antigen delivery vectors from the plurality of hybrid vectors by mixing the plurality of hybrid vectors with a separation enzyme solution; activating the separated plurality of COVID-19 antigen delivery vectors by mixing the separated plurality of COVID-19 antigen delivery vectors with an activation enzyme solution; and transferring the plurality of activated COVID-19 antigen delivery vectors into a plurality of host genomes of Arthrospira platensis by an electroporation technique.

29. The method of claim 8, wherein forming the plurality of hybrid vectors further comprises multiplying the plurality of hybrid vectors by incubating the plurality of hybrid vectors at a temperature in a range of 4°C to 16ºC for a time period in a range of 2 hours to 12 hours.

10. The method of claim 8, wherein forming the plurality of hybrid vectors comprises loading the plurality of COVID-19 antigen delivery vectors into a plurality of plasmid vectors.

11. The method of claim 8, wherein forming the plurality of hybrid vectors comprises loading the plurality of COVID-19 antigen delivery vectors into a plurality of pBluescript II SK(+) vectors with a nucleotide sequence identical to nucleotide sequence of SEQ ID NO.

4.

12. The method of claim 8, wherein separating the plurality of COVID-19 antigen delivery vectors from the plurality of hybrid vectors comprises mixing the plurality of hybrid vectors with the separation enzyme solution with a volume ratio of the plurality of hybrid vectors to the separation enzyme solution in a range of 20:0.4 to 20:4 (the plurality of hybrid vectors: the separation enzyme solution).

13. The method of claim 12, wherein mixing the plurality of hybrid vectors with the separation enzyme solution comprises mixing the plurality of hybrid vectors with a solution comprising a separation enzyme dissolved in a buffered aqueous glycerol solution with a concentration of the separation enzyme solution in a range of 10 U / µl to 50 U / µl.

14. The method of claim 8, wherein separating the plurality of COVID-19 antigen delivery vectors from the plurality of hybrid vectors comprises mixing the plurality of hybrid vectors 3with at least one of an EcoRI solution, a HindIII solution in a buffered aqueous glycerol solution, and combinations thereof for a time period in a range of 1 hour to16 hours.

15. A method of preparing an oral vaccine for COVID-19, the method comprising: forming a plurality of COVID-19 antigen delivery vectors by loading a plurality of antigens of COVID-19 into a plurality of transposon vectors; activating the plurality of COVID-19 antigen delivery vectors by mixing the plurality of COVID-19 antigen delivery vectors with an activation enzyme solution; and transferring the plurality of activated COVID-19 antigen delivery vectors into a plurality of host genomes of Arthrospira platensis by an electroporation technique.

16. The method of claim 15, wherein loading the plurality of COVID-19 antigens into the plurality of transposon vectors comprises: isolating the plurality of COVID-19 antigens by a method comprising at least one of synthesizing the plurality of COVID-19 antigens, digestion ligation, and combinations thereof; forming a ligation reaction mixture by mixing the plurality of isolated COVID- 19 antigens, at least a ligase enzyme, at least a ligase buffer solution, and the plurality of transposon vector together; and incubating the ligation reaction mixture for a time period in a range of 4 hours to 16 hours at a temperature in a range of 4°C to 37°C.

17. The method of claim 15, wherein activating the plurality of COVID-19 antigen delivery vectors comprises mixing the plurality of COVID-19 antigen delivery vectors with the 4activation enzyme solution with a volume ratio of the plurality of COVID-19 antigen delivery vectors to the activation enzyme solution in a range of 8:2 to 6:8 (the plurality of COVID-19 antigen delivery vectors: the activation enzyme solution).

18. The method of claim 17, wherein mixing the plurality of COVID-19 antigen delivery vectors with an activation enzyme solution comprises mixing the plurality of COVID-19 antigen delivery vectors with an activation enzyme dissolved in water thereof with a concentration of the activation enzyme solution in a range of 1 U / µl to 4 U / µl.

19. The method of claim 15, wherein activating the plurality of COVID-19 antigen delivery vectors comprises mixing the plurality of COVID-19 antigen delivery vectors with at least one of a DNA transposon solution, a Transposase solution, and combinations thereof.

20. The method of claim 15, wherein transferring the plurality of activated COVID-19 antigen delivery vectors into a host genome of Arthrospira platensis comprises applying a voltage to the Arthrospira platensis in a range of 1 KV / cm to 7.5 KV / cm for a time period in a range of 0.5 ms to 2 ms. 5

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