Oral vaccine for hepatitis b

Abstract

An oral vaccine for Hepatitis B is disclosed. The oral vaccine for Hepatitis B includes a delivery platform including Arthrospira platensis with a plurality of host genomes and a plurality of Hepatitis B surface antigen delivery vectors coupled to the plurality of host genomes. Each Hepatitis B surface antigen delivery vector of the plurality of Hepatitis B surface antigen delivery vectors has a nucleotide sequence identical to nucleotide sequence of SEQ ID NO. 2. Each respective Hepatitis B surface antigen delivery vector of the plurality of Hepatitis B surface antigen delivery vectors includes at least one surface antigen of Hepatitis B with a weight ratio of the delivery platform to the at least one surface antigen of Hepatitis B in a range of 1: 10-4 to 1: 2x10- 3 (delivery platform: at least one surface antigen of Hepatitis B).

Description

ORAL VACCINE FOR HEPATITIS BTECHNICAL FIELD

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

[0002] Hepatitis is a group of viral infections that primarily affect the liver, causing inflammation and potentially leading to severe health complications. The most common types of viral hepatitis are hepatitis A, B, C, D, and E. Each type is caused by a different virus, with hepatitis B and C being of particular concern due to their chronic nature and potential to cause long-term liver damage. Hepatitis A and E are typically acute and self-limiting, often transmitted through contaminated food and water, while hepatitis B, C, and D are primarily spread through contact with infected blood or other bodily fluids. Hepatitis can lead to symptoms such as jaundice, fatigue, abdominal pain, and, in some cases, progress to chronic liver disease or liver cancer.

[0003] Vaccination plays a crucial role in preventing hepatitis, especially hepatitis B, which is a vaccine-preventable disease. Hepatitis B vaccines are effective in preventing infection and subsequent complications, including chronic liver disease and liver cancer. The vaccine is typically administered as a series of shots, providing long-lasting protection against the virus. In addition to hepatitis B vaccines, there are also vaccines available for hepatitis A, offering protection against this acute form of the virus. Vaccination is a key public health measure, as it not only protects individuals from the potentially severe consequences of hepatitis but also contributes to the overall reduction of the spread of these viral infections in the community.

[0004] There are several categories of vaccines for hepatitis B, each designed to provide immunity against the virus. The most common and widely used is the recombinant hepatitis B vaccine, which contains a small, harmless part of the hepatitis B virus. Additionally, there are combination vaccines that offer protection against multiple diseases in a single shot, such as vaccines that include both hepatitis B and hepatitis A protection. Another category includes vaccines specifically designed for certain populations, such as those formulated for infants or healthcare workers who may be at an increased risk of exposure. The availability of various formulations and combination vaccines contributes to the flexibility and effectiveness ofhepatitis B vaccination strategies, helping to tailor immunization efforts to specific populations and public health needs. For example, Edward Tabor et al. presented a patent on “Hepatitis B core antigen vaccine” (US4547367A). Edward Tabor et al. disclosed a vaccine against hepatitis B virus (HBV) using purified hepatitis B core antigen (HBcAg) for Chimpanzees vaccination. Ulrike protzer et al. presented a patent on “means and methods to treat HBV” (ES2834698T3). Ulrike protzer et al. formed a recombinant vector for vaccination against hepatitis B virus expressing a coat protein of serotype adw of hepatitis B virus. While vaccines are essential tools in preventing and controlling hepatitis, there are some drawbacks associated with their use. One concern is the potential for adverse reactions, although serious side effects are rare. Additionally, some individuals may not respond adequately to vaccination, leaving them susceptible to infection. Vaccine hesitancy and misinformation can also contribute to lower vaccination rates, reducing the overall effectiveness of immunization programs. Despite these challenges, the benefits of hepatitis vaccines in preventing illness, reducing the spread of the virus, and ultimately saving lives underscore their importance in public health strategies aimed at controlling and eliminating hepatitis infections worldwide.

[0005] There is, therefore, a need for a non-toxic, available, easy to use, and highly effective vaccine for hepatitis B. There is further a need for a simple, scalable, and cost-effective method to produce the aforementioned vaccine for hepatitis B.SUMMARY OF THE DISCLOSURE

[0006] 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.

[0007] According to one or more exemplary embodiments, the present disclosure is directed to an oral vaccine for Hepatitis B. In an exemplary embodiment, an exemplary oral vaccine may include a delivery platform including Arthrospira platensis with a plurality of host genomes and a plurality of Hepatitis B 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 Hepatitis B antigendelivery vector may have a nucleotide sequence identical to nucleotide sequence of SEQ ID NO. 2. In an exemplary embodiment, an exemplary each respective Hepatitis B antigen delivery vector may include at least one antigen of Hepatitis B with a weight ratio of an exemplary delivery platform to an exemplary at least one antigen of Hepatitis B in a range of 1: 10’4to 1: 2xl0-3(delivery platform: at least one antigen of Hepatitis B).

[0008] In an exemplary embodiment, each respective Hepatitis B antigen may include Hepatitis B Surface antigen. In an exemplary embodiment, each respective Hepatitis B Surface antigen delivery vector of an exemplary plurality of Hepatitis B Surface antigen delivery vectors may include a transposon vector. In an exemplary embodiment, each respective Hepatitis B Surface antigen delivery vector of an exemplary plurality of Hepatitis B Surface antigen delivery vectors may include an exemplary at least one Surface antigen of Hepatitis B with a nucleotide ratio of each respective Hepatitis B Surface antigen delivery vector to an exemplary at least one Surface antigen of Hepatitis B in a range 10: 2 to 10: 6 (each Hepatitis B Surface antigen delivery vector: an exemplary Surface antigen of Hepatitis B).

[0010] In an exemplary embodiment, an exemplary surface antigen of Hepatitis B 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 pg of an exemplary at least one Surface antigen of Hepatitis B. 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.

[0011] According to one or more exemplary embodiments, the present disclosure is directed to a method for preparing an oral vaccine for Hepatitis B . In an exemplary embodiment, an exemplary method may include forming a plurality of Hepatitis B Surface antigen delivery vectors by loading a plurality of Surface antigens of Hepatitis B into a plurality of transposon vectors, forming a plurality of hybrid vectors by loading an exemplary plurality of Hepatitis B Surface antigen delivery vectors into a plurality of vectors, separating an exemplary plurality of Hepatitis B Surface antigen delivery 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 Hepatitis B Surface antigen delivery vectors by mixing an exemplary separated plurality of Hepatitis B Surface antigen delivery vectors with an activation enzyme solution, and transferring an exemplary plurality of activated Hepatitis B Surface antigen delivery vectors into a plurality of host genomes of Arthrospira platensis by an electroporation technique.

[0012] 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 tol6°C for a time period in a range of 2 hours to 12 hours.

[0013] In an exemplary embodiment, forming an exemplary plurality of hybrid vectors may include loading an exemplary plurality of Hepatitis B Surface antigen delivery vectors into a plurality of plasmid vectors.

[0014] In an exemplary embodiment, forming an exemplary plurality of hybrid vectors may include loading an exemplary plurality of Hepatitis B Surface antigen delivery vectors into a plurality of pBluescript II SK(+) vectors with a nucleotide sequence identical to nucleotide sequence of SEQ ID NO. 4.

[0015] In an exemplary embodiment, separating an exemplary plurality of Hepatitis B Surface 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).

[0016] 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 a buffered aqueous glycerol solution with a concentration of an exemplary separation enzyme solution in a range of 10 U / pl to 50 U / pl. In an exemplary embodiment, separating an exemplary plurality of Hepatitis B Surface 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 Hindlll solution in a buffered aqueous glycerol solution, and combinations thereof for a time period in a range of 1 hour to 16 hours.

[0017] According to one or more exemplary embodiments, the present disclosure is directed to a method of preparing an oral vaccine for Hepatitis B. In an exemplary embodiment, an exemplary method may include forming a plurality of Hepatitis B Surface antigen delivery vectors by loading a plurality of Surface antigens of Hepatitis B into a plurality of transposon vectors, activating an exemplary plurality of Hepatitis B Surface antigen delivery vectors by mixing an exemplary plurality of Hepatitis B Surface antigen delivery vectors with an activation enzyme solution, and transferring an exemplary plurality of activated Hepatitis B Surface antigen delivery vectors into a plurality of host genomes of Arthrospira platensis by an electroporation technique.

[0018] In an exemplary embodiment, loading an exemplary plurality of Hepatitis B Surface antigens into an exemplary plurality of transposon vectors may include isolating an exemplary plurality of Hepatitis B Surface antigens by a method including at least one of synthesizing an exemplary plurality of Hepatitis B Surface antigens, digestion ligation, and combinations thereof, forming a ligation reaction mixture by mixing an exemplary plurality of isolated Hepatitis B Surface antigens, at least a ligase enzyme, and at least a ligase buffer solution, and an exemplary plurality of transposon vectors together, 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.

[0019] In an exemplary embodiment, activating an exemplary plurality of Hepatitis B Surface antigen delivery vectors may include mixing an exemplary plurality of Hepatitis B Surface antigen delivery vectors with an exemplary activation enzyme solution with a volume ratio of an exemplary plurality of Hepatitis B Surface antigen delivery vectors to an exemplary activation enzyme solution in a range of 8:2 to 6:8 (an exemplary plurality of Hepatitis B Surface antigen delivery vectors: an exemplary activation enzyme solution).

[0020] In an exemplary embodiment, mixing an exemplary plurality of Hepatitis B Surface antigen delivery vectors with an activation enzyme solution may include mixing an exemplary plurality of Hepatitis B Surface antigen delivery vectors with an activation enzyme dissolved in water. In an exemplary embodiment, an exemplary activation enzyme solution may have a concentration in a range of 1 U / pl to 4 U / pl. In an exemplary embodiment, activating an exemplary plurality of Hepatitis B Surface antigen delivery vectors may include mixing an exemplary plurality of Hepatitis B Surface antigen delivery vectors with at least one of a DNA transposon solution, a Transposase solution in water, and combinations thereof.

[0021] In an exemplary embodiment, transferring an exemplary plurality of activated Hepatitis B Surface antigen delivery vectors into a host genome of Arthrospira platensis may include applying a 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

[0022] 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.

[0023] FIG. 1 illustrates a schematic view of genetic components of a Hepatitis B Surface antigen delivery vector, consistent with one or more exemplary embodiments of the present disclosure;

[0024] FIG. 2A illustrates a flowchart of a method of producing an exemplary oral vaccine for Hepatitis B, consistent with one or more exemplary embodiments of the present disclosure;

[0025] FIG. 2B illustrates a flowchart of a method of producing an exemplary oral vaccine for Hepatitis B using a plasmid vector, consistent with one or more exemplary embodiments of the present disclosure;

[0026] FIG. 2C illustrates a flowchart of a method of loading a plurality of exemplary Surface antigens of Hepatitis B into an exemplary plurality of transposon vectors, consistent with one or more exemplary embodiments of the present disclosure;

[0027] FIG. 2D illustrates a flowchart of a method for loading an exemplary plurality of Hepatitis B Surface antigen delivery vectors into a plurality of plasmid vectors, consistent with one or more exemplary embodiments of the present disclosure;

[0028] 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;

[0029] FIG. 4 illustrates a fluorescent image of an exemplary modified Arthrospira platensis, consistent with one or more exemplary embodiments of the present disclosure;

[0030] FIG. 5 illustrates an image of expression of an exemplary Hepatitis B Surface protein in 4 replicates of transgenics and negative control by Western blot method, consistent with one or more exemplary embodiments of the present disclosure;

[0031] FIG. 6 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;

[0032] FIG. 7 illustrates an image of comparison of mean and standard divination of Hepatitis B Surface Ag titers, consistent with one or more exemplary embodiments of the present disclosure; and

[0033] FIG. 8 illustrates animal tests for an exemplary ice cream containing algae-coated expressing HBsAg as a vaccine in ice cream, consistent with one or more exemplary embodiments of the present disclosure.DESCRIPTION OF EMBODIMENTS

[0035] 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.

[0036] 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.

[0009] Hepatitis B is a widespread disease which may require a vaccine for prevention and protection against Hepatitis B virus. In one general aspect of the present disclosure, an oral vaccine may be produced for Hepatitis B. In an exemplary embodiment, an exemplary oral vaccine may include a delivery platform and a plurality of Hepatitis B antigen delivery vectors. In an exemplary embodiment, an exemplary plurality of Hepatitis B antigen delivery vectors may include a plurality of Hepatitis B surface antigen delivery vectors. In anexemplary embodiment, an exemplary surface antigen may stimulate immune system to produce antibodies, providing protection against future infections. 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. In an exemplary embodiment, an exemplary plurality of Hepatitis B Surface 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 1015. 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 Hepatitis B Surface antigen delivery vector may include at least one Hepatitis B Surface antigen. In an exemplary embodiment, a weight ratio of an exemplary delivery platform to an exemplary Hepatitis B antigen may be in a range of 1: 10’4to 1: 2xl0-3(delivery platform: Hepatitis B antigen). In an exemplary embodiment, a weight ratio of an exemplary delivery platform to an exemplary Hepatitis B Surface antigen may be in a range of 1 : 10’4to 1 : 2x 10’3(delivery platform: Hepatitis B Surface antigen). In an exemplary embodiment, each respective Hepatitis B Surface antigen delivery vector of an exemplary plurality of Hepatitis B Surface antigen delivery vectors may include a nucleotide sequence identical to nucleotide sequence of SEQ ID NO. 2. In an exemplary embodiment, Hepatitis B vaccination using an exemplary oral vaccine may require a booster for prolonging immunity to Hepatitis B. In an exemplary embodiment, an exemplary oral vaccine may be used as a booster for prolonging immunity to Hepatitis B.

[0037] 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 Hepatitis B 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, spiralshaped, 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 nutrientcontent. 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 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 be environmentally 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 platensisA 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 Hepatitis B 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 platensisA properties of producing a specific protein in abundance may increase expression of an exemplary Hepatitis B Surface antigen while an exemplary Hepatitis B Surface antigen may be coupled to an exemplary host genome of Arthrospira platensis.

[0038] In an exemplary embodiment, each of an exemplary Hepatitis B Surface antigen delivery vector of an exemplary plurality of Hepatitis B Surface antigen delivery vectors may include one EcoRI restriction site, one Left ME sequence, one C-Phycocyanin operon, one Right ME sequence, and one Hindlll restriction site. In an exemplary embodiment, an exemplary C-Phycocyanin operon may include one specific promoter of C-Phycocyanin, one Ribosomal Binding Site (RBS) sequence, at least one Hepatitis B Surface 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 C-Phycocyanin operon may include a distance between an exemplary Hepatitis B Surface antigen and an exemplary Green FlorenceProtein (GFP). In an exemplary embodiment, an exemplary Hepatitis B Surface 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 Hindlll 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 C-Phycocyanin may include a nucleotide sequence identical to nucleotide sequence of SEQ ID NO. 9. In an exemplary embodiment, an exemplary 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 Hepatitis B Surface antigen sequence. In an exemplary embodiment, an exemplary histidine nucleotide sequence may be incorporated for purpose of an exemplary Hepatitis B Surface antigen purification. In an exemplary embodiment, an exemplary Surface antigen of Hepatitis B may include a Hepatitis B Surface Antigen with Receptor Binding Domain (Hepatitis B Surface - Ag). In an exemplary embodiment, an exemplary Surface Ag may refer to Surface protein of Hepatitis B virus. In an exemplary embodiment, an exemplary hepatitis B surface antigen may be a crucial protein found on surface of a hepatitis B virus, serving as a structural component and a primary diagnostic marker for hepatitis B infections. In an exemplary embodiment, high concentrations of hepatitis B surface antigen in blood and other bodily fluids may indicate potential infectiousness, allowing for identification of individuals currently infected with an exemplary hepatitis B virus. In an exemplary embodiment, an exemplary hepatitis B surface antigen may play a pivotal role in development of an exemplary oral vaccine of hepatitis B.

[0039] In an exemplary embodiment, each exemplary respective Hepatitis B Surface antigen delivery vector of an exemplary plurality of Hepatitis B Surface 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 Hepatitis B Surface antigen delivery vector of an exemplary plurality of Hepatitis B Surface antigen delivery vectors may include an exemplary at least one surface antigen of Hepatitis B with a nucleotide ratio of each exemplary respective Hepatitis B surface antigen delivery vector to an exemplary at least one Surface antigen of Hepatitis B in a range 10: 2 to 10: 6 (each exemplary Hepatitis B Surface antigen delivery vector: an exemplary Surface antigen of Hepatitis B).

[0040] 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 exemplary tablet 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 Hepatitis B Surface 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 an additive in a food. In an exemplary embodiment, an exemplary food may include at least one of yogurt, dessert, gum, ice cream, pastilles, and combinations thereof.

[0041] In an exemplary embodiment, an exemplary oral vaccine may be consumed for at least one portion for vaccination against Hepatitis B. 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 pg of an exemplary Hepatitis B Surface antigen. In an exemplary embodiment, an exemplary at least one portion may include at least 10 pg of an exemplary Hepatitis B Surface antigen. 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 aprophylactic food. In an exemplary embodiment, an exemplary food with incorporation of an exemplary oral vaccine may be a preventive food.

[0042] FIG. 1 illustrates a schematic view 100 of genetic components of a Hepatitis B Surface antigen delivery vector 102, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embodiment, Hepatitis B Surface antigen delivery vector 102 may include EcoRI restriction site 104, left ME sequence 106, C-Phycocyanin operon 108, Right ME sequence 110, and Hindlll 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, Hindlll restriction site 112 may include a nucleotide sequence identical to nucleotide sequence of SEQ ID NO. 8.

[0043] In an exemplary embodiment, C-Phycocyanin operon 108 may include specific promoter of C-Phycocyanin 114, Ribosomal Binding Site (RBS) sequence 116, at least one Hepatitis B Surface antigen 118, Green Florence Protein (GFP) 122, and terminator sequence 124. In an exemplary embodiment, C-Phycocyanin operon 108 may include a gene distance 120 between at least one Hepatitis B Surface antigen 118 and GFP 122. In an exemplary embodiment, Specific promoter of C-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, Hepatitis B Surface 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, Hepatitis B Surface antigen delivery vector 102 and an exemplary delivery platform may be used for producing an exemplary oral vaccine for Hepatitis B. In an exemplary embodiment, Hepatitis B Surface antigen delivery vector 102 and an exemplary Arthrospira platensis may be used for producing an exemplary oral vaccine for Hepatitis B. In an exemplary embodiment, an exemplary plurality of Hepatitis B Surface antigen delivery vector 102 may be coupled to an exemplary plurality of host genome of anexemplary Arthrospira platensis. In an exemplary embodiment, methods of producing an exemplary oral vaccine for Hepatitis B are illustrated in FIG. 2A and FIG. 2B.

[0044] FIG. 2A illustrates a flowchart of a method 200 of producing an exemplary oral vaccine for Hepatitis B, 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 Hepatitis B Surface antigen delivery vectors by loading a plurality of Surface antigens of Hepatitis B into a plurality of transposon vectors, a step 204 of activating the plurality of Hepatitis B Surface antigen delivery vectors by mixing the plurality of Hepatitis B Surface antigen delivery vectors with a activation enzyme solution, and a step 206 of transferring the plurality of activated Hepatitis B Surface antigen delivery vectors into a host genome of Arthrospira platensis by an electroporation technique.

[0045] In further detail with respect to step 202, step 202 of forming a plurality of Hepatitis B Surface antigen delivery vectors may include loading a plurality of antigens of Hepatitis B into a plurality of transposon vectors. In an exemplary embodiment, an exemplary Surface antigen of Hepatitis B may be isolated by at least a method of synthesizing an exemplary Surface antigen of Hepatitis B, digestion ligation method, and combinations thereof. In an exemplary embodiment, an exemplary Surface antigen of Hepatitis B may include a nucleotide sequence identical to nucleotide sequence of SEQ ID NO. 3. In an exemplary embodiment, loading an exemplary plurality of Surface antigens of Hepatitis B into an exemplary plurality of transposon vectors may be performed by method 230 illustrated in FIG. 2C herein below.

[0046] FIG. 2C illustrates a flowchart of method 230 of loading plurality of Surface antigens of Hepatitis B 118 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 Hepatitis B Surface antigens by at least a method of synthesizing an exemplary plurality of Hepatitis B Surface antigens, digestion ligation method, and combinations thereof, a step 234 of forming a first ligation reaction mixture by mixing the plurality of Hepatitis B Surface 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.

[0047] In further detail with respect to step 232, step 232 of isolating plurality of Hepatitis B Surface antigens 118 may include performing at least a method of synthesizing an exemplary plurality of Hepatitis B Surface antigens, digestion ligation method, and combinations thereof.In an exemplary embodiment, an exemplary plurality of Hepatitis B Surface 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, Hepatitis B Surface antigen 118 may include a nucleotide sequence identical to nucleotide sequence of SEQ ID NO. 3. In an exemplary embodiment, an exemplary plurality of Hepatitis B Surface 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 Hepatitis B virus at specific sites using enzymes. In an exemplary embodiment, an exemplary enzyme may be a restriction enzyme.

[0048] In further detail with respect to step 234, step 234 of forming a first ligation reaction mixture may include mixing plurality of Hepatitis B Surface antigens 118 with at least a first ligase enzyme, at least a first ligase buffer solution, and an exemplary plurality of transposon vectors together. In an exemplary embodiment, an exemplary plurality of Hepatitis B Surface 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 Hepatitis B Surface 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 Sad, Xbal, 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 Hepatitis B Surface antigens 118 and an exemplary plurality of transposon vectors to be joined together; thereby, resulting in forming an exemplary plurality of Hepatitis B Surface antigen delivery vector.

[0049] 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, anexemplary first ligation reaction mixture may be incubated for a time period in a range of 4 hours to 16 hours.

[0050] In an exemplary embodiment, loading plurality of Surface antigens of Hepatitis B 118 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 65°C to 80 °C for such inactivation purpose.

[0051] 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. coll). 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 1633 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, Hepatitis B Surface antigen 118 may be coupled to an exemplary transposon vector to form Hepatitis B Surface antigen delivery vector 102. In an exemplary embodiment, Hepatitis B Surface antigen delivery vector 102 may have a nucleotide sequence identical to nucleotide sequence of SEQ ID NO. 2. In an exemplary embodiment, each Hepatitis B Surface antigen delivery vector 102 of an exemplary plurality of Hepatitis B Surface antigen delivery vectors may include at least one Surface antigen of Hepatitis B 118. In an exemplary embodiment, a weight ratio of an exemplary delivery platform to Surface antigen of Hepatitis B 118 may be in a range of 1: 10’4to 1: 2xl0-3(delivery platform: Surface antigen of Hepatitis B). In an exemplary embodiment, Hepatitis B Surface antigen delivery vector 102 may include EcoRI cutting site 104, Left ME sequence 106, C-Phycocyanin operon 108, Right ME sequence 110, and Hindlll restriction site 112. In an exemplary embodiment, C-Phycocyanin operon 108 may include Specific promoter of C-Phycocyanin 114, Ribosomal Binding Site (RBS) sequence 116, at least one Hepatitis B Surface antigen 118, Green Florence Protein (GFP) 122, and Terminator sequence 124. In an exemplary embodiment, C- Phycocyanin operon 108 may include a gene distance 120 between at least one Hepatitis B Surface antigen 118 and GFP 122. In an exemplary embodiment, Specific promoter of C-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, Hepatitis B Surface antigen delivery vector 102 and an exemplary delivery platform may be used for producing an exemplary oral vaccine for Hepatitis B. In an exemplary embodiment, Hepatitis B Surface antigen delivery vector 102 and an exemplary Arthrospira platensis may be used for producing an exemplary oral vaccine for Hepatitis B Surface. In an exemplary embodiment, an exemplary plurality of Hepatitis B Surface 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 Surface antigen of Hepatitis B 118. In an exemplary embodiment, an exemplary histidine sequence may be incorporated for purpose of purifying Hepatitis B Surface antigen 118. In an exemplary embodiment, an exemplary histidine tag may have a nucleotide sequence identical to nucleotide sequence of SEQ ID NO. 14.

[0052] 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, Surface antigen of Hepatitis B 118 may be purified using IMAC.

[0053] Referring back to FIG. 2A, in further detail with respect to step 204, step 204 of activating plurality of Hepatitis B Surface antigen delivery vectors 102 may include mixing plurality of Hepatitis B Surface antigen delivery vectors 102 with an activation enzyme solution. In an exemplary embodiment, an exemplary activation enzyme solution may activateLeft ME sequence 106 and Right ME sequence 110. In an exemplary embodiment, activating plurality of Hepatitis B Surface antigen delivery vectors 102 may include mixing plurality of Hepatitis B Surface 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 Hepatitis B Surface antigen delivery vectors 102 may include mixing plurality of Hepatitis B Surface 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 Hepatitis B Surface antigen delivery vectors 102 may include mixing plurality of Hepatitis B Surface 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 Hepatitis B Surface antigen delivery vectors 102 may include mixing plurality of Hepatitis B Surface antigen delivery vectors 102 with an exemplary activation enzyme solution with a weight ratio of plurality of Hepatitis B Surface antigen delivery vectors 102 to an exemplary activation enzyme solution in a range of 8:2 to 6:8 (an exemplary plurality of Hepatitis B Surface 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 / pl to 4 U / pl.

[0054] In further detail with respect to step 206, step 206 of transferring an exemplary plurality of activated Hepatitis B Surface 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 Hepatitis B Surface 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 Hepatitis B Surface antigen delivery vectors. In an exemplary embodiment, for preparing an exemplary Arthrospira platensis suspension, an exemplary Arthrospira platensis may be suspended in afirst 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 Hepatitis B Surface 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 Hepatitis B Surface antigen delivery vectors in an exemplary second solution may have a weight ratio of an exemplary plurality of activated Hepatitis B Surface antigen delivery vectors to an exemplary second solution in a range of 1.0 pg / ml to 4.0 pg / ml (an exemplary plurality of activated Hepatitis B Surface 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 Hepatitis B Surface antigen delivery vectors 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 Hepatitis B Surface antigen delivery vectors in a range of 1:100 to 1:10 (suspension of Arthrospira platensis'. suspension of an exemplary plurality of activated Hepatitis B Surface 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 Hepatitis B Surface 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 platensisA cell membrane. In an exemplary embodiment, an exemplary electrical pulse may create pores in Arthrospira platensisA cell membrane. In an exemplary embodiment, during brief moment when an exemplary cell membrane is permeabilized, an exemplary plurality of activated Hepatitis B Surface antigen delivery vectors may enter an exemplary Arthrospira platensisA cell through exemplary pores. In an exemplary embodiment, once an exemplary electrical field is removed, an exemplary Arthrospira platensisA cell membrane may reseal. In an exemplary embodiment, transferring an exemplary plurality of activated Hepatitis B Surface antigendelivery vectors into a host genome of Arthrospira platensis may include applying a voltage to an exemplary Arthrospira platensis in a range of 1 KV / cm to 8 KV / cm. In an exemplary embodiment, transferring an exemplary plurality of activated Hepatitis B Surface 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.

[0055] 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 Hepatitis B 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 Hepatitis B Surface antigen delivery vectors by loading a plurality of Surface antigens of Hepatitis B into a plurality of transposon vectors, a step 214 of forming a plurality of hybrid vectors by loading the plurality of Hepatitis B Surface antigen delivery vectors into a plurality of vectors, a step 216 of separating the plurality of Hepatitis B Surface 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 Hepatitis B Surface antigen delivery vectors by mixing the separated plurality of Hepatitis B Surface antigen delivery vectors with an activation enzyme solution, and a step 220 of transferring the plurality of activated Hepatitis B Surface antigen delivery vectors into a plurality of host genomes of Arthrospira platensis by an electroporation technique.

[0056] In further detail with respect to step 212, step 212 of forming a plurality of Hepatitis B Surface antigen delivery vectors may include loading a plurality of Surface antigens of Hepatitis B 118 into a plurality of transposon vectors. In an exemplary embodiment, antigen of Hepatitis B 118 may be isolated by at least a method of synthesizing Surface antigen of Hepatitis B 118, digestion ligation, and combinations thereof. In an exemplary embodiment, Surface antigen of Hepatitis B 118 may include a nucleotide sequence identical to nucleotide sequence of SEQ ID NO. 3. In an exemplary embodiment, loading an exemplary plurality of Surface antigens of Hepatitis B 118 into an exemplary plurality of transposon vectors may be performed by a method 230 illustrated in FIG. 2C herein below.

[0057] FIG. 2C illustrates a flowchart of method 230 of loading plurality of Surface antigens of Hepatitis B 118 into the plurality of transposon vectors, consistent with one or moreexemplary embodiments of the present disclosure. In an exemplary embodiment, method 230 may include a step 232 of isolating the plurality of Hepatitis B Surface antigens by at least a method of synthesizing Surface antigen of Hepatitis B Surface 118, digestion ligation, and combinations thereof, a step 234 of forming a ligation reaction mixture by mixing the plurality of Hepatitis B Surface 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.

[0058] In further detail with respect to step 232, step 232 of isolating plurality of Hepatitis B Surface antigens 118 may include using at least a method of synthesizing surface antigen of Hepatitis B 118, digestion ligation, and combinations thereof. In an exemplary embodiment, an exemplary plurality of Hepatitis B Surface 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, Hepatitis B Surface antigen 118 may include a nucleotide sequence identical to nucleotide sequence of SEQ ID NO. 3. In an exemplary embodiment, an exemplary plurality of Hepatitis B Surface 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 Hepatitis B virus at specific sites using enzymes. In an exemplary embodiment, an exemplary enzyme may be a restriction enzyme.

[0059] In further detail with respect to step 234, step 234 of forming a first ligation reaction mixture may include mixing plurality of Hepatitis B Surface 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 Hepatitis B Surface 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 Hepatitis B Surface 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 Sad, Xbal, 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 allowplurality of Hepatitis B Surface antigens 118 and an exemplary transposon vector be joined together forming an exemplary plurality of Hepatitis B Surface antigen delivery vector.

[0060] 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 first ligation reaction mixture may be incubated for a time period in a range of 4 hours to 16 hours.

[0061] In an exemplary embodiment, loading plurality of Surface antigens of Hepatitis B 118 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 65°C to 80 °C.

[0062] 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. coll). 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 1633 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, Hepatitis B Surface antigen 118 may be coupled to an exemplary transposon vector to form Hepatitis B Surface antigen delivery vector 102. In an exemplary embodiment, Hepatitis B Surface antigen delivery vector 102 may have a nucleotide sequence identical to nucleotide sequence of SEQ ID NO. 1. In an exemplary embodiment, each Hepatitis B Surface antigen delivery vector 102 of an exemplary plurality of Hepatitis B Surface antigen delivery vectors may include at least one Surface antigen of Hepatitis B 118. In an exemplary embodiment, a weight ratio of an exemplary delivery platform to Hepatitis B Surface antigen 118 may be in a range of 1: 10’4to 1: 2xl0-3(delivery platform: Hepatitis B Surface antigen). In an exemplary embodiment, an exemplary Hepatitis B Surface antigen delivery vector may include EcoRI cuttinzg site 104, Left ME sequence 106, C-Phycocyaninoperon 108, Right ME sequence 110, and Hindlll restriction site 112. In an exemplary embodiment, C-Phycocyanin operon 108 may include Specific promoter of C-Phycocyanin 114, Ribosomal Binding Site (RBS) sequence 116, at least one Hepatitis B Surface antigen 118, Green Florence Protein (GFP) 122, and Terminator sequence 124. In an exemplary embodiment, C-Phycocyanin operon 108 may include a gene distance 120 between at least one Hepatitis B Surface antigen 118 and GFP 122. In an exemplary embodiment, Specific promoter of C-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, Hepatitis B Surface antigen delivery vector 102 and an exemplary delivery platform may be used for producing an exemplary oral vaccine for Hepatitis B. In an exemplary embodiment, Hepatitis B Surface antigen delivery vector 102 and an exemplary Arthrospira platensis may be used for producing an exemplary oral vaccine for Hepatitis B. In an exemplary embodiment, an exemplary plurality of Hepatitis B Surface antigen delivery 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 Surface antigen of Hepatitis B 118. In an exemplary embodiment, an exemplary histidine sequence may be incorporated for purpose of purifying Hepatitis B Surface antigen 118. In an exemplary embodiment, an exemplary histidine tag may have a nucleotide sequence identical to nucleotide sequence of SEQ ID NO. 14.

[0063] 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 may 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 mixtureof cellular proteins. In an exemplary embodiment, antigen of Hepatitis B 118 may be purified using IMAC.

[0064] 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 Hepatitis B Surface antigen delivery vectors 102 into a plurality of vectors. In an exemplary embodiment, loading an exemplary plurality of Hepatitis B Surface antigen delivery vectors 102 into an exemplary plurality of vectors may be performed using method 240 illustrated in FIG. 2D.

[0065] FIG. 2D illustrates a flowchart of method 240 for loading an exemplary plurality of Hepatitis B Surface antigen delivery vectorsl02 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 Hepatitis B Surface 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.

[0066] In further detail with respect to step 242, step 242 of forming an exemplary second ligation reaction mixture may include mixing plurality of Hepatitis B Surface 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 in a mixer. In an exemplary embodiment, plurality of Hepatitis B Surface 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 Hepatitis B Surface 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 Sad, Xbal, 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 Hepatitis B Surface 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. Inan exemplary embodiment, forming an exemplary plurality of hybrid vectors may include loading plurality of Hepatitis B Surface 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 Hepatitis B Surface antigen delivery vector 102, preserving Hepatitis B Surface 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 bacterium. In an exemplary embodiment, an exemplary bacteria may be an E. coli bacterium.

[0067] 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. coll). In an exemplary embodiment, an exemplary pBluescript 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 Hepatitis B Surface antigen delivery vector 102.

[0068] 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 Hepatitis B Surface antigen 118 that may be included in an exemplary transposon vector. In an exemplary embodiment, Hepatitis B Surface antigen delivery vector 102 incorporating Hepatitis B Surface antigen 118 may be expressed within an exemplary host organism, allowing for production of a larger quantity of an exemplary Hepatitis B Surface antigen 118. In an exemplary embodiment, Secondly, usingan exemplary plasmid vector incorporating an exemplary transposon vector may serve as a means of preserving an exemplary Hepatitis B Surface antigen delivery vector 102 for an extended period of time.

[0069] 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, Hepatitis B Surface antigen delivery vector 102 may be replicated by a polymerase chain reaction (PCR) method. In an exemplary embodiment, an exemplary Hepatitis B Surface antigen delivery vector 102 may be replicated using at least one primer. In an exemplary embodiment, replicating Hepatitis B Surface antigen delivery vector 102 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 Hepatitis B Surface antigen delivery vector 102. In an exemplary embodiment, an exemplary PCR method may be employed to amplify an exemplary Hepatitis B Surface 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 Hepatitis B Surface antigen delivery vector 102 that carry Hepatitis B Surface antigen 118 may be replicated. In an exemplary embodiment, an exemplary primer 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 nucleotide sequence of SEQ ID NO. 16.

[0070] In an exemplary embodiment, an exemplary PCR method may include a series of temperature-dependent steps. In an exemplary embodiment, for multiplying an exemplary Surface antigen of Hepatitis B 118 using an exemplary PCR method, an exemplary primer, an exemplary host organism, and an exemplary Surface 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 Surface 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 anexemplary 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 temperaturedependent 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 Surface antigen of Hepatitis B 118, which may be used for an exemplary oral vaccine.

[0071] Referring back to FIG. 2B, in further detail with respect to step 216, step 216 of separating an exemplary plurality of Hepatitis B Surface 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 enzyme solution 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 Hindlll solution, and combinations thereof. In an exemplary embodiment, an exemplary EcoRI solution and an exemplary Hindlll solution may include two respective restriction enzymes that may cut an exemplary plurality of Hepatitis B Surface antigen delivery vectors 102 at specific recognition sites. In an exemplary embodiment, exemplary recognition sites may be EcoRI restriction site 104 and Hindlll restriction site 112. In an exemplary embodiment, an exemplary plurality of Hepatitis B Surface antigen delivery vectors 102 may be separated from an exemplary plurality of hybrid vectors at EcoRI restriction site 104 and Hindlll restriction site 112. In an exemplary embodiment, a concentration of an exemplary separation enzyme solutionmay be in a range of 10 U / |il to 50 U / |il. In an exemplary embodiment, separating an exemplary plurality of Hepatitis B surface 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).

[0072] In further detail with respect to step 218, step 218 of activating an exemplary plurality of Hepatitis B Surface antigen delivery vectors 102 may include mixing an exemplary plurality of Hepatitis B Surface 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 Hepatitis B Surface antigen delivery vectors 102 may include mixing an exemplary plurality of Hepatitis B Surface 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 Hepatitis B Surface antigen delivery vectors 102 may include mixing an exemplary plurality of Hepatitis B Surface 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 Hepatitis B Surface antigen delivery vectors 102 may include mixing an exemplary plurality of Hepatitis B Surface antigen delivery vectors 102 with an exemplary activation enzyme solution with a weight ratio of an exemplary plurality of Hepatitis B Surface antigen delivery vectors 102 to an exemplary activation enzyme solution in a range of 8:2 to 6:8 (an exemplary plurality of Hepatitis B Surface 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 / pl to 4 U / pl. 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.

[0073] In further detail with respect to step 220, step 220 of transferring an exemplary plurality of activated Hepatitis B Surface antigen delivery vectors into a host genome of Arthrospiraplatensis may include electroporating Arthrospira platensis in the presence of activated Hepatitis B Surface antigen delivery vectors. In an exemplary embodiment, electroporating Arthrospira platensis in the presence of activated Hepatitis B Surface antigen delivery vectors may include forming a suspension of Arthrospira platensis, forming a suspension of an exemplary plurality of activated Hepatitis B Surface antigen delivery vectors, mixing an exemplary suspension of Arthrospira platensis with an exemplary suspension of an exemplary plurality of activated Hepatitis B Surface 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 Hepatitis B Surface 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 Hepatitis B Surface antigen delivery vectors.

[0074] 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 of 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 Hepatitis B Surface 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 Hepatitis B Surface antigen delivery vectors in an exemplary second solution may have a concentration in a range of 1.0 pg / ml to 4.0 pg / ml. In an exemplary embodiment, an exemplary suspension of Arthrospira platensis and an exemplary suspension of an exemplary plurality of activated Hepatitis B Surface 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 Hepatitis B Surface antigen delivery vectors 102 in a range of 1:100 to 1:10 (suspension of Arthrospira platensis', suspension of an exemplary plurality of activated Hepatitis B Surface 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 Hepatitis B Surface antigen delivery vectors 102 may be placed in an electroporation cuvette or chamber. In an exemplaryembodiment, 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 Hepatitis B Surface 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 Hepatitis B Surface 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 Hepatitis B Surface 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 1 KV / cm to 8 KV / cm. In an exemplary embodiment, transferring an exemplary plurality of activated Hepatitis B Surface 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 Hepatitis B Surface 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 1 KV / cm to 8 KV / cm. In an exemplary embodiment, transferring an exemplary plurality of activated Hepatitis B Surface 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.

[0075] Example 1: Transferring an exemplary Hepatitis B Surface antigen delivery vector into a host genome

[0076] An exemplary Hepatitis B Surface 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 pl of DOTAP Liposomal Transfection Reagent was mixed with a transposon. The complex of Roch and transposon was mixed with 40 pl 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 1 KV / cm to 8 KV / cm, at 1000 Q and 25 pF for 5.0 ms. Transgenic cells' accumulation was observed after 3 weeks of incubation at 30°C

[0077] Example 2: Replicating an exemplary Hepatitis B Surface antigen delivery vector

[0078] An exemplary Hepatitis B Surface 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 CaCh method of Sambrook (according to Sambrook’s book, specifically the 1989 edition). Competent cell Preparation using CaC12 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 CaC12 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) CaC12 solution and 20% glycerol. Competent cells were stored in the freezer until use the final competent cells were kept at -80 °C until use. In the next step, melting component was mixed with 5 pl 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 pl 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 with Ampicillin 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 Hepatitis B Surface 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 Hepatitis B Surface 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.

[0079] 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 Hepatitis B Surface antigen delivery vector, which can be used for producing an exemplary oral vaccine.

[0080] Example 3: Activating an exemplary Hepatitis B Surface antigen delivery vector

[0081] In order to activate an exemplary Hepatitis B Surface antigen delivery vector, known as the Tn5 transposon, within an in vivo environment, a transposase enzyme was employed. To achieve this, 4 pl of transposase and 2 pl of pure glycerol were added to 2 pl of transposon DNA without Mg2+. The mixture was thoroughly vortexed and allowed to incubate at room temperature (25 °C) 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.

[0082] Example 4: Analyzing protein expression

[0083] 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 pg of the lysates were loaded onto a 12% SDS-PAGE gel. The separated proteins were transferred onto a 0.2 pm 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), 2500 bp (314), 3000 bp (316), 4000 bp (318), 5000 bp (320), 6000 bp (322), 8000 bp (324), 12000 bp (326).

[0084] 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.

[0085] Example 5: Analyzing expression of Hepatitis B antibody in animals

[0086] 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:

[0087] Table 1. Recommended vaccine doses for oral administration in an Arthrospira platensis-based dietary supplement.Dose of Ag Name CodeControl sample Negative control (C‘) Hl(Arthrospira platensis)30 pg 1 dose 11280 pg 2 doses H3150 pg 3 doses H410 pg A Hepatitis B vaccine (C+) H5

[0088] As shown in Table 1, Hl is a control sample without transgenic Arthrospira platensis, H2 is a sample using 30 pg of Surface antigen of Hepatitis B, H3 is a sample using 80 pg of Surface antigen of Hepatitis B, H4 is a sample using 150 pg of Surface antigen of Hepatitis B, and H5 is a sample that used 10 pg of Surface antigen of Hepatitis B via injection.

[0089] FIG. 5 illustrates an image 500 of expression of an exemplary Hepatitis B Surface 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 Hepatitis B Surface Ag cultured under white light condition and 508 and 510 are two repeats of transgenic Spirulina including Hepatitis B Surface Ag cultured under blue light condition.

[0090] The summary of the findings for the humoral immune response to different doses of the oral Hepatitis B subunit vaccine is as follows:

[0091] Significant differences have been demonstrated in the negative control group and positive control group with respect to all oral doses. There is practically no significant difference between doses of 30 and 80 micrograms. The dose of 150 micrograms has shown the highest level of antibody expression in the oral form, and although it still differs significantly from the positive control (injectable vaccine), the expression level is close.

[0092] The significance of the difference in HBsAb expression is clearly evident in FIG. 6 and FIG. 7. FIG. 6 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. An equation of the measured line is y=0.0089X+0.0485 with R2= 0.9991.

[0093] FIG. 7 illustrates an image of comparison of mean and standard divination of Hepatitis B Surface Ag titers, consistent with one or more exemplary embodiments of the present disclosure.

[0094] Table 2 illustrates examining changes in various factors (excluding Ab) under the influence of vaccination between the control group and treatments.Studied factors interpretation Sig.Significant difference withALT(U / L)nesat™contro1°"ly Wlth0 007 injectable vaccine (positive control).Significant difference withAST(U / L) negative control only with the 0.027150|iig oral vaccine dose.No significant difference wasALP(U / L) observed between any of the groups. No significant difference wasUrea(mg / dl) observed between any of the groups. No significant difference wasCr(mg / dl) observed between any of the groups.Significant difference with„ _z, negative control only with the „n_ .T.P(g / dl) ° , 0.035150pg oral vaccine dose. Difference compared to others.No significant difference wasAlb(mg / dl) observed between any of the groups.No significant difference wasGGT(U / L) observed between any of the groupsNegative control does not have , ... a significant difference withGlucose(mg / dl) .,” any of the groups. Difference compared to others.

[0095] The result of comparing the mean of all studied factors in the animal test is performed by the one-way Analysis of Variance (ANOVA) method. ANOVA is a statistical method used to assess whether there are any statistically significant differences between the means of three or more independent (unrelated) groups. It is an extension of the t-test, which is used for comparing two groups.

[0096] Table 3. Shows the result of comparing the mean of all studied factors in the animal test using the one-way ANOVA method.. . . . . 1 j 33333 | .49171 j .513 j -.7623 j 1.4289 2 | -1,00000 ) .49171 ,069 -2,0956 | ,0956 j 3 j -1-20000* | .49171 [ .035 | -2.2956 ] -.10445 | -.43333 .49171 .399 | -1.5289 .6623 ! 1 | .76667 | .49171 [ .150 j -.3289 | 1.8623 | 2 -.56667 .49171 1 .276 -1.6623 ! .5289 . t . . 1. . i 3 i -.76667 .49171 .150 i -1.8623 .3289i4 .43333 .49171 .399 -.6623 1.52892 36667 34833 317 4095 1.14283 46667 34833 210 3095 1.24284 .33333 .34833 .361 -.4428 1.10955 .63333 .34833 .099 -.1428 1.40951 -.36667 .34833 .317 1.1428 .40953.10000 .34833 .780 -.6761 .87614 | -.03333 .34833 .926 | -.8095 .7428 5 | .26667 .34833 .462 | -.5095 1.04281 | -.46667 .34833 .210 | -1.2428 .3095 !2 -.10000 .34833 .780 -.8761 .67614 -.13333 .34833 .710 -.9095 .64285 .16667 .34833 .643 -.6095 .94281 -.33333 .34833 .361 -1.1095 .4428 2 | .03333 .34833 .926 | -.7428 .80953 | .13333 .34833 .710 | -.6428 .90955.30000.34833.409-.47611.0761

[0097] Example 6: Preparing a therapeutic ice cream containing an exemplary oral vaccine of Hepatitis B

[0098] First, 78% pasteurized milk, 8.8% sugar, 0.4% vanilla, 0.8 teaspoons, and 7% dry milk were mixed, then pasteurized and cooled. Then C-PC protein with a concentration of 20 mg per 100 cc of milk was added to the mixture, and ice cream was prepared. Finally, the ice cream samples were stored at a temperature of -20 °C.

[0099] FIG. 8 illustrates animal tests for an exemplary ice cream containing algae-coated expressing HBsAg as a vaccine in ice cream, consistent with one or more exemplary embodiments of the present disclosure. Group 1 (802): Negative control (plain ice cream). Group 2 (804): Ice cream vaccine treatment - approximately 500 milligrams of algae-coated with about 80 micrograms of antigen in the ice cream. Group 3 (806): Conventional vaccine in injectable form - 10 micrograms. Group 4 (808): Algae-coated oral vaccine alone - antigen dose approximately 80 micrograms. Group 5 (810): Algae-coated oral vaccine alone - antigen dose approximately 150 micrograms. The negative control shows a significant difference at a 95% confidence level across all groups.

[0100] The ice cream group containing approximately 500 milligrams of algae-coated with 80 micrograms of HBsAg (Ice Cream Vac. 80pg) has a significant difference compared to its equivalent group in the form of an algae-coated oral vaccine without combination with ice cream (Oral Vac. 80pg) (P=0.536). Even the average of the ice cream group containing the vaccine 4U is higher than the oral vaccine above. In other words, the consumption of the hepatitis B vaccine derived from spirulina-coated algae in ice cream does not result in a reduction in the stimulatory effect of Ag on the stimulation and expression of Ab.

[0101] Table 4. Shows results of the test using therapeutic ice cream.*. The mean difference is significant at the 0.05 level.

[0103] Industrial Applicability

[0104] Industrial applicability of an oral vaccine for hepatitis B using Arthrospira platensis as a carrier component holds significant promise, particularly when incorporated into various food products. Arthrospira platensis, a nutrient-rich cyanobacterium, can serve as an effective carrier for oral vaccines. This approach offers several advantages, including ease of incorporation into different food matrices. By integrating the hepatitis B vaccine into popular consumables such as yogurt, dessert, gum, ice cream, and pastilles, an exemplary vaccine becomes more accessible and acceptable to diverse populations. Versatility of Arthrospira platensis allows for seamless integration of an exemplary vaccine without compromising stability or efficacy of immunization. The present disclosure delivery system not only enhancesthe convenience of vaccine administration but also addresses challenges associated with traditional injection-based methods. The industrial utilization of Arthrospira platensis as a carrier for oral hepatitis B vaccines thus represents a promising avenue for expanding immunization coverage and mitigating burden of hepatitis B viral infection.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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 orimplying 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 nonexclusive 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.

[0110] 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.

[0111] 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 in combination 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 Hepatitis B, 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 Hepatitis B antigen delivery vectors coupled to the plurality of host genomes, each Hepatitis B antigen delivery vector of the plurality of Hepatitis B surface antigen delivery vectors has a nucleotide sequence identical to nucleotide sequence of SEQ ID NO. 2, each respective Hepatitis B antigen delivery vector comprising at least one antigen of Hepatitis B with a weight ratio of the delivery platform to the at least one antigen of Hepatitis B in a range of 1: 10’4to 1: 2xl0-3(delivery platform: at least one antigen of Hepatitis B).

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

3. The oral vaccine of claim 1, wherein each respective Hepatitis B antigen comprises a Hepatitis B surface antigen.

4. The oral vaccine of claim 1 , wherein each respective Hepatitis B antigen delivery vector of the plurality of Hepatitis B antigen delivery vectors comprises the at least one antigen of Hepatitis B with a nucleotide ratio of each respective Hepatitis B antigen delivery vector to the at least one antigen of Hepatitis B in a range of 10: 2 to 10: 6 (each Hepatitis B antigen delivery vector: the at least one antigen of Hepatitis B).

5. The oral vaccine of claim 1, wherein the at least one antigen of Hepatitis B has a nucleotide sequence identical to nucleotide sequence of SEQ ID NO. 3.

6. 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.

7. 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.

8. The oral vaccine of claim 1, wherein the oral vaccine comprises at least 10 pg of the at least one antigen of Hepatitis B .

9. A method of preparing an oral vaccine for Hepatitis B, the method comprising: forming a plurality of Hepatitis B surface antigen delivery vectors by loading a plurality of surface antigens of Hepatitis B into a plurality of transposon vectors; forming a plurality of hybrid vectors by loading the plurality of Hepatitis B surface antigen delivery vectors into a plurality of vectors; separating the plurality of Hepatitis B surface 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 Hepatitis B surface antigen delivery vectors by mixing the separated plurality of Hepatitis B surface antigen delivery vectors with an activation enzyme solution; andtransferring the plurality of activated Hepatitis B surface antigen delivery vectors into a plurality of host genomes of Arthrospira platensis by an electroporation technique.

10. The method of claim 9, 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 3 °C to 18°C for a time period in a range of 1 hour to 14 hours.

11. The method of claim 9, wherein forming the plurality of hybrid vectors comprises loading the plurality of Hepatitis B surface antigen delivery vectors into a plurality of plasmid vectors.

12. The method of claim 9, wherein forming the plurality of hybrid vectors comprises loading the plurality of Hepatitis B surface antigen delivery vectors into a plurality of pBluescript II SK(+) vectors with a nucleotide sequence identical to nucleotide sequence of SEQ ID NO. 4.

13. The method of claim 9, wherein separating the plurality of Hepatitis B surface 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).

14. The method of claim 13, 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 / pl to 50 U / pl.

15. The method of claim 9, wherein separating the plurality of Hepatitis B surface antigen delivery vectors from the plurality of hybrid vectors comprises mixing the plurality of hybrid vectors with at least one of an EcoRI solution, a Hindlll solution in a buffered aqueous glycerol solution, and combinations thereof for a time period in a range of 1 hour to 16 hours.

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

17. The method of claim 16, wherein loading the plurality of Hepatitis B surface antigens into the plurality of transposon vectors comprises: isolating the plurality of Hepatitis B surface antigens by a method comprising at least one of synthesizing the plurality of Hepatitis B surface antigens, digestion ligation, and combinations thereof;forming a ligation reaction mixture by mixing the plurality of isolated Hepatitis B surface antigens, at least a ligase enzyme, at least a ligase buffer solution, and the plurality of transposon vectors 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.

18. The method of claim 16, wherein activating the plurality of Hepatitis B surface antigen delivery vectors comprises mixing the plurality of Hepatitis B surface antigen delivery vectors with the activation enzyme solution with a volume ratio of the plurality of Hepatitis B surface antigen delivery vectors to the activation enzyme solution in a range of 8:2 to 6:8 (the plurality of Hepatitis B surface antigen delivery vectors: the activation enzyme solution), the activation enzyme solution comprises an activation enzyme dissolved in water thereof with a concentration of the activation enzyme solution in a range of 1 U / pl to 4 U / pl.

19. The method of claim 16, wherein activating the plurality of Hepatitis B surface antigen delivery vectors comprises mixing the plurality of Hepatitis B surface antigen delivery vectors with at least one of a DNA transposon solution, a Transposase solution, and combinations thereof.

20. The method of claim 16, wherein transferring the plurality of activated Hepatitis B surface 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 8 KV / cm for a time period in a range of 0.5 ms to 2 ms.

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