DESC:DESCRIPTION:
Field of the invention:
[0001] The present disclosure generally relates to the technical field of vaccine delivery and, in particular, relates to probiotic vaccines for oral delivery using lateral gene transfer (LGT) to all age groups with low to negligible side effects.
Background of the invention:
[0002] Vaccines are one of the most effective public health interventions ever developed, preventing millions of deaths from infectious diseases each year. However, traditional vaccines have several limitations. First, they are typically delivered through multiple painful injections. This can be invasive and can deter people, especially young children, from getting vaccinated. Second, traditional vaccines may not always be effective in boosting immunity, which can require multiple booster shots. Third, traditional vaccines may be associated with side effects, such as skin allergies and fever.

[0003] Traditional vaccines are typically delivered through multiple painful injections. This can be invasive and can deter people, especially young children, from getting vaccinated. Traditional vaccines may not always be effective in boosting immunity, which can require multiple booster shots. Traditional vaccines may be associated with side effects, such as skin allergies and fever.

[0004] Traditional therapeutic agents are pharmacological medications and treatments that are derived from nature or synthesized to treat a wide range of diseases and medical problems in living beings. Furthermore, therapeutic chemicals can be injected into an organism to prevent the emergence of a disease or medical condition, such as immunization. Drug distribution while there are several strategies for delivering medicinal chemicals to living creatures, the majority have substantial drawbacks. Furthermore, more advanced ways for delivering larger, more complicated molecules are required. Existing, delivery methods has severe limitations and do not provide all of the attributes of an optimum therapeutic agent delivery vehicle.

[0005] In existing technology, a mucin comprising vehicle for the transport of biologically-active agents is known. The biologically-active agents comprise a mucin that is present in a natural or modified form. The mucin enhances the transport of biologically active agents, such as therapeutic agents into living organisms, improves the delivery of biologically active agents to cells, tissues, organs or organelles, increases the level of specificity in targeting particular cells or cells types, and further enhance the activity of such therapeutic agents once they enter an organism.

[0006] The biologically-active agents are used for the delivery biochemical, therapeutic, clinical, or other applications in organisms and cells including delivery of DNA (deoxyribonucleic acid), polynucleotides and proteins into cells, tissues or organisms, gene delivery applications. However, the biologically-active agents that might infection children, elderly, and people with needle phobias. Moreover, the biologically-active agents that potentially damage the immune system while delivery the bio-logically agents.

[0007] Therefore, there is a need for probiotic vaccines for oral delivery using lateral gene transfer (LGT) to all age groups. There is also a need for probiotic vaccines that transfer antigens to other bacteria over time, which can help to boost vaccine immunity without the need for multiple booster shots. Further, there is also a need for probiotic vaccines that has a potential to deliver vaccines with low to negligible side effects.
Objectives of the invention:
[0008] The primary objective of the invention is to develop probiotic vaccines for oral delivery using lateral gene transfer (LGT) to all age groups.

[0009] Another objective of the invention is to develop probiotic vaccines that are beneficial for children, the elderly, and people with needle phobias.

[0010] The other objective of the invention is to develop probiotic vaccines that utilises lateral gene transfer (LGT) allows for targeted antigen delivery to specific gut microbiome bacteria, potentially enhancing immune response.

[0011] The other objective of the invention is to develop probiotic vaccines that adapted to deliver antigens from various pathogens for different diseases.

[0012] The other objective of the invention is to develop probiotic vaccines that utilises sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) techniques for analysing the protein expression.

[0013] Yet another objective of the invention is to develop probiotic vaccines that are delivered in a variety of food and beverage products, which makes them more palatable and appealing to patients.

[0014] Further objective of the invention is to develop probiotic vaccines that provide a delicious and painless way to deliver vaccines to all age groups with minimal to no side effects.
Summary of the invention:
[0015] The present disclosure proposes probiotic vaccines for oral delivery using lateral gene transfer and method thereof. The following presents a simplified summary in order to provide a basic understanding of some aspects of the claimed subject matter. This summary is not an extensive overview. It is not intended to identify key/critical elements or to delineate the scope of the claimed subject matter. Its sole purpose is to present some concepts in a simplified form as a prelude to the more detailed description that is presented later.

[0016] In order to overcome the above deficiencies of the prior art, the present disclosure is to solve the technical problem to provide probiotic vaccines for oral delivery using lateral gene transfer (LGT) to all age groups with low to negligible side effects.

[0017] According to an aspect, the invention discloses a method for formulating a probiotic vaccine. At one step, culturing donor bacterial cells comprising a plasmid containing a coronavirus spike protein gene. At another step, co-culturing the donor bacterial cells with recipient bacterial cells under conditions suitable for lateral gene transfer. At another step, selecting the recipient bacterial cells that acquired the plasmid containing the coronavirus spike protein gene. Further, at another step, formulating the selected recipient bacterial cells into the probiotic vaccine. The probiotic vaccine is formulated in a pharmaceutically acceptable carrier that delivers targeted spike protein expression in gut.

[0018] In one embodiment herein, the pharmaceutically acceptable carrier is suitable for oral administration. The oral administration is in the form of at least one of yogurt, fermented products and capsules, etc. In one embodiment herein, the co-culturing is performed for a period of time of 1 hour to 2 hours at temperature of 36° C to 38° C.

[0019] In one embodiment herein, the donor bacterial cells are Lactobacillus species. In one embodiment herein, the recipient bacterial cells are at least one of Escherichia coli (E. coli), helicobacter pylori (H. pylori) and gut microbiota-related organisms. In one embodiment herein, the co-culturing is performed in a liquid medium under anaerobic or microaerophilic conditions or similar conditions. In one embodiment herein, the selection for recipient bacterial cells that have acquired the plasmid is performed using a medium containing ampicillin including eosin methylene blue (EMB) medium including EMB broth and EMB agar.

[0020] In one embodiment herein, the presence of the coronavirus spike protein gene in the selected recipient bacterial cells is confirmed using at least one of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and gram staining. In one embodiment herein, the growth of E. coli and H. pylori cells on the EMB medium is confirmed by the formation of purple colored colonies or colonies with a green metallic sheen.

[0021] In one embodiment herein, the EMB broth and EMB agar plates are prepared by 4 to 6 grams of eosin methylene blue (EMB) powder is added with 250 ml of deionized water to form a first solution. Next, 4 to 6 grams of the EMB powder and 2 to 4 grams of bacteriological agar are added with 250 ml of deionized water to form a second solution. Next, the first solution and the second solution are stirred gently without exposing to direct light due to photosensitivity.

[0022] Next, the first solution and the second solution are autoclaved at a pressure of at least 15 lbs and at a temperature of 120° C to 122° C for a time period of at least 15 minutes. Next, the first solution and the second solution are cooled at a temperature of 45° C and 50 µg/mL of ampicillin is added to each solution. Finally, at least 25 ml of each solution is poured into sterile petri dishes and allowed to solidify each solution at a room temperature, and sterile packing the prepared media plates and storing at a temperature of 4° C.

[0023] Further, objects and advantages of the present invention will be apparent from a study of the following portion of the specification, the claims, and the attached drawings.
Detailed description of drawings:
[0024] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate an embodiment of the invention, and, together with the description, explain the principles of the invention.

[0025] FIG. 1 illustrates a flowchart of a method for formulating a probiotic vaccine, in accordance to an exemplary embodiment of the invention.

[0026] FIG. 2 illustrates a graphical representation of an expression of a coronavirus spike protein by SDS-PAGE (Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis), in accordance to an exemplary embodiment of the invention.

[0027] FIG. 3 illustrates a pictorial representation of bacterial culture plates before and after performing lateral gene transfer (LGT), in accordance to an exemplary embodiment of the invention.
Detailed invention disclosure:
[0028] Various embodiments of the present invention will be described in reference to the accompanying drawings. Wherever possible, same or similar reference numerals are used in the drawings and the description to refer to the same or like parts or steps.

[0029] The present disclosure has been made with a view towards solving the problem with the prior art described above, and it is an object of the present invention to provide probiotic vaccines for oral delivery using lateral gene transfer (LGT) to all age groups with low to negligible side effects.

[0030] According to another exemplary embodiment of the invention, FIG. 1 refers to a flowchart 100 of a method for formulating a probiotic vaccine. At step 102, cultures donor bacterial cells comprising a plasmid containing a coronavirus spike protein gene. At step 104, co-cultures the donor bacterial cells with recipient bacterial cells under conditions suitable for lateral gene transfer.

[0031] At step 106, selects the recipient bacterial cells that acquired the plasmid containing the coronavirus spike protein gene. Further, at step 108, formulates the selected recipient bacterial cells into the probiotic vaccine. The probiotic vaccine is formulated in a pharmaceutically acceptable carrier that delivers targeted spike protein expression in gut.

[0032] In one example embodiment herein, the pharmaceutically acceptable carrier is suitable for oral administration. The oral administration is in the form of at least one of yogurt, fermented products and capsules, etc. In another example embodiment herein, the co-culturing is performed for a time period of 1 hour to 2 hours at temperature of 36° C to 38° C. In one example embodiment herein, the delivery agents is usually a fermented food products such as kombucha, yogurt, aged/raw cheeses, sauerkraut, pickles, miso, tempeh, natto and kimchi.

[0033] According to another exemplary embodiment of the invention, FIG. 2 refers to a graphical representation 200 of a coronavirus spike protein by SDS-PAGE (Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis). In one embodiment herein, the analysing the quantified levels of spike protein expression in lactobacillus, E. coli, and H. pyorli. After performing the LGT, the spike protein expression was detected in both recipient strains (E. coli and H. pylori). This indicates successful transfer of the relevant gene from the donor (Lactobacillus).

[0034] In another embodiment herein, the expression levels varied among the strains, the lactobacillus shows the highest level of spike protein expression. Both E. coli (pink line) and H. pylori (blue line) exhibit less expression compared to Lactobacillus. The Lactobacillus expressed the spike protein at 1.09 times more than H. pylori and 1.7 times more than E. coli. The LGT can effectively transfer the coronavirus spike protein gene to other bacteria like E. coli and H. pylori. The donor strain (Lactobacillus) shows significantly higher expression efficiency compared to the recipients.

[0035] According to an exemplary embodiment of the invention, FIG. 3 refers to a pictorial representation 300 of bacterial culture plates before and after performing a lateral gene transfer (LGT). In one embodiment herein, the probiotic vaccines utilises the lateral gene transfer (LGT) allows for targeted antigen delivery to specific gut microbiome bacteria, potentially enhancing immune response. The probiotic vaccines that adapted to deliver antigens from various pathogens for different diseases.

[0036] In one embodiment herein, the LGT is capability of the Lactobacillus (donor) from the probiotic vaccine was tested by co-culturing them with both E. coli and H. pylori (recipients), respectively, and analyses the expression of the spike protein. The ampicillin-resistance gene present in the plasmid (containing spike protein) was used as a selectable marker to identify the cells with the plasmid after performing the LGT. The bacterial cultures were initially tested for ampicillin resistance before the LGT experiment, which showed that neither of the bacterial species possess ampicillin resistance without the plasmid.

[0037] The presence of the spike protein plasmids can only give ampicillin resistance to the bacteria. The final product for human usage will not include the ampicillin resistance gene in the plasmid since the gene is purely for laboratory screening purposes. The LGT co-culture experiments were performed twice to analyze the expression of spike protein in Lactobacillus, E. coli, and H. pylori. The co-cultures, when plated on 50 µg/ml ampicillin-containing LB agar or BHI blood agar plates, yielded colonies of both donors. In one embodiment herein, the pharmaceutically acceptable carrier is suitable for oral administration. The oral administration is in the form of at least one of yogurt, fermented products and capsules, etc. In one embodiment herein, the co-cultures is performed for a period of time of 1 hr to 2 hr at temperature of 36° C to 38° C.

[0038] Lactobacillus and the recipient (E. coli and H. pylori). However, the co-cultures were plated on 50 µg/ml ampicillin-containing EMB agar, only the recipient colonies were seen, as the EMB suppressed the growth of gram-positive Lactobacillus (donor). Both the recipients were confirmed as gram-negative bacteria using a gram staining, where the colonies were found to display a metallic green sheen (H. pylori) and a dark purple coloration (E. coli). The presence of a metallic green sheen and dark purple coloration of the colonies is a characteristic of the gram-negative bacteria when grown on an EMB agar plate.

[0039] The expression of coronavirus spike protein in the co-cultures was tested using SDS-PAGE. The protein bands obtained were pixel-quantified and represented as a horizontal line graph, with the standard deviations representing the error bars. The spike protein expression in the donor cells (Lactobacillus) was 1.09- and 1.7-fold higher compared to the recipients, H. pylori and E. coli, respectively. These results suggest that the recipients are capable of producing at least half, if not equal, amounts of coronavirus spike protein in vitro from LGT in co-cultures. We predict a similar trend in vivo and propose a model for the potential efficacy-boosting of probiotic vaccines through LGT.

[0040] In one embodiment herein, the preparation of the selective growth media. An EMB (eosin methylene blue) agar is selective for gram-negative bacteria against gram-positive bacteria. The EMB agar is useful in isolation and differentiation of the various gram-negative bacilli and enteric bacilli, which is generally known as coliforms and feculent coliforms respectively. Eosin-Y and methylene blue components of the EMB agar will inhibit the growth of gram positive bacteria. The growth of the gram negative bacterium on the EMB agar plate be corroborated by the formation of purple colour colonies or colonies with a green metallic sheen. The current protocol includes lactobacillus (gram positive bacteria), Escherichia coli (gram negative bacteria), and helicobacter pylori (gram negative bacteria).

[0041] The EMB broth is suggested for the isolation and differentiation of the gram negative enteric bacteria from lactobacillus in co-cultures and nonclinical specimens after the lateral gene transfer. The bacteria which ferment lactose in the medium form colour colonies, while those that do not ferment lactose appear as colourless colonies. In one embodiment herein, the EMB agar contains peptone, lactose, sucrose, and the dyes eosin Y and methylene blue; it is commonly used as both a selective and a differential medium. EMB agar is selective for gram-negative bacteria. The dye methylene blue in the medium inhibits the growth of gram-positive bacteria; small amounts of this dye effectively inhibit the growth of most gram-positive bacteria. Eosin is a dye that responds to changes in pH, working from colourless to black under acidic conditions. EMB agar medium contains lactose and sucrose, but not glucose, as energy sources.

[0042] In one embodiment herein, the preparation of the EMB broth and the EMB agar plate. At one step, take two clean sanitized 500 ml of glass storage bottles and fill each with at least 250 ml of deionised water. Next, weight 5.615 gr of EMB powder and combine with 250 ml of deionised water in the glass storage bottle. Weight of 5.615 gr of EMB powder and 3.75 gr of bacteriological agar and combine them in the glass storage bottle with at least 250 ml of deionised water to form a first solution.

[0043] Next, 4 to 6 grams of the EMB powder and 2 to 4 grams of bacteriological agar are added with at least 250 ml of deionized water to form a second solution. Next, the first solution and the second solution are stirred gently without exposing to direct light due to photosensitivity. Ensure that the media was not exposed to direct light throughout the preparations, as it is photosensitive. Gently swirl the solution to thoroughly combine it.

[0044] Next, the autoclave the obtained solution at 15 lb of pressure and 121° C for at least 15 min, and after chills the obtained solution to at a temperature of 45° C, adds 50 g/ml of ampicillin to the solution. Next, pours 25 ml of LB agar solution into each sterile petri dish and set aside to cool at a room temperature. Finally, at least 25 ml of each solution is poured into sterile petri dishes and allowing each solution to solidify at a room temperature, and sterile packing the prepared media plates and storing at a temperature of 4° C.

[0045] In one embodiment herein, the lateral gene transfer of genetic material between two species, typically via conjugation. The current approach employs lactobacillus (which carrying a spike protein plasmid) as the donor bacteria cells to assess the efficacy of lateral gene transfer to other gut microbiota related organisms such as Escherichia coli and Helicobacter pylori.

[0046] In one embodiment herein, the recipient bacterial cells is at least one of Escherichia coli (E. coli), helicobacter pylori (H. pylori), and gut microbiota-related organisms. In one embodiment herein, the co-cultures is performed in a liquid medium under anaerobic or microaerophilic conditions or similar conditions. In one embodiment herein, the selection for recipient bacterial cells that have acquired the plasmid is performed using a medium containing ampicillin including eosin methylene blue (EMB) medium including EMB broth and EMB agar.

[0047] In another embodiment herein, the preparation of the co-culture on LB agar. First, inoculate 100 µl of E. coli cells from the vial and incubate the culture tubes at a temperature of 37° C overnight. Ensure the chosen solution for supports growth of E. coli. Next, from a brain heart infusion (BHI) Blood Agar plate containing H. pylori colonies, pick a loopful of colonies and inoculate it into 5 ml of BHI broth (or appropriate medium) under microaerophilic conditions.

[0048] Incubate the inoculated broth in a microaerophilic box at a temperature of 37° C for 20 hr. Ensure the chosen medium supports H. pylori growth under microaerophilic conditions. Next, inoculate a loopful of Lactobacillus cells cultured from the probiotic vaccine prototype into 5 ml of MRS broth with ampicillin (50 mcg/ml) and incubate the culture tubes at a temperature of 37° C overnight. Ensure MRS broth is suitable for Lactobacillus growth.

[0049] After overnight incubation, mix equal volumes of the respective media broths containing each bacterial culture (5 ml each of H. pylori, E. coli, and Lactobacillus cultures). Incubate the mixed cultures in their respective optimal conditions (e.g., E. coli and H. pylori at a temperature of 37° C, Lactobacillus at a temperature of 37° C with anaerobic or microaerophilic conditions) for at least 3 hr. After incubation, prepare fresh sterile 15 ml culture tubes. To each tube, add 0.2 ml of the Lactobacillus culture (donor cells) and 0.2 ml of the respective recipient culture (H. pylori or E. coli) in separate tubes. Incubate the co-culture tubes at a temperature of 37° C for 1.5 hr.

[0050] After incubation, add 2 ml of the respective recipient cell broth (H. pylori broth for H. pylori co-culture and E. coli broth for E. coli co-culture) to each co-culture tube. Incubate the co-cultures for an additional 1.5 hr at a temperature of 37° C. Spread 200 µl of each co-culture on separate EMB Agar plates with ampicillin for H. pylori and E. coli. For H. pylori, also spread 200 µl on a BHI Agar plate with ampicillin.

[0051] For E. coli, spread 200 µl on an LB Agar plate with ampicillin. In one embodiment herein, the spread recipient cells (H. pylori and E. coli) not co-cultured on separate EMB agar plates with ampicillin. Incubate all plates under their respective optimal conditions (e.g., E. coli and H. pylori at a temperature of 37° C. Lactobacillus at a temperature of 37° C with anaerobic or microaerophilic conditions) overnight.

[0052] In one embodiment herein, the confirmation of corona virus spike protein. Mini cultures from co-culture plates, prepares fresh of sterile tubes with EMB broth (selective medium for gram-negative bacteria) are prepared. Inoculates the mini cultures, a loopful of bacteria from the overnight co-culture plates is added to each broth tube. These co-culture plates likely contained a mix of the donor Lactobacillus cells and the recipient cells (E. coli and H. pylori) after performes LGT to transfer the gene for the spike protein.

[0053] In one embodiment herein, the presence of the coronavirus spike protein gene in the selected recipient bacterial cells is confirmed using at least one of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and gram staining. In one embodiment herein, the growth of E. coli and H. pylori cells on the EMB medium is confirmed by the formation of purple colored colonies or colonies with a green metallic sheen.

[0054] The incubation of the mini cultures are incubated overnight at a temperature of 37° C. The whole cell extract preparation start by the next day, the bacteria from the mini cultures are used to prepare whole cell extracts. These extracts are likely analyzed using SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), a technique that separates proteins based on their size and charge.

[0055] Consequently, the mini cultures serves as a small-scale test to check if the recipient bacteria in the co-cultures actually produced the coronavirus spike protein after receives the gene from the donor Lactobacillus. By analyzing the whole cell extracts using SDS-PAGE, the researchers can see if the protein band corresponding to the spike protein is present, indicates successful expression.

[0056] In one embodiment herein, the preparation of whole cell extracts, the whole cell extracts are bacterial cell extracts which are prepared by lysis (breaking down) of bacterial cells to collect the total protein content present in the bacterial cells. At one, the centrifuge the mini culture tubes are first centrifuged at 5000 rpm for 10 min. This spins the contents of the tube, causing the heavier bacterial cells to pellet at the bottom and the liquid supernatant to rise to the top.

[0057] Next, a supernatant removal and lysis buffer addition, the supernatant contains mostly media and smaller molecules, is discarded. Instead, 0.1 ml of cell lysis buffer is added to the pellet. This buffer contains substances that break down the cell walls and membranes of the bacteria, releases their internal contents.

[0058] Next, boil the re-suspended pellet and lysis buffer are then boiled in a water bath at a temperature of 60° C for at least 10 min. This further helps in lysing the bacterial cells and releases all their proteins. After, boil the tubes are centrifuged again at a higher speed of 8000 rpm for at least 10 min. The separates remaining cell debris from the released proteins, which remains in the supernatant. Next, post centrifugation of the extracted the supernatant of 200 µl by using a micro pipette for easily transfer, sterile polypropylene tubes. These tubes are labelled for easy identification and stored at a temperature of -20° C.

[0059] In another embodiment herein, the whole cell extract preparation process essentially creates a concentrated solution of all the proteins that were present inside the bacteria in the mini cultures. These extracts are then used for further analysis, likely with SDS-PAGE, to detect the presence of the coronavirus spike protein produced by the bacteria.

[0060] In one embodiment herein, the SDS-PAGE for whole cell extract samples, where the SDS-PAGE is an electrophoretic technique used to separate the proteins available in a sample. The proteins are separated and analysed based on their mass (in kDa). The protocol below is used to identify the expression of spike protein from the cultures used in LGT. The usage of the SDS-PAGE as the final test to verify whether the LGT experiment successfully transferred the gene for the coronavirus spike protein and induced its expression in the recipient bacteria.

[0061] In another embodiment herein, the preparation of a polyacrylamide gel casting apparatus by assemble the gel casting chamber by fixes the glass plates and plate holder in the correct positions. Ensure the assembled apparatus doesn't leak, pipette distilled water between the glass plates and wait for at least 2-3 min. Any leakage could compromise the gel and affect the analysis. If no leakage is observed, discard the water and carefully clean the glass plates. Finally, reassemble the apparatus for making the gel.

[0062] In another embodiment herein, the preparation of the separating gel solution, by labellers tubes of two 50 ml tubes as "separating gel" and "stacking gel" to differentiate between the two layers of the gel. The preparation of the 12% separating gel solution involves several specific components as outlined in Table 1. These components include 6 ml of 30% Acrylamide-Bisacrylamide Solution, 3 ml of distilled water, 6 ml of 2.5X Tris-SDS Buffer (pH 8.8), 125 µL of 10% APS solution, and 18 µL of TEMED.

[0063] These components are combined in the appropriate volumes and carefully mixed to ensure that the gel solution is properly formed. The 5% of stacking gel is prepared using 1.3 ml of 30% Acrylamide-Bisacrylamide Solution, 5.1 ml of distilled water, 1.6 ml of 2.5X Tris-SDS Buffer (pH 8.8), 75 µL of 10% APS solution, and 10 µL of TEMED.

[0064] Table 1:
12% Separating Gel
S. No Component Volume
1 30% Acrylamide-Bisacrylamide Solution 6 ml
2 Distilled Water 3 ml
3 2.5X Tris-SDS Buffer (pH 8.8) 6 ml
4 10% APS solution 125 µL
5 TEMED 18 µL
5% Stacking Gel
1 30% Acrylamide-Bisacrylamide Solution 1.3 ml
2 Distilled Water 5.1 ml
3 2.5X Tris-SDS Buffer (pH 8.8) 1.6 ml
4 10% APS solution 75 µL
5 TEMED 10 µL

[0065] The functional gel casting apparatus and prepare the first layer of the polyacrylamide gel (separating gel) for further processing in the subsequent steps of gel electrophoresis. Next, pour the prepared separated gel solution quickly between the glass plates till the marked line using a serological pipette. The de-ionised water is poured above the separated gel solution to allow the gel to solidify at the top with a smoother surface. The gel is allowed to solidify for at least 45-50 min, which can be monitored by observes the tube contains the remaining gel solution.

[0066] Next, remove water and prepares the stacking gel, the water on top is discarded. The stacking gel is prepared. The stack gel solution is quickly poured between the plates until the brim. The comb is immediately placed between the plates at the top of the gel to create sample wells. The gel is allowed to solidify again for at least 45-50 min, monitored as before. The gel plate are carefully transferred from the cast tray to the electrophoresis chamber. The gel running buffer is added to the chamber, fillies it up to the indicated level. The comb is gently removed from the solidified gel.

[0067] The prepared whole cell extracts from your LGT experiment are loaded into each well using a pipette. A protein ladder is also loaded in one well to serve as a reference for protein size based on known markers. The samples are run on the gel for at least 45-60 min at 120 volts. After the proteins have separated as desired, the electrophoresis chamber is disconnected from the power supply. The gel is washed with distilled water to remove excess SDS (detergent used in the process). The gel is then placed in a staining solution for one hour to visualize the separated proteins.

[0068] Further, the gel is rinsed with water to remove excess stain. It is then transferred to a de-staining solution to remove background staining and further clarify the protein bands. The gel is left to de-stain overnight for maximum clarity. Finally, the gel image is captured and documented for further analysis of the protein bands, specifically looking for the presence of the coronavirus spike protein in the lanes containing extracts from the co-cultured bacteria.

[0069] In one embodiment herein, the gram staining technique is used to differentiate between gram positive (for example, Lactobacillus) and gram negative (for example, E. coli and H. pylori) bacterial cells based on coloring. Gram staining technique uses crystal violet and safranin staining solutions, where, gram positive bacterial cells retain crystal violet in their cell walls due to which they appear in violet/purple color and gram negative bacterial cells retain safranin and appear in pink color.

[0070] In one embodiment herein, the crystal violet dye is used to initially stain all bacteria. The gram-positive bacteria retain the crystal violet and appear purple. The gram-negative bacteria lose the crystal violet during de-colorization and then stain pink with safranin. Preparation of slides and smear, sterile slides are labelled using a single colony diluted with water. The smear is heat-fixed. The crystal violet solution is added for at least 30 sec and then washed off with water. The safranin solution is added for at least 20 sec and then washed off with water. The slide is dried, a coverslip is added and the strained bacteria are observed under a microscope.

[0071] Numerous advantages of the present disclosure may be apparent from the discussion above. In accordance with the present disclosure, the probiotic vaccines are beneficial for children, the elderly, and people with needle phobias. The proposed probiotic vaccines utilises the lateral gene transfer (LGT) allows for targeted antigen delivery to specific gut microbiome bacteria, potentially enhanced the immune response. The proposed probiotic vaccines that adapted to deliver antigens from various pathogens for different diseases.

[0072] The proposed probiotic vaccines that utilises sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) techniques for analysing the protein expression. The proposed probiotic vaccines that are delivered in a variety of food and beverage products, which makes them more palatable and appealing to patients. The proposed probiotic vaccines that provide a delicious and painless way to deliver vaccines to all age groups with minimal to no side effects.

[0073] It will readily be apparent that numerous modifications and alterations can be made to the processes described in the foregoing examples without departing from the principles underlying the invention, and all such modifications and alterations are intended to be embraced by this application.
,CLAIMS:CLAIMS:
I/We Claim:
1. A method for formulating a probiotic vaccine, comprising:
culturing donor bacterial cells comprising a plasmid containing a coronavirus spike protein gene;
co-culturing the donor bacterial cells with recipient bacterial cells under conditions suitable for lateral gene transfer;
selecting the recipient bacterial cells that acquired the plasmid containing the coronavirus spike protein gene; and
formulating the selected recipient bacterial cells into the probiotic vaccine,
whereby, the probiotic vaccine is formulated in a pharmaceutically acceptable carrier that delivers targeted spike protein expression in gut.
2. The method for formulating a probiotic vaccine as claimed in claim 1, wherein the pharmaceutically acceptable carrier is suitable for oral administration, wherein the oral administration is in the form of at least one of yogurt and fermented products or capsules.
3. The method for formulating a probiotic vaccine as claimed in claim 1, wherein the co-culturing is performed for a period of time of 1 hr to 2 hr at temperature of 36° C to 38° C.
4. The method for formulating a probiotic vaccine as claimed in claim 1, wherein the donor bacterial cells is a Lactobacillus species.
5. The method for formulating a probiotic vaccine as claimed in claim 1, wherein the recipient bacterial cells is at least one of Escherichia coli (E. coli), helicobacter pylori (H. pylori), and gut microbiota-related organisms.
6. The method for formulating a probiotic vaccine as claimed in claim 1, wherein the co-culturing is performed in one of a liquid medium under anaerobic and microaerophilic conditions.
7. The method for formulating a probiotic vaccine as claimed in claim 1, wherein the presence of the coronavirus spike protein gene in the selected recipient bacterial cells is confirmed using at least one of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and gram staining.
8. The method for formulating a probiotic vaccine as claimed in claim 1, wherein the selection for recipient bacterial cells that have acquired the plasmid is performed using a medium containing ampicillin including eosin methylene blue (EMB) medium including EMB broth and EMB agar.
9. The method for formulating a probiotic vaccine as claimed in claim 8, wherein the growth of E. coli and H. pylori cells on the EMB medium is confirmed by the formation of purple colored colonies or colonies with a green metallic sheen.
10. The method for formulating a probiotic vaccine as claimed in claim 8, wherein the EMB broth and EMB agar plates are prepared by:
adding 4 to 6 gr of eosin methylene blue (EMB) powder to 250 ml of deionized water to form a first solution;
adding 4 to 6 gr of the EMB powder and 2 to 4 gr of bacteriological agar to 250 ml of deionized water to form a second solution;
stirring the first solution and the second solution gently without exposing to direct light due to photosensitivity;
autoclaving the first solution and the second solution at a pressure of at least 15 lbs and at a temperature of 120° C to 122° C for a time period of at least 15 min;
cooling the first solution and the second solution at a temperature of 45° C and adding 50 µg/ml of ampicillin to each solution; and
pouring at least 25 ml of each solution into sterile petri dishes and allowing each solution to solidify at room temperature, and sterile packing the prepared media plates and storing at a temperature of 4° C.