Description:DESCRIPTION:
Field of the invention:
[0001] The present disclosure generally relates to the technical field of synthetic biology and protein engineering, and in specific relates to a probiotic vaccine that can target all the existing mutant forms of coronavirus.
Background of the invention:
[0002] Currently, coronavirus infection (CoV) has emerged as a replacement for influenza, which is a classic respiratory viral infection and has been identified as a large problem and a risk to human health worldwide. The risk of respiratory viral infection is because of its rapid and various routes of transmission, such as via droplets and air in a closed environment.

[0003] It is known that the main causes of epidemic respiratory diseases in mammals, including humans and other species, are influenza viruses. Influenza viruses are transmitted almost every winter, and influenza virus infections pose social and economic risks in many countries. Severe influenza infection increases the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), which are inflammatory cytokines. Increases in the levels of these cytokines have often been observed during the lethal course of infection.

[0004] Moreover, influenza virus infection is dangerous to high-risk groups, including pregnant women, patients with diabetes, infants, and the elderly. Such high-risk groups do not have a sufficient immune response against viral infections and frequently show severe and lethal conditions. Thus, proper maintenance of the immune system, which forms a barrier to foreign antigens, is essential to prevent the severe and lethal conditions of influenza.

[0005] Coronaviruses, which belong to the Orthocoronavirinae family, infect mammalian species, including humans and other species. Various CoV have threatened human society over the many years. In particular, severe acute respiratory syndrome CoV (SARS-CoV), Middle East respiratory syndrome CoV (MERS-CoV), and SARS-CoV-2, which causes coronavirus disease 2019 (COVID-19) have adversely affected global health. The mild symptoms of SARS-CoV-2 infection are similar to those of influenza and common cold-like illness, such as cough, fever, and fatigue, whereas the severe symptoms of SARS-CoV-2 include severe dyspnea, multifocal pneumonia, acute respiratory distress syndrome, and multi-organ dysfunction syndrome. Immune-naive populations across the world are the cause of the efficient SARS-CoV-2 transmission.

[0006] For influenza and CoV infections, prevention through vaccination is very important; however, if the inoculated virus vaccine does not match the vaccine-produced antibodies and the current pandemic virus strains, the spread of the virus cannot be prevented. Therefore, it is important to increase immunity, maintain personal hygiene, and eat nutritious food in daily life.

[0007] Probiotics from dairy products are consumed worldwide every day. Probiotics include many lactic acid bacteria, a group of bacteria belonging to many genera, including Lactobacillus and Bifidobacterium. They are also reported to have beneficial effects in gut disorders, diabetes, and cancer, and to improve defenses against infectious diseases through immune regulation.

[0008] In existing technology, an engineered bacteria for use in vaccine compositions is disclosed. The composition bacteria (e.g., Lactobacillus) is engineered to express immunogenic polypeptides and immunogenicity-enhancing adjuvant polypeptides to treat and/or prevent infection from a pathogenic organism. The exogenous polypeptide is a coronavirus antigen is derived from a spike protein (S). The genetically engineered bacteria can be administered as part of a food product (e.g., Yogurt). However, the vaccine composition does not contain genetic circuits that can get activated based on sensing signals either from pathogens within the body or manually from the outside.

[0009] There are one or more technical problems with the composition for the treatment of SARS-CoV-2 in the prior art. Further, in the existing art, various compositions such as inactivated vaccines, viral vector vaccines, mRNA vaccines, protein subunit vaccines are proposed to treat the SARS-CoV-2 virus. All these vaccines are delivered through an intramuscular injection in most of the cases. Some can be in the form of subcutaneous injection or as a nasal spray in very few cases. However, these existing vaccines which were designed to provide immunity against SARS-CoV-2 virus but not all mutants were targeted by these vaccines. Moreover, for getting vaccinated, the person needs to go to a hospital or a vaccination center to get their dose of vaccine which is a dangerous, hazardous task, the person may effect by the virus on the go. Furthermore, the existing vaccines have one or more side effects such as pain, swelling, and redness in the arm.

[0010] Therefore, there is a need for a probiotic vaccine that can target all the existing mutant forms of coronavirus. There is also a need for a vaccine that can consume orally to reduce the side effects like pain, swelling, and redness in the arm. There is also a need for a vaccine that does not have mercury content and is safe for all ages. Further, there is also a need to prepare the vaccine using natural ingredients to generate antibodies against a wide spectrum of mutant strains of SARS-CoV-2.
Objectives of the invention:
[0011] The primary objective of the invention is to provide a probiotic vaccine that can target all the existing mutant forms of coronavirus.

[0012] Another objective of the invention is to provide a non-invasive probiotic vaccine that eliminates the side effects such as pain, swelling, and redness in the arm.

[0013] The other objective of the invention is to provide a yoghurt vaccine that can directly go into the gut and it is safe for all ages.

[0014] Yet another objective of the invention is to provide a yoghurt vaccine that contains higher levels of proteins, vitamins and minerals to improve nutritional status and health of older adults and possibly healthy and active ageing.

[0015] Another objective of the invention is to provide yoghurt as a vaccine that exhibit potent antimicrobial activity against viruses and bacteria causing respiratory tract infections.

[0016] Further objective of the invention is to provide a non-invasive probiotic vaccine to enhance resistance to upper respiratory tract infections.
Summary of the invention:
[0017] The present disclosure proposes a probiotic vaccine for preventing coronavirus infection and preparation 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.

[0018] In order to overcome the above deficiencies of the prior art, the present disclosure is to solve the technical problem to provide a probiotic vaccine that can target all the existing mutant forms of coronavirus.

[0019] According to an aspect, the invention provides a method for the preparation of a probiotic vaccine for preventing coronavirus infection. In the first step, a normal RNA polymerase transcribes DNA to mRNA without causing any changes to the mRNA sequence of the bacteria. The bacteria is a probiotic bacterium such as lactobacillus. The gene coding for the SARS CoV-2 spike protein and the gene coding for the faulty RNA Polymerase is genetically engineered into a probiotic bacterium such as lactobacillus by using the techniques like Polymerase Chain Reaction (PCR), restriction digestion, and ligation.

[0020] In the next step, a faulty RNA polymerase creates the mutated spike protein of the coronavirus in the mRNA sequence of the bacteria to challenge the host immune system. The transformed bacterial cells are cultured and tested for the production of SARS-CoV-2 spike protein mutants. In the next step, the milk of 500 ml is pasteurized to form curding. In the next step, the mutated spike protein of the coronavirus in the pasteurized milk is inoculated and stirred in the pasteurized milk containing the mutated spike protein to form transformed bacterial cells. The inoculate of the mutated spike protein of the coronavirus in pasteurized milk is between 0.5 millilitres to 1.0 millilitres.

[0021] Later in the next step, the transformed bacterial cells containing pasteurized milk are incubated at a temperature between 35°C and 40°C for a time period between 10 hours and 12 hours. Further, in the final step, the probiotic vaccine containing the mutated spike protein of the coronavirus is obtained to generate a wide spectrum of mutant strains of the coronavirus variant such as SARS-CoV-2.

[0022] 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:
[0023] 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.

[0024] FIG. 1A illustrates an example view of the production of mutated spike protein from Faulty RNA Polymerase, in accordance to an exemplary embodiment of the invention.

[0025] FIG. 1B illustrates a flowchart of a method for preparation of a probiotic vaccine for preventing coronavirus infection, in accordance to an exemplary embodiment of the invention.
Detailed invention disclosure:
[0026] Various embodiments of the present invention will be described in reference to the accompanying drawings. Wherever possible, the same or similar reference numerals are used in the drawings and the description to refer to the same or like parts or steps.

[0027] 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 a probiotic vaccine that can target all the existing mutant forms of coronavirus.

[0028] Probiotics are live microorganisms that confer a beneficial physiological effect on the host when administered in adequate amounts. Some lactic acid bacteria that can be found in different fermented foods such as yogurt, cheese, and pickles are generally recognized as safe and classified as probiotics because of their health benefits. It was suggested that probiotics should be consumed daily at doses of 108 to 1010 CFU (colony-forming unit) to produce health benefits in humans. The approved health benefits include reducing symptoms of lactose intolerance by improving lactose digestion, inhibiting the initiation of allergic diseases, maintaining intestinal pH, preventing or treating ischemic heart syndromes, reducing blood cholesterol levels, producing vitamins B, improving the bioavailability of dietary calcium, and boosting immune activity. Meanwhile, other potential health benefits such as the treatment of acute diarrheal diseases and prevention of cancer and tooth decay require additional research for validation.

[0029] In an embodiment herein, the Probiotics include bacteria such as Lactobacillus acidophilus, L. amylovorus, L. brevis, L. bulgaricus, L. casei, L. cellobiosus, L. crispatus, L. curvatus, L. delbrueckii spp. bulgaris, L. fermentum, L. gallinarum, L. helveticus, L. johnsonii, L. lactis, L. paracasei, L. plantarum, L. reuteri, L. rhamnosus; Streptococcus thermophilus, Lactococcus lactis, Leuconostoc mesenteroides, Pediococcus pentosaceus, P. acidilactici, Bifidobacterium adolescentis, B. animalis, B. bifidum, B. breve, B. essensis, B. infantis, B. laterosporum, B. thermophilum, B. longum, Propionibacterium acidipropionici, P. freudenreichii, P. jensenii, P. thoenii, Enterococcus fecalis, E. faecium, Bacillus alcolophilus, B. cereus, B. clausii, B. coagulans, B. subtilis, Escherichia coli, Sporolactobacillus inulinus; as well as yeast such as Saccharomyces boulardii and S. cerevisiae. Probiotics are proposed as antimicrobial agents against a large number of pathogenic and spoilage bacteria.

[0030] According to an exemplary embodiment of the invention, FIG. 1A and FIG. 1B refers to an example method for the preparation of a probiotic vaccine for preventing coronavirus infection. At step 102, a normal RNA polymerase transcribes DNA to mRNA without causing any changes to the mRNA sequence of the bacteria. The bacteria is a probiotic bacterium such as lactobacillus. Herein one embodiment, The gene coding for the SARS CoV-2 spike protein and the gene coding for the faulty RNA Polymerase is genetically engineered into a probiotic bacterium such as lactobacillus by using techniques such as but not limited to, Polymerase Chain Reaction (PCR), restriction digestion, and ligation.

[0031] At step 104, a faulty RNA polymerase creates mutations in the mRNA produced in step 102. The produced mRNA of the coronavirus results in the mutated spike protein. The produced random mutated spike protein from the faulty RNA polymerase challenges the host immune system. The transformed bacterial cells are cultured and tested for the production of SARS-CoV-2 spike protein mutants.

[0032] At step 106, milk is pasteurized to form curding. For that purpose, 500m millilitres of milk is boiled at a temperature between 80°C to 85°C. The is pasteurized for a time period of at least 10 minutes for thicker yogurt and at least 20 minutes for thicker yogurt to prepare the probiotic vaccine. Herein one embodiment, the fermented milk containing probiotic strains significantly reduced the incidence of upper respiratory tract infections and existing all mutant forms of SARS-CoV-2 virus among healthy infants, children, adults, and the elderly.

[0033] At step 108, the mutated spike protein of the coronavirus in the pasteurized milk is inoculated and stirred in the pasteurized milk containing the mutated spike protein to form transformed bacterial cells. The inoculate of the mutated spike protein of the coronavirus in pasteurized milk is between 0.5 millilitres to 1.0 millilitres.

[0034] At step 110, the transformed bacterial cells containing pasteurized milk are incubated at a temperature between 35°C and 40°C for a time period between 10 hours and 12 hours. Finally, at step 112, the probiotic vaccine containing the mutated spike protein of the coronavirus is obtained to generate a wide spectrum of mutant strains of the coronavirus variant such as SARS-CoV-2. The probiotic vaccine which when prepared as a yoghurt vaccine, can be possibly consumed orally as plain yoghurt.

[0035] Numerous advantages of the present disclosure may be apparent from the discussion above. In accordance with the present disclosure, a probiotic vaccine can target all the existing mutant forms of coronavirus. The proposed composition is a non-invasive probiotic vaccine that eliminates the side effects such as pain, swelling, and redness in the arm. The yoghurt vaccine can directly go into the gut and it is safe for all ages. The proposed yoghurt vaccine contains higher levels of proteins, vitamins and minerals to improve the nutritional status and health of older adults and possibly healthy and active ageing. The yoghurt vaccine exhibits potent antimicrobial activity against viruses and bacteria causing respiratory tract infections. Further, the non-invasive probiotic vaccine enhances resistance to upper respiratory tract infections.

[0036] 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 preparation of a probiotic vaccine for preventing coronavirus infection, comprising:
transcribing, by using a RNA polymerase, DNA to mRNA without causing any changes to the mRNA sequence of bacteria;
creating, by using a faulty RNA polymerase, mutated spike protein of the coronavirus in the mRNA sequence to challenge the host immune system;
pasteurizing milk of at least 500 milliliters at a temperature between 80°C to 85°C;
inoculating the mutated spike protein of the coronavirus in the pasteurized milk and stirring the pasteurized milk containing the mutated spike protein to form transformed bacterial cells;
incubating the transformed bacterial cells containing pasteurized milk at a temperature between 35°C and 40°C for a certain time; and
obtaining the probiotic vaccine containing the mutated spike protein of the coronavirus to generate a wide spectrum of mutant strains of coronavirus infection.
2. The method as claimed in claim 1, wherein the bacteria is a probiotic bacterium such as lactobacillus.
3. The method as claimed in claim 1, wherein the gene coding for the mutated spike protein of the coronavirus and the gene coding for the faulty RNA polymerase are genetically engineered into a probiotic bacterium such as lactobacillus.
4. The method as claimed in claim 1, wherein the mutated spike protein of bacteria is genetically engineered into a probiotic bacterium by using the techniques like Polymerase Chain Reaction (PCR), restriction digestion, and ligation.
5. The method as claimed in claim 1, wherein the inoculating the mutated spike protein of the coronavirus in the pasteurized milk is between 0.5 millilitres to 1.0 millilitres.
6. The method as claimed in claim 1, wherein the certain time for incubating the transformed bacterial cells containing pasteurized milk in between 10 hours and 12 hours.
7. The method as claimed in claim 1, wherein the milk is pasteurized for a time period of at least 10 minutes for thicker yogurt and at least 20 minutes for thicker yogurt to prepare the probiotic vaccine.