Patent application filed on Sept. 27, 2020. Published on January 15, 2021. Status : pending.
Abstract
The invention belongs to the field of nanomaterials and biomedicine, and relates to a vaccine, in particular to the development of 2019-nCoV coronavirus nuclear recombinant nanovaccine. The invention also includes a method of preparation of the vaccine and application of the vaccine in animal experiments. The novel coronavirus vaccine contains graphene oxide, carnosine, CpG and novel coronavirus RBD; binding of carnosine, CpG and neocoronavirus RBD on the backbone of graphene oxide; the CpG coding sequence is represented as SEQ ID NO 1; the novel coronavirus RBD refers to a novel coronavirus protein receptor binding region that can generate a high-titer specific antibody targeting the RBD in a mouse body.
Description
Nano coronavirus recombinant vaccine using graphene oxide as a carrier
Technical area
The vaccine is an ultimate weapon for the elimination of major infectious diseases, has the advantages of the lowest cost and more advantages of previous enemy than other therapies, undoubtedly becomes hopeful for the public, smallpox is eliminated by humans by vaccination, the cases of poliomyelitis are reduced by 99 percent, the infectious diseases such as diphtheria are rare, and the incidence of diseases such as measles, neonatal tetanus and the like is remarkably reduced. The effect of vaccines on human health cannot be overstated, and the birth of each new vaccine is a great victory for humans to overcome an infectious disease! To date, no medical treatment has been able to have as significant, lasting and profound an effect on human health as a vaccine; nor is there a therapy available by which a disease of the earth can be eliminated at the minimal cost of a vaccine.
After the outbreak of the SARS-CoV-2 epidemic, several laboratories in China completed the isolation of virus strains, and in order to make a great step forward in vaccine development, we believe that we will soon have a definitive weapon for the eradication of SARS-CoV-2, but so far there is no approved vaccine or drug for the treatment of CoV infection, and there is a great need for the development of an effective drug for the treatment or prevention of coronavirus infection and outbreak.
According to the research of coronavirus vaccines such as against SARS and MERS, the main target of the existing coronavirus vaccine is the S protein of the coronavirus. Vaccines must induce not only humoral and cellular immune responses, but also mucosal immune responses, and with adjuvants, induce balanced Th1 and Th2 pathways to produce truly effective vaccines. Currently, research into more SARS and MERS vaccines focuses primarily on viral vector vaccines and subunit vaccines, and a large body of research shows that the problem with SARS and MERS is that long-term memory B cells cannot be stimulated to produce the long-term memory cells in cured SARS and MERS patients can only last for 2-3 years, immunological memory cannot be generated, and vaccine development fails, and only 6 potential coronavirus vaccines are currently entering the clinical research phase, but no 1 effective vaccine has been approved for marketing.
Disclosure of the invention.
An object of the invention is to develop a recombinant vaccine against coronavirus. Another object of the invention is to provide a preparation method of the virus recombinant vaccine.
Given the various problems of the conventional vaccines at present, how to change the problems of the existing vaccines and improve the immune response is a problem that is always considered, in order to improve the immunocompetence of the immunogen and increase the immune response ability of the body, the most fundamental method is to mix the immunogen with an adjuvant, and the immune adjuvant is a promoter that can improve the immune response of the body to the immunogen. CpG oligodeoxynucleotide (ODN) is a promising adjuvant that has been discovered in recent years. CpG ODN appears to have better adjuvant activity in vivo, in vitro and clinical studies in animals, and the best studies are CpG7909 and CpG 1018. 11/9.2017 United States of America FDA approved hepatitis B vaccine with CpG1018 as adjuvant is on the market, the first approved CpG ODN adjuvant vaccine in the world and is used for the prevention of HBV infection of adults aged 18 years and older, and a multitude of different types of CpG ODN is used as adjuvant in a multitude of clinical trials. CpG is combined with TLR9 to activate immature pDC cells and induce a natural and adaptive immune response, but a single CpG structure has a limited activation effect on immune cells and is easily hydrolyzed rapidly by exonuclease, so its stability in vivo is inadequate, and side effects are also caused; CpG-oligodeoxynucleotide (ODN) synthesized in the sequence can also enhance the stimulation effect, and after the CpG is coupled with other proteins such as antigen and the like, the CpG-oligodeoxynucleotide is combined, so that the CpG-oligodeoxynucleotide has a very clear immune activation effect.
Graphene is a two-dimensional carbon nanomaterial consisting of carbon atoms in sp-hybridized orbitals in a hexagonal honeycomb lattice. The fundamental structural unit of the material is the most stable hexagonal benzene ring in the organic material, and the material is currently the most ideal two-dimensional material. Graphene oxide (GO) is a derivative of graphene oxide, and is an exfoliated product. Because of the characteristics of unique SP2 hybridization, perfect two-dimensional structure and high edge reactivity, the treatment platform based on the hybrid structure can be used as an ideal charge and graft carrier in design and development, and plays an important role in aspects of nano-drug delivery systems, biological detection, tumor treatment, cell imaging and the like.
The present invention is completed based on the aforementioned studies.
The invention discloses a brand new vaccine development method based on a graphene oxide material that serves as a framework for loading CpG molecules and recombinant proteins. Based on the technical platform, a novel nano-new corona vaccine is prepared by combining the recombinant protein of the RBD region of the Spike protein of the SAR-CoV-2. The prepared nano-new corona vaccine has stronger immunogenicity in mouse experiments and can generate antibodies with high titer.
In one aspect, the invention provides a coronavirus vaccine comprising graphene oxide, carnosine, CpG and RBD. Preferably, the vaccine is called GO-Carnosine-CpG-RBD vaccine.
Graphene oxide (GO) is an oxide of graphene, and after oxidation, oxygenated functional groups on the graphene oxide are increased, so that the graphene oxide is more active than graphene. Thus, hydroxyl groups and epoxy groups are randomly distributed throughout a graphene oxide monolith, while carboxyl groups and carbonyl groups are attached to the edge of the monolith. Common commercial products of graphene oxide exist in the form of powder, flakes and solutions, and are brownish yellow in color.
Carnosine, known by its scientific name β -alanyl-L-histidine, is a crystalline solid consisting of a dipeptide composed of two amino acids, β -alanine and L-histidine. Carnosine has strong antioxidant capacity, and can trap Reactive Oxygen Species (ROS) and alpha-beta unsaturated aldehydes formed by over-oxidation of fatty acids in cell membranes during oxidative stress.
CpG motifs activate the body's immune system and can be used as adjuvants. The CpG coding sequence is preferably represented as SEQ ID NO 1.
The coronavirus vaccine described by the invention is obtained by combining carnosine, CpG and a novel coronavirus RBD on activated graphene oxide.
The dosage of GO in the coronavirus vaccine provided by the invention is used as a framework base, the dosage is usually excessive, and the dosage of carnosine may be about twice that of GO. CpG and novel coronavirus RBD are used as biological macromolecules, and the dosage of CpG and novel coronavirus RBD is less, and is usually one ten-thousandth of that of GO in mass ratio. And RBD is used in an amount more than 2 times that of CpG, such as CpG:RBD = 1: 2-10, preferably, the dosage of RBD is 3-6 times of that of CpG.
In another aspect, the present invention provides a method of preparing coronavirus vaccine, the method comprising the steps of:
obtaining CpG, RBD recombinant protein and carnosine;
In a preferred embodiment of the present invention, the ratio of graphene oxide, carnosine, CpG, and RBD is: 26 mg: 40 mg: 1.2. mu.g: (3-6) μ g.
The reaction temperature is preferably 20-28 ℃. For example, room temperature is used.
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The invention creates a technical platform for the preparation of nano-recombinant protein vaccines that can rapidly excite the human immune system and rapidly produce a large quantity of preventive vaccines after infectious viruses are confirmed. The technical platform makes full use of the feature that the surface of graphene oxide has COOH, hydroxyl and other groups, and takes advantage of the interaction between pi-pi bonds to combine the screened RBD recombinant protein, CpG molecules and carnosine to prepare the nano recombinant protein vaccine based on the graphene oxide as the framework. The vaccine can stimulate an organism to produce a high-titer RBD-neutralizing antibody against SAR-CoV-2, and lays a technical foundation for the prevention and treatment of coronavirus infection and future major outbreaks of similar epidemics.
In another aspect, the invention provides for an application of the GO-Car-Carnosine-CpG-RBD vaccine, namely an application of the GO-Car-Carnosine-CpG-RBD vaccine in the preparation of a drug for the prevention of a new coronavirus.
This patent application makes it appear that graphene oxide is not yet being used in vaccines. Nothing could be further from the truth. A Spanish study shows that there is a high dose of graphene oxide in the Pfizer mRNA vaccine.
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