CN113876941A - Design and application of novel rapid, efficient and low-cost artificially synthesized vaccine - Google Patents

Abstract

The invention provides a novel artificially synthesized vaccine combined by multiple polypeptides. Based on the advantages of the vaccine of synthetic peptide, the vaccine carries one or more B and/or T cell epitope composite polypeptides through artificial optimization design synthesis, chitosan nanoparticles are used as carriers, and the vaccine generates good cellular and humoral immunity on a human body by adopting an intranasal administration mode. The vaccine is simple and rapid to prepare, low in cost, safe and reliable, can be produced in a large scale in a short time, flexibly copes with various outbreak epidemics and viruses, and does not need cold chain transportation and storage. The vaccine injection mode is convenient and fast, can be independently completed, and does not need to go to professional places such as hospitals.

Description

Design and application of novel rapid, efficient and low-cost artificially synthesized vaccine

Technical Field

The invention relates to design and application of a novel, rapid, efficient and low-cost artificially synthesized vaccine.

Background

Most leading experts show that reliable detection will help control pandemics and personal infections, but the ultimate goal is an effective vaccine. Unfortunately, vaccines for this virus are not available outside of the small clinical trials, and the U.S. experts show that if all successful commercial vaccines will be available before 2021 s. This document describes the principles, design, formulation of SARS-CoV-2 vaccine and intranasal self-administration. Since 3 months 2020, our group followed these protocols based on previous publications and preprinted literature on the rapid growth of SARS-CoV-2 to produce and self-manage multiple generations of progressively improved vaccines.

There is a large amount of published information on all aspects of vaccine production, testing and route of administration, some of which are directed against the SARS-CoV-2 virus. The published information is sufficient to establish vaccines and to conduct pharmacodynamic tests in a much shorter time than commercial vaccines. Vaccines are the safest of all treatments. Due to their safety and effectiveness, they have the highest probability of success (over 40%, approximately twice the next highest level) throughout the clinical trial. Because of this and the impact of this epidemic, accelerated commercial trials have been planned and have begun to bypass expanded animal studies, some of which will be conducted directly in human trials (e.g., modena, BioNTech, currevac, etc.).

However, commercial vaccine designs must be capable of large-scale production and deployment, and they require regulatory approval to be sold, which greatly limits and slows progress. Critically, certain properties of commercial vaccines do not require research-level production and testing; thus, for smaller scale, self-administered vaccines, shorter times are possible. There have been many advances in vaccine research and technology development, but not in commercial products, in part because commercial vaccine design and production is limited by different factors, unlike small-scale research vaccines. Key attributes of vaccines are high safety, low cost and ease of production and management. We discuss these issues herein and consider issues such as short-term and long-term safety.

The key issues and some differentiating factors of commercial and research vaccines:

recent safety the recent safety of intranasal vaccines should be excellent at the time of preparation. Intranasal vaccines have long-lasting safety. The vaccine formulations described herein may also be used for other routes of delivery, including inhalation, oral administration or injection, but these (particularly the latter) should only be tried by the skilled practitioner. Generally, vaccines are safer to administer by inhalation, orally, or nasally than by injection. The focus of this document is intranasal administration, but the expert will know how to adapt the information here to another route of delivery. For any route of administration, purchasing high quality materials and careful preparation are critical to maintaining vaccine safety.

Long-term safety the long-term safety of any vaccine is currently difficult or unpredictable, and even widely deployed commercial vaccines have led to serious and unpredictable complications. Vaccines that show severe side effects are injectable whole virus or subunit formulations. At least three potentially serious complications may occur in the long term: tolerance, vaccine-enhanced disease, and adjuvant-triggered immune or neurologic injury complications.

Immune tolerance is a term meaning a decrease in immunity due to exposure to an antigen. This attenuated immune response is commonly observed in food antigens and "spontaneous" antigens in the body. In general, it is believed that extreme and/or frequent irradiation and oral doses lead to tolerability.

The vaccine enhances the disease. A small injection of vaccine resulted in an increase in disease, which means that the infectivity of vaccinated people was increased, or the disease became more severe, relative to unvaccinated controls. This is in response to respiratory syncytial virus, dengue, Zika virus and atypical vaccines. One mechanism is antibody-dependent enhancement (ADE), in which antibodies of the systemic immune system augment immunopathology (in the case of SARS, lung tissue in particular) or otherwise enhance the disease. This highlights an important advantage of intranasal vaccines: a powerful mucosal immune response should greatly reduce or prevent this systemic response by eliminating the initial infection. An early report on SARS-CoV-2 suggests that neutralizing antibodies to the SARS-CoV-2 Receptor Binding Domain (RBD) do not exhibit this enhancement. We mention ADE and other possible negative consequences of the vaccine against SARS-CoV-2 to provide a sufficient risk background, but some experts believe that ADE is not critical in developing a vaccine against this virus.

Adjuvant hyperstimulation or toxicity: adjuvants help to stimulate a strong immune response to the vaccine; however, certain adjuvants cause over-stimulation and other serious side effects. For example, alum produced a strong Th2 immune response, but Th 2: the proportion of Th1 is unbalanced. Th2 is hyperstimulated and is associated with immunopathology, including ADE. The additive may also be toxic. For example, intranasal administration of detoxified mutant forms of E.coli heat labile toxin has resulted in transient Bell's palsy, or facial nerve palsy. One of the reasons for using strong adjuvants in commercial vaccines is that a single administration can elicit a strong immune response and avoid boosting.

And (3) stabilizing: stability is a key determinant of commercial vaccines. Formulations that are both safe and effective in research environments, but have limited shelf life, are often excluded from commercial products. We have found that the formulation of the vaccine is very simple, safe and effective, but has only short term stability. For example, chitosan gel nanoparticles have proven to be very effective and very simple formulations, but their short shelf life has led to their limited use in commercial vaccines.

Intranasal administration: intranasal vaccines offer advantages over other methods of delivery, including the most common mode of delivery by injection. Nasal administration has proven to be very safe with the same minor side effects as after placebo treatment. Importantly, it can not only elicit systemic immunity, but also mucosal immunity at the site of respiratory viral infection. Commercial intranasal influenza vaccines are available, with efficacy for systemic immunity equivalent to administration by injection, but greater efficacy for mucosal immunity at the site of respiratory virus entry (nose, lungs). This is crucial for SARS-CoV-2, since earlier studies have shown that most infections are initiated from the nasal cavity. By 6 months 2020, most or all commercial SARS-CoV-2 vaccines under development were designed for injection, a route unlikely to provide mucosal immunity to infection.

Prime-boost: intranasal injections may be as effective as injections, but multiple injections are often required to achieve this level of immune response and protection, particularly with milder adjuvants. The initial dose is primary and the subsequent doses are to enhance or augment the immune response. The effect is similar to the main dose of a vaccine before exposure to a pathogen or closely related pathogen. The only commonly used intranasal vaccine is the influenza vaccine. Nasal vaccines with attenuated viruses are inherently booster since essentially everyone is naturally exposed to influenza. The need for booster vaccines limits the commercial production of vaccines, which have not previously been widely available.

Commercial vaccines are difficult to administer intranasally for a variety of reasons, including those mentioned above. However, it is not only relatively easy to study vaccines, but in some cases it is the preferred mode of administration for certain pathogen types (e.g. respiratory viruses). There is no risk of needle stick injury or blood-borne infection relative to injection. Immunization by the intranasal route not only prevents viral infection through the nasal mucosa, but also effectively stimulates mucosal immune responses in the lungs and upper respiratory tract. For example, Gai and colleagues have demonstrated that a SARS vaccine delivered intranasally elicits a powerful mucosal immune response, preventing initial infection, whereas injection of the same vaccine did not. This difference is important because the area of mucosal surfaces (nasal, pulmonary, gastrointestinal, urogenital, etc.) is very large, about 200 times the surface area of the skin, and about 70% of pathogens enter through these routes. Single administration compliance is also high because intranasal administration does not involve a needle or cause pain. High safety and manageability are expected to increase the immunization rate. For a recent review of nasal nano-vaccine studies, see Bernocchi et al, table 1.

Synthetic peptide-based vaccines have advantages over the most widely used vaccine designs based on full-length Open Reading Frames (ORFs) of attenuated viruses and even key epitope proteins. Coronavirus spike and nucleolar full length proteins have been associated with ADE in animal and human cell studies. Yasui and his colleagues showed that the vaccination with nuclear chlamydia did not provide protective immunity, but enhanced immunopathology. Vaccination with certain epitopes of Spike proteins does provide protection, but the use of full spikes is not recommended. For example, from Tais and coworkers: the full-length S protein should be used with caution. Kam, etc. It was reported that, although the recombinant trimeric SARS-CoV S protein vaccine elicited a protective immune response in mice, anti-S antibodies also mediated an enhanced effect of antibody-dependent virus entry into human B cells in vitro. In another study, ferrets vaccinated with the recombinant modified vaccinia Ankara-expressed SARS-CoV full-length S protein vaccine grown in BHK21 and Vero E6 cells showed enhanced SARS-CoV-induced hepatitis virulence. In addition, the use of SARS S protein vaccine may lead to disease and immunopathology enhancement, rather than protection as with feline coronavirus, feline infectious peritonitis virus. In view of these problems, SARS vaccine strategies using full-length S protein may not be the optimal choice for humans. Thus, the best approach may be to use small S protein epitopes, which are the main neutralizing determinants. "full length constructs of vaccines against similarly used other pathogens also cause enhancement of viral susceptibility or disease. The vaccine design described herein is based on such B cell peptide epitopes of the S or spike proteins, which are expected to be the major neutralizing determinant, as well as the effective T cell epitopes predicted and experimentally tested. Some are combined B and T cell epitopes.

The synthesis of synthetic peptides provides freedom to design epitopes long enough, but is not expected to cause these serious side effects. This approach also allows the use of multiple epitope peptides, which can be combined into a single poly-linear peptide or as a collection of individual peptides. The methods employed herein use a single B cell epitope peptide of the S protein, as well as T cell epitopes of S and other proteins. Certain B cell epitope amino acid sequences comprise predicted T cell epitopes. We used some of these combined epitopes. Newer versions of the epitope were selected based on data on T cell responses of neutralizing antibodies and convalescent patients previously infected with SARS-CoV-2. s

The choice of adjuvant is important for safely enhancing the immune response. Many adjuvants have been compared for their ability to elicit various aspects of the immune response. These include alum, chitosan, inactivated Cholera Toxin (CT), CpG DNA, monophosphoryl lipid A (MPL), poly IC, plus ground-up-mod and E.coli heat labile toxin. Alum is an aluminium salt (potassium aluminium sulphate, AlK (SO 4) 2) and is the most widely used adjuvant in commercial vaccines. It has been used for nearly a century as an effective adjuvant. In direct comparison, alum at higher doses was superior to most other adjuvants, including intranasal vaccine. In animal studies, alum was found to be equal to or superior to most other animals in eliciting systemic immunity (in IgG1, IgG2a, IgG2 b), and superior to or superior to most others in eliciting secretory iga (siga) mucosal immunity and antiviral challenge. The poly (I: C) can be used as an adjuvant; however, it showed weaker activity relative to alum and already elicited autoimmunity in animal models.

Chitosan-based therapeutics have been developed for many biomedical applications. In the aspect of vaccine application, the chitosan can be used as nanoparticles and adjuvants and used for intranasal administration, parenteral injection, oral administration, sublingual administration and the like. It has found widespread use in animal testing and has been safely used in human clinical and preclinical testing (some using commercial products such as viscogels). Guro Gafvelin and HansGr nano in "molecular vaccine: from prophylaxis to treatment-volume 2, edited by mathias Giese "; page 39, part of the human test reviewed on page pp) 624-; see table 39.1 for Springer.

Nasal instillation of chitosan to healthy volunteers with influenza vaccine resulted in systemic (IgG) protection, although less than alum-adjuvanted parenteral vaccines (despite the low dose of 15 μ g); in addition, unlike parenteral vaccines, it can also induce mucosal immune responses.

Chitosan as an adjuvant has been compared to alum and other adjuvants in injectable and intranasal forms, alone or as part of an adjuvant mixture. Chitosan alone is a potent mucosal and systemic adjuvant that has a synergistic effect with alum and other adjuvants. Importantly, nasal administration of chitosan-based vaccines can stimulate both mucosal and systemic immunity.

Disclosure of Invention

The technical problem to be solved by the invention is as follows:

commercial vaccines must be designed for large-scale production and deployment, requiring regulatory approval for sale, which greatly limits and slows progress. Importantly, some of the functions of commercial vaccines are not required for research-grade production and testing. Thus, for smaller scale, self-administered vaccines, shorter times are possible. Herein we describe vaccines formulated using mature, cheap and mostly ready-made ingredients. The SARS-CoV-2 vaccine described can be produced quickly and inexpensively in a variety of laboratory and physician office environments. Our team rapidly deployed vaccine cooperative organization (ravlac) produced and injected our own vaccine for the first time in late 4 months of 2020. By the middle of 6 months, we have designed and self-administered a sixth generation (Gen 6) vaccine. This would be a versioned live file, enabling a gradual improvement in vaccine design and testing.

Drawings

The following detailed description of embodiments of the invention refers to the accompanying drawings in which:

(ii) a safe component. A long history of published results;

simplicity and robustness of production. Simple to manufacture but may compromise short term stability. Under a variety of conditions and ingredient concentrations;

③ the chitosan nano-particle can be spontaneously and repeatedly formed;

fourthly, intranasal immunization. Preferred modes of delivery for respiratory and rhinoviruses. The highest security. Potentially stimulating protective mucosal and systemic immunity at the site of infection;

polypeptide epitope antigen. Short peptides of linear epitopes are most readily available or produced and are predicted to function optimally without specific structural constraints. These can be extracted from abundant literature or newly generated. The antigen incorporated into the vaccine may be produced synthetically or as a recombinant expressed protein;

sixthly, the plan progress is accelerated. Allows for intranasal administration and lower or milder doses of adjuvant, but potentially produces an equivalent immune response to a single dose of a highly irritating adjuvant;

figure 1 is a schematic showing the pathway of acquired immunity for Th1 and Th2 (a.k.a. adaptive immunity), as well as the role of mhc class i and class II.

Detailed Description

The present invention will now be described in further detail with reference to the accompanying drawings. The figures are simplified schematic drawings only

The schematic diagrams illustrate the technical principles of the present invention, and therefore show only the configurations related to the present invention.

Key technical features and specifications

(ii) intranasal administration is probably the safest route of administration. For respiratory viruses, intranasal delivery has the advantage of eliciting a mucosal immune response at the site of viral entry. Intranasal administration may also elicit systemic immunity, although generally not as rapidly as parenteral administration after a single dose;

② chitosan nano-particles and adjuvant. Successful vaccines depend on successful delivery and immunostimulating adjuvants. Many adjuvants have been validated and available, each with its advantages and disadvantages. Leading nanoparticle nasal delivery and adjuvant combination systems based on chitosan and Sodium Triphosphate (STP) or Tripolyphosphate (TPP) were the first choice for intranasal vaccines. Chitosan is a partially or fully deacetylated derivative of chitin, a linear polysaccharide found in the shells of crustaceans (e.g., shrimp). Nanoparticles of various sizes can be created using simple adjustable parameters. Ideally, the size of the nanoparticles should be adjusted to between 100 nm and 200 nm. As mentioned above, chitosan has proven to be safe and well tolerated, and intranasal delivery elicits mucosal and systemic immune responses;

(iii) possible other adjuvants/immunostimulants. Chitosan is a Th1 immune-triggered, self-adjuvanting polysaccharide. Thus, no additional adjuvant or immunostimulant is absolutely required, and no adjuvant or immunostimulant is incorporated into the initial formulation. However, if chitosan alone does not produce sufficient immune stimulation, we are considering attempts to add other adjuvants that enhance Th1 or specific T cell targeting responses.

Peptide antigen: peptide-based vaccines are the approach we chose.

Phi, a poly/multi-epitope vaccine has been demonstrated to be effective. The approach taken here is to use a plurality of peptides, each of which will carry one or more B and/or T cell epitopes.

② the synthetic peptides are cheap and can be ordered rapidly from many peptide manufacturers. They allow the synthesis of many possible chemical modifications that have been reported to increase immunogenicity.

③ an early version of the ravlac vaccine contains simple linear epitopes, the conformation of which is not specifically considered. Starting with Gen 3, most B cell epitope peptides are constrained by disulfide bond conformations. Ideally, the 3D structure of viral proteins should be imaged and linear epitopes selected that do not require special conformational constraints, or the native structural conformation of the epitope peptide should be attempted to achieve.

Other antigens are also possible and may be delivered intranasally by chitosan, including DNA or RNA.

Epitope selection is crucial. B-cell and T-cell epitopes have been selected and published by others. Multiple epitopes of both types should be selected, preferably in highly conserved regions of the virus. In this way, the likelihood of each epitope successfully stimulating immunity is increased, rather than relying on a single epitope.

B cell epitope: antibody profiling studies from convalescent patients have helped identify the portion of the virus available for antibody binding to B cell epitopes, some of which have been shown in human cell studies to neutralize viral infection. In convalescent sera, some B-cell epitopes predicted to be high by common machine learning have not been reported with high frequency. This may be due to the fact that linear peptide-based methods can map epitopes, extensive glycan shielding around the virus or other complicating factors. Whatever the interpretation used, we believe that convalescent antibody data outperforms purely computational predictions.

T cell epitopes: empirical methods similar to, but more complex than, those used for B cell epitope selection have been used to select for superior T cell epitopes.

And (3) testing: successful acquisition of mucosal and systemic immune stimulation will be assessed by testing nasal washes, saliva and serum for antibody titers. Immunoassays will be performed using standard detection methods and reagents currently under development, as well as using new technologies, such as transcriptome profiling of Peripheral Blood Mononuclear Cells (PBMCs).

Claims (6)

1. A synthetic polypeptide vaccine against a pathogenic agent (virus, bacteria, fungi, etc.): the polypeptide contains ten polypeptides, combines a plurality of epitopes of T cells and B cells, covers conservative virus contact and target points entering cells, can generate human immunity most effectively, and prevents COVID-19.

2. The vaccine of claim 1, which can prevent not only new coronavirus that is currently circulating, but also new coronavirus that is later circulating, including SARS and MERS.

3. The immunity generated by the vaccine comprises antibody immunity generated by B cells and cell immunity generated by T cells

The method for designing polypeptide by combining B cell and T cell can generate comprehensive immunity of human body to any virus and bacteria.

4. The vaccine mode can be flexibly used for other disease-treating bodies, including viruses (influenza, RSV and the like), bacteria (strep).

5. The vaccine mode can be flexibly used for various immunization routes, including nasal spray, nasal drip method and intramuscular injection.

6. The vaccine mode can flexibly add various modifications, including DNA, RNA, recombinant protein and even inactivated virus.

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