KR102211077B1 - A pseudo type rabies virus vaccine using virus-like particles - Google Patents

Abstract

Rabies virus-like particles containing vesicular stomatitis virus (VSV) into which a gene encoding the G protein, which is a major antigenic protein and surface protein of rabies virus, has been introduced, rabies vaccine composition containing the same, and prevention of rabies virus infectious diseases using the same Or a method of treatment, and a method of preparing rabies virus-like particles.

Description

A pseudo-type rabies virus vaccine using virus-like particles

It relates to a pseudo-type rabies vaccine using virus-like particles (VLPs) for the prevention or treatment of diseases caused by rabies virus infection.

Rabies is a disease that occurs in all warm-blooded animals, and is a common contagious disease that is transmitted from infected animals to animals and humans through bite. It is also called rabies because people are afraid to drink water. When infected with this disease, it is a fatal disease that causes central nervous system lesions such as neurological symptoms and encephalitis, and most of them die. In Korea, it is classified as a type 2 legal infectious disease (international OIE standard list B). It is a common disease that is transmitted by saliva and blood. It is mainly infected by the bite of most mammals such as dogs, cats, skunks, and bats infected with the disease. Therefore, in many countries, rabies vaccination is mandatory for pets including dogs. In addition, in order to prevent rabies infection of wild animals and the damage caused by residents, a vaccine is put in the food of wild animals, and a bait vaccine to have antibodies when ingested is sprayed outdoors.

The causative agent of rabies is a bullet-shaped rabies virus (RV: Rabies virus) belonging to the Lyssavirus of the Rhabdoviridae family, and exhibits pathogenicity in all mammals, and proliferates mainly in nerve tissue, salivary glands, corneal epithelial cells, etc. It is known to do. In order to suppress the onset of rabies in animals and prevent rabies in humans, there is a need to develop more effective, safe and inexpensive vaccines. Accordingly, many new vaccines, including DNA vaccines and other recombinant vaccines, are being developed and tested.

Specifically, a purified vaccine strain produced by serial passage and purification of ERA (Evelyn-Rokitnicki-Abelseth) strain as a live venom vaccine using various cells, and cloning the virus by limiting dilution method was developed. Although it is permitted to inoculate dogs, inoculation to other mammals such as cats and cows is prohibited, and mice are known to die when inoculated into the brain. In addition, in the case of oral vaccination, safety cannot be guaranteed, and since it is only approved for injection, it cannot be used in wild animals. It produces effective immune responses and antibodies in such a small amount, but has safety issues.

Zadok vaccine (inactivated vaccine) was developed and approved in Korea since the 1980s, and can be inoculated into various animals such as dogs, cats, and cattle, but it is known to have insignificant effect in forming antibodies since it is inactivated.

As a DNA vaccine, a vaccine method using a DNA vector expressing the surface protein of the rabies virus is known to stimulate the production of helper T cells and neutralizing antibodies. However, induction of an immune response by a DNA vaccine takes a long time, and the degree of an immune response is lower than that of a general vaccine method.

The vaccinia vaccine is made to express the G protein, the surface protein of the rabies virus, in live vaccinia virus.Since 2000, it is a bait vaccine in the form of a bait vaccine to prevent rabies transmitted by wild animals. Has been sprayed on. However, when a person comes into contact, the problem of being infected with the vaccinia virus has been reported to the Centers for Disease Control and Prevention in the United States, so safety for humans is a problem.

The adenovirus vaccine is a recombinant adenovirus type 5 (Ad5) expressing the G protein and nucleocapsid protein of the rabies virus. It is easy to proliferate and can be mass-produced, and shows high immune response and protective performance. However, since most mammals have antibodies to Ad5, it has been reported that the infection rate of Ad5 is reduced (Patent Publication No. 10-2014-0017253).

As such, various types of rabies vaccines are being developed, but problems with the stability of live rabies vaccines have been presented, and there are limitations in the types and inoculation methods of the inoculated individuals. Therefore, various mammals including wild animals can be safely and effectively inoculated. There is a demand for the development of a new vaccine for preventing rabies.

Under this background, the present inventors produced rabies virus-like particles (VSV pseudotyped RV) based on vesicular stomatitis virus expressing G protein of rabies virus in order to develop a vaccine with excellent immunological efficacy and high safety, and the above The prepared virus-like particles were injected into mice to confirm excellent immunogenicity.

It provides rabies virus-like particles, including vesicular stomatitis virus, into which a gene encoding the G protein of rabies virus has been introduced.

It provides a rabies vaccine composition for preventing or treating a rabies virus infectious disease comprising the virus or its virus-like particles as an active ingredient.

It provides a method of preparing the rabies virus-like particles.

It provides a method of preventing or treating a rabies virus infectious disease comprising administering the vaccine composition to an individual other than a human.

It provides rabies virus-like particles, including vesicular stomatitis virus, into which a gene encoding the G protein of rabies virus has been introduced.

The term "G protein" refers to a glycoprotein, which is a surface protein of rabies virus, and is known as a surface protein that induces neutralizing antibodies important for rabies virus infection. In the present specification, the terms may be used interchangeably with GP protein or glycoprotein.

The gene encoding the G protein may be composed of the nucleotide sequence of SEQ ID NO: 1. Not only the nucleotide sequence composed of SEQ ID NO: 1, but also 80% or more, specifically 90% or more, more specifically 95% or more, more specifically 98% or more, most specifically 99% or more homology with the sequence The nucleotide sequence is included without limitation as long as it is a gene sequence that is substantially the same as each gene or exhibits a corresponding efficacy. In addition, in the case of a nucleotide sequence having such homology, it is obvious that a nucleotide sequence in which some sequences are deleted, modified, substituted or added are also included within the scope of the present invention.

The term "homology" refers to the percent of identity between two polynucleotide or polypeptide moieties. Homology between sequences from one moiety to another can be determined by known art techniques. For example, homology can be determined by aligning sequence information and directly aligning sequence information between two polynucleotide molecules or two polypeptide molecules using readily available computer programs. The computer program may be BLAST (NCBI), CLC Main Workbench (CLC bio), MegAlignTM (DNASTAR Inc), or the like, but any program capable of determining homology may be used without limitation. In addition, homology between polynucleotides can be determined by determining the number of hybridizations of polynucleotides under conditions that form a stable double-stranded between homologous regions and the size of the fragments decomposed by decomposition with single-stranded-specific nucleases, but are limited thereto. no.

The term "vesicular stomatitis virus" is a negative-stranded RNA virus that causes vesicular stomatitis, and is classified in the family Rhabdovirus. In the present specification, the term may be abbreviated as VSV.

The term "rabies virus" is a virus of the genus Rhabdovirus family and lyssavirus, is a pathogen of rabies, and is known to be pathogenic to almost all mammals. Rabies virus has a bullet-shaped envelope. Nucleocapsid is a single-stranded RNA stacked with N (nucleoprotein), L (polymerase) and P (phosphoprotein) proteins, and the outer envelope is composed of a double-layered lipid membrane including G (glycoprotein) and M (matrix protein) proteins. do. In the present specification, the term may be abbreviated as RV.

The term “Virus-like particles” is a viral structural protein having a natural self-assembly property, mimicking the shape of an actual pathogen virus, and showing very similar biochemical properties. And is commonly known. Unlike a live virus, it does not have the genetic material that causes infection, so only one infection occurs but does not have the ability to replicate, causing an immune response due to infection, but it does not have the ability to replicate, so the virus causes disease or other This system has a very important advantage in that there are no problems related to propagation to individuals. Specifically, virus-like particles are prepared with a structure similar or identical to that of the original pathogen, and present an epitope exhibiting antigenicity in a highly repeatable form. This allows cross-linking with B cell immunoglobulin receptors and induces B cell activation. As an externally provided antigen (exogenous antigen), VLP enters the cell efficiently by specialized antigen presenting cells, especially dendritic cells (DC), and undergoes antigen processing to form MHC II type molecules. And induced strong humoral and cellular immunity by DC activation, maturation, and stimulation of CD4+ helper T cells by increasing co-stimulatory molecule and cytokine production. Furthermore, similar to the virus in its natural state, virus-like particles are processed in the cytoplasm of DC and presented on the cell surface by MHC type I molecules. In addition, it is presented to apoptotic CD8+ T cells by a cross-presentation mechanism to induce a strong apoptotic immune response. In the present specification, the term may be abbreviated as VLP. In addition, it may be used in combination with a pseudotyped virus or a pseudotyped virus. The pseudo-type virus refers to a virus or virus particle produced by combining a foreign viral envelope protein and a virus or viral vector. The foreign viral membrane protein is used to change host dysfunction or increase or decrease the stability of viral particles.

In one embodiment, host cells expressing rabies virus G protein were infected with vesicular stomatitis virus (VSVluc-VSV). A pseudo-type rabies virus (VSVluc-RV) expressing rabies virus G protein on the vesicular stomatitis virus envelope was prepared from the infected host cells. Therefore, the rabies virus G protein may be expressed in the envelope of the bullous stomatitis virus.

Another aspect provides a rabies vaccine composition for preventing or treating a rabies virus infection disease comprising the rabies virus-like particles as an active ingredient.

In one embodiment, it was confirmed that a large amount of rabies virus G protein was expressed in the rabies virus-like particles. In addition, as a result of inoculating the virus-like particles into mice, it was confirmed that a high antibody production rate and virus neutralizing ability were shown.

The term “vaccine” refers to a pharmaceutical composition containing at least one immunologically active ingredient that induces an immunological response in an animal. The immunologically active component of the vaccine may contain the appropriate elements of a live or dead virus (subunit vaccine), whereby these elements destroy the entire virus or its growth culture, followed by the desired structure(s). By a purification step that yields, or by means of a synthetic process induced by the appropriate manipulation of an appropriate system such as, but not limited to, bacteria, insects, mammals or other species, followed by isolation and purification, or by a suitable pharmaceutical composition. By induction of this synthetic process in animals in need of a vaccine by direct incorporation of genetic material using (polynucleotide vaccination). The vaccine may comprise one or more of the elements described above or at the same time.

The term "prevention" refers to any action of inhibiting or delaying the onset of a disease caused by infection with the rabies virus due to administration of the rabies vaccine composition.

The term "treatment" refers to any action in which the symptoms of a disease already caused by infection with the rabies virus are improved or benefited by the administration of the rabies vaccine composition.

The rabies virus-like particles can be administered as a vaccine to induce immunity against diseases caused by infection with rabies virus, and the rabies virus-like particles may be appropriately formulated together with a pharmaceutically acceptable carrier, excipient or diluent. I can.

Suitable carriers for vaccines are known to those skilled in the art, and include, but are not limited to, proteins, sugars, and the like. The carrier may be an aqueous solution, or a non-aqueous solution, a suspension or an emulsion. As an adjuvant for increasing immunogenicity, a typical or atypical organic or inorganic polymer may be used. Adjuvants are generally known to promote immune responses through chemical and physical binding to antigens. As an adjuvant, an atypical aluminum gel, an oil emulsion, or a double oil emulsion and an immunosol may be used. In addition, various plant-derived saponins, levamisol, CpG dinucleotides, RNA, DNA, LPS, and various types of cytokines may be used to promote the immune response. The immune composition as described above can be used as a composition for inducing an optimal immune response by a combination of various adjuvants and immune response promoting additives. In addition, as a composition to be added to the vaccine, stabilizers, inactivators, antibiotics, preservatives, and the like may be used. Depending on the route of administration of the vaccine, the vaccine antigen may be mixed with distilled water or a buffer solution.

The formulation of the composition may be variously prepared by mixing with a pharmaceutically acceptable carrier as described above. For example, when administered orally, it may be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like, and in the case of injections, it may be prepared in the form of unit dosage ampoules or multiple dosage forms.

The administration route of the vaccine composition may be administered through any general route as long as it can reach the target tissue.

In one embodiment, the vaccine composition is selected from the group consisting of a composition for intramuscular administration, subcutaneous administration, intraperitoneal administration, intravenous administration, oral administration, dermal administration, ocular administration, and intracranial administration. I can.

It is understood that of the terms or elements mentioned in the above rabies virus-like particles, such as those mentioned in the description of the claimed vaccine composition, as mentioned in the description of the claimed vaccine composition.

Another aspect provides a method of preparing the rabies virus-like particles.

In one embodiment, the method of preparing the rabies virus-like particles comprises the steps of transfecting a host cell expressing the G protein of the rabies virus with a vesicular stomatitis virus; And obtaining rabies virus-like particles from the transfected host cell.

The term "transfection" refers to a phenomenon in which a gene is introduced into a host so that the gene can be replicated as a factor of a chromosome or by the completion of chromosomal integration, and an external gene is introduced into a cell to artificially cause a genetic change. Any transformation method may be used as the transfection method, and may be easily performed according to a conventional method in the art.

The "obtaining step" refers to a step of culturing the transfected cells according to a known microbial culture method and then separating the rabies virus-like particles from the obtained product culture. The culture may contain or contain cells, including both culture supernatant or cell lysate. The separating step may be performed by a method known in the art. Specifically, but not limited thereto, centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution, chromatography, etc. may be performed.

Among the terms or elements mentioned in the above rabies virus-like particles, such as those mentioned in the description of the method for preparing the claimed rabies virus-like particles, it is understood that they are as mentioned in the description of the above-claimed manufacturing method. .

Another aspect provides a method of preventing or treating a rabies virus infectious disease comprising administering the vaccine composition to an individual other than human.

The term "administration" means introducing a predetermined substance to a patient by any suitable method, and is formulated for human or veterinary use and administered by various routes. The rabies virus-like particles may be administered by a parenteral route, such as intravascular, intravenous, intraarterial, intramuscular or subcutaneous, orally, nasal, rectal, transdermal, or by inhalation via aerosol. Alternatively, it may be administered by a bolus or may be slowly injected, but as an example, it may be administered through a nasal route.

The vaccine composition is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" refers to an amount sufficient to exhibit a vaccine effect and an amount that does not cause side effects or serious or excessive immune responses, and the effective dose level is the disorder to be treated, the severity of the disorder, Activity of a particular compound, route of administration, rate of removal of the rabies virus-like particles, duration of treatment, drugs used in combination or concurrent with the virus-like particles, age, weight, sex, diet, general health condition of the subject, And factors known in the pharmaceutical industry and in the medical field. Various general matters to be considered when determining a “therapeutically effective amount” may take into account those known to those skilled in the art.

The vaccine composition may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent. And can be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all the above factors, and can be easily determined by a person skilled in the art.

The term “individual” refers to a living organism capable of infecting a rabies virus and developing a disease due to the infected rabies virus, and may preferably be a mammal, but is not particularly limited thereto. The mammal may be selected from the group consisting of bats, red foxes, skunks, raccoons, badgers, dogs, wolves, mongooses, coyotes, ferrets, dogs and cats.

In one embodiment, the prevention or treatment of the rabies virus infectious disease may be achieved by inducing a protective immune response against the rabies virus. The immune response is the production of antibodies, B cells, helper T cells, inhibitory T cells, cytotoxic T cells, gamma-delta T cells, specifically directed against antigens or antigens contained in a vaccine composition or a vaccine comprising the same. Or activation, exhibiting a therapeutic or protective immunological response in the host, thereby enhancing resistance to new infections or reducing the clinical severity of the disease, but is not limited thereto.

Among the terms or elements mentioned in the above rabies virus-like particles, the same as those mentioned in the description of the method of preventing or treating the claimed viral infectious disease are mentioned in the description of the method of preventing or treating the claimed viral infectious disease. It is understood to be the same.

All technical terms used in the present application, unless otherwise defined, are used in the same sense as those of ordinary skill in the art generally understand in the related field of the present invention. In addition, although preferred methods or samples are described in the present specification, those similar or equivalent are included in the scope of the present application. The contents of all publications referred to herein by reference are incorporated herein by reference in their entirety.

Rabies virus-like particles including vesicular stomatitis virus into which the gene encoding the rabies virus G protein according to one aspect is introduced, rabies vaccine composition comprising the same, and methods for preventing or treating rabies virus infectious diseases using the same are conventional Unlike ERA-based live vaccine methods, stability is secured and excellent immunogenicity is shown, thereby effectively preventing rabies virus infection.

1 shows the results of Western blotting confirming the expression of the G protein of the vector in which the G protein gene was cloned.
Figure 2 shows the Western blotting results confirming the expression of G protein in 293 T cells transformed with the vector.
3 shows a method of preparing rabies virus-like particles by infecting 293 T cells, which are host cells expressing G protein, with VSVluc-VSVG.
FIG. 4 shows a luminase assay in which the 293 T cells were infected with VSVluc-VSVG, and a virus-like result confirmed the expression of the rabies virus G protein in the particles.
Figure 5 shows a mouse inoculation scheme of PBS, RV, VSV-Bald or VSV-RV and a method for evaluating immunogenicity thereafter.
6 shows the immunogenicity evaluation results of VSV virus-like particles containing rabies virus G protein.

It will be described in more detail through the following examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

<Example 1> Rabies virus glycoprotein (GP) gene production

In order to express the rabies virus G protein (GP: glycoprotein) gene, a G protein gene (ERA strain) was synthesized. In addition, in order to increase the expression rate of the gene in eukaryotic cells, Kozak sequence was added and codon-optimization was performed. As a result, the nucleotide sequence of SEQ ID NO: 1 encoding the G protein was prepared. The amino acid sequence encoded from the nucleotide sequence of SEQ ID NO: 1 is the same as the natural variant in which L, which is the 27th amino acid sequence of the G protein of the known rabies virus (ERA strain) (UniProtKB-P03524), is substituted with P.

<Example 2> Preparation of rabies virus-like particles using vesicular stomatitis virus (VSV)

In order to prepare rabies virus-like particles (VLP), a vesicular stomatitis virus (VSV) system known to have excellent immunogenicity was used. Specifically, in order to produce rabies virus-like particles, the G protein gene prepared in Example 1 was introduced into a cloning vector. After the gene was introduced at the BamHI and XhoI restriction enzyme sites, in order to confirm the introduction of the G protein gene, the gene was cut out using BamHI and XhoI restriction enzymes.

As a result, as shown in FIG. 2, it was confirmed that the gene size and position of the gene on the agarose gel were confirmed, and the introduction of the rabies virus G protein gene was successfully performed.

<Example 3> Confirmation of the expression of the cloned rabies virus G protein

In order to express the G protein from the rabies virus G protein gene cloning vector introduced in Example 2, 293 T cells were transformed. In order to confirm the expression of the G protein in the transformed cells, the cell line was crushed after 2 days, and Western blotting was performed to confirm the expression of the rabies virus G protein in the crushed cell contents.

As a result, as shown in FIG. 3, it was confirmed that the rabies virus G protein corresponding to about 58 kDa was expressed.

< Example 4> Confirmation of the presence of rabies virus G protein in rabies virus-like particles

As shown in Figure 4, for the production of rabies virus-like particles using vesicular stomatitis virus (VSV), which express rabies virus G protein, 293 T cells expressing rabies virus G protein were infected with VSVluc-VSVG. Made it. 1-2 days after infection, the supernatant was collected. In order to confirm the virus in the supernatant, the virus was purified using an ultra-high speed centrifuge, and only pure virus-like particles were purified. To confirm the rabies virus G protein in the purified virus-like particles, western blotting was performed.

As a result, as shown in Fig. 5A, it was confirmed that the rabies virus G protein corresponding to about 58 kDa was present.

In addition, BHK21 cells susceptible to rabies virus were infected with the supernatant, and after 18 hours, the amount of protein reported in the cell was measured by luciferase assay.

As a result, as shown in Fig. 5B, it was confirmed that a large amount of luciferase was expressed compared to the BALD group not expressing the rabies virus G protein. Through this, it can be confirmed that a large amount of rabies virus G protein is present in rabies virus-like particles based on vesicular stomatitis virus (VSV) into which the rabies virus G protein gene has been introduced.

<Example 5> Evaluation of immunogenicity of VSV virus-like particles containing rabies virus G protein

For the immunogenicity evaluation of VSV virus-like particles containing rabies virus G protein, Balb/c mice were purchased and divided into the following groups. Each group used 5 mice. The first group was inoculated with PBS only (PBS), the second group was inoculated with rabies virus (RV), the third group was inoculated with VSV virus-like particles (VSV-Bald), and the fourth group was inoculated with rabies virus G protein. VSV rabies virus-like particles containing rabies were classified into groups inoculated (VSV-RV).

As shown in FIG. 6, inoculation was performed according to each experimental group and control group, and 2 weeks after the first inoculation, boosting was performed. Two weeks after the second inoculation, serum was collected from all mice, and the serum antibody production rate (antibody titer) and neutralizing ability (virus 　 neutralizing 　 capacity) were compared.

As a result, as shown in FIG. 7, the antibody generation rate was measured as the highest value in the VSV-RV group, which indicates a similar value to the group inoculated with the rabies virus itself. In addition, the virus neutralizing ability was also measured as the highest value in the VSV-RV group, which was 16 times higher than that of the group inoculated with the rabies virus itself, indicating excellent immunogenicity.

In other words, when a VSV-based pseudo-type rabies virus containing G protein, which is the main antigenic protein of rabies virus, is produced, and rabies virus-like particles produced therefrom are used, excellent immunogenicity is shown, and stability is secured and novel It can be seen that the limitations of the existing rabies vaccine (live ERA vaccine) can be overcome by preparing for defense against the emergence of outdoorism.

So far, the present invention has been looked at around its preferred embodiments. Those of ordinary skill in the art to which the present invention pertains will be able to understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative point of view rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the above description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

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Claims (10)

As a rabies virus-like particle, produced by a vesicular stomatitis virus into which a gene encoding the G protein of rabies virus consisting of the nucleotide sequence of SEQ ID NO: 1 is introduced, the number Rabies virus-like particles, wherein the rabies G protein is expressed in a follicular stomatitis virus envelope.
delete delete A rabies vaccine composition for preventing or treating rabies virus infectious diseases, comprising the rabies virus-like particles of claim 1 as an active ingredient.
The rabies vaccine composition according to claim 4, further comprising a pharmaceutically acceptable excipient, diluent or carrier.
The method of claim 4, wherein the vaccine composition is selected from the group consisting of a composition for intramuscular administration, subcutaneous administration, intraperitoneal administration, intravenous administration, intravenous administration, dermal administration, ocular administration, and brain administration, Rabies vaccine composition.
Transfecting a host cell expressing the G protein of the rabies virus encoded by the gene consisting of the nucleotide sequence of SEQ ID NO: 1 with a bullous stomatitis virus; And
A method for producing a rabies virus-like particle comprising the step of obtaining rabies virus-like particles from the transfected host cell.
delete A method of preventing or treating a rabies virus infection disease comprising administering the composition of any one of claims 4 to 6 to an individual other than a human.
The method of claim 9, wherein the individual is a mammal selected from the group consisting of bats, red foxes, skunks, raccoons, badgers, dogs, wolves, mongooses, coyotes, ferrets, dogs, and cats, preventing or treating rabies virus infectious diseases. How to.

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