KR20140036610A - Vaccine against rsv - Google Patents

Abstract

The present invention relates to: recombinant baculovirus transformed to express G protein of respiratory syncytial virus (RSV) into a form which is contained in the capsid of baculovirus; and a vaccine composition against respiratory syncytial virus containing the recombinant baculovirus or virus particles thereof as an active ingredient. The vaccine composition of the present invention uses G protein of RSV with excellent immunogenicity as an antigen while improving safety, thereby being widely applied for prevention and treatment of diseases caused by infection of RSV.

Description

Vaccine against RSV

The present invention relates to a vaccine against respiratory syncytial virus, and more particularly, the present invention is transformed so that the G protein of Respiratory syncytial virus (RSV) is expressed in a form contained in the envelope of the baculovirus. It relates to a recombinant baculovirus and a vaccine composition against respiratory syncytial virus comprising the recombinant baculovirus or a virus particle thereof as an active ingredient.

Baculovirus is a type of circular double-stranded DNA, 88-135 Kbp long, enclosed in a rod-shaped nucleocapsid. Baculoviruses are generally utilized as carriers for high levels of recombinant protein production, but they can also be used to cause gene transfer into mammalian cells. Several research groups have attempted to inoculate with recombinant baculoviruses capable of eliciting high levels of humoral and cellular immunity against various antigens, taking note that baculoviruses can be used as vaccine carriers. In addition, it has been reported that immunization with recombinant baculoviruses expressing the hemagglutinin gene of influenza virus elicited a strong innate immune response and protected mice against influenza virus sensitization. Furthermore, there is no baculovirus specific immunity that would inhibit the activity of baculovirus based vaccines in mammals.

Meanwhile, respiratory syncytial virus (RSV) is a virus having non-segmented RNA as a genome and belongs to paramyxoviridae. Infected host cells synthesize 10 proteins, such as NS1, NS2, P, N, M, SH, G, F, M2, and L. Surface antigens are known to be F proteins (fusion proteins), G proteins, and SH proteins. have. G protein and F protein in the outer membrane of the virus with relatively high molecular weight are glycosylated glycosylated and plays the most important role in the immune and antigenic variation of RSV infection. G proteins are involved in attachment to host cells to be infected, F proteins are involved in the formation of giant cells and the penetration and attachment of viruses, and F and G proteins produce neutralizing antibodies. However, since the G protein has the advantage of having very good immunogenicity and low safety, there is a possibility of causing fatal damage to the host animal when the G protein is used as an antigen for immunization. On the other hand, while the F protein has the advantage of excellent safety, but has the disadvantage of very low immunogenicity, there is a possibility that immunity cannot be imparted when the F protein is used as an antigen for immunization. Therefore, G protein and F protein of RSV have not been used as a vaccine for preventing RSV infection.

RSV is a major pathogen of pediatric lower extremity infections and is the major virus causing pneumonia and bronchitis (Brandt et al., 1973; Selwyn et al., 1990). In particular, pulmonary dysplasia, congenital heart disease, cystic fibrosis, infants with cancer or various immunodeficiencies, and adults with immunocompromised conditions before bone marrow transplantation are high-risk groups that can cause fatal infections (McDonald et al., 1982; Chandwani et al., 1990; Bruhn et al., 1977). In the elderly, the infection is similar to that of the influenza virus, and excess mortality is reported in the RSV epidemic than in the influenza epidemic (Lancet 1993 Flemming et al).

There are 100,000 to 200,000 high-risk patients in the United States, with hospitalizations of more than 90,000 RSV infections each year, with about 4,500 deaths reported. In Korea, RSV admitted to pediatric hospitals is prevalent every year, and 60% of all viral lower limb infections isolated from Seoul National University Pediatric Hospital are caused by RSV. In Korea, hospitalization and death due to RSV infection is estimated to be very high. do.

Despite these serious problems, no vaccines or specific therapies are currently available. In particular, formalin-inactivated RSV (FI-RSV) vaccines have been developed in the past and clinical trials have been carried out. Currently, Ribavirin (Virazole) is used as an approved treatment, but its clinical efficacy is not high, and only a few of the infected patients are effective. In addition, it has a high toxicity and has the inconvenience of spraying in the form of aerosols and is only used in some limited cases. On the other hand, studies have been actively attempted to prepare a vaccine using the F protein or the G protein of the RSV, F protein is relatively safe but low immunogenicity, G protein is relatively good immunogenicity but safety There was a problem of being low. If it is possible to improve the immunogenicity of the F protein or improve the safety of the G protein, it is expected to be able to effectively prepare a vaccine against RSV, but no results have been reported yet.

Under these circumstances, the present inventors have diligently researched to prepare a vaccine against RSV. As a result, using recombinant baculovirus or viral particles expressing the G protein of RSV in the viral envelope, the inherent immunogenicity of G protein is maintained. While maintaining, it was confirmed that the safety could be improved, and the present invention was completed.

One object of the present invention is to provide a recombinant baculovirus transformed such that the G protein of RSV is expressed in a form contained in the envelope of the baculovirus.

Another object of the present invention is to provide an RSV vaccine composition comprising the recombinant baculovirus or viral particles thereof as an active ingredient.

According to an embodiment of the present invention for achieving the above object, there is provided a recombinant baculovirus transformed so that the G protein of RSV is expressed in a form contained in the envelope of the baculovirus.

As used herein, the term "G protein of RSV" is a major adhesion protein of RSV, which plays an important role in the defense of RSV infection, and does not have an MHCI species-restricted epitope, resulting in cytotoxic T lymphocytes. Does not induce an immune response, but has a I-Ed-limited immunodominant epitope region (amino acids 183-195) capable of partially inducing CD4 T cells, wherein the RSV Wild type nucleotide and amino acid sequences of G proteins are known in the art (Wertz et al., Proc. Natl. Acad. Sci. USA 92: 4075-4079, 1985; Satake et al., Nucl.Acids Res. 13) 21): 7795-7810, 1985). For the purposes of the present invention, the G protein of RSV may be used as an antigen expressed in baculovirus to generate or enhance immunity to RSV, wherein the G protein of RSV is preferably a whole wild type protein, a fragment thereof, or an amino acid thereof. It may be a sequence variant or a multimer to which they are linked, more preferably the entire wild type protein, most preferably a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, but is not particularly limited thereto.

Wild type G protein of RSV (SEQ ID NO: 1)

MSKNKDQRTAKTLERTWDTLNHLLFISSCLYKLNLKSVAQITLSILAMIISTSLIIAAIIFIASANHKVTPTTAIIQDATSQIKNTTPTYLTQNPQLGISPSNPSEITSQITTILASTTPGVKSTLQSTTVKTKNTTTTQTQPSKPTTKQRQNKPPSKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPNKKPGKKTTTKPTKKPTLKTTKKDPKPQTTKSKEVPTTKPTEEPTINTTKTNIITTLLTSNTTGNPELTSQMETFHSTSSEGNPSPSQVSTTSEYPSQPSSPPNTPRQ

In the present invention, the G protein of RSV is expressed in a form inserted into the envelope of the baculovirus to improve the safety of the G protein, thereby preventing the safety problem that is a problem that occurs when the G protein of the existing RSV is used as an antigen for immunity. There is an advantage to overcome.

According to the prior art (patent registration No. 938105) to which the inventor has applied for and patented, it is disclosed that the use of a fragment of G protein of RSV or repeated use of the fragment three times can induce immunity more effectively. Therefore, the fragment of the G protein of the RSV is not particularly limited thereto, but preferably, a fragment comprising the amino acid sequence 120 to 230 from the N-terminus of the polypeptide comprising the amino acid sequence of SEQ ID NO: 1 may be used. More preferably, fragments containing the 130-230 amino acid sequences of the polypeptide can be used.

As used herein, the term "amino acid sequence variant" refers to a protein in which the natural amino acid sequence of the protein and one or more amino acid residues have a sequence that differs by deletion, insertion, non-conservative or conservative substitution, or a combination thereof. For the purposes of the present invention, the amino acid sequence variant refers to an amino acid sequence variant of the G protein of RSV, which variant enhances the glycosylated form, the lipidated form, antigen presentation of the G protein of RSV and the antigen. It may be a form derivatized to include a molecule that enhances the target to the antigen-presenting cells of, but is not particularly limited thereto.

Finally, in order to further enhance the immunogenicity of the G protein of RSV, the multimer of the G protein of RSV has an amino acid sequence of 2 or more, preferably 3 to 8, full length or fragment of G protein of RSV, More preferably, three to six may be in the form of covalently linked directly or through a linker, and more preferably in the form of a trimer linked to three, but is not particularly limited thereto. In the multimer, when connected through a linker, it is not particularly limited, but preferably glycine, alanine, leucine, isoleucine, proline, serine, threonine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid , Amino acids such as lysine and arginine acid, and the like, and more preferably valine, leucine, aspartic acid, glycine, alanine, proline, and the like, may be linked to each other. In consideration of ease, one to five amino acids such as glycine, valine, leucine, and aspartic acid may be linked to each other.

The term "baculovirus" of the present invention refers to a virus comprising 130-200 kbp cyclic double-stranded genomic DNA in a rod-shaped nucleocapsid, which mainly infects insect cells and causes a fatal disease. Polynucleotides encoding proteins derived from eukaryotic cells are introduced into host eukaryotic cells (eg, insect cells) and used as gene vectors for performing a series of experiments for expressing the proteins in the host cells.

As used herein, the term "recombinant baculovirus" refers to a baculovirus that is transformed by inserting a polynucleotide encoding the G protein of RSV into the genomic DNA of the baculovirus to be operable. The G protein of RSV is expressed in the form inserted into the envelope of the baculovirus. Typically, baculovirus is a series of experiments for introducing polynucleotides encoding proteins from eukaryotic cells into host eukaryotic cells (eg, insect cells) by gene recombination methods to express the proteins in the host cells. Although it is used as a gene vector for carrying out the present invention, the present invention is not used as a vector for transferring genes to eukaryotic cells, but a recombinant produced using the baculovirus as a host expressing the G protein of RSV in its envelope. Provide baculovirus. The recombinant baculovirus of the present invention can use itself or its viral particles as an active ingredient of a vaccine for preventing or treating infection of RSV. The G protein of RSV expressed in the envelope of the recombinant baculovirus typically provides enhanced safety for the host, unlike G protein, which is low in safety. Therefore, the G protein was not used for vaccine development of RSV due to safety issues. The conventional problem can be solved.

According to one embodiment of the present invention, a base sequence encoding the RSV G protein of the RSV A2 strain is introduced to produce a recombinant baculovirus Bac-RSV / G capable of expressing the RSV G protein (FIGS. 1A and 1B). This was immunized by inoculating BALB / c mice (FIG. 2A), and it was confirmed that RSV G-specific serum IgG and intratracheal IgA were effectively induced in the immunized BALB / c mice (FIG. 2B). In addition, G protein-specific CD4 + cells and IFN-γ + cells were detected in lung tissue of RSV-sensitized mice after immunization (FIG. 3), and the frequency of G-specific IL-17-producing CD4 T cells was increased. Since it was confirmed that (Fig. 4), the mixed G-specific Th1 / Th17-cell responses were induced by Bac-RSV / G vaccination. In addition, as a result of measuring the concentration of eosinophils contained in bronchial alveolar fluid of RSV-sensitized mice after immunization, eosinophilia was not induced (FIG. 6a), and weight loss and disease index were not changed (FIG. 6b). It was confirmed that replication of RSV was prevented in the lung (FIG. 7).

According to another embodiment of the present invention for achieving the above object, the present invention comprises the recombinant baculovirus or viral particles thereof as an active ingredient, RSV for preventing or treating a disease caused by infection of RSV Provide a vaccine composition.

RSV vaccine composition provided by the present invention includes the recombinant baculovirus as an active ingredient, it can exhibit the advantages of the recombinant baculovirus as it is. In other words, G protein of RSV expressed in the envelope of recombinant baculovirus typically provides enhanced safety for the host, unlike G protein, which is less safe. Therefore, it has excellent immunogenicity and can enhance immunity when used as a vaccine. In addition to the advantages of conventional G proteins, it may exhibit the advantages of recombinant baculoviruses that can increase stability to the host. Since the characteristics of the recombinant baculovirus completely solve the conventional problems that provided difficulties in the preparation of a vaccine against RSV, it can be used to effectively prevent or treat diseases caused by RSV infection.

As used herein, the term "disease caused by infection of RSV" refers to a respiratory disease caused by infection of RSV in an animal including a person equipped with a respiratory system, but is not limited thereto, but cough, Sneezing, high fever, wheezing, bronchitis, bronchiolitis, pneumonia, asthma, bronchitis, croup and respiratory failure.

As used herein, the term "prevention" means any action that inhibits or delays the onset of a disease caused by RSV infection by administration of a composition.

The term "treatment" of the present invention means any action that improves or benefits the symptoms of a disease already caused by RSV infection due to administration of the vaccine composition of the present invention.

The recombinant baculovirus or viral particles thereof of the present invention can be administered as a vaccine to induce immunity against respiratory diseases caused by RSV infection, wherein the recombinant baculovirus or viral particles thereof are suitably combined with a pharmaceutically acceptable carrier. Can be formulated. Pharmaceutically acceptable carriers can be used as oral administration binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments and flavors, and in the case of injections, physiological saline And non-aqueous solvents such as aqueous solvents such as ring gel solution, vegetable oils, higher fatty acid esters (e.g., oleic acid, etc.), and alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.). Buffers, preservatives, analgesic agents, solubilizers, isotonic agents, stabilizers and the like can be used in combination. In the case of propellants, suitable propellants, such as compressed air, nitrogen, carbon dioxide, or hydrocarbon-based low boiling point solvents, etc., may be conveniently delivered in the form of aerosol spray presentation from pressurized packs or nebulizers.

Formulations of the pharmaceutical compositions of the present invention may be prepared in a variety of ways by mixing with pharmaceutically acceptable carriers as described above. For example, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers and the like in the case of oral administration. In the case of injections, it can be prepared in unit dosage ampoules or in multiple dosage forms.

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

The term "administration" of the present invention means introducing any substance into a patient by any suitable method and is formulated for human or veterinary administration and administered by various routes. The recombinant baculovirus or viral particles thereof of the present invention can be administered by parenteral routes such as intravascular, intravenous, intraarterial, intramuscular or subcutaneous and can be administered orally, nasal, rectal, transdermal or aerosol. It may be administered by inhalation route, may be administered by bolus, or may be injected slowly, but preferably the vector is administered by nasal route.

The vaccine composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to exhibit a vaccine effect and an amount not to cause side effects or serious or excessive immune responses, and an effective dose level refers to the disorder or disorder to be treated. Severity, activity of a particular compound, route of administration, rate of removal of recombinant baculovirus or viral particles thereof, duration of treatment, drugs used in combination or concurrently with recombinant baculovirus or viral particles thereof, age, weight, sex of the subject And various factors including dietary habits, general state of health, and factors known in the pharmaceutical and medical arts. Various general considerations considered in determining a "therapeutically effective amount" are known to those skilled in the art and are described, for example, in Gilman et al., Eds., Goodman And Gilman's: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press, 1990 And Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., 1990. In the case of administering the recombinant baculovirus or viral particles thereof of the present invention, the number to be administered in one administration is usually about 1 × 10 ⁷ to 1 × 10 ¹¹ , more preferably 1 × 10 ⁸ to 5 × 10 ¹⁰ , most preferably 5 × 10 ⁸ to 2 × 10 ¹⁰ .

Vaccine compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. And can be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art.

On the other hand, in order to further improve immunogenicity against RSV, the vaccine composition of the present invention may further include a recombinant baculovirus or viral particles thereof containing all or part of the RSV F protein, and all or part of the RSV M2 protein. It may further include a recombinant baculovirus or viral particles thereof, and may further include a recombinant baculovirus or viral particles thereof including all or a portion of RSV F protein and all or a portion of RSV M2 protein. As long as the purpose of improving the immunogenicity against RSV can be achieved, the kind of RSV-derived protein further included is not particularly limited.

According to another embodiment of the present invention for achieving the above object, the present invention comprises the step of administering the RSV vaccine composition to a subject in anticipation of infection or disease caused by RSV infection Provided are methods for preventing or treating a disease caused by an infection.

The term "individual" of the present invention means a living organism that can infect RSV and develop respiratory diseases due to infected RSV. Preferably, the term "individual" includes higher respiratory organs. It may be a vertebrate, more preferably a mammal, most preferably a primate, but is not particularly limited thereto.

Since the vaccine composition of the present invention improves safety while using the G protein of RSV having excellent immunogenicity as an antigen, it may be widely used for the prevention and treatment of diseases caused by RSV infection.

Figure 1 shows the structure and characteristics of the Bac-RSV / G vaccine. (A) The shuttle vector pFastBac-RSV / G was designed and produced by the method of the example. The vector was used to prepare Bac-RSV / G recombinant baculovirus. (B) The presence of RSV G protein in Bac-RSV / G was confirmed by Western blot analysis. That is, virus particles were prepared by the method of the example, and Western blot analysis was performed using RSV G-specific monoclonal antibody.
2 shows the humoral immune response induced by the Bac-RSV / G vaccine. (A) Inoculated 2 × 10 ⁸ PFU of recombinant baculovirus (Bac-RSV / G or Bac-control) into BALB / c mice twice by nasal route, boosted by serum ELISA at 2 weeks The titer of the structural anti--RSV IgG antibody was measured. (B) Secretory IgA titers were measured in BAL solution of each immune group 5 days after RSV sensitization. The results were expressed as Log2 dot values of 4-5 mice. ND indicates that it was not detected.
3 shows G-specific Th1-cell responses in Bac-RSV / G-immunized mice. (A) Mice were nasal immunized with Bac-RSV / G, Bac-control or PBS, and RSV A2 was sensitized 4 weeks after boosting. Five days after sensitization, lung monocytes were obtained from the lungs of the same group of mice, and stimulated with or without addition of G183-195 peptide (+). Cells were stained with CD4, CD44 and IFN-γ and analyzed by flow cytometry. Cells in which CD4 was detected are each plotted and the ratio is expressed as the frequency of G-specific IFN-γ-positive cells. (B) The mean value of the data is expressed as mean ± standard deviation.
4 shows G-specific Th17-cell responses in Bac-RSV / G-immunized mice. (A) Mice were nasal immunized with Bac-RSV / G, Bac-control or PBS, and RSV A2 was sensitized 4 weeks after boosting. Five days after sensitization, lung monocytes were obtained from the lungs of the same group of mice, and stimulated with or without addition of G183-195 peptide (+). Cells were stained with CD4, CD43 and IL-17A and analyzed by flow cytometry. Cells from which CD4 was detected are each plotted and the ratio is expressed as the frequency of G-specific IL-17A-positive cells. (B) The mean value of the data is expressed as mean ± standard deviation.
5 shows that Bac-RSV / G immunization in the lung does not induce Th2 cytokines. Mice were immunized and sensitized in the same manner as in FIG. 3. Five days after sensitization of RSV, lungs were removed from mice, and cytokine levels were assessed in the supernatants of the lungs using multiplex antibody-based assays (FlowCytomix). Bars represent mean values of 4-5 mice per group and data were obtained by two independent experiments.
FIG. 6 shows that Bac-RSV / G immunization does not promote lung eosinophilia as well as adverse symptoms that may be increased by the vaccine. (A) Mice were immunized and sensitized in the same manner as in FIG. 3. Five days after BAL RSV sensitization, BAL cells were stained with antibodies against CD45, Siglec-F, and CD11c, and eosinophils were quantified in CD45 + − cells. The mean ratio of Siglec-F + CD11c-cells in total CD45 + cells was expressed as mean ± SD (n = 6). (B) RSV was sensitized to the same group of immunized mice and evaluated daily. Results were expressed as mean ± SEM from 5 mice in each group.
7 shows immunoprotection by Bac-RSV / G vaccination against RSV sensitization by respiration. Each group of immunized mice was sensitized with 1 × 10 ⁶ PFU of RSV A2 after 4 weeks, and at 5 days, virus replication levels in the lungs were determined by plaque assay. Results are expressed as mean ± SEM of 4-5 mice per group. The detection limit is 200 PFU per gram of lung. MD represents an experimental group that was not detected.
8 is a graph showing changes in the level of eosinophils after RSV sensitization after treatment of the vaccine of the present invention.

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

Example  One: Baculovirus  And RSV Preparation of

Baculovirus was propagated using Sf9 (Spodoptera frugiperde 9) cell line cultured in serum free medium (SF-900, Invtrogen) at 27 ° C. RSV A2 strains were grown in DMEM medium (Life Technologies) with 3% heat-inactivated fetal bovine serum, 2 mM glutamine, 20 mM HEPES, non-essential amino acids, penicillin and streptomycin, and using known methods. The infectivity of the strain was titrated (Park SH, et al., Immune Netw., 7; 186-196, 2007).

Example  2: recombination Baculovirus  Production and production

The base sequence encoding the RSV G protein of the RSV A2 strain was amplified from cDNA by PCR and cloned into a pFastBac-1 vector (EcoRI / Xho I), a shuttle vector capable of introducing the amplified sequence into baculovirus. The recombinant vector pFastBac-RSV / G was obtained, and the obtained recombinant vector was applied to the Bac-to-Bac ^® system to produce a recombinant baculovirus Bac-RSV / G capable of expressing RSV G protein (FIG. 1A). . 1A is a cleavage map of the recombinant vector pFastBac-RSV / G.

The culture supernatant of sf9 insect cells infected with the Bac-RSV / G was placed in a 25% (w / v) sucrose solution containing 5 mM NaCl and 10 mM EDTA, and centrifuged using a SW28 rotor (Beckman, USA). (4 ° C., 24,000 rpm, 75 minutes) was carried out to obtain a precipitate, which was suspended in PBS and centrifuged again (4 ° C., 24,000 rpm, 4 hours) to obtain a precipitate to obtain Bac-RSV / G. Purified. The precipitate was suspended in PBS and titrated by plaque analysis using sf9 insect cells.

In order to confirm whether the purified Bac-RSV / G expresses RSV G in the virus particles, Western blot analysis using RSV G protein-specific monoclonal antibody was performed (FIG. 1B). At this time, a wild type baculovirus without a recombinant vector was used as a negative control, and a RSV particle purified in HEp-2 cells was used as a positive control. Figure 1b is a photograph showing the results of Western blot analysis to confirm the expression of RSV G protein in Bac-RSV / G. As shown in FIG. 1B, no band was detected in the negative control, but an RSV G protein of about 70 kDa to 90 kDa was detected in Bac-RSV / G, and a larger RSV G protein was detected in the positive control.

The difference between the experimental group and the positive control group was analyzed to be due to the difference in glycation patterns in insect cells and human cells.

Example  3: immunization and inoculation

Female BALB / c mice were purchased from Charlse River Laboratories Inc. (Yokohama, Japan) and maintained SPF (specific-pathogen-free) status. For immunization, 6 to 8 week old mice were inoculated with baculovirus intranasally. For intranasal inoculation, mice were lightly anesthetized with ether / chloroform and inoculated into the left nasal cavity by titrating 70 × volume of Bac-RSV / G or Bac-control of 2 × 10 ⁸ PFU. 3-4 weeks after the second immunization, mice were inoculated intranasally with 1 × 10 ⁶ PFU of live RSV A2 strain. All animal experiments were conducted according to the guidelines of the Animal Experimentation Ethics Committee.

RSV G-specific antibody concentrations in the serum of mice were measured by ELISA from 2 weeks after priming to determine immunogenicity. Blood for ELISA experiments was obtained from retro-orbital plexus using heparinized capillary tubes, collected in Effendorf tubes, and serum was stored at -20 ° C after centrifugation. Titers of RSV G-specific antibodies were measured by direct ELISA method. Briefly, 0.5 µg / ml of purified G protein fragment diluted in PBS was coated in a 96 well plate at 100 µl / well for overnight, followed by 2 ml of PBS containing 1% skim milk powder and 0.05% Tween 20. Blocked for time. Then, serial serum dilutions were added and incubated for 2 hours. Plates were washed five times with PBS containing 0.05% Tween 20, and with various dilutions of HRP-conjugated affinity purified rabbit anti-mouse total IgG secondary antibodies bound to HRP. Incubate for minutes. The plate was washed five times and developed by treatment with 3,3 ', 5,5'-tetramethylbenzadine, and then the color reaction was stopped by addition of H ₃ PO ₄ and analyzed at 450 nm with a Thermo plate reader. It was. At this time, the wells containing no serum were used to determine the cutoff value.

As a result, no RSV G-specific antibody responses were detected in the sera of all mouse groups at 14 days post-priming, but after booster immunization, the mean titer of serum antibodies significantly increased only in the mice immunized with Bac-RSV / G. (FIG. 2A).

On the other hand, because secretory IgA directly mediates local immunity against atmospheric pathogens, it plays an important role for antibodies in the defense of the upper respiratory tract, and therefore, on airway mucosal surfaces to develop effective RSV vaccines. It is necessary to induce secretion of IgA. To determine whether Bac-RSV / G vaccination elicited IgA responses in the airways, we performed bronchoalveolar lavage 5 days after RSV inoculation and measured the amount of IgA via RSV-specific ELISA ( 2B). Bac-control or PBS immunized mice did not show a specific IgA response, but Bac-RSV / G-immunized mice showed a significant increase in specific IgA responses, indicating that Bac-RSV / G Intranasal immunization was analyzed to effectively induce both RSV G-specific serum IgG and intratracheal IgA.

Example  4: pulmonary lymphocyte preparation and Flow cell  analysis

A syringe fitted with a 25 gauge needle was used to perfuse 5 ml of PBS containing 10 U / ml heparin (Sigma-Aldrich, St. Louis. MO) into the right ventricle of the rat, and lung tissue was extracted to obtain glutamine, It was immersed in RPMI medium containing gentamycin, penicillin G and 10% FBS (Hyclone, Logan, UT). The isolated lung tissue was passed through a steel screen to obtain a single cell suspension, and particulate matter was removed from the suspension using a 70 μm Falcon cell strainer (BD Labware, Franklin Lakes, NJ). Removed. The particulate-free suspension was subjected to concentration gradient centrifugation to obtain lung cells, and the obtained lung cells were resuspended in PBS containing 3% FBS, 0.09% NaN ₃ , followed by phosphor-binding antibodies. Staining was performed using. In this case, an anti-CD4 antibody (clone RM4-5), an anti-CD44 antibody (clone IM7), or an anti-CD43 antibody (clone 1B11) (BD PharMingen, USA) was used. The stained lung cells were fixed using PBS containing 2% paraformaldehyde and subjected to flow cytometry by application to a flow cytometer (FACSCalibur, BD Bioscience, USA).

In addition, to determine whether Bac-RSV / G immunization induced specific CD4 T-cells, RSV was sensitized to mice immunized with Bac-RSV / G, Bac-control, or PBS and then I-Ed. ^- by measuring the production level of IFN-γ in the lung lymphocytes were stimulated with G-limiting epi top peptides were evaluated CD4 cellular response. In order to measure the production level of IFN- [gamma], IFN- [gamma] present in lung cells was stained by a known method. That is, ² × 10 ⁶ pulmonary lymphocytes were incubated at 5% CO ₂ and 37 ° C. for 5 hours with or without 10 μM G (183-195) protein (WAICKRIPNKKPG), and Brefeldin A (5 μg / mL) (Sigma-Aldrich) was added to accumulate IFN-γ in the cells. The cells were then stained, washed and fixed with surface markers, immersed in fluorescence-activated cell assay buffer containing 5% saponin (Sigma-Aldrich, Seoul, Korea), followed by anti-IFN-γ antibody (clone XMG1.2) was used to stain IFN-γ present in the cells. At this time, the killed cells were determined by the anterior and site dispersion pattern, which was removed. Data was collected with CELLQuest software (BD Bioscience) and analyzed using CELLQuest and WinMDI version 2.9 software (Scripps Research Institute, La Holla, Calif.) (FIG. 3). As shown in FIG. 3, G-specific CD4 + IFN-γ + cells were found in the lungs of the Bac-RSV / G-immunized group (mean ˜2.5% CD4 cells), whereas the lungs of the Bac-control or PBS-immunized group Almost no specific cells were detected (<0.2% of CD4 cells).

Next, Bac_RSV / G immunization was examined to induce other CD4 + T cell subsets such as Th2 and Th17 (FIG. 4). As shown in FIG. 4, mice treated with Bac-RSV / G-vaccine increased the frequency of G-specific IL-17-producing CD4 T cells in the lung after RSV inoculation (mean ˜3.7% CD4 T cells). ). However, the amount of Th2 cytokines such as IL-4, IL-5, IL-10 and IL-13 was not significantly increased in all mouse groups by multiplex antibody-based assays (FIG. 5). ).

Taken together, it can be seen that mixed G-specific Th1 / Th17-cell responses are induced by Bac-RSV / G vaccination.

Example  5: for eosinophilia Bac - RSV Effect of / G Immunity

It has been reported that immunization with a vaccine virus expressing the whole RSV G glycoprotein results in eosinophilia in the lungs following subsequent sensitization to live RSV. These studies indicate that vaccines expressing RSV G have the potential to develop pulmonary eosinophilia. To determine if the intranasal immune activity of Bac-RSV / G is potentially potential for eosinophilia, the levels of eosinophils in BAL fluid of immune mice were discussed as previously described 5 days after RSV sensitization, Siglec-F, CD45. And CD11c antibody using a flow cytometer (FIG. 6).

As shown in FIG. 6A, Bac-RSV / G-immunized mice showed high levels of CD11c-Siglec-F + in bronchial alveolar compared to Bac-control or PBS groups, and Bac-RSV / G immunized mice The amount of eosinophil influx at was relatively low (~ 2% of total CD45 + BAL). From the above results, it can be seen that intranasal Bac-RSV / G immunization does not increase the risk of vaccine-induced eosinophilia.

As shown in FIG. 6B, in order to evaluate RSV-induced pathology by Bac-RSV / G immunization, weight loss was observed in mice immunized after RSV inoculation, and after live RSV was sensitized, Bac-RSV / G No significant weight loss was observed in the immunized mice, and the disease index did not show any significant variation.

From the above results, it was found that intranasal Bac-RSV / G vaccination caused protective immunity, but the vaccine did not cause other diseases.

Example  6. In the lungs RSV  Threshold and RSV In sensitization  About Bac - RSV / G vaccine's protective effect

To assess whether Bac-RSV / G is protective against RSV sensitization, mice were inoculated with live RSV A2 virus 4 weeks after booster immunization. Specifically, on days 4-5 of RSV inoculation, mice were euthanized a subset and lungs were removed from Eagle's modified essential medium (EMEM). Single cell suspensions were obtained from tissues via a still screen, passed through a 70um cell steiner (BD Labware, Franklin Lakes, NJ) to remove particulate matter, and RSV thresholds were determined via standard plaque assay in a subconfluent Hep2 single cell layer. . Data was then expressed as PFU per gram of lung tissue (FIG. 7). As shown in FIG. 7, active RSV was present in the lungs of mice immunized with BAC-control or PBS, but mice immunized with Bac-RSV / G were found to prevent RSV replication in the lungs when infectivity was at its highest. .

Bac-control immunization resulted in a partial decrease in RSV replication, and previous studies have shown that intranasal inoculation of wild-type baculovirus induces a strong innate immune response, which confers protection against influenza virus infection. Therefore, the results were analyzed to be due to a nonspecific innate immune response caused by inoculation of baculovirus.

Example  7: Variation of Eosinophil Level by Vaccine

It has been reported that immunization with vaccinia virus expressing whole RSV G glycoprotein causes pulmonary eosinophilia after inoculation with live RSV. These studies indicate that RSV G expressed vaccines can cause pulmonary eosinophilia. To determine whether intranasal immunization of Bac-RSV / G can cause eosinophilia, 5 days after RSV inoculation with antibodies to Siglec-F, CD45, and CD11c as previously described Eosinophil levels in bronchial alveolar larvage (BAL) fluid of immune-reacted mice were measured using a flow cytometer.

Specifically, on day 5 after inoculation, tracheotomy was performed at the expense of each group of mice to extract the degree of obstruction from each mouse. The isolated wastewater was washed with 0.8 ml of PBS containing 1% FBS, which was chopped and filtered to obtain bronchial alveolar (BAL) cells. The suspension comprising the obtained cells was treated with anti-Fc receptor antibody (2.4G2) to prevent nonspecific binding, and then the cells were suspended in PBS containing 3% FBS, 0.09% NaN ₃ to obtain a suspension. PE-binding anti-Siglec-F antibody (E50-2440) (BD Pharmingen, USA), APG-binding anti CD-45 antibody (30-F11) (BD Pharmingen, USA) ) And FITC-binding anti-CD-11c antibody (HL3) (BD Pharmingen, USA). After staining, the cells were treated with PBS containing 2% paraformaldehyde and fixed, and flow cytometry was performed using a FACSCallibur flow cytometer (BD Biosciences, San Diego, Calif.) (FIG. 8). 8 is a graph showing changes in the level of eosinophils after RSV sensitization after treatment of the vaccine of the present invention. As shown in Figure 8, the level of eosinophils did not increase when treated with the vaccine of the present invention, the level of eosinophils increased when treated with other vaccines.

These results suggest that the vaccine of the present invention can secure safety without causing additional problems that can be caused by vaccination.

<110> Ewha University-Industry Collaboration Foundation <120> Vaccine against RSV <130> PA120416 / KR <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 298 <212> PRT <213> Respiratory syncytial virus G protein <400> 1 Met Ser Lys Asn Lys Asp Gln Arg Thr Ala Lys Thr Leu Glu Arg Thr   1 5 10 15 Trp Asp Thr Leu Asn His Leu Leu Phe Ile Ser Ser Cys Leu Tyr Lys              20 25 30 Leu Asn Leu Lys Ser Val Ala Gln Ile Thr Leu Ser Ile Leu Ala Met          35 40 45 Ile Ile Ser Thr Ser Leu Ile Ile Ala Ala Ile Ile Phe Ile Ala Ser      50 55 60 Ala Asn His Lys Val Thr Pro Thr Thr Ala Ile Ile Gln Asp Ala Thr  65 70 75 80 Ser Gln Ile Lys Asn Thr Thr Pro Thr Tyr Leu Thr Gln Asn Pro Gln                  85 90 95 Leu Gly Ile Ser Pro Ser Asn Pro Ser Glu Ile Thr Ser Gln Ile Thr             100 105 110 Thr Ile Leu Ala Ser Thr Thr Pro Gly Val Lys Ser Thr Leu Gln Ser         115 120 125 Thr Thr Val Lys Thr Lys Asn Thr Thr Thr Thr Gln Thr Gln Pro Ser     130 135 140 Lys Pro Thr Thr Lys Gln Arg Gln Asn Lys Pro Pro Ser Lys Pro Asn 145 150 155 160 Asn Asp Phe His Phe Glu Val Phe Asn Phe Val Pro Cys Ser Ile Cys                 165 170 175 Ser Asn Asn Pro Thr Cys Trp Ala Ile Cys Lys Arg Ile Pro Asn Lys             180 185 190 Lys Pro Gly Lys Lys Thr Thr Thr Lys Pro Thr Lys Lys Pro Thr Leu         195 200 205 Lys Thr Thr Lys Lys Asp Pro Lys Pro Gln Thr Thr Lys Ser Lys Glu     210 215 220 Val Pro Thr Thr Lys Pro Thr Glu Glu Pro Thr Ile Asn Thr Thr Lys 225 230 235 240 Thr Asn Ile Thr Thr Thr Leu Leu Thr Ser Asn Thr Thr Gly Asn Pro                 245 250 255 Glu Leu Thr Ser Gln Met Glu Thr Phe His Ser Thr Ser Ser Glu Gly             260 265 270 Asn Pro Ser Pro Ser Gln Val Ser Thr Thr Ser Glu Tyr Pro Ser Gln         275 280 285 Pro Ser Ser Pro Pro Asn Thr Pro Arg Gln     290 295

Claims (10)

A recombinant baculovirus transformed such that the G protein of Respiratory syncytial virus (RSV) is expressed in a form contained in the envelope of the baculovirus.
The method of claim 1,
Wherein said G protein is a wild type G protein, fragment or multimer thereof.
3. The method of claim 2,
The wild type G protein is a recombinant baculovirus comprising the amino acid sequence of SEQ ID NO: 1.
3. The method of claim 2,
Wherein said multimer is in the form of three to eight wild type G proteins or fragments thereof covalently linked directly or via a linker.
5. The method of claim 4,
The linker comprises 1 to 5 amino acids selected from the group consisting of glycine, alanine, leucine, isoleucine, proline, serine, threonine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, lysine and arginine acid. Recombinant baculovirus.
An RSV vaccine composition for preventing or treating a disease caused by RSV infection, comprising the recombinant baculovirus of any one of claims 1 to 5 or a virus particle thereof as an active ingredient.
The method according to claim 6,
Vaccine composition further comprising a pharmaceutically acceptable carrier.
The method according to claim 6,
The disease caused by the infection of RSV is selected from the group consisting of cough, sneeze, high fever, wheezing, bronchitis, bronchiolitis, pneumonia, asthma, bronchitis, croup and respiratory failure.
The method according to claim 6,
The vaccine composition further comprising a recombinant baculovirus or viral particles thereof comprising all or part of the RSV F protein.
The method according to claim 6,
The vaccine composition further comprising a recombinant baculovirus or viral particles thereof comprising all or part of the RSV M2 protein.

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