TWI522469B - Novel multivalent vaccine against enterovirus, influenza virus, and/or japanese encephalitis virus - Google Patents

Description

Novel multivalent vaccine against enterovirus, influenza virus and/or Japanese encephalitis

The present invention relates to a multivalent vaccine comprising a deactivated enterovirus antigen, more particularly to a deactivated enterovirus antigen and selected from: deactivated influenza virus; deactivated Japanese encephalitis virus; or deactivated influenza virus mixed A multivalent vaccine that activates the antigen of Japanese encephalitis virus, and the preparation and use of such multivalent vaccines.

Enterovirus belongs to the family Picaraviridae and is a general term for a group of viruses. Before 1997, the known and classified enteroviruses shared a total of type 3 (types 1 to 3) of poliovirus (Poliovirus), Keshaqi. Virus (Coxsackievirus), containing 23 types of A (A1 to A22, A24) and 6 B (B1-B6), Echovirus (type 30 to 1), but 8, 10 And type 28 (except type 28) and Enterovirus (type 68 ~) and other 60 types, in recent years, have repeatedly found a variety of types, according to the results of gene sequence analysis, reclassified into human enterovirus A, B, C, D (Human enterovirus A, B, C, D) type, wherein enterovirus 71 is classified as human enterovirus type A.

Among all enteroviruses, in addition to poliovirus, enterovirus type 71 (Enterovirus Type 71) is the most susceptible to neurological complications. This virus was first isolated in a 1969 California epidemic. Cases that cause many cases of aseptic meningitis and encephalitis. Since then, there have been popular reports in Australia, Japan, Sweden, Bulgaria, Hungary, France, Hong Kong, Malaysia and other places. Taiwan has also been popularized more than a decade ago. It can be seen that the distribution of this type of enterovirus is worldwide.

Enterovirus can cause a variety of diseases clinically, many of which are asymptomatic, while others have only fever or similar cold symptoms, but some have special clinical manifestations, such as hand-foot-mouth disease, herpes. Apharyngitis (herpangina), aseptic meningitis, viral encephalitis, limb paralysis syndrome, acute hemorrhagic conjunctivitis, infant acute myocarditis and adult pericarditis, epidemic rib pain, acute lymph nodes Acet lymphonodular pharyngitis, febrile illness with rash, etc.

According to clinical statistics, the most common and the highest mortality rate is enterovirus 71. If children under the age of five are infected with enterovirus, there will be more serious complications, about 1000 to 10000. The gene of human immunity is very complicated, so it is impossible to know for sure why young children under the age of five have a high mortality rate. In addition, cases are also regionally concentrated, so the living environment should be one of the important factors for enterovirus disease. . At present, there is still no vaccine to prevent enterovirus infection, and there is no specific medicine after infection, which can only strengthen personal health care. At present, a number of companies or research institutions have obtained government approval to start human clinical trials. However, there have been no previous proposals for the mixing of enteroviruses with other antigens, in particular, the mixing of antigens such as enterovirus, influenza virus, and Japanese encephalitis virus into a multivalent vaccine.

Regarding the prevention of seasonal influenza and new influenza, the current vaccination is based on the "flu vaccination program" of the Department of Health's Disease Management Bureau. However, based on the statistics of seasonal influenza vaccines and the experience of new influenza vaccination in 2009, the willingness of parents to bring flu vaccine to their children is very low, which makes it difficult to prevent influenza.

The current flu vaccine is still mixed with three strains that are most likely to cause a pandemic, and the virus strain is replaced annually. In the future, for the issue of virus variability, international pharmaceutical companies such as Novartis Pharmaceuticals, Dutch merchants GlaxoSmithKline (GSK), and Sanofi-Pasteur are targeting new adjuvants. R&D is the main axis, and an immune adjuvant that can initiate an innate immune response is added to the candidate vaccine, starting from the source of the immune response, thereby achieving the effect of cross-protection of new and old viruses.

As for the vaccination of the Japanese encephalitis vaccine, there are many restrictions that must be imposed. For example, it is necessary to vaccinate the vaccine containing the adjuvant for more than one month to avoid allergic "post-vaccination encephalitis"; in addition, Taiwan is concentrated in 3 per year. Inoculation from month to May, many small babies have delayed vaccination; and the number of times of smuggling is the highest (the child vaccination time is up to four times: two doses of vaccine are given at intervals of two weeks, and one dose is added one year later. The first grade of the national junior school adds another dose). The reason is that the current vaccine is derived from rat brain tissue extracting virus, purified and treated with formaldehyde inactivated, and the purification process requires the use of protamine sulfate, so the residual small amount of impurities and The role of the agent will improperly strengthen its allergic reaction.

Furthermore, the Japanese encephalitis antigen produced by infecting the mouse brain is limited in that it cannot be combined with other antigens alone, and thus cannot be used for preparing a multivalent vaccine. However, the vaccine technique used in the present invention is to produce a Japanese encephalitis virus antigen by cell culture, and after being purified by fumarin, it is purified by using benzonase nuclease, thereby effectively reducing the side effects produced, and It can overcome the problem of Japanese encephalitis virus antigen inoculation with other antigen vaccines.

Accordingly, one aspect of the present invention relates to a multivalent vaccine comprising a deactivated enterovirus antigen and at least one selected from the group consisting of: (1) deactivating an influenza virus; (2) deactivating a Japanese encephalitis virus; or 3) Deactivate the influenza virus to deactivate the antigen of Japanese encephalitis virus, and a pharmaceutically acceptable carrier.

In a specific embodiment of the present invention, the Japanese encephalitis virus used for deactivation of the multivalent vaccine of the present invention is obtained by cell culture.

In a particular embodiment of the invention, the multivalent vaccine of the invention further comprises a pharmaceutically acceptable adjuvant. In a specific embodiment, the adjuvant is a vaccine delivery system. In another specific embodiment, the vaccine delivery system is an aluminum salt or emulsion.

In a specific embodiment, the adjuvant is a complex adjuvant comprising a vaccine delivery system and an immunostimulating substance as described above. In another specific embodiment, the immunostimulatory substance is a CpG oligodeoxynucleotide capable of enhancing viral neutralizing effects. In yet another embodiment, the immunostimulatory substance is a short-chain antibacterial peptide capable of enhancing a T cell response. In still another embodiment, the short-chain antibacterial peptide is an antibacterial peptide having a left-handed-rotor amino acid sequence. In still another embodiment, the short chain antimicrobial peptide is an antimicrobial peptide having a lipidated fragment.

The specific terms used in the present invention have their original meanings, and certain specific terms are used as follows to provide those skilled in the art to further understand the present invention. Unless otherwise specified, the vocabulary used in the science and technology of the present invention is the same as the vocabulary used in the general general skill. If there is a conflict, the present invention will give a new definition of the noun.

As used herein, the term "multivalent vaccine" means a different subtype or serotype containing the same bacteria or virus, or a mixture of other antigens from different bacteria or strains, for a single or oral administration. At the same time, it can prevent multiple diseases at the same time or can control different bacteria or virus strains. For example, these infectious diseases are prevented by injectable diphtheria, tetanus, acellular pertussis, inactivated polio, and Haemophilus influenzae type 5 vaccine.

"Adjuvant" is a component of the vaccine that enhances the immune response elicited by the antigen in the vaccine and achieves better results. With the addition of an adjuvant, the amount of antigen in the vaccine can be reduced. For example, in the manufacture of a novel H1N1 influenza vaccine, it is possible to manufacture a fixed amount of antigen into a larger number of vaccines, and to manufacture a large amount in a short period of time. Vaccines are available in a timely manner.

Commonly used adjuvants can be roughly divided into four categories: (1) inorganic adjuvants, which are prepared by mixing aluminum hydroxide or alum with antigen; (2) organic adjuvants, mostly using microorganisms and their products such as pertussis, bacterial cell wall Extracts, etc.; (3) synthetic adjuvants, artificially synthesized, such as double-stranded polynucleotide chains, isoproterenol, etc.; and (iv) oil agents, such oils, such as mineral oil, Peanut oil, etc.

Among them, aluminum salt has been widely recognized as a convenient and safe adjuvant. However, due to its unsatisfactory adjuvant efficacy, heterogeneity and reproducibility, it is limited in use. Three potential oil-in-water adjuvants were developed: MF59 (Tween) 80/Span 85-stabilized squalene emulsion, Focetria , Novartis), AS03 (Tween 80-stabilized squalene/α-tocopherol emulsion, Pandemrix , GSK) and AF03 (polyoxyethylene cetostearyl ether/Span 85-stabilized squalene/mannitol emulsion, Humenza , Sanofi-Pasteur). Emulsions have better adjuvant efficacy than aluminum salts, and WHO recommends the use of oil-in-water adjuvants as a tool to reduce antigen use.

Oligodeoxynucleotides (ODN) with CpG motifs can stimulate animals, including large livestock animals such as cattle, pigs, and sheep. Lymphocytes and antigens exhibit cell activity and increase dendritic cells (DC). The antigen exhibits activation and maturation, and promotes the immune system's response to specific antigens toward Th1 cells. However, the present invention has unexpectedly discovered that the addition of the Japanese encephalitis vaccine in the presence or absence of an adjuvant can further enhance the antigenic immunopotency of influenza and enterovirus, indicating that the deactivated encephalitis antigen has an adjuvant-like adjuvant. utility.

Example

The other features and advantages of the present invention are further exemplified and illustrated in the following examples, which are intended to be illustrative only and not to limit the scope of the invention.

Example 1: Preparation of multivalent vaccine Vaccine antigen composition

The virions used in this example were obtained by cell culture, and the vaccine composition was a whole virus vaccine activated by formalin. The source and preparation of each antigen are as follows: (1) Deactivated H5N1 subtype influenza virus vaccine (NIBRG-14), derived from reassorted H5N1 vaccine strain A/Vietnam/1194/2004, and in serum-free medium Proliferation in Madine-Darby canine kidney (MDCK) cells by microcarrier cell culture technique; (2) Deactivated H1N1 subtype influenza virus vaccine (NIBRG-121), derived from recombination H1N1 vaccine strain A/California/7/2009, and proliferation in Madine-Darby canine kidney (NDCK) cells by roller bottle cell culture technique in serum-free medium; (3) Deactivation of the intestine The virus EV71 vaccine (EV71vac) is derived from the enterovirus type 71 E59, and is propagated in Vero cells by cell culture technique of serum-free perfusion bioreactor in culture medium; (4) Deactivation of Japanese brain The inflammatory virus vaccine (JEV) is derived from the Japanese encephalitis virus strain Kitasato Beijing-1 and is propagated in Vero cells by a roller bottle cell culture technique in serum-free medium.

Adjuvant preparation

Mouse CpG demethylated oligodeoxynucleotide [5'-TCC ATG ACG TTC CTG ACG TT-3', SEQ ID NO. 1] was synthesized by Invitrogen Taiwan Ltd and contained 10 μg per dose. Among the candidate vaccines. The aluminum salt (aluminum phosphate) suspension was supplied by Taiwan CDC and was dissolved in an acidic buffer solution (pH = 6) at 300 μg per dose. PELC emulsion is a kind via Span A composite phase nanoemulsion emulsified with 85 (sorbitan trioleate, Sigma-Aldrich, Steinheim, Germany) and a bioabsorbable polymer PEG- b- PLACL. Wherein, the overall molecular weight of PEG- b- PLACL is 7,000 amps, the hydrophilic end portion (75 wt-%) is water-soluble polyethylene glycol (PEG), and the hydrophobic end portion (25 wt-%) has Copolymer of polylactic acid and polycaprolactone (PLACL) of biodegradable nature.

Preparation of PELC emulsion, using a homogenizer (PT3100 polytron; Kinematica AG, Switzerland) at 6,000 rpm, an aqueous solution containing PEG- b- PLACL polymer (120 mg of polymer sample dissolved in 0.8 ml PBS buffer) Solution) with oil (0.165 ml Span) 85 was dissolved in 0.935 ml of squalene and homogenized for 5 minutes. The vaccine prepared in PELC emulsion was prepared by dissolving 200 μL of stock emulsion in 1800 μL PBS buffer solution and mixing at 5 rpm with a tube rotator (Labinco LD-79, New Zealand) before injection. At least 1 hour. The deactivated virus and/or CpG are separately added to the buffer solution.

Example 2: Vaccination and immunopotency analysis

Five-week-old female BALB/c mice were purchased from the Experimental Animal Center of the National Experimental Research Institute (Taipei, Taiwan) and were acclimated for at least one week prior to use in the National Institutes of Health (NHRI) animal room. When vaccination is carried out, a candidate combination vaccine (multivalent vaccine, with or without adjuvant) is administered to the experimental mice by intramuscular injection in a single dose or a second dose schedule. Serum samples were collected from immunized mice by blood collection from the submandibular artery, and antibody titers were determined by enzyme-linked immunosorbent assay (ELISA), hemagglutination inhibition assay (HI), and virus neutralization assay (VN). Significant differences in statistics ( p < 0.05) were determined by using Microsoft Excel, numerically logarithmically transformed, and then two-tailed Student's t -test. The results of immunopotency analysis of each exemplary vaccine combination are described below.

H5N1 and H1N1 combination vaccine

In this experiment, we first test whether each of the monovalent influenza vaccines (H5N1 or H1N1) will produce cross-neutralizing antibodies, or it is necessary to apply a combination vaccine to activate the two different subtypes. Influenza virus neutralizing antibodies. As shown in Figure 1, mice that received two doses of H5N1 deactivated virus vaccine failed to produce cross-neutralizing antibodies against H1N1. Similarly, mice that were exposed to the H1N1 virus were unable to produce cross-neutralizing antibodies against H5N1. However, vaccine compositions prepared by mixing and deactivating viruses, whether using an aluminum salt or an emulsion as an adjuvant, can produce virus neutralizing antibodies induced by the two deactivated influenza viruses. In addition, the results of this experiment also show that the multivalent vaccine composition with PELC emulsion will not affect the immune effect induced by the individual deactivation of influenza virus as the antigen composition dose increases (p ~ 1). .

H5N1 and EV71 combination vaccine

In this experiment, a depleted H5N1 virus was combined with a deactivated EV71 enterovirus vaccine to prepare a multivalent vaccine, and the immunogenicity of the resulting combination vaccine was studied. The results are shown in FIG. Deactivation of EV71 virus alone (no combination antigen) did not produce an H5N1-specific antibody immune response, even with adjuvant addition. When the deactivated H5N1 virus is incorporated into the activated EV71 virus vaccine, an anti-H5N1 specific immune response is produced.

This experiment also found that PELC emulsion can significantly increase the H5N1-specific antibody immune response (blood agglutination inhibition, HI>40). As for the EV71-specific response, vaccines without adjuvants could not induce an effective response, while PELC emulsions enhanced the -EV71-specific immune response. The vaccine formulated from PELC emulsion, which induced an anti-EV71-specific immune response, did not differ significantly before and after incorporation into the virus, indicating that the PELC emulsion can avoid antigen competition.

In this experiment, a composite adjuvant (CpG and emulsion and CpG and aluminum salt) was added to deactivate the combined vaccine of H5N1 influenza virus and deactivated EV71 enterovirus, and the anti-EV71 specificity of the mouse was observed after the application. Sexual immune response. From the results of Fig. 3, the mice receiving the vaccine composition prepared by adding PELC/CpG can produce good influenza and enterovirus immune antibodies together, and add CpG and add aluminum salt/CpG as compared with the hitting. The animal group of the vaccine composition, adding a combination of CpG and emulsion adjuvant, can significantly enhance the immune effect of the mouse against the mixed vaccine of influenza and enterovirus.

H5N1, EV71 and JEV combination vaccine

In this experiment, mice were inoculated to deactivate H5N1 influenza virus, deactivated EV71 enterovirus and JEV to activate Japanese encephalitis vaccine combination to prepare multivalent vaccine, and observe the immune response induced in mice. The results in Figure 4 show that at 12 weeks after receiving a single injection, the vaccine composition of the present invention allows the immunized mice to simultaneously produce an antigen-specific immune response against H5N1, EV71 and JEV. Moreover, it was found that the immune response was significantly increased after the addition of the adjuvant (p<0.05).

In order to further evaluate the addition of deactivated Japanese encephalitis virus, it will have an additive or inhibitory effect on the mixed vaccine. The deactivated JEV will be added to the 2-in-1 vaccine combination formed by H5N1 and EV71, and administered to mice. The immune response was observed and the results are shown in Figure 5. In the absence of adjuvant, the addition of deactivated Japanese encephalitis virus to the mixed vaccine of influenza and enterovirus can enhance the immune-inducing effect of the mixed vaccine of influenza and enterovirus on enterovirus. If a PELC emulsion is used as an adjuvant, the deactivated Japanese encephalitis virus is added to the influenza and enterovirus mixed vaccine, and the immune effect induced by the mixed vaccine against the enterovirus and the influenza virus can be enhanced at the same time.

T-cell immune response of combination vaccine

BALB/c mice were injected intramuscularly (im) with a vaccine candidate with or without adjuvant. After the mice were immunized with the vaccine, the spleen on the 7th day was taken for T cell analysis. Briefly, after spleen cells were removed, single suspension cells were purified, and the cell concentration was adjusted to 1×10 ⁶ /mL using RPMI medium supplemented with 10% fetal bovine serum (FBS, HyClone, Perbio), and 200 μL cells were added to 96-well plates. The suspension was added to the deactivated virus and cultured at 37 ° C for 4 days. The supernatant was collected and analyzed for cytokine concentration using a cytokine ELISA quantification kit (R&D, Abingdom). The cytokine secretion of the third day of cell culture was measured using an enzyme-linked immunosorbent spot (ELISpot) quantification kit (eBioscience).

Figure 6 shows that mice are vaccinated with a deactivated virus vaccine supplemented with a PELC emulsion, which is more effective in inducing cytokine secretion than a vaccine without the adjuvant; and the virus vaccine is activated from the injection of PELC emulsion. The T-cells purified from the immunized mice were significantly higher in the number of cells produced than in the no-adjuvant, and immunized with the aluminum salt vaccine. In addition, Figure 6 also shows spleen cells collected from mice immunized with the H5N1/EV71/JEV combination vaccine, and the cells were incubated in vitro with H5N1 deactivated virus-stimulated spleen cell supernatant, IFN. The amount of γ secretion was significantly higher than that of the H5N1/EV71 vaccine immunization group, indicating that the increased cellular response was due to the presence of JEV, and it was again demonstrated that the deactivated Japanese encephalitis vaccine had an adjuvant-like effect in enhancing the immune response.

According to the results of Fig. 7, the mice were vaccinated with PELC and indolicidin monopalmitic acid complex adjuvant (PELC/Pam-indo), or with L-dextrorotatory amino acid sequence antibacterial peptide indolicidin and hBD2 (human β -defensin II) The H5N1 deactivated virus vaccine, after the spleen cells are stimulated by the same virus, a large number of T cells capable of secreting Th1 cell secretin IFN-γ are produced.

Other specific aspects

All of the features disclosed in this specification can be combined in any combination. Thus, the individual features disclosed in this specification can be replaced by alternative features that are the same, equivalent, or similar. Therefore, the various features disclosed are merely examples of a series of equivalents or similar features, unless otherwise clearly indicated.

From the foregoing description, those skilled in the art can readily determine the essential features of the invention, and various changes and modifications of the invention can be made to adapt to various different uses and conditions without departing from the scope thereof. Therefore, other specific aspects are included in the scope of patent application.

<110> National Institute of Health Research

<120> Novel multivalent vaccine against enterovirus, influenza virus and/or Japanese encephalitis

<160> 1

<170> FastSEQ for Windows Version 4.0

<210> 1

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> CpG oligodeoxynucleotides

<400> 2

Figure 1 shows virus neutralizing antibodies elicited in mice after immunization with a deactivated virus vaccine. Mice were vaccinated intramuscularly on day 0 and day 14 with a candidate vaccine formulation. Blood was collected on day 21 and serum antibodies were tested with H5N1 or H1N1 strain. The data shown is the geometric mean plus standard deviation of each group of six mice. The amount that cannot be detected is determined by the antibody titer equal to 20.

Figure 2 shows (a) H5N1-specific HI, and (b) EV71-specific IgG antibodies elicited in mice following immunization with a deactivated virus vaccine. Serum samples were collected from immunized mice and antibody titers were determined by immunoassay. The data shown is the geometric mean plus standard deviation of each group of six mice. The parallel dashed line represents an HI titer of 40. *p<0.05: compared to the experimental group without adjuvant.

Figure 3 shows (a) H5N1-specific HI, and (b) EV71-specific VN antibody elicited in mice following immunization with a deactivated EV71/H5N1 combination vaccine. Serum samples were collected from immunized mice and antibody titers were determined by immunoassay. The data shown is the geometric mean plus standard deviation of each group of eight mice. *p<0.05: compared to the experimental group containing CpG adjuvant. #p<0.05: compared with the experimental group containing CpG/alum adjuvant.

Figure 4 shows (a) H5N1-specific VN induced in mice after immunization with a candidate combination vaccine containing EV71 deactivated virus 0.2 μg, H5N1 deactivated virus 0.5 μg HA, and JEV deactivated virus 0.5 μg, ( b) EV71-specific VN, and (c) JEV-specific IgG antibodies. Serum samples were collected from immunized mice and antibody titers were determined by immunoassay. The data shown is the geometric mean (GMT) plus standard deviation (STD) obtained for each group of six mice. *p<0.05: compared to the experimental group without adjuvant.

Figure 5 shows (a) EV71-specific VN, and (b) H5N1-specific VN antibody elicited in mice after immunization with a deactivated EV71/H5N1 combination vaccine. Serum samples were collected from immunized mice and antibody titers were determined by immunoassay. The data shown is the geometric mean plus standard deviation of each group of six mice. *p<0.05: compared to the experimental group of EV71+H5N1/PBS. #p<0.05: Compared with the experimental group of EV71+H5N1+JEV/PBS.

Figure 6 shows the spleen of mice after immunization with EV71/H5N1 combination vaccine (containing EV71 deactivated virus 0.2 μg and H5N1 deactivated virus 0.5 μg HA) without adjuvant or addition of alum, PELC or JEV. Cytokine response elicited in cells. Additional doses were administered at week 12 and spleen cell suspensions from six mice per group were pooled seven days later. The data shown is the mean plus standard deviation of the three replicates.

Figure 7 shows that after immunization with H5N1 deactivated virus vaccine in mice, 3 mice per group took the spleen on day 7 to purify 2 x 10 ⁵ T cells and stimulated with deactivated virus at a concentration of 0.5 μg HA/mL. After three days of culture, the IFN-γ secretion reaction of T cells was measured by the ELISpot method. The data shown is the mean plus standard deviation of the three replicates.

Claims (8)

A multivalent vaccine composition comprising an antigen for deactivating enterovirus 71 and a deactivated H5N1 subtype influenza virus to deactivate an antigen of Japanese encephalitis virus, and a pharmaceutically acceptable carrier. The multivalent vaccine composition according to the first aspect of the patent application, wherein the deactivated enterovirus 71 is obtained by cell culture. The multivalent vaccine composition according to claim 1, wherein the deactivated Japanese encephalitis virus is obtained by cell culture. The multivalent vaccine composition according to claim 1, wherein the deactivated influenza virus antigen comprises a deactivated H5N1 subtype influenza virus mixed to deactivate the H1N1 subtype influenza virus. The multivalent vaccine composition according to claim 1 of the patent application, further comprising a pharmaceutically acceptable adjuvant, wherein the adjuvant is a complex adjuvant consisting of a vaccine delivery system and an immunostimulating substance. A multivalent vaccine composition according to claim 5, wherein the vaccine delivery system is an aluminum salt or a PELC emulsion. A multivalent vaccine composition according to claim 5, wherein the immunostimulatory substance is a CpG oligodeoxynucleotide capable of enhancing virus neutralization. The multivalent vaccine composition according to claim 5, wherein the immunostimulating substance is a short-chain antibacterial peptide capable of enhancing a T cell reaction, and the short-chain antibacterial peptide is a sequence having a left-handed or trans-lateral amino acid sequence or An antibacterial peptide having a lipidated fragment.