Patent Grant
9603919
The present invention relates to a vaccine for the prophylaxis of influenza, and a method for the prophylaxis of influenza.
Influenza is a viral infectious disease that repeatedly becomes epidemic with different antigen every year. The vaccines approved in Japan for the prophylaxis of influenza are those for subcutaneous administration. Since the vaccination induces an IgG antibody having a neutralizing activity in the serum, it is highly effective for preventing progression of the condition into a more severe one such as pneumonia and the like. In the upper airway mucosa, which is the infection site, however, IgA is the main prophylactic component. Since IgA is not induced by subcutaneous administration, the infection-preventive effect is not sufficient. Therefore, the development of an infection-preventive vaccine has been desired for a long time.
The present inventors have developed an influenza vaccine for mucosal administration comprising a double-stranded RNA and an influenza virus antigen, which is superior in the infection-preventive effect (WO2005/014038).
It is therefore an object of the present invention to provide a novel vaccine which is useful for the prophylaxis of influenza, and a method for the prophylaxis of influenza.
To achieve the above-mentioned object, the present inventors have tried to examine the effects of vaccines by adding various components, and completed the present invention.
Accordingly, the present invention provides the following.
The characteristics and the advantages of the present invention will be apparent from the detailed description of the invention below.
In the present invention, the influenza virus includes any subtype known at present, and subtypes which will be isolated and identified in the future. Since no epidemic has been heretofore observed in human and human infection needs to be effectively prevented hereafter, the influenza virus is preferably a subtype consisting of a combination of a type selected from H1-H16 excluding H1 and H3 (i.e., H2 and H4-16) and a type selected from N1-N9. These subtypes are also called new type of influenza virus. The aforementioned subtype is more preferably a subtype consisting of a combination of a type selected from H5, H7 and H9 and a type selected from N1-N9. The influenza virus may be one type of strain belonging to the same subtype, or two or more types of strains belonging to the same subtype, or two or more types of strains belonging to different subtypes.
The influenza virus antigen contained in the vaccine composition of the present invention is largely divided into an inactivated antigen and a subunit antigen.
The term “inactivated antigen” as used herein refers to an antigen deprived of infectivity, used as a vaccine antigen; such antigens include, but are not limited to, complete virus particle virions, incomplete virus particles, virion-constituting particles, virus non-structural proteins, antigens that protect against infections, neutralizing reaction epitopes and the like. The term “inactivated antigen” as used herein refers to an antigen deprived of infectivity, but retaining immunogenicity; when such an antigen is used as a vaccine, it is called an “inactivated vaccine.” Examples of the inactivation methods of antigens include, but are not limited to, physical (e.g., X-ray irradiation, heat, ultrasound), chemical (formalin, mercury, alcohol, chlorine) or other procedures. Subunit antigen per se also falls within the definition of inactivated antigen because they have usually lost infectivity. Alternatively, a killed virus may be used.
The term “subunit antigen” as used herein refers to a component derived from an influenza virus. The subunit antigen includes hemagglutinin (HA), neuraminidase (NA), matrices (M1, M2), non-structures (NS), polymerases (PB1, PB2: basic polymerases 1 and 2, acidic polymerase (PA)), nuclear proteins (NP) and the like, with preference given to HA or NA, which is exposed to the surface of the virus particle. Currently known types of HA are HA1 to HA16, and known types of NA are NA1 to NA9. The subunit antigen may be purified from a pathogen such as a naturally occurring virus, or may be prepared by a synthetic or recombinant technology. Such methods are well known and in common use in the art, and can be performed using commercially available equipment, reagents, vectors and the like.
The amount of the influenza virus antigen to be contained in the vaccine composition of the present invention is not particularly limited as long as it is sufficient to produce secretary IgA, and can be appropriately determined in consideration of the ratio to the below-mentioned poly (I:C) or a derivative thereof. When HA is used as an antigen, for example, its concentration is preferably 10-500 μg HA/mL (based on HA), more preferably 30-400 μg HA/mL (based on HA). The aforementioned concentration is obtained by measuring the concentration of HA protein.
Poly(I:C) as contained in the vaccine composition of the present invention is a double-stranded RNA (dsRNA) comprising polyinosinic acid (pI) and polycitidic acid (pC).
A derivative of poly (I:C) refers to a mismatched dsRNA obtained by modifying the specific configuration of poly (I:C) through the introduction of unpaired bases thereinto, and includes poly (I:CxU), poly (IxU:C) (where x is on average a number from 3 to 40) and the like. Preferably, a derivative of poly (l:C) is poly (I:C12U) or poly (C:I12U), which is commercially available under the trade name Ampligen™.
Poly (l:C) or a derivative thereof is supplied in a size sufficient to produce secretory IgA. Examples of such sizes include 100 bp or more, with preference given to sizes of 300 bp or more, which sizes, however, are not to be construed as limiting. Examples of the upper limit of size include, but are not limited to, 108 bp.
Poly (I:C) or a derivative thereof is present at a concentration sufficient to produce secretory IgA. Such a concentration of poly (I:C) or a derivative thereof is, for example, 0.1 to 10 mg/mL, more preferably 0.5 to 2 mg/mL, and still more preferably about 1 mg/mL (e.g., 0.8 to 1.2 mg/mL).
The weight ratio of the influenza virus antigen and poly (I:C) or a derivative thereof to be contained in the vaccine composition of the present invention is recommended to be 1:1-1:50.
A carboxyvinyl polymer (CVP) contained in the vaccine composition of the present invention is a hydrophilic polymer which is produced by polymerization of acrylic acid as the main monomer component and includes conventional ones such as Carbopol™ commercially available from Lubrizol Advanced Materials, Inc. US and the like. The concentration of CVP used in the present invention is generally in the range of 0.1-2.0% by weight.
The vaccine composition of the present invention may contain a water-soluble basic substance for the purpose of thickening CVP. The water-soluble basic substance includes, for example, inorganic bases (e.g., sodium hydroxide, potassium hydroxide, ammonia, etc.), and organic bases such as alkylamines (e.g., methylamine, ethylamine, propylamine, etc.), dialkylamines (e.g., dimethylamine, diethylamine, dipropylamine, etc.), trialkylamines (e.g., trimethylamine, triethylamine, tripropylamine, etc.), alkanolamines (e.g., methanolamine, ethanolamine, propanolamine, etc.), dialkanolamines (e.g., dimethanolamine, diethanolamine, dipropanolamine, etc.), trialkanolamines (e.g., trimethanolamine, triethanolamine, tripropanolamine, etc.), amino acids (e.g., arginine, lysine, ornithine, etc.) and the like. These water-soluble bases are used in an amount which is necessary for neutralization to adjust the pH value of CVP aqueous solution to a desired pH.
The pH value of the vaccine composition of the present invention is adjusted to the desired pH with a water-soluble basic substance or other pH adjustors taking into consideration the stability or absorption of an influenza virus antigen. Preferable pH range is 6.0-8.0.
Adjustment of the viscosity can be performed depending on the vaccine dosage form. For example, CVP corresponding to 0.1-2.0% by weight is thickened with a water-soluble basic substance and the viscosity is adjusted by applying an outside shearing force, or the viscosity is adjusted with a viscosity modulating agent and an outside shearing force, whereby a base managed to suit the spray angle and spray density from a sprayer is prepared. Thereafter, an influenza virus antigen and poly (I:C) are admixed.
The vaccine composition of the present invention may contain a suitable active medicament, diluent, bactericide, preservative, surfactant, stabilizer and the like which can be used together with the vaccine.
The present invention relates to a method of preventing influenza, comprising a step of administering a vaccine composition comprising an effective amount of an influenza virus antigen and poly (I:C) or a derivative thereof, and a carboxyvinyl polymer at least once to the nasal mucosa of a subject in need thereof.
The subject of administration of the vaccine composition of the present invention includes, but is not limited to, mammals including human, birds and the like.
Nasal mucosal administration of the vaccine composition can be performed in an appropriate form. Various methods such as spraying, coating, or direct dripping of a vaccine liquid can be used.
Administration frequency of the vaccine composition of the present invention is at least once, preferably at least twice, in view of the effectiveness. Additional administration is sometimes called booster immunization. Booster immunization makes it possible to achieve a higher infection-protective effect. The interval of booster immunization is recommended to be at least 1 week, preferably 1 to 4 weeks.
The present invention is hereinafter described in more detail by means of the following examples, which, however, are not to be construed as limiting the present invention.
In the development of an influenza vaccine for nasal administration, a vaccine corresponding to pandemic influenza is highly important and highly urgent as compared to vaccine for seasonal influenza, and needs to be preferentially developed. Therefore, a sample vaccine containing a vaccine for new type of influenza (PR8-IBCDC-RG2 strain: attenuated virus strain of A/Indo/5/2005 (H5N1)) and Ampligen in a 10-fold amount of HA antigen was prepared in the same manner as for seasonal influenza HA vaccine. The sample vaccine contained, as an additive for enhancing an immune response in the nasal mucosa by delaying the clearance of vaccine from the nasal mucosa, a CVP base [mixture of 0.55% CVP, 1.2% L-arginine, 1% glycerin] also applied to allergy medicines.
For a nasal administration test, 7-week-old female BALB/c mice were used (5 per group), and antigen was administered at 0.033-1 μg HA/head. As a comparison control, a group for which Ampligen was added to vaccine in a 10-fold amount of HA antigen and CVP base was not added, a group for which Ampligen was added to vaccine in a 20-fold amount of HA antigen and CVP base was not added, a group for which CVP alone was added to vaccine, a group to which vaccine without addition was nasally administered, and a group to which vaccine without addition was intramuscularly administered were prepared. The vaccine was administered twice at 3-week intervals, and nasal washings and serum were recovered 2 weeks after booster. Specific IgA-ELISA antibody titer of the nasal washings was measured, and the antibody titers of hemagglutination inhibition (HI), neutralizing antibody and specific IgG-ELISA of the serum were measured. Since the sample vaccine had high viscosity, the administration method was changed from conventional nasal drip using a micropipette to injection by inserting about 4 mm of a blunt needle (27G, 0.4 mm×38 mm) set to a microsyringe.
The results are shown in Tables 1-9.
2 μl to each
(Results)
Nasal Administration Test of Whole Virion Vaccine for New Type of Influenza, Containing Ampligen and CVP Base in Combination, and Using Mouse
A remarkable immunoenhancing effect was confirmed in the mucosa and serum in the group administered with a vaccine containing Ampligen and a CVP base in combination, which was not found in the group administered with Ampligen alone. The specific IgG, HI and neutralizing antibody titer (NT) of the serum increased to levels equivalent to those of the intramuscular injection group (no adjuvant).
A sample pandemic influenza vaccine containing, as an additive for enhancing an immune response in the nasal mucosa, a CVP base also applied to allergy medicines in combination, was prepared and used for a nasal administration test.
We investigated the convenience of nasal administration method at this time and obtained unexpected effects of a remarkably improved immune response of the nasal mucosa, a specific antibody titer of the serum, which is equivalent to that of intramuscular injection without adjuvant, and the like. Hence, a more practical vaccine was obtained.
(Consideration of Optimal Dose and Administration Frequency for Mouse)
The optimal dose and administration frequency for mouse were examined based on the measurements of serum specific IgG antibody titer and HI antibody titer after addition of Ampligen, CVP and the like to an antigen, and the comparison with existing vaccines having known composition and known administration method, and using a “new type influenza virus antigen at high concentration”.
(Materials)
A vaccine containing A/Indo/5/2005 (H5N1)/PR8-IBCDC-RG2 virus antigen (obtained by adding Ampligen to a 20-fold concentration of HA antigen, and adding CVP base (1.1% carboxyvinyl polymer, 2.4% L-arginine, 2.0% glycerol) to 50.0 v/v % of the total amount) was transnasally administered to BALB/c mice and samples (nasal swab and serum) were taken 3 weeks later. In this case, samples were not taken from a part of the mice, and booster was given thereto one to three times. The interval between the boosters for mice subjected to two or three times of boosters was 2 weeks. Samples were taken at 2 weeks from the last booster, and immune response was confirmed when all samples were taken. As comparison controls for the confirmation of the effects of Ampligen and CVP base added, a non-addition vaccine transnasal administration group (high dose, medium dose, low dose) with the same antigen dose but free of Ampligen and CVP base and a negative control group (transnasal administration (once) of saline) were set, and a non-addition vaccine intramuscular administration group (high dose) was set for comparison with vaccines having known dosage form and known administration method. From the above-mentioned test, the relationship between dose and administration frequency, and immune response was evaluated. In addition, immune response with a virus strain having different antigenicity was also investigated to obtain findings as to the cross-reactivity.
As negative control, a saline administration group was set. In addition, for confirmation of effects of vaccine addition, non-addition vaccine transnasal administration group (high dose, medium dose, low dose) with the same antigen dose but free of Ampligen and CVP base was set, and an intramuscular administration group (high dose) was set for the comparison with conventional vaccines (known dosage form•administration method).
The transnasal administration group was administered with 6 μL into one nostril, and the control non-addition vaccine subcutaneous administration group was administered with 50 μL to unilateral femoral area.
BALB/c mice (♀, 6.5 w when test was started) were used, and immune response was examined when the antigen (A/Indonesia/5/2005 (H5N1)) supplemented with ampligen at 20-fold ratio of HA antigen, and CVP was transnasally administered 1-4 times, using, as a comparison control, an antigen+Ampligen+CVP transnasal administration group for the first half of the test group and transnasal administration of antigen alone/intramuscular administration of antigen alone/saline transnasal administration for the latter half. The results are shown in Tables 12-19 and
In the antigen+Ampligen+CVP group, several to dozen-fold mucous membrane IgA/serum IgG and NT antibody titer were observed as compared to the antigen alone transnasal administration group, and the difference was remarkable in the groups with lower dose and less administration frequency.
Conventional common knowledge holds that, as compared to vaccines for subcutaneous or intramuscular administration, transnasal administration-type vaccines show lower specific neutralizing antibody, serum specific IgG antibody titer and HI antibody titer. However, the vaccine evaluated at this time was found to acquire specific neutralizing antibody, serum specific IgG antibody titer and HI antibody titer equivalent to or above those of the vaccines with conventional composition and administration method, by the addition of Ampligen® and the like.
Furthermore, the immune response of the samples obtained by this test with the same strain as the administered antigen and a strain with different antigenicity was examined to confirm cross-reactivity.
Based on the relationship between the administration dose, use and immune response obtained by the experiments using mice in Example 2, administration tests were performed using Cynomolgus monkeys (Macaca fascicularis) as an animal close to human, and the dosage form and composition optimal as a transnasal administration-type vaccine were searched and the usefulness from the aspect of infection defense was investigated.
To be precise, (1) search of optimal dosage form/composition by comparison of immune responses after administration of transnasal administration-type vaccine candidates, and (2) evaluation of practicality from the ability to defend against infection by measurement of serum and mucous membrane antibody titers after vaccine inoculation were performed.
The search was performed by immuno-chemical test of serum (influenza HI antibody titer, IgG-ELISA antibody titer and neutralizing antibody titer) and immuno-chemical test of nasal swab (influenza specific IgA-ELISA antibody titer).
(Test Method)
Anesthesized Cynomolgus monkeys (Macaca fascicularis) was transnasally administered with a vaccine containing A/Bar-headed Goose/Qinghai/1A/2005 (H5N1) strain virus antigen or saline at 3 weeks intervals, using a transnasal administration device with trial actuator (test actuator: manufactured by Toko Yakuhin Kogyo Kabushiki Kaisya, Spray Pump; Apta Pharma, VP-7 type, spraying use) six times by 150 μl (50 μl×3) to each nasal cavity (total 300 μl), and samples (nasal swab and serum) were taken at the time of each administration, and every 2 weeks from the booster up to 12 weeks from the booster. Furthermore, where necessary, blood samples nasal swab were taken every 2 weeks thereafter.
Animal and Vaccine:
The nasal swab was measured for the total IgA concentration and the specific IgA-ELISA antibody titer calculated in the same manner as in Example 2 was amended to be the numerical value per total IgA concentration of 1 μg/mL. serum: specific IgG-ELISA antibody titer, specific, cross-reactive HI (hemagglutination inhibition) antibody titer, specific/cross-reactive neutralizing antibody titer
Among the above-mentioned measurement items, the outline of the measurement method of the ELISA antibody titer was as described below.
Vaccine antigen (diluted with 100 mM carbonate buffer (pH 9.6) to protein concentration of 1 μg/mL) was solid phased (4° C., overnight) on a 96-well ELISA plate at 100 μL/well, and washed 3 times with PBS containing 0.1% Tween 20. A sample diluted 2-fold series or negative control was added at 100 μL/well. Then, the plate was incubated at 37° C. for 1 hr, and washed 3 times with PBS containing 0.1% Tween 20. An antibody for detection (alkaliphosphatase-labeled anti-monkey IgG or biotin-labeled anti-monkey IgA) was added at 100 μL/well. The plate was incubated at 37° C. for 1 hr, and washed 3 times with PBS containing 0.1% Tween 20. The substrate solution (4-NPP or TMB) was added at 100 μL/well and the mixture was shaded and incubated at room temperature for 30 min.
The maximum dilution rate that affords an absorbance of sample exceeding the average absorbance of negative control+2SD was taken as the antibody titer of the sample. For specific antibody titer, the same strain as the administration vaccine antigen was used and, for cross-reactive antibody titer, vaccine antigen of a different strain was used. In the following, HI antibody titer and neutralizing antibody titer were measured in the same manner.
The results are shown in Tables 21-30 and
It was confirmed that transnasal spray administration of a vaccine containing Ampligen and CVP to monkey induces specific IgA production in the mucous membrane, and derivation of specific neutralizing antibody in the serum. Since these effects were insufficient with an additive-free vaccine, the usefulness of addition of Ampligen and CVP was confirmed.
While this invention has been described with an emphasis upon preferred embodiments, it will be obvious to those of ordinary skill in the art that variations of the preferred embodiments may be used and that it is intended that the invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the invention as defined by the following claims.
All of the references cited herein, including patents, patent applications, and publications, are hereby incorporated in their entireties by reference.
This application is based on a provisional patent application No. 61/165,098 filed in USA (filing date: Mar. 31, 2009), the contents of which are incorporated, in full herein by this reference.
| Filing Document | Filing Date | Country | Kind | 371c Date |
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| PCT/JP2010/056274 | 3/31/2010 | WO | 00 | 12/21/2011 |
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| WO2010/114169 | 10/7/2010 | WO | A |
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