CN1153064A - Adjuvants for viral vaccines - Google Patents

Abstract

The present invention discloses mammalian vaccine compositions having an effective amount of an adjuvant, the adjuvant comprising squalene or squlane, one or more phospholipids and a surfactant. These compositions also optionally contain an aluminium salt and one or more pharmaceutically acceptable buffers.

Description

Adjuvants for viral vaccines

The present invention relates to be used to increase the new adjuvant of mammal vaccine antigen.Especially, the present invention relates to utilize the new adjuvant of Squalene or squalane or its mixture, phospholipid and surfactant.

Utilize the main vaccine of four classes to increase mammal, especially people's anti-disease antibody.They are live vaccine, killed vaccine or inactivated vaccine, subunit vaccine and toxoid.Wherein, live vaccine makes the host produce the strongest immunne response.Usually with these live vaccine attenuations, make them can increase its time, and do not produce relevant usually disease antigen immune response.

Come the deactivation killed vaccine, the antigenic factor that exists in wherein said not deactivation of the method host immune system by chemistry or other method.Pertussis and Sol gram poliomyelitis vaccine are the examples of killed vaccine.

For some disease epidemic disease mediator, can not stop it to cause the unwanted effect of receptor even kill and wound body.In this case, reagent must be split into itself is not the subunit or the inferior part of pathogen.The example of this subunit vaccine comprises some influenza vaccine, some experimental herpesvirus and hepatitis vaccine.

At last.Toxoid vaccine is the excretory toxin of body to be become avirulent, stimulates the immunne response of contratoxin then as any other antigenic factor.Tetanus toxoid is the example of this vaccine.

Unfortunately, subunit and the toxoid vaccine antigen that is difficult to provide enough triggers the host and produces secular immunity.Therefore, by nonactive vaccine, the inductive immunity of especially highly purified subunit vaccine only continues 1-2.

Describe various adjuvants and strengthened the immunogenicity of nonactive vaccine.Freund's complete adjuvant is wherein arranged, and it comprises mineral oil, water, emulsifying agent and fast knot pyrenomycetes.Be used for mammiferous a lot of adjuvant effectively, having only a seldom part, comprising that aluminium hydroxide and aluminum phosphate have been widely used for the mankind.Because several adjuvants cause serious local response and general reaction, have refused they are used for the mankind.Some of them, as the Freund's complete adjuvant that contains mineral oil is can not be metabolic and owing to can cause the cancer of laboratory animal, being considered to may be carcinogenic.Therefore, need effective and safe adjuvant, it can strengthen vaccine, especially at present effectively and the effect of the subunit type vaccine that can develop.

The invention provides the mammal vaccine combination, it comprises complete or subunit vaccine or the toxoid and the adjuvant of deactivation.

Adjuvant of the present invention comprises Squalene, phospholipid and surfactant mixtures.Especially, first adjuvant comprises the mixture of these components and mammal vaccine, and wherein, Squalene accounts for about 1%-40% of amount of the mixture, and phospholipid accounts for about 0.5%-4% of amount of the mixture, and about 0.1%-4.0% of surfactant comprise amount of the mixture.The listed percent of this paper is that the residue percent of the percentage by volume of every kind of component and known total mixture produces by vaccine itself or by vaccine and dispensable aluminum salt and buffer agent as herein described.

The preferred aspect of the present invention is, about 5%-20% of adjuvant zamene ingredients constitute amount of the mixture, and phospholipid accounts for about 0.7%-2% of amount of the mixture, and about 0.15%-1.6% of surfactant comprise amount of the mixture.The further preferred aspect of the present invention is, about 8%-12% of the Squalene ingredients constitute mixture amount of accounting for of adjuvant, and phospholipid fraction accounts for about 0.8%-1.2% of amount of the mixture, and surface active agent composition accounts for about 0.18%-0.22% of amount of the mixture.

The term Squalene is meant Merck Index, the chemical compound described in the 1383rd page of the 11th Edition, be called (complete-E)-2,6,10,15,19, and 23-hexamethyl-2,6,10,14,18, the 22-tetracosene also claims Sinacene and Supraene.Squalane is also at MerckIndex, mentions among the 1383rd page of the 11th Edition, is called 2,6,10,15,19,23-hexamethyl lignocerane; The perhydro Squalene; Ten dihydro Squalenes; And Spinacane, can be used for replacing the Squalene component of adjuvant mixture as herein described or mixing with it.

Can be used for surfactant in this prescription comprises but is not restricted to polysorbate20 (polyoxyethylene 20 sorbitan monolaurates), polysorbate acid 60, sorbester p17 ^Sorbitan oleate, ICI Americas Wilmington, the product of DE, by BASF Corporation, Parsippany, the CremophoI that NJ produces ^Surfactant, and polysorbate80, known poly-(the oxygen-1 of anhydrosorbitol list-9-vaccenic acid acid esters that is called again, 2-second two bases) derivant, polyoxyethylene (20) dehydrating sorbitol monooleate, dehydrating sorbitol monooleate polyoxyethylene, Sorlate, Tween 80, wherein and the oleate polysorbate80 that is indicated as the sorbitol of every mole of Sorbitol or sorbitan and about 20 moles of ethylene oxide copolymerizationization and its acid anhydride be the surfactant that the present invention preferably uses.

Many phospholipid can be used in this prescription.Phospholipid in preferred this prescription comprises lecithin.Lecithin is ester phatidylcholine or the common name that is connected to various stearic acid, Palmic acid and oleic diglyceride mixt on the phosphocholine ester.Various types of lecithin or lecithin source product (as isolating phospholipid) can be used as the final component of above-mentioned prescription.For example these lecithin fractions can comprise Alcole ^Lecithin (by American LecithinCompany Danbury, CT produces), the medium of Phosal 50MCT phosphatldylcholine and chain lock triglyceride and Phospholipan 90 ^Lecithin (they all are by Natlermann Phospholipid GMBH, Colone, Germany. produces), by Central Soya, Fort Wayne, the Controplil that IN produces ^And Centrophase ^Lecithin.

Non-imposed ground, adjuvant of the present invention also can contain one or more pharmaceutically acceptable aluminum salt, and as aluminium hydroxide, aluminum phosphate or aluminum sulfate, its concentration is about 0.1%-2.0%, preferably be about 0.3%-0.7%, most preferably from about the 0.5%. aluminium hydroxide is preferred aluminum salt.

Non-imposed ground, adjuvant of the present invention also can contain one or more pharmaceutically acceptable pH buffer agents, as phosphate buffered saline (PBS) (PBS), Tris-HCL, structure same regimen acid salt-phosphate buffer, Tricine buffer agent, Hepes and maleate buffer agent.Ooze under the condition if final solution remains on basic grade, so, other salt such as KCL can replace the sodium chloride in the PBS buffer agent.Preferred these buffer agents that use keep the pH of adjuvant mixture between 6.0～8.0.

Employed adjuvant shows significant immunoregulatory activity and supposes that the assosting effect of these chemical compounds is its direct results of regulating the immunne response ability in the present composition.And because these adjuvant components lack aitiogenic ability, so they are as the adaptability of vaccine adjuvant even further reinforcement.Should be noted that also these adjuvants are made up of known injectable substance and can see in the injection that the commercial channel is buied.

Can be by the specified any way of employed specific vaccine be used employed vaccine in the present composition.Preferably by muscle or subcutaneous injection.Described vaccine can be used as for animals or vaccine for man, and comprises complete vaccine and the subunit vaccine and the toxoid of deactivation.And the vaccine of use is that those are used for antibacterium, the rickettsia immune vaccine for the viral pathogen of body.For example, Shi Yi vaccine for man can comprise influenza, poliomyelitis, arbovirus (arbovirsus) infection, typhoid fever and paratyphoid fever, ekolcar, the plague, pertussis, typhus fever, Rocky Mountain Spotted Fever, Type B hemophilus influenza, the scorching coccus polysaccharide of multivalence, C group meningococcus and the new human diploid cell rabies vaccine who produces and the complete vaccine and the subunit vaccine of hepatitis vaccine.

For example, Shi Yi veterinary comprises equine influenza virus, equine herpes virus, horse brain ridge poliovirus, Verrucosis poison, sufficient stomatosis virus, rabies, the minimizing of cat total leukocyte, Feline Rhinotracheitis, cat calicivirus, infectious bovine rhinotracheitis with vaccine.Para-influenzal complete vaccine of parainfluenza-3, bovine viral diarrhoea, cattle celo virus, pseudorabies, Transmissible gastroenteritis virus, pig parvoviral, hepatitis infectiosa canis virus, canine distemper virus and dog and subunit vaccine.Strangles, brucellosis, vibriosis, hook end spiral shell are executed complete vaccine and subunit vaccine, vaccine and the toxoid of body disease, clostridial infection, salmonellosis, colibacillosis, Anaplasmosis, Pasteurella infection, hemophilus infection, Erysipelothrix etc.Further, fully take into account,, therefore require to strengthen, and can find that system of the present invention is to suit very much by suitable and acceptable adjuvant because following vaccine (especially virus and antibacterial subunit type) may weaken immunogen similarly.

The benefit of using disclosed effective adjuvant and vaccine is tangible.By regulating immune certain zone, adjuvant can increase the immunoreactivity of humoral immunization, and the result is when giving receptor adjuvant and bacterin preparation, and the production of antibodies of contained antigenic substance increases in the bacterin preparation.Certainly, this increase will obtain stronger and more permanent immune level, even even immunoreagent may be weak immunogen, as in complete vaccine, the especially subunit vaccine of a lot of deactivations and toxoid, seeing this effect.This reinforcement to antigen immune response directly produces this effect, and further benefit is to produce effectiveness serious and the strong immunization reaction to reduce the host by using less immunizing antigen material.In the teenager inoculation, this point is even more important.And, use the pure antigen that reduces dosage except can producing more successful and safer immunization, the preparation vaccine can be more economical and preferably.

By with the adjuvant substance dissolves or be suspended in the antigen diluent agent and release in the agent assist agent solution of appropriate volume and antigenic solution mixed and prepare the present composition at suitable antigen.The antigen diluent agent is those of this area routine, as phosphate buffered saline (PBS), minimum minimal medium, peptone or the like.

Can adjuvant of the present invention be mixed with concentration, then before using or storing, with vaccine and the dilution of dispensable buffer agent greater than ultimate density.The preferred sterilization adjuvant component or in some cases used is used sterilization filtrate.Then, with before the vaccine of selecting mixes, the complete mixing of mixture with the adjuvant component that obtains preferably uses homogenizer, guarantees that mixture is even with the speed and the time that suit, and for example the time is about 1-10 minute.

Especially, can come production adjuvant of the present invention through the following steps:

1) at first, all adjuvant components should be transferred to temperature and be about 37 ℃.

2) should with buffer agent (if use), Squalene and phospholipid fraction be admixed together and make it reach room temperature.This mixture should be remained on room temperature about 15 minutes.

3) while mixing surface active agent composition is added in the mixture then.With the whole mixture autoclaving and the sonication of adjuvant component, obtain sterilization, uniform adjuvant mixture.Can in ice bath,, finish sonication in about No. 6 positions with Heat System sonde-type acoustic processor.The time of requirement sound was at least 5 minutes, and will be divided into the sonication pulse time and be approximately 30 seconds, then through 15 seconds interruption, kept adjuvant mixture in ice bath.More consistent and the preferred selection of this kind sonication is with adjuvant mixture microfluidization or homogenization, up to its filter membrane by 0.22 μ m, if desired, can under mixing aluminum salt as herein described be added in the adjunvant composition.

4) dosage as required is added to viral vaccine or vaccine concentrate in the adjuvant mixture.

5) adjuvant/vaccine mixture appropriateness is mixed.During mixing, add a certain amount of buffer agent in addition, make final mixture meet the requirements of volume.

Embodiment 1

Prepare adjuvant of the present invention and adjuvant relatively, and mix with tervalent Wyeth-Ayerstl993 influenza virus vaccine (, describing among the 2578th page of the 47th Edition) at 1993 Physician ' s Desk Reference.The CD-1-mice in, 7 week ages female to 6 of test group gives each in three kinds of contained in 1.5 μ g vaccines hemagglutinins, A/BeiJing/32/92, A/Texas/36/91 and B/Panama/45/90.Every Mus intramuscular injection 0.2ml Wyeth-Ayerst vaccine.In inoculation in the time of back 28 days, blood sampling is also measured the antibody of A/BeiJing component in the serum with hemagglutination inhibition method.Following Table I is listed the result that 6 mice average geometric of every test group are tired.Should be noted that and use an amount of PBS buffer agent adjuvant as herein described and comparison adjuvant to be transferred to the volume of requirement.

Employed adjuvant is as follows:

Adjuvant No.1) contains the adjuvant mixture of 5% Squalene, 1% lecithin and 0.2% Tween 80.

Adjuvant No.2) contains the adjuvant mixture of 0.5% aluminium hydroxide (weight/volume), 5% Squalene, 1% lecithin and 0.2% Tween 80.

Comparison adjuvant No.1) Syntex Laboratories is as Syntex Adjuvant Formulation Microfluidized carrier.This adjuvant contains 5% Squalene, 1.25%Pluronic L-121 (can buy from BASF Wyandotle Corp. by the commercial channel) and 0.2% Tween 80.

Adjuvant No.2 relatively) refers to the adjuvant mixture of 5% Squalene/0.2% Tween 80.

Comparison adjuvant No.3) adjuvant mixture of 5% Squalene/1.0% Tween 80.

Comparison adjuvant No.4) adjuvant mixture of 5% Squalene/2.0% Tween 80.

Comparison adjuvant No.5) adjuvant mixture of 5% Squalene/4.0% Tween 80.

Comparison adjuvant No.6) adjuvant mixture of 5% Squalene, 0.2% lecithin, 0.2% Tween 80.

Comparison adjuvant No.7) adjuvant mixture of 5% Squalene, 5% glycerol, 0.5% lecithin.

Comparison adjuvant No.8) the adjuvant temperature compound of 5% Squalene, 0.2% polyethylene pyrrole alkane ketone, 0.2% Tween 80.

Table I

Influenza adjuvant adjuvant HI relatively adjuvant 1 5.793 adjuvant 2 1.825 adjuvant 3 1.625 adjuvant 4 2.048 adjuvant 5 2.048 adjuvant 6 1.29 adjuvant 7 1.149 adjuvant 8 0.406 relatively relatively relatively relatively relatively relatively relatively of (thousand) adjuvant 1 2.8966 adjuvants 2 4.096 of tiring

Embodiment 2

Finish another adjuvant test with Cynomolgus monkey test group.Every group of 3 monkeys, by intramuscular inoculation 0.5ml with 50: 50 ratio and blended trivalent Wyeth-Ayerst 1993 influenza virus vaccines of adjuvant hereinafter described (, describing among the 2578th page of the 47th Edition) at 1993 Physician ' s DeskReference.After the beginning vaccination 28 days the time, with identical inoculum reinforcement.At the 28th day and 42 days, get 5ml blood from every monkey, analyze creatine phosphokinase (cpk), the following table II shows the analysis result of every group of 3 monkeys.As mentioned above, with an amount of PBS buffer agent adjuvant/vaccine mixture is hereinafter described transferred to the volume of requirement.

The test adjuvant mixture

Adjuvant A: the adjuvant mixture of the present invention that contains 10% Squalene, 1% lecithin and 0.2% Tween 80.

Compare adjuvant A: the vaccine mixture that only in the PBS buffer agent, contains vaccine.

Compare adjuvant B: contain inclined to one side succinate of 2mg cholesterol (CHS) or succinic acid hydrogen cholesterol ester liposome (purchasing the St.Louis in Sigma Chemical Company, Missouri, Catalog no.C6013) by the commercial channel.

Compare adjuvant C: the adjuvant mixture that contains 5% Squalene, 30% malonic acid and 0.2% Tween 80.

Table II

The intravital adjuvant of Cynomolgus monkey

The relative HI of A/BeiJing (GMT) relatively adjuvants 88 adjuvants 51 102 adjuvants 102 256 relatively relatively of 28 days 42 days adjuvant A 323 2580 of adjuvant of tiring

Claims (14)

1. mammal vaccine combination said composition contains complete or the subunit vaccine and the effective dose adjuvant of deactivation, wherein the adjuvant amount that contains Squalene about 1%-40% of accounting for compositions cumulative volume amount of containing phospholipid accounts for about 0.5%-4% of compositions cumulative volume, and the amount that contains surfactant accounts for about 0.1%-4.0% of compositions cumulative volume.

2. the mammal vaccine combination of claim 1, wherein the adjuvant amount that contains Squalene accounts for about 5%-20% of compositions cumulative volume, the amount that contains phospholipid accounts for about 0.7%-2% of compositions cumulative volume, and the amount that contains surfactant accounts for about 0.15%-1.6% of compositions cumulative volume.

3. the suckling campaign vaccine combination of claim 1, wherein the amount of adjuvant Squalene accounts for about 8%-12% of compositions cumulative volume, the amount that contains phospholipid accounts for about 0.8%-1.2% of compositions cumulative volume, and the amount that contains surfactant accounts for the about 0.18%-0.22% of combination cumulative volume.

4. the mammal vaccine combination of claim 1, wherein adjuvant further contains pharmaceutically acceptable pH buffer agent.

5. the mammal vaccine of claim 4, wherein pharmaceutically acceptable pH buffer agent is PBS.

6. the mammal vaccine combination of claim 1, wherein adjuvant further contains the aluminum salt that concentration is about 0.1%-2.0%.

7. the mammal vaccine combination of claim 5, wherein aluminum salt is aluminium hydroxide.

8. the mammal vaccine combination of claim 1, wherein vaccine is a subunit influenza A vaccine.

9. the mammal vaccine combination of claim 1, wherein phospholipid is lecithin.

10. the mammal vaccine combination of claim 1 wherein uses the squalane of equivalent to replace Squalene.

11. the mammal vaccine combination of claim 1 wherein replaces Squalene with the Squalene of equivalent and the mixture of squalane.

12. mammal vaccine combination, said composition contains deactivation complete or subunit vaccine and the adjuvant of effective dose in pharmaceutically acceptable buffer agent, wherein the adjuvant amount that contains Squalene accounts for about 8%-12% of compositions cumulative volume, and the amount that contains phospholipid accounts for about 0.8%-1 of compositions cumulative volume.1.2%, and the amount that contains surfactant accounts for about 0.18%-0.22% of compositions cumulative volume, and said composition further contains the aluminum salt of about 0.1%-2.0%.

13. the mammal vaccine combination of claim 12, wherein phospholipid is lecithin.

14. the mammal Seedling compositions of claim 12, wherein aluminum salt is aluminium hydroxide.