Patent Application
20230052315
The present invention relates to a specific vaccine adjuvant, the preparation thereof and the vaccine comprising same.
A vaccine composition generally consists of an antigen, an immunogenic compound which induces protection against a disease of interest, and a vaccine adjuvant, which enables the amplification of the immune response of the vaccinated animal against the antigen. The use of adjuvants in vaccine compositions makes it possible in particular to increase the intensity of the humoral or cellular immune response conferred by a dose of vaccine, making it possible to ensure a better level of protection; to prolong the duration of protection conferred by a dose of vaccine; to obtain, with a lower antigen dose, an efficacy equivalent to that conferred by a full dose used without adjuvant; to reduce the number of immunizations needed to ensure vaccine protection.
Immunity adjuvants of various types have been developed in the past. Among the existing technological solutions for obtaining immunity adjuvants, mention may be made of emulsions comprising at least one oily phase and at least one aqueous phase (such as for example Freund's adjuvants), liposomes, synthetic immunostimulatory polymers, adjuvants of biological origin (saponin, chitosan, cytokines, oligonucleotides, etc.) or water-insoluble mineral salts (for example aluminum hydroxide, which is very commonly used).
Oily vaccine adjuvants are composed of oil and surfactants and make it possible to formulate vaccines in the form of emulsions, the aqueous phase of which contains the vaccine antigen. Among the oils used, mention may be made of oils of plant origin, mineral oils, synthetic oils and oils of animal origin. The surfactants present in the oily adjuvants are emulsifying surfactants, having a hydrophilic character characterized by a hydrophilic-lipophilic balance (HLB) value of between 8 and 19, more particularly between 8 and 15. Such a hydrophilic surfactant may consist, for example, of an alkyl polyglycoside or a mixture of alkyl polyglycosides; of saponins; of lecithins; of polyoxyethylated alkanols; of polymers comprising polyoxyethylene and polyoxypropylene blocks; of esters obtained by condensation of a fatty acid, advantageously a fatty acid that is liquid at 20° C., with a sugar polyol, such as for example sorbitol, mannitol or glycerol; of esters obtained by condensation of a fatty acid, advantageously a fatty acid that is liquid at 20° C., with an ethoxylated sugar.
The surfactants present in the oily adjuvants can also be emulsifying surfactants of the “water-in-oil” type, denoting surfactants that have a low enough HLB value, preferably greater than or equal to 1 and less than 8.0, for obtaining water-in-oil emulsions, for which the aqueous phase is dispersed in the lipophilic fatty phase. Among water-in-oil surfactants, mention may be made of anhydrohexitol esters of saturated or unsaturated, linear or branched, aliphatic carboxylic acids comprising from 12 to 22 carbon atoms, optionally substituted with one or more hydroxyl groups, or a mixture of these esters.
The vaccine emulsions obtained can be of water-in-oil, oil-in-water or water-in-oil-in-water type, depending in particular on the nature of the surfactant system used. In particular, adjuvants of the water-in-oil emulsion type make it possible to significantly increase the humoral and cellular response against the vaccine antigen over a prolonged period compared to the non-adjuvanted vaccine or compared to the vaccine adjuvanted with an aqueous adjuvant such as aluminum hydroxide. Such a long-term response can make it possible to reduce the number of vaccine injections. Adjuvants of the water-in-oil emulsion type are notably used for the preparation of vaccine compositions intended for the vaccination of cattle, sheep, goats, fish and avian species against viral, bacterial or parasitic pathogens.
Some synthetic polymers also possess immunostimulatory properties and have been used as vaccine adjuvants.
Among the immunostimulatory polymers used as veterinary vaccine adjuvants, mention may notably be made of block copolymers of polyoxyethylene and polyoxypropylene (POE-POP), polyethyleneimines, acrylic acid homopolymers in the sodium form thereof, copolymers of acrylic acid and acrylic acid esters (also called carbomers). The polymers obtained from acrylic acid, methacrylic acid, acrylic acid esters or methacrylic acid esters can be synthesized according to a precipitation polymerization process in a suitable solvent or by inverse emulsion polymerization, as described in the patent application published under the number FR2922767A1. Among the polymers of acrylic acid, mention will be made, for example, of the polymers sold by the company Lubrizol under the brand name CARBOPOL™ described in particular in the US patents published under the numbers U.S. Pat. Nos. 5,373,044, 2,798,053 and in European patent application EP 0301532A2.
Carbomers (or acrylic acid polymers) are used at weight contents of the order of one percent for applications as a vaccine adjuvant, and the dilution thereof is in the form of an easily injectable, fluid and translucent vaccine.
These polymeric adjuvants have very good safety and induce a strong short-term response against the associated antigen, and are notably used for the vaccination of pigs, as described for example in the patent application published under the number WO2007094893).
A promising avenue of research consists in formulating these polymeric adjuvants in combination with emulsion-type oily adjuvants, with the aim of combining the immunostimulatory properties of the two types of adjuvants to obtain an adjuvant of improved performance, as described for example in the US patent published under the number U.S. Pat. No. 3,919,411.
However, the combination of these two technologies (i.e. oily adjuvants and polyacrylate gels) in order to obtain a ready-to-use polymeric oily immunity adjuvant that contains a polyacrylate, is stable and can be directly emulsified by the user is a galenic challenge, notably with regard to the stability and sterilization of the adjuvant.
For the purposes of the present invention, a “ready-to-use polymeric oily immunity adjuvant” is understood to mean a mixture consisting of an oily phase containing at least one surfactant and one polymer, which mixture is already sterilized and can immediately be used by mixing with the aqueous antigen medium in an emulsification step. When said mixture comes into contact with an aqueous phase (containing an antigen and/or an active principle), an emulsion is formed owing to the use of a low- or high-shear stirring system. This type of adjuvant (hereinafter referred to as “polymeric oily adjuvant”) is used with the aim of obtaining prophylactic or therapeutic vaccine emulsions which are stable over time.
Several strategies can be considered and are identified in order to prepare a polymeric oily adjuvant, but each one poses some technical problems:
The difficulties in formulating a ready-to-use polymeric oily adjuvant are also linked to the steps of sterilizing each compound that is incorporated into the composition of the vaccine. Specifically, vaccine compositions dedicated to administration by injection must be sterilized before formulating with the antigen under aseptic conditions. Among the sterilization methods that can be used, note can be made of heat sterilization of the product in an autoclave, followed by a step of filtration sterilization on a filter with a pore diameter of 0.2 micrometers or a step of gamma-ray irradiation.
Since the vaccine compositions are in the form of an emulsion that cannot be sterilized, it is therefore necessary to sterilize the oily adjuvants, by filtration or by heat by passing through an autoclave, before the step of emulsifying with the antigen medium. It should also be noted that the surfactants contained in the oily adjuvants are generally not compatible with irradiation sterilization.
As regards the present polymeric vaccine adjuvants on the market, the crosslinked structures thereof and the thickening properties thereof make both filtration operations on sterilizing filters with a pore diameter of 0.2 micrometers, and irradiation sterilization operations, impossible. Therefore, exposure to heat by passing through an autoclave is the only sterilization technique suitable for polymeric vaccine adjuvants. Since this technique requires the preparation of a dilute solution of polymeric adjuvant in water, said polymeric adjuvants cannot be sterilized by this route when they are combined with oily adjuvants.
It follows from the elements indicated above that to prepare a sterile vaccine, containing a combination of a polymeric adjuvant and an oily adjuvant, it is necessary for:
This method is therefore considered by those skilled in the art to be expensive since it comprises many steps, consumes energy, and does not allow direct marketing of the mixture of the combination of the polymeric adjuvant and the oily adjuvant.
Thus, there is a need for a solution which consists in providing a polymeric oily adjuvant containing at least one oil and at least one polymer such as for example a polyacrylate, said polymeric oily adjuvant being stable over time for at least 1 year and more. particularly at least 2 years at 20° C. (“stable” is understood to mean the absence of phase separation, of solidification of the polymer during storage), easily sterilizable and which makes it possible to achieve emulsions that are stable over time at +4° C. for 1 year and for at least 1 month at +37° C. (i.e. exhibiting neither sedimentation or phase separation). The polymeric oily adjuvant according to the invention must make it possible to obtain vaccine compositions that are effective from an immunological point of view.
A solution of the present invention is a vaccine adjuvant comprising at least one inverse microlatex.
For the purposes of the present invention, an inverse microlatex denotes an inverse microemulsion comprising at least one polyelectrolyte-type polymer.
For the purposes of the present invention, a “microemulsion” denotes a mixture of two immiscible liquids which is thermodynamically stable, stabilized by the presence of a surfactant system comprising at least one emulsifying surfactant. A microemulsion is generally transparent because the size of the droplets of the dispersed phase is characterized by a mean particle diameter of less than or equal to 200 nanometers, and preferably less than or equal to 100 nanometers.
For the purposes of the present invention, an “inverse microemulsion” denotes a microemulsion as defined above, for which the dispersed phase is an aqueous phase and the continuous phase is an oily phase.
For the purposes of the present invention, a “polyelectrolyte-type polymer” denotes a polymer in which all or some of the monomer units present in the polymer bear an ionized chemical function. Thus, an anionic polyelectrolyte-type polymer predominantly comprises monomer units possessing an anionic function and a cationic polyelectrolyte-type polymer predominantly comprises monomer units possessing a cationic function.
For the purposes of the present invention, an “anionic and crosslinked polyelectrolyte-type polymer” denotes an anionic polyelectrolyte-type polymer as defined above and which comprises, via its constituent monomer units, at least one monomer unit possessing at least two reactive functions that may be employed during the polymerization reaction and that thus make it possible to link together at least two polymer chains.
Inverse microlatexes are prepared by implementing a process which comprises the following steps:
Such a process for preparing an inverse microlatex is described in European patent application published under the number EP 1 371 692 A1, which is incorporated by reference in the present patent application.
Depending on the case, the vaccine adjuvant according to the invention may have one or more of the following characteristics:
with: R1 chosen from —H, —CH3, —C2H5 and —C3H7, preferably —CH3, n between 0 and 50, and m between 8 and 22;
Preferentially, the inverse microlatex included in the vaccine adjuvant according to the invention will comprise, per 100% of its weight:
it being understood that the sum of the weight contents a′)+b′)+c′)+d′) is equal to 100%.
The oil (H1) included in the vaccine adjuvant that is the subject of the present invention is identical to or different from the oil (H2) included in the inverse microlatex.
According to one particular aspect, the oil (H1) included in the vaccine adjuvant that is the subject of the present invention is identical to the oil (H2) included in the inverse microlatex. The oils (H2) and the oils (H1) are chosen in particular from:
The water-in-oil surfactant (E1) included in the vaccine adjuvant that is the subject of the present invention is identical to or different from the water-in-oil surfactant (E′1) included in the inverse microlatex.
According to one particular aspect, the water-in-oil surfactant (E1) included in the vaccine adjuvant that is the subject of the present invention is identical to the water-in-oil surfactant (E′1) included in the inverse microlatex.
For the purposes of the present invention, a “surfactant” denotes a compound which modifies the surface tension between two surfaces and which is an amphiphilic molecule, that is to say that it has in its structure a lipophilic part and another hydrophilic part. Thus, a surfactant makes it possible to solubilize and/or disperse a phase of a certain polarity in another phase of different polarity.
The term “water-in-oil surfactant” denotes surfactants that have a low enough HLB value, preferably greater than or equal to 1 and less than 8.0, for obtaining water-in-oil emulsions, for which the aqueous phase is dispersed in the lipophilic fatty phase.
Among the water-in-oil surfactants (E1) and (E′1), mention may be made of anhydrohexitol esters of saturated or unsaturated, linear or branched, aliphatic carboxylic acids comprising from 12 to 22 carbon atoms, optionally substituted with one or more hydroxyl groups, or a mixture of these esters.
The term “hexitol” denotes hexols derived from hexoses such as sorbitol, mannitol, dulcitol (also known as galactitol) or iditol.
The term “anhydrohexitol” denotes the products resulting from the dehydration of hexitols. Examples of anhydrohexitols include, for example, anhydrosorbitols, anhydromannitols, anhydrodulcitols or anhydroiditols. The term “anhydrohexitols” denotes monoanhydrohexitols (such as for example sorbitan, mannitan, dulcitan, iditan), optionally as a mixture with dianhydrohexitols (such as for example isosorbide, isomannide, isodulcide, isoidide) obtained as by-products during the same dehydration reaction.
The term “mixture of esters” denotes the esters obtained either from a single acid and from a single hexitol, or from a single acid and from a mixture of several hexitols, or from a mixture of several acids and from a single hexitol, or from a mixture of several acids with several hexitols.
The expression “anhydrohexitol ester of saturated or unsaturated, linear or branched, aliphatic carboxylic acids comprising from 12 to 22 carbon atoms, optionally substituted with one or more hydroxyl groups” denotes for example the esters of acids chosen from dodecanoic acids, dodecenoic acids, tetradecanoic acids, tetradecenoic acids, hexadecanoic acids, hexadecenoic acids, octadecanoic acids, octadecenoic acids, octadecadienoic acids, octadecatrienoic acids, octadecatetraenoic acids, eicosanoic acids, eicosenoic acids, eicosadienoic acids, docosanoic acids, docosenoic acids, hydroxyhexadecanoic acids, hydroxyoctadecanoic acids, dihydroxydocosanoic acids or dihydroxyoctadecanoic acids.
The expression “anhydrohexitol ester of saturated or unsaturated, linear or branched, aliphatic carboxylic acids comprising from 12 to 22 carbon atoms, optionally substituted with one or more hydroxyl groups” denotes for example the esters of acids chosen from lauric acid, isolauric acid, 4-dodecenoic acid, 5-dodecenoic acid, myristic acid, palmitic acid, hypogeic acid, stearic acid, isostearic acid, oleic acid, isooleic acid, linoleic acid, isogeranic acid, linolenic acid, arachidic acid, 10,13-eicosadienoic acid, behenic acid, erucidic acid, cetoleic acid, brassic acid, 3-hydroxyhexadecanoic acid, 4-hydroxyhexadecanoic acid, 11-hydroxyhexadecanoic acid, 16-hydroxyhexadecanoic acid, 12-hydroxystearic acid, brasileic acid or 8,9-dihydroxystearic acid.
These esters are obtained by esterification of the corresponding acids and anhydrohexitols. The esterification reaction is known to those skilled in the art; it is described in numerous patents and reference books.
Among the anhydrohexitol esters of saturated or unsaturated, linear or branched, aliphatic carboxylic acids comprising from 12 to 22 carbon atoms, optionally substituted with one or more hydroxyl groups, mention may be made of sorbitan laurate (sold under the brand name Montane™20), mannitan laurate, dulcitan laurate, sorbitan isolaurate, mannitan isolaurate, ducitan isolaurate, sorbitan palmitate, mannitan palmitate, dulcitan palmitate, sorbitan stearate (sold under the brand name Montane™60), mannitan stearate, dulcitan stearate, sorbitan isostearate (sold under the brand name Montane™70), mannitan isostearate, dulcitan isostearate, sorbitan oleate (sold under the brand name Montane™80), mannitan oleate (sold under the brand name Montanide™80), dulcitan oleate, sorbitan sesquioleate (sold under the brand name Montane™83), mannitan sesquioleate, sorbitan trioleate (sold under the brand name Montane™85), mannitan trioleate, sorbitan benenate, mannitan behenate, sorbitan arachinate, and mannitan arachinate.
Among the water-in-oil surfactants (E1) and (E′1), mention may also be made of sorbitan oleate ethoxylated with 5 mol of ethylene oxide (5 EO) sold by the applicant under the name Montanox™ 81, diethoxylated (2 EO) oleocetyl alcohol sold by the applicant under the name Simulsol™ 0072.
The term “oil-in-water surfactant” (E2) and (E′2) denotes surfactants that have a high enough HLB value, preferably greater than or equal to 8.0 and less than or equal to 20, preferably greater than or equal to 8.0 and less than or equal to 15.0, for obtaining oil-in-water emulsions, for which the lipophilic fatty phase is dispersed in the aqueous phase.
Among the oil-in-water surfactants (E2) and (E′2), mention may be made of anhydrohexitol esters of saturated or unsaturated, linear or branched, aliphatic carboxylic acids comprising from 12 to 22 carbon atoms, optionally substituted with one or more hydroxyl groups, or a mixture of these esters, which are subsequently subjected to a step of adding ethylene oxide to a variable degree of between 2 and 30 molar equivalents of ethylene oxide.
Among the anhydrohexitol esters of saturated or unsaturated, linear or branched, aliphatic carboxylic acids comprising from 12 to 22 carbon atoms, optionally substituted with one or more hydroxyl groups, and ethoxylated, mention may in particular be made of ethoxylated sorbitan esters, and more particularly sorbitan oleate ethoxylated with 20 mol of ethylene oxide (20 EO), sold by the applicant under the name Montanox™ 80, ethoxylated sorbitan oleate containing 15 moles of ethylene oxide (15 EO), sorbitan oleate ethoxylated with 10 mol of ethylene oxide (10 EO), sorbitan oleate ethoxylated with 5 mol of ethylene oxide (5 EO), mannitan oleate ethoxylated with 20 mol of ethylene oxide (20 EO), mannitan oleate ethoxylated with 15 mol of ethylene oxide (15 EO), mannitan oleate ethoxylated with 10 mol of ethylene oxide (10 EO), mannitan oleate ethoxylated with 5 mol of ethylene oxide (5 EO), sorbitan stearate ethoxylated with 20 mol of ethylene oxide (20 EO), sorbitan stearate ethoxylated with 10 mol of ethylene oxide (10 EO), sorbitan stearate ethoxylated with 5 mol of ethylene oxide (5 EO), mannitan stearate ethoxylated with 20 mol of ethylene oxide (20 EO), mannitan stearate ethoxylated with 10 mol of ethylene oxide (10 EO), mannitan stearate ethoxylated with 5 mol of ethylene oxide (5 EO), sorbitan laurate ethoxylated with 20 mol of ethylene oxide (20 EO) sold by the applicant under the name Montanox™ 20, sorbitan laurate ethoxylated with 10 mol of ethylene oxide (10 EO), sorbitan laurate ethoxylated with 5 mol of ethylene oxide (5 EO), mannitan laurate ethoxylated with 20 mol of ethylene oxide (20 EO), mannitan laurate ethoxylated with 10 mol of ethylene oxide (10 EO), mannitan laurate ethoxylated with 5 mol of ethylene oxide (5 EO), sorbitan trioleate ethoxylated with 5 mol of ethylene oxide, sorbitan trioleate ethoxylated with 10 mol of ethylene oxide, sorbitan trioleate ethoxylated with 20 mol of ethylene oxide, sorbitan trioleate ethoxylated with 25 mol of ethylene oxide sold by the applicant under the name Montanox™ 85, mannitan trioleate ethoxylated with 5 mol of ethylene oxide, mannitan trioleate ethoxylated with 10 mol of ethylene oxide, mannitan trioleate ethoxylated with 20 mol of ethylene oxide, mannitan trioleate ethoxylated with 25 mol of ethylene oxide.
Among the oil-in-water surfactants (E2) and (E′2), mention may be made of ethoxylated plant oils such as, for example, castor oil ethoxylated with 25 EO sold by the applicant under the name Simulsol™ 1292, castor oil ethoxylated with 40 EO sold by the applicant under the name Simulsol™ OL 50, corn oil ethoxylated with 3 mol of ethylene oxide (3 EO), corn oil ethoxylated with 8 mol of ethylene oxide (8 EO), corn oil ethoxylated with 10 mol of ethylene oxide (10 EO), corn oil ethoxylated with 20 mol of ethylene oxide (20 EO), corn oil ethoxylated with 30 mol of ethylene oxide (30 EO), corn oil ethoxylated with 40 mol of ethylene oxide (40 EO), rapeseed oil ethoxylated with 3 mol of ethylene oxide (3 EO), rapeseed oil ethoxylated with 10 mol of ethylene oxide (10 EO), rapeseed oil ethoxylated with 20 mol of ethylene oxide (20 EO), rapeseed oil ethoxylated with 30 mol of ethylene oxide (30 EO), rapeseed oil ethoxylated with 40 mol of ethylene oxide (40 EO), sunflower oil ethoxylated with 3 mol of ethylene oxide (3 EO), sunflower oil ethoxylated with 10 mol of ethylene oxide (10 EO), sunflower oil ethoxylated with 20 mol of ethylene oxide (20 EO), sunflower oil ethoxylated with 30 mol of ethylene oxide (30 EO), sunflower oil ethoxylated with 40 mol of ethylene oxide (40 EO).
Among the oil-in-water surfactants (E2) and (E′2), mention may be made of alkyl polyglycosides, more particularly alkyl polyglucosides and alkyl polyxylosides, or a mixture of alkyl glycosides, lecithins, saponins, polyoxyethylated alkanols, polymers comprising polyoxyethylene and polyoxypropylene blocks, lauryl alcohol ethoxylated with 7 mol of ethylene oxide (7 EO) sold by the applicant under the name Silmulsol™ P7, pentaethoxylated (5 EO) oleocetyl alcohol, octaethoxylated (8 EO) oleocetyl alcohol, decaethoxylated (10 EO) oleocetyl alcohol sold by the applicant under the name Simulsol™ OC 710, or polyethoxylated sorbitan hexaoleates sold under the names G-1086™ and G1096™.
A crosslinking monomer unit denotes a unit derived from a monomer possessing at least two reactive functions by which covalent bonds can be established between the extending polymer chains and said crosslinking monomer. For example, a crosslinking monomer unit may be derived from a monomer which may comprise at least two ethylenic functions in its structure and, when engaged in a radical polymerization reaction with acrylic monomers, said crosslinking monomer can bond covalently to two chains of acrylic polymer during the propagation step, to obtain a crosslinked polymer.
A crosslinked polymer denotes a nonlinear polymer that is in the form of a three-dimensional network which is insoluble in water but which can swell in water and which therefore results in a chemical gel being obtained.
A crosslinked polymer denotes a polymer composed of at least one crosslinking monomer unit and of at least one other monomer unit, and more particularly of a hydrophilic monomer unit. For the purposes of the present invention, a hydrophilic monomer unit is understood to mean a monomer unit resulting from a monomer that is soluble in water and more particularly that is soluble in water at a temperature above or equal to 5° C., more particularly at a temperature above or equal to 10° C., more particularly at a temperature above or equal to 20° C.
The crosslinked polymer may comprise hydrophilic monomer units derived from: 2-methyl-2-[(1-oxo-2-propenyl)amino]-1-propanesulfonic acid in free acid form or partially or totally salified form; acrylic acid in free acid form or partially or totally salified form, methacrylic acid in free acid form or partially or totally salified form, itaconic acid in free acid form or partially or totally salified form, 2-carboxyethyl acrylic acid in free acid form or partially or totally salified form, maleic acid in free acid form or partially or totally salified form, acrylamide, N,N-dimethylacrylamide, methacrylamide, N-isopropylacrylamide, 2-hydroxyethyl acrylate, 2,3-dihydroxypropyl acrylate, 2-hydroxyethyl methacrylate, 2,3-dihydroxypropyl methacrylate, vinyl pyrrolidone.
A crosslinking monomer unit denotes a monomer unit derived from a diethylenic or polyethylenic monomer, notably chosen from ethylene glycol dimethacrylate, diethylene glycol diacrylate, ethylene glycol diacrylate, diallylurea, triallylamine, trimethylolpropane triacrylate, methylenebis(acrylamide) or a mixture of these compounds, diallyloxyacetic acid or a salt thereof such as sodium diallyloxyacetate, or a mixture of these compounds.
The surfactants present in the adjuvant according to the invention are water-in-oil surfactants (E1) or water-in-oil surfactants (E′1), as defined and described above, having a lipophilic nature characterized by an HLB value greater than or equal to 1 and less than 8 and oil-in-water surfactants (E2) or oil-in-water surfactants (E′2), as defined and described above, having a hydrophilic nature characterized by an HLB value greater than or equal to 8 and less than or equal to 20.
Another subject of the present invention is the use of an inverse microlatex as defined previously, for the preparation of a vaccine adjuvant.
The process for preparing a vaccine adjuvant according to the invention comprises a step of sterilizing the vaccine adjuvant by sterilizing filtration or autoclaving.
The filtration will preferably be carried out on a filter having pores with a mean diameter of less than or equal to 0.22 microns (see standard ISO 13408-2:2018(en)).
Before being filtered, the adjuvant may be prefiltered. By way of example, the adjuvant can be prefiltered using hydrophobic filters having pores with a mean diameter of 0.45 μm. The prefiltration and filtration steps can be carried out in a single step, which involves the use of double-membrane hydrophobic filter, of which a first membrane has pores with a mean diameter of 0.45 μm and the second membrane has pores with a mean diameter of 0.2 μm, or the combination of a first hydrophobic filter having pores with a mean diameter of 0.45 μm and a second hydrophobic filter having pores with a mean diameter of 0.2 μm. This means that the first membrane or the first filter has larger pores than the second membrane or the second filter. Ideally, the first membrane or the first filter have pores with a diameter greater than or equal to 0.3 μm, preferably pores with a diameter less than or equal to 0.6 μm and more particularly equal to 0.45 μm. The second membrane has pores with a diameter less than or equal to 0.22 μm in order to obtain a sterilizing action.
The filters and the membranes used for the filtration and/or the prefiltration of the adjuvant can consist of polymeric supports of PTFE (polytetrafluoroethylene) or PP (polypropylene) type.
Preferentially, said process for preparing an adjuvant according to the invention comprises the following steps:
For the purposes of the present invention, ambient temperature is understood to mean a temperature above or equal to 15° C. and below or equal to 30° C.
Another subject of the present invention is a vaccine comprising the adjuvant according to the invention and also at least one aqueous solution (S) of at least one antigen or of at least one in vivo generator of a compound comprising an amino acid sequence.
Preferably the vaccine will comprise:
Preferably, said vaccine is in the form of a water-in-oil emulsion or an oil-in-water emulsion. An antigen or at least one in vivo generator of a compound comprising an amino acid sequence denotes either killed microorganisms, such as viruses, bacteria or parasites, or purified fractions of these microorganisms, or living microorganisms, the pathogenicity of which has been reduced. Examples of viruses which can constitute an antigen according to the present invention include orthomyxoviruses such as the influenza virus, paramyxoviruses such as the Newcastle disease virus, coronaviruses such as the infectious bronchitis virus, herpes viruses such as Aujeszky's disease virus or Marek's disease virus. As a microorganism of bacterial type which can constitute an antigen according to the present invention, mention may be made E. coli, and those of the Pasteurella, Avibacterium, Staphylococcus and Streptococcus genera. Examples of parasites include those of the Eimeria, Trypanosoma, and Leishmania genera. Mention may also be made of recombinant viruses, in particular nonenveloped viruses, such as adenoviruses, the vaccinia virus, the canarypox virus, herpes viruses or baculoviruses. Also denoted is a live, nonenveloped, recombinant viral vector, the genome of which contains, preferably inserted into a part that is not essential for replication of the corresponding enveloped virus, a sequence encoding an antigen subunit which induces antibody synthesis and/or a protective effect against the abovementioned enveloped virus or pathogenic microorganism; these antigen subunits may, for example, be a protein, a glycoprotein, a peptide or a fraction which is a peptide fraction and/or protective against an infection with a living microorganism such as an enveloped virus, a bacterium or a parasite. The exogenous gene inserted into the microorganism may, for example, be derived from an Aujeszky virus. Mention may in particular be made of a recombinant plasmid made up of a nucleotide sequence, into which an exogenous nucleotide sequence, originating from a pathogenic microorganism or virus, is inserted. The aim of the latter nucleotide sequence is to allow the expression of a compound comprising an amino acid sequence, the aim of this compound itself being to trigger an immune reaction in a host organism.
The vaccine as defined above comprises an antigen concentration which depends on the nature of this antigen and on the nature of the individual treated. It is, however, particularly noteworthy that an adjuvant according to the invention makes it possible to significantly decrease the usual antigen dose required. The suitable antigen concentration can be determined conventionally by those skilled in the art. Generally, this dose is about 0.1g/cm3 to 1 g/cm3, more generally between 1
g/cm3 and 100 mg/cm3. The concentration of said in vivo generator in the composition according to the invention depends, here again, in particular on the nature of said generator and on the host to which it is administered. This concentration can be readily determined by those skilled in the art, on the basis of routine experiment. By way of indication, when the in vivo generator is a recombinant microorganism, its concentration in the composition according to the invention is in general between 102 and 1015 microorganisms/cm3 and preferably between 105 and 1012 microorganisms/cm3. When the in vivo generator is a recombinant plasmid, its concentration in the composition obtained according to the process which is the subject of the invention can be between 0.01 g/dm3 and 100 g/dm3. The vaccine, as defined above, is prepared by mixing the adjuvant phase and the antigen phase, optionally adding water or a pharmaceutically acceptable diluent medium. The process for preparing the vaccine according to the invention comprises the following steps:
Preferably the antigen medium is intended to form a vaccine, reference will be made to a vaccine antigen medium.
An antigen medium denotes an aqueous medium comprising at least one antigen or at least one in vivo generator of a compound comprising an amino acid sequence, as described above. Preferably the mixture will be such that the vaccine will comprise, per 100% of its weight, between 10% and 80% by weight of adjuvant and between 20% and 90% by weight of antigen medium, preferably between 50% and 80% by weight of adjuvant and between 20% and 50% by weight of antigen medium, and even more preferably between 50% and 70% by weight of adjuvant and between 30% and 50% by weight of antigen medium.
During step b), an immunostimulant chosen from saponins, animal and/or plant and/or mineral and/or synthetic oils, surfactants, aluminum hydroxide, lecithins and lecithin derivatives may optionally be added to the mixture.
Preferentially, in step b) the antigen medium will be added to the adjuvant gradually with high-shear stirring to form an emulsion.
At the end of the emulsification process, a vaccine in the form of a stable and homogeneous emulsion, preferably of water-in-oil type, is obtained.
The final vaccine can be administered as soon as it is manufactured and can be stored for at least 1 year, at a temperature of +4° C. (depending on the nature of the antigen(s) present in the vaccine and their physicochemical stability over time).
The vaccine is intended to be administered in human or veterinary therapy by injection and by oral, parenteral, mucosal or in ovo routes.
Examples of adjuvants according to the invention are presented below.
1.1 Preparation of Inverse Microlatexes (A), (B), (C) and (D)
Inverse microlatexes comprising, as polymer, a crosslinked sodium polyacrylate, are prepared according to the teachings of the European patent published under the number 1 371 692 B1, which is incorporated here by reference. More particularly the teachings of paragraph [0021], paragraphs [0025] and [0026], paragraphs [0033] to [0048], and even more particularly paragraphs [0039] to [0041] (example 2) of the European patent published under the number 1 371 692 B1, are used to prepare the inverse microlatexes. For each of the microlatexes prepared, a fluid white mineral oil, Marcol™52 sold by the company Exxon Mobil, is used. The same process for preparing inverse microlatexes is carried out in the presence of various weight concentrations of surfactants, and makes it possible to obtain:
The inverse microlatexes (A), (B), (C) and (D), obtained after radical polymerization, are in the form of opalescent to translucent oily compositions. These inverse microlatexes contain 60% by weight of a mixture of oily phase and surfactants, 15% by weight of crosslinked sodium polyacrylate and 25% by weight of water.
1.2 Preparation of Inverse Microlatexes (E), (F), (G) and (H)
The process for preparing the inverse microlatex (A) is carried out using mannitan oleate as water-in-oil surfactant instead of sorbitan oleate in order to obtain the inverse microlatex (E).
The process for preparing the inverse microlatex (B) is carried out using mannitan oleate as water-in-oil surfactant instead of sorbitan oleate in order to obtain the inverse microlatex (F).
The process for preparing the inverse microlatex (C) is carried out using mannitan oleate as water-in-oil surfactant instead of sorbitan oleate in order to obtain the inverse microlatex (G).
The process for preparing the inverse microlatex (D) is carried out using mannitan oleate as water-in-oil surfactant instead of sorbitan oleate in order to obtain the inverse microlatex (H).
The inverse microlatexes (E), (F), (G) and (H) are in the form of an opalescent to translucent oily composition, containing 60% by weight of a mixture of oily phase and surfactants, 15% by weight of crosslinked sodium polyacrylate and 25% by weight of water.
The process for preparing the inverse microlatexes (A), (B), (C), and (D), described in example 1.1, is carried out in the presence of a mixture of acrylic acid and tetraethoxylated lauroyl methacrylate (2 mol %), in order to obtain respectively the inverse microlatexes (A′), (B′), (C′), and (D′).
The inverse microlatexes (A′), (B′), (C′), and (D′) are in the form of opalescent to translucent oily compositions, containing 60% by weight of a mixture of oily phase and surfactants, 15% by weight of crosslinked copolymer of acrylic acid and tetraethoxylated lauroyl methacrylate and 25% by weight of water.
The process for preparing the inverse microlatex (B) of example 1 is carried out in the presence of a larger amount of water, so as to obtain an inverse microlatex (B″) that is in the form of opalescent to translucent oily compositions, containing 49% by weight of a mixture of oily phase and surfactants, 15% by weight of crosslinked sodium polyacrylate and 36% by weight of water.
The process for preparing the inverse microlatex (C) of example 1 is carried out in the presence of a larger amount of water, so as to obtain an inverse microlatex (C″) that is in the form of opalescent to translucent oily compositions, containing 49% by weight of a mixture of oily phase and surfactants, 15% by weight of crosslinked sodium polyacrylate and 36% by weight of water.
The polymeric oily adjuvants are prepared according to the following process:
For the purposes of the present invention, ambient temperature is understood to mean a temperature above or equal to 15° C. and below or equal to 30° C.
In this way, the ADJ1, ADJ2, ADJ3, ADJ′1 adjuvants are prepared and are characterized by the compositions described in table 1:
(1): Sodium polyacrylate that is in the form of an inverse latex, the preparation of which is described in patent FR2922767 B1.
5.1 Filterability
The filterability of the adjuvants according to the invention and of the comparative adjuvants is evaluated according to the following protocol:
The amount of adjuvant filtered is measured as a function of time and the results obtained for each adjuvant tested are recorded in the tables below:
The results recorded in tables 2 to 5 show that the filtration of the adjuvants according to the invention ADJ1, ADJ2, ADJ3 is faster than that of the comparative adjuvant ADJ′1 on a hydrophobic filter, in particular made of PTFE, with a mean pore diameter of 0.2 micrometers.
5.2 Study of the Stability of the Adjuvants According to the Invention and of the Comparative Adjuvants
The stability of the adjuvants according to the invention ADJ1, ADJ2, ADJ3 and of the comparative adjuvant ADJ′1 is evaluated according to the following protocol:
i) An amount of 90 ml of the composition to be tested, contained in a 100 ml flask, is introduced into a climatic chamber regulated at 20° C., for a period of one year. The visual appearance of the compound tested is evaluated before being placed in the stability test in the chamber and after a period of one month (M1), three months (M3), six months (M6) and one year (Y1).
ii) An amount of 90 ml of the composition to be tested, contained in a 100 ml flask, is introduced into a climatic chamber regulated at 37° C., for a period of one month (M1). The visual appearance of the composition tested is evaluated before being placed in the stability test in the chamber and after a period of one month.
Stability is understood to mean the absence of phase separation and/or observed sedimentation. The results of the observations are recorded in table 6 below.
(Clear): homogeneous and clear, a single phase
(Heterogeneous): heterogeneous, two or three phases observed, presence of deposit.
The adjuvants ADJ1, ADJ2, ADJ3, ADJ′1 according to the invention have homogeneous appearances under the storage conditions described below, whereas heterogeneous appearances (phase separation and sedimentation) are observed over time and at various temperatures for the comparative adjuvant ADJ′1.
5.3. Characterization of the Stability Properties of Vaccine Compositions Containing Adjuvants According to the Invention
The stability properties of the placebo vaccine emulsions containing the adjuvants according to the invention ADJ1, ADJ2, ADJ3, are evaluated on an amount of 200 grams (prepared in a 250 ml low form beaker) according to the following protocol, using a Silverson L4 or L5 rotor-stator mixer fitted with its standard head provided with a standard emulsion screen:
i/ 60 grams of aqueous phase are added to 140 grams of adjuvant with mechanical stirring using a Silverson L4 or L5 mixer at a rotational speed of 1000 rpm (during this step the stirring head should be placed 0.5 cm from the bottom of the beaker);
ii/ the emulsion is produced by subjecting the mixture obtained in step i/ to high shear (with the Silverson L4 or L5 mixer) for a period of 3 minutes at a rotational speed of 4000 rpm (or 7 m/s).
The emulsions obtained at the end of step ii/ are fluid, homogeneous and injectable. The term “fluid” more particularly means a liquid emulsion, the dynamic viscosity of which, measured using a Brookfield LVDV1+ equipped with an M62 spindle at a rotational speed of 60 rpm, at 20° C., is between 30 and 40 mPa·s.
The stability of the emulsions obtained is characterized as follows:
i) An amount of 25 ml of the composition to be tested, contained in a 30 ml flask, is introduced into a climatic chamber regulated at 4° C., for a period of one year. The visual appearance of the compound tested is evaluated before being placed in the stability test in the chamber and after a period of one month (M1), three months (M3), six months (M6) and one year (Y1).
ii) An amount of 25 ml of the composition to be tested, contained in a 30 ml flask, is introduced into a climatic chamber regulated at 20° C., for a period of one year. The visual appearance of the compound tested is evaluated before being placed in the stability test in the chamber and after a period of one month (M1), three months (M3), six months (M6) and one year (A1).
iii) An amount of 25 ml of the composition to be tested, contained in a 30 ml flask, is introduced into a climatic chamber regulated at 37° C., for a period of one month (M1). The visual appearance of the composition tested is evaluated before being placed in the stability test in the chamber and after a period of one month.
Stability is understood to mean the absence of phase separation and/or observed sedimentation. The results of the observations are recorded in table 7 below.
5.4 Characterization of the Immunological Properties of Vaccines Containing Adjuvants According to the Invention
The adjuvant properties of the polymeric oily adjuvants ADJ1, ADJ2 and ADJ3 according to the invention and as described in the preceding examples were characterized on several vaccine models and several animal species.
During a first test, the adjuvant according to the invention ADJ3 was formulated with a solution of ovalbumin to obtain a vaccine intended to be injected into mice.
In a second test, the adjuvant according to the invention ADJ2 was formulated with a bacterial antigen medium consisting of inactivated Pasteurella multocida bacteria to obtain a vaccine intended to be administered to avian species.
In a third test, the adjuvant according to the invention ADJ1 was used for the formulation of a viral vaccine intended for avian species against Newcastle disease and H9N2 influenza. These tests demonstrate the vaccine adjuvant properties of the adjuvants according to the invention in several species and several antigen models.
The results obtained are presented below.
5.4.1 Trial 1: Trial in Mice, with Ovalbumin as Antigen
The trial is carried out on OF1 mice in a 90-day vaccination protocol with ovalbumin (OVA) as an antigen model. The vaccines were prepared from an antigen solution of OVA prepared in physiological serum at 10 mg/ml, and sterilized by filtration on a 0.22 μm filter. The formulation of the vaccine comprising the OVA antigen and the ADJ3 adjuvant according to the invention is carried out by emulsification through an i-connector in an adjuvant according to the invention ADJ3/antigen medium ratio of 70/30 (volume/volume).
The safety of the vaccines is assessed by observing local reactions at the injection sites. Vaccine efficacy is assessed by detection of IgG1 and IgG2a antibodies in the blood by the ELISA method. This detection is carried out on the day of vaccination (“primo-vaccination” on DO), then after 14 days (D14), at the time of the booster vaccination on the twenty-eighth day (D28), then on the forty-second day (D42), then on the fifty-sixth day (D56), then on the ninetieth day (D90) at the time of euthanasia.
No local reaction was observed among the members of the test group, thus signifying that the vaccine comprising the ADJ3 adjuvant according to the invention was well tolerated.
Significantly higher antibody titers are observed for the vaccine comprising the ADJ3 adjuvant according to the invention compared to the non-adjuvanted vaccine comprising the antigen, for the two classes of antibodies, which confirms the vaccine adjuvant properties of the formula developed.
5.4.2 Trial 2: Vaccine Trial in Chickens, Pasteurella multocida Bacterial Antigen
This trial was carried out on chickens in a 42-day vaccination protocol against the Pasteurella multocida bacterial pathogen. The animals used in this experiment are red chickens aged 36 days at the time of vaccination (DO). Each vaccine dose contains 1 dose (0.5 ml)=0.5×108 CFU or 1.108 CFU/ml of inactivated Pasteurella multocida bacteria. The vaccine groups consist of 11 male and female chickens randomly distributed between the groups.
The formulation containing the ADJ2 adjuvant according to the invention is prepared using a Tube Drive emulsifier (sold by the company Ika) in sterile DT50 tubes in an ADJ2 adjuvant according to the invention/antigen medium ratio of 70/30 (weight/weight): speed 3 for 2 minutes (1100 rpm) for the pre-emulsion then speed 9 for 6 minutes (4000 rpm).
The animals are vaccinated on DO. Local reactions are observed at slaughter on D42. Blood samples will be taken on DO, D14, D42. Antigen-specific ELISA assays will be performed for the detection of antibody levels in serums using a commercial detection kit (ID Screen Pasteurella multocida chicken and turkey indirect kit sold by ID-VEt).
The vaccine comprising the ADJ2 adjuvant according to the invention is well tolerated in chickens, since no critical local reaction is observed at slaughter. Significantly higher antibody titers are also observed for the vaccine adjuvanted with the ADJ2 adjuvant compared with the non-adjuvanted vaccine in chickens, as shown in table 10 below.
5.4.3 Trial 3: Vaccine Trial in Chickens, Newcastle Disease/Avian Influenza Viral Antigen
This trial was carried out on chickens in a 28-day vaccination protocol with a bivalent inactivated viral vaccine against Newcastle disease LaSota strain (NDV) and H9N2 avian influenza (AIV). The animals used in this experiment are SPF (specific pathogen free) chickens aged 28 days at the start of the experiment (DO). The vaccine groups are formed as follows in table 11:
The test vaccine is formulated using the polymeric oily adjuvant ADJ1 according to the invention by emulsification in an ADJ1 adjuvant according to the invention/antigen medium ratio of 70/30 (weight/weight) with the antigen medium containing the two AIV and NDV inactivated viral valencies. The control group is not vaccinated.
The vaccines are injected intramuscularly on DO. Blood samples are taken on D0, D7, D14, D21 and D28 after vaccination and analyzed by hemagglutination inhibition test to determine the specific antibody titers against each valency (AIV and NDV). In the AIV group, a protection challenge is carried out on D28 after vaccination to measure the viral load after challenge (2.106 EID50 in 0.2 ml intravenously of XZ strain H9N2 AIV virus). Oropharyngeal and cloacal swabs are collected 5 days after viral challenge and inoculated into SPF chicken embryos to measure viral presence after two generations of transmission.
Strong antibody titers are observed in the vaccinated group after vaccination for the two valencies (
5.5 Experimental Conclusion
The adjuvants according to the invention are characterized by:
| Number | Date | Country | Kind |
|---|---|---|---|
| 2001698 | Feb 2020 | FR | national |
This application is the U.S. national phase of International Application No. PCT/EP2021/053873 filed Feb. 17, 2021 which designated the U.S. and claims priority to French Patent Application No. 2001698 filed Feb. 20, 2020, the entire contents of each of which are hereby incorporated by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/053873 | 2/17/2021 | WO |
