ES2367194T3 - CHANGING THE TH1 / TH2 BALANCE IN VACCINES AGAINST THE FLU FRACTIONED WITH ADJUSTERS. - Google Patents

Abstract

An immunogenic composition comprising a fractionated influenza virus antigen and a Th1 adjuvant, in which the antigen is prepared from a virus developed in a cell culture and does not include any egg protein and the Th1 adjuvant is in the form of (i) an oil-in-water emulsion that includes squalene, a tocopherol and polysorbate 80, or (ii) a submicrometric emulsion of squalene, polysorbate 80, sorbitan trioleate and an immunostimulatory oligonucleotide.

Description

Technical field

The present invention belongs to the field of vaccines to protect against infection by the influenza virus and, in particular, from fractional vaccines.

Background Technique

Chapters 17 and 18 of reference 1 describe flu vaccines. They are based on live viruses or inactivated viruses, and inactivated vaccines can be based on whole viruses, "fractionated" viruses or purified surface antigens (including hemagglutinin and neuraminidase). Hemagglutinin (HA) is the main immunogen of inactivated influenza vaccines, and vaccine doses are standardized based on HA levels, typically containing approximately 15 µg of HA vaccines per strain.

"Fractionated" vaccines are obtained by treating virions with detergents to produce subvirion preparations, using procedures such as the "Tween-ether" fractionation procedure. Fractionated vaccines generally include multiple influenza virion antigens. BEGRIVAC ™, FLUARIX ™, FLUZONE ™ and FLUSHIELD ™ products are fractional vaccines. WO 01/80836 combines fractional vaccines with a vector comprising a non-liquid hydrophilic core.

During the 2000-01 season in Canada, a newly identified oculorespiratory syndrome (SOR) was observed in patients who had received fractional vaccines. The SOR has been associated with incomplete fractionation of virions during manufacturing, providing compositions with a high proportion of microaggregates of unfractionated virions [2].

It is an object of the invention to minimize the risk that a fractional flu vaccine can induce SOR.

Disclosure of the invention

There is no causal explanation for the association between fractional vaccines and SOR, but the clinical and epidemiological characteristics of SOR suggest hypersensitivity, and therefore it has been proposed that the vaccine may alter the natural Th1 / Th2 balance, causing virions not fractionated as a deviation towards a Th2 phenotype. In reference 3, for example, it was found that the presence of aggregates in fractional flu vaccines diverted the immune response to a larger Th2 cytokine pattern. However, in reference 4, no association between the SOR and the Th1 / Th2 equilibrium can be confirmed.

Despite the absence of any reasonable evidence that associates the SOR and a Th2 deviation, the invention aims to minimize the possibility that a fractionated vaccine may produce an excessive Th2 response. In a situation where flu vaccines have to be produced quickly (for example, after a pandemic outbreak) pressures on manufacturers could cause unintentionally to provide vaccines that had the same problems as the added Canadian lots partially unfractionated from the 2000-01 season. In fact, reference 2 indicates that "it is possible that unfractionated virions and aggregates may not be completely eliminated," and that "some low level risk to trigger ocular and respiratory symptoms may be inevitable." Accordingly, the invention aims to avoid components in fractional vaccines that may cause an excessive Th2 response. Th2 responses are not necessarily completely avoided, as they may be important for protection, but a strong Th2 polarization. If incomplete fractionation occurs unintentionally during manufacturing, or if a fractionated vaccine undergoes aggregation during storage, some adverse effects (for example SOR) related to Th2 polarization can be avoided.

To avoid Th2 polarization, two approaches are followed, preferably in combination. First, when a fractional vaccine includes an adjuvant, then the adjuvant is chosen to stimulate at least a partial Th1 type response, for example, it is preferred to avoid adding only aluminum salts as an adjuvant to a fractionated influenza vaccine. Secondly, the presence of allergens is avoided in fractional vaccines. Since influenza vaccines are special since they are administered at any station, patients receiving multiple doses are sensitized to any impurity that is present, and allergens have been reported to produce Th2 responses in sensitized patients [5]. To avoid allergens, current egg-based procedures for developing influenza viruses are replaced by procedures that use cell cultures, thereby avoiding the presence of contaminating egg allergens such as ovalbumin and ovomucoid.

Thus, the invention provides an immunogenic composition as defined in claim 1.

The invention also provides a kit as defined in claim 17.

The fractionated influenza virus antigen

The compositions of the invention include an antigen obtained by fractionation of influenza virions that are obtained by viral growth in cell cultures. The fractionated virion will typically include multiple influenza virion antigens, including hemagglutinin, neuraminidase, matrix and nucleoprotein. The invention does not include live virus vaccines (such as the FLUMIST ™ product), inactivated whole virion vaccines (such as the INFLEXAL ™ product), purified surface antigen vaccines (which include only hemagglutinin and neuraminidase surface glycoproteins, such such as FLUVIRIN ™, AGREPPAL ™ and INFLUVAC ™ products) or virosomal vaccines (which take the form of viral-type liposomal particles without nucleic acids [6], as in INFLEXAL V ™ and INVAVAC ™ products).

Virions can be collected from fluids containing viruses by various procedures. For example, a purification procedure may involve zonal centrifugation using a linear sucrose gradient solution that includes detergent to break virions.

Fractioned virions can be obtained by treating purified virions with detergents (e.g., ethyl ether, polysorbate 80, deoxycholate, tri-N-butyl phosphate, Triton X-100, Triton N101, cetyltrimethylammonium bromide, Tergitol NP9, etc.) to produce preparations of subvirions, including the “Tween-ether” fractionation process. Procedures for fractionating influenza viruses are well known in the art, for example, see references 7-12, etc. Virus fractionation is typically performed by breaking or fragmenting the entire virus, either infectious or non-infectious, with a concentration of dissociation of a fractionating agent. Dissociation results in a partial or complete solubilization of the virus proteins, altering the integrity of the virus. Preferred fractionation agents are nonionic and ionic surfactants (for example cationic), for example, alkylglucosides, alkylthioglucosides, acyl sugars, sulfobetaines, betaines, alkyl polyoxyethylene ethers, N, N-dialkyl Glucamides, Hecameg, alkylphenoxy compounds of quaternary ammonium, sarcosyl, CTAB (cetyl trimethyl ammonium bromides), tri-N-butyl phosphate, Cetavlon, myristyltrimethylammonium salts, lipofectin, lipofectamine and DOT-MA, octyl-or nonylphenoxy polyoxyethanes (for example, Triton surfactants, such as Triton X-100 or Triton N101), polyoxyethylene sorbitan esters (Tween surfactants), polyoxyethylene ethers, polyoxyethylene esters, etc. A useful fractionation procedure uses the consecutive effects of sodium deoxycholate and formaldehyde, and fractionation can be performed during the initial purification of virions (for example, in a gradient solution of sucrose density). Fractional virions can be resuspended in an isotonic solution of sodium chloride buffered with sodium phosphate.

The flu virus can be attenuated. The flu virus can be temperature sensitive. The flu virus can adapt to the cold.

The influenza virus strains used in vaccines change from one season to another. In the current inter-pandemic period, trivalent vaccines are typical, including two strains of influenza A (H1N1 and H3N2) and one strain of influenza B. The invention can be used with interpandemic strains of this type, but can also be used with pandemic strain viruses. (i.e. strains against which the vaccine recipient and the general human population are immunologically virgins), such as subtype strains H2, H5, H7 or H9 (in particular influenza A virus), and Flu vaccines for pandemic strains may be monovalent or, for example, may be based on a normal trivalent vaccine supplemented with a pandemic strain. Depending on the season and nature of the antigen included in the vaccine, however, the invention may protect against one or more of the HA subtypes of the influenza virus A H1, H2, H3, H4, H5, H6, H7 , H8, H9, H10, H11, H12, H13, H14, H15 or H16. The invention can protect against one or more NA subtypes of the influenza virus A N1, N2, N3, N4, N5, N6, N7, N8 or N9.

In addition to being suitable for immunization against interpandemic strains, the compositions of the invention are particularly useful for immunizing against pandemic strains. The characteristics of a flu strain that give it the possibility of producing a pandemic outbreak are: (a) it contains a new HA compared to the HA present in currently circulating human strains, that is, one that has not been seen in the human population for a decade (for example H2) or previously not seen at all in the human population (for example H5, H6 or H9, which have generally been found only in bird populations), such that the human population it will be immunologically virgin to the HA of the strains; (b) is capable of transmitting horizontally in the human population; and (c) it is pathogenic in humans. A virus with the H5 hemagglutinin type is preferred to immunize against a pandemic flu, such as an H5N1 strain. Other possible strains include H5N3, H9N2, H2N2, H7N1 and H7N7, and any other potentially emerging pandemic strain. Within the subtype H5, a virus can be classified into HA clade 1, HA clado 1 ’, HA clado 2 or HA clado 3 [13], clades 1 and 3 being particularly relevant.

The influenza virus strains used with the invention may be resistant to antiviral therapy (for example, resistant to oseltamivir [14] and / or zanamivir), including resistant pandemic strains [15].

Compositions of the invention may include antigens of one or more (for example, 1, 2, 3, 4 or more) influenza virus strains, including influenza A and / or influenza B viruses. When a Vaccine includes more than one strain of influenza, the different strains typically develop separately and mix after the viruses have been collected and fractionated. Thus, a disclosure procedure may include the step of mixing fractional antigens from more than one flu strain. A trivalent vaccine, which includes two strains of influenza A virus and one strain of influenza B virus, is preferred.

In some embodiments of the invention, the compositions may include antigens from a single strain of influenza.

A. In some embodiments, the compositions may include antigens from two strains of influenza A, provided that these two strains are not H1N1 and H3N2. In some embodiments, the compositions may include antigens from more than two strains of influenza A.

The influenza virus may be a reordering strain and may have been obtained by inverse genetic techniques. Inverse genetic techniques [eg 16-20] allow to prepare influenza viruses with desired genomic segments in vitro using plasmids. Typically, they involve the expression of (a) DNA molecules encoding desired viral RNA molecules, for example, from poly promoters, and (b) DNA molecules encoding viral proteins, for example, from promoters of polII, so that the expression of the two types of DNA in a cell leads to the assembly of a complete intact infectious virion. The DNA preferably provides all the viral proteins and RNAs, but it is also possible to use an auxiliary virus to provide some of the RNAs and proteins. Plasmid-based procedures using separate plasmids to produce each viral RNA are preferred [21-23], and these procedures will also involve the use of plasmids to express all or some (eg, only PB1, PB2, PA and NP proteins) of viral proteins, using 12 plasmids in some procedures.

To reduce the number of plasmids needed, a recent approach [24] combines a plurality of RNA polymerase I transcription cassettes (for viral RNA synthesis) in the same plasmid (eg, sequences encoding 1, 2, 3, 4, 5, 6, 7 or the 8 RNAv segments of influenza A) and a plurality of protein coding regions with RNA polymerase II promoters in another plasmid (e.g., sequences encoding 1, 2, 3, 4 , 5, 6, 7 or the 8 mRNA transcripts of influenza A). Preferred aspects of the procedure of reference 24 involve: (a) mRNA coding regions of PB1, PB2 and PA in a single plasmid; and (b) the 8 segments that encode mRNA in a single plasmid. It may also facilitate the matter to include the NA and HA segments in one plasmid and the other six segments in another plasmid.

As an alternative to the use of poll promoters to encode viral RNA segments, it is possible to use bacteriophage polymerase promoters [25]. For example, promoters for SP6, T3 or T7 polymerases can be conveniently used. Due to the species specificity of poly promoters, bacteriophage polymerase promoters may be more convenient for many cell types (e.g., MDCK) although a cell must also be transfected with a plasmid encoding the exogenous polymerase enzyme.

In other techniques it is possible to use double poly and poly II promoters to simultaneously encode viral RNAs and for expressible mRNAs from a single template [26,27].

Thus, an influenza A virus can include one or more RNA segments of an A / PR / 8/34 virus (typically 6 segments of A / PR / 8/34, the HA and N segments of a strain being of vaccine, that is, a reordering 6: 2). It may also include one or more RNA segments of an A / WSN / 33 virus, or of any other virus strain useful for generating reordering viruses for vaccine preparation. Typically, the composition of the invention protects against a strain that is capable of being transmitted from one human being to another, and in this way the genome of the strain will normally include at least one segment of RNA that originated from a flu virus. mammal (for example, in a human being). It can include an NS segment that originated from an avian influenza virus.

Viruses used as a source of antigens develop in cell culture. The viral growth substrate will typically be a cell line from a mammal. Suitable mammalian cells of origin include, but are not limited to, hamster, cow, primate (including humans and monkeys) and dog cells. Various cell types can be used, such as renal cells, fibroblasts, retinal cells, lung cells, etc. Examples of suitable hamster cells are cell lines that have the names BHK21

or HKCC. Suitable monkey cells are, for example, African green monkey cells, such as renal cells as in the Vero cell line. Suitable dog cells are, for example, renal cells, as in the MDCK cell line. Thus, suitable cell lines include, but are not limited to: MDCK; CHO; 293T; BHK; Vero; MRC-5; PER.C6; WI-38; etc. Preferred mammalian cell lines for developing influenza viruses include: MDCK cells [28-31], derived from Madin Darby canine kidney; Vero cells [32-34], derived from African green monkey kidney (Cercopithecus aethiops); or PER.C6 cells [35], derived from human embryonic retinoblasts. These cell lines are widely available, for example, in the American Type Culture Collection (ATCC) [36], in the Coriell Cell Warehouse [37], or from the European Cell Culture Collection (ECACC). For example, the ATCC supplies several different Vero cells with catalog numbers CCL-81, CCL-81.2, CRL-1586 and CRL-1587, and supplies MDCK cells with catalog number CCL-34. PER.C6 is available at ECACC with deposit number 96022940. As a less preferred alternative to mammalian cell lines, viruses can develop in avian cell lines [eg, refs. 38-40], including cell lines derived from ducks (for example, duck retina) or chickens, for example, chicken embryonic fibroblasts (CEF), etc. Examples include avian embryonic stem cells [38, 41], including the

EBx cell line derived from chicken embryonic stem cells, EB45, EB14 and EB14-074 [42].

The most preferred cell lines for developing influenza viruses are MDCK cell lines. The original MDCK cell line is available in the ATCC as CCL-34, but derivatives of this cell line can also be used. For example, reference 28 discloses an MDCK cell line that was adapted for growth in suspension culture ("MDCK 33016", deposited as DSM ACC 2219). Similarly, reference 43 discloses an MDCK-derived cell line that develops in suspension in a serum-free culture ("B-702", deposited as FERM BP-7449). Reference 44 discloses non-tumorigenic MDCK cells, including "MDCK-S" (ATCC PTA6500), "MDCK-SF101" (ATCC PTA-6501), "MDCK-SF102" (ATCC PTA-6502) and "DCK-SF103" ( PTA-6503). Reference 45 discloses MDCK cell lines with high susceptibility to infection, including "MDCK.5F1" cells (ATCC CRL-12042). Any of these MDCK cell lines can be used.

Viruses can develop in cells in suspension [28, 46, 47] or in adherent cultures. Alternatively, a culture on micro-support can be used. A suitable MDCK cell line for suspension culture is MDCK 33016 (deposited as DSM ACC 2219).

Cell lines that support influenza virus replication are preferably grown in a culture medium without serum and / or medium without proteins. A medium is said to be a serum-free medium in the context of the present invention when there are no additives from serum from a human or animal being. It is understood that no protein means cultures in which cell multiplication occurs with the exclusion of proteins, growth factors, other protein additives and non-serum proteins, but may optionally include proteins such as trypsin or other proteases that may be necessary. for viral growth The cells that develop in these cultures naturally contain proteins by themselves.

The cell culture used for growth, and also the viral inoculum used to initiate the culture, preferably lacks (ie it will have been tested and will have given a negative result for contamination by) herpes simplex virus, respiratory syncytial virus, paragipal virus 3, SARS coronavirus, adenovirus, rhinovirus, reovirus, polyomavirus, bimavirus, circovirus and / or parvovirus [48]. The absence of herpes simplex virus is particularly preferred.

Cell lines that support influenza virus replication preferably develop below 37 ° C [49], for example, at 30-36 ° C during viral replication.

The vaccines of the invention preferably contain less than 10 ng (preferably less than 1 ng and more preferably less than 100 pg) of residual DNA from the host cell per dose, although very small amounts of host cell DNA may be present. In general, the host cell DNA that is desirable to exclude from the compositions of the invention is DNA that is greater than 100 bp.

The measurement of the residual DNA of the host cell is currently a routine regulatory requirement for biological products and is within the normal competence of the person skilled in the art. The assay used to measure DNA will typically be a validated assay [50,51]. The behavioral characteristics of a validated trial can be described in mathematical and quantifiable terms, and its possible sources of error will have been identified. The test will generally have been tested for characteristics such as accuracy, precision and specificity. Once an assay has been calibrated (for example, against known standard amounts of host cell DNA) and tested, then quantitative DNA measurements can be routinely performed. Three main techniques can be used for DNA quantification: hybridization procedures, such as Southern blots or slot transfers [52]; immunoassay procedures such as the Threshold ™ system [53]; and quantitative PCR [54]. The person skilled in the art will be familiar with all these procedures, although the precise characteristics of each procedure may depend on the host cell in question, for example, the choice of probes for hybridization, the choice of primers and / or probes for amplification. , etc. The Threshold ™ system of Molecular Devices is a quantitative assay for peak DNA levels of total DNA, and has been used to monitor levels of contaminating DNA in biopharmaceuticals [53]. A typical assay involves the non-specific sequence formation of a reaction complex between a biotinylated mRNA (single stranded) mDNA binding protein, a urease-conjugated anti-mDNA antibody, and DNA. All components of the assay are included in the complete total DNA assay kit available from the manufacturer. Various commercial manufacturers offer quantitative PCR assays to detect residual DNA from the host cell, for example, AppTec ™ Laboratory Services, BioReliance ™, Althea Technologies, etc. A comparison of a chemiluminescent hybridization assay and the Threshold ™ total DNA system to measure DNA contamination of the host cell of a human viral vaccine can be found in reference 55.

The contaminating DNA can be removed during vaccine preparation using conventional purification procedures, for example, chromatography, etc. The removal of residual DNA from the host cell can be increased by nuclease treatment, for example, using DNase. In references 56 and 57 a convenient method for reducing the contamination by host cell DNA that involves a two-stage treatment is disclosed, using first a DNase (for example Benzonase), which can be used during viral development, and then a cationic detergent (for example CTAB), which can be used during dissociation of virions. Treatment with an alkylating agent, such as β-propiolactone, can also be used to remove DNA from the

host cell, and advantageously can also be used to inactivate virions [58].

Vaccines containing <10 ng (for example <1 ng, <100 pg) of host cell DNA per 15 µg of hemagglutinin are preferred, such as vaccines containing <10 ng (for example <1 ng, <100 pg) of host cell DNA per volume of 0.25 ml. Vaccines containing <10 ng (for example <1 ng, <100 pg) of host cell DNA per 50 µg of hemagglutinin are more preferred, as are vaccines containing <10 ng (for example <1 ng, <100 pg) of host cell DNA per volume of 0.5 ml.

The method for propagating virus in cultured cells generally includes the steps of inoculating the cultured cells with the strain to be cultured, culturing the infected cells for a desired period of time for the propagation of the virus, such as, for example, as determined by the Virus titer or antigen expression (for example between 24 and 168 hours after inoculation) and collect the spread virus. Cultured cells are inoculated with a virus (measured by PFU or TCID50) at a cell ratio of 1: 500 to 1: 1, preferably 1: 100 to 1: 5, and more preferably 1:50 to 1:10 . The virus is added to a suspension of the cells or applied to a monolayer of the cells, and the virus is absorbed into the cells for at least 60 minutes, but usually less than 300 minutes, preferably between 90 and 240 minutes at a temperature from 25 ° C to 40 ° C, preferably from 28 ° C to 37 ° C. The infected cell culture (for example monolayers) can be removed by freezing-thawing or by enzymatic action to increase the viral content of the collected culture supernatants. The collected fluids are then inactivated or stored frozen. Cultured cells can be infected at a multiplicity of infection ("mdi") of about 0.0001 to 10, preferably 0.002 to 5, and more preferably 0.001 to 2. Even more preferably, the cells are infected at a mdi of approximately 0.01. Infected cells can be collected 30 to 60 hours after infection. Preferably, the cells are collected 34 to 48 hours after infection. Even more preferably, the cells are collected 38 to 40 hours after infection. Proteases (typically trypsins) are generally added during cell culture to allow viral release, and proteases can be added at any suitable stage during culture.

Hemagglutinin (HA) is the main immunogen of inactivated influenza vaccines, including fractional vaccines, and vaccine doses are standardized by reference to HA levels, typically measured by a simple radial immunodiffusion (SRID) assay. . Existing fractional vaccines typically contain approximately 15 µg of HA per strain, although lower doses are also used, for example, for children or in pandemic situations. Fractional doses such as 1/2 (i.e. 7.5 µg of HA per strain, and 1/8 [63.64]) have been used, as well as higher doses (for example, 3x or 9x [59,60]) Thus, vaccines can include between 0.1 and 150 µg of HA per strain of influenza, preferably between 0.1 and 50 µg, for example, 0.1-20 µg, 0.115 µg, 0.1- 10 µg, 0.1-7.5 µg, 0.5-5 µg, etc. Particular doses include, for example, about 45, about 30, about 15, about 10, about 7.5, about 5, about 3.8, about 1.9, about 1.5, etc. per strain The inclusion of an adjuvant in the vaccine can compensate for the lower intrinsic immunogenicity of these lower doses.

The HA used with the invention may be a natural HA such as that found in a virus, or it may have been modified. For example, it is known how to modify HA to remove determinants (for example, hyperbasic regions around the cleavage site between HA1 and HA2) that make a virus very pathogenic in avian species.

The compositions of the invention may include a detergent, for example, a polyoxyethylene sorbitan ester surfactant (known as "Tween"), an octoxynol (such as octoxynol-9 (Triton X-100) or toctylphenoxypolyethoxyethanol), a cetyl bromide trimethyl ammonium ("CTAB"), or sodium deoxycholate, particularly for a surface or fractional antigen vaccine. The detergent may be present only in very small amounts. In this way, the vaccine may include less than 1 mg / ml of each of octoxynol-10 and polysorbate 80. Other residual components in very small amounts could be antibiotics (eg, neomycin, kanamycin or polymyxin B).

Adjuvants

The use of adjuvants with influenza vaccines is known. For example, the FLUAD ™ product, which is based on purified surface antigens, includes an MF59 emulsion adjuvant. In addition, references 61 to 64 disclose the use of aluminum salts to act as an adjuvant in influenza vaccines of whole virions, to reduce the amount of antigen required for a single dose of influenza vaccine (thus allowing to produce a higher number of doses from a fixed amount of antigen). There are currently no fractional flu vaccines with adjuvants in the market.

Since aluminum salts (such as those used in references 61 to 64) promote a Th2 type immune response when used by themselves, the invention does not use this adjuvant for fractionated influenza viruses. Instead, alternative adjuvants such as those described below are used.

The distinction between Th1 and Th2 helper T lymphocytes is well known. The Th1 and Th2 adjuvants produce an immune response against a co-administered antigen that will deviate, respectively, towards a Th1 or Th2 type response. Thus, Th1 adjuvants result in the production of antigen-specific T lymphocytes that release cytokines such as IL-2 and interferon-γ (which lead to IgG2a antibodies) and TNF-α, while Th2 adjuvants result in the production of antigen-specific T lymphocytes that release cytokines such as IL-4 (which lead to IgG1) and IL-5. The Th1 / Th2 balance of a particular adjuvant can be assessed by known assays (see below), but may vary depending on factors such as the route of administration or the presence of co-administered substances. The adjuvants used with the invention can induce an exclusively Th1 type response against influenza antigens when administered to a patient, but preferably they will induce a mixed Th1 / Th2 type response. Th0 lymphocytes can also be induced, but a polarized Th2 response will be avoided.

Th1 type responses are naturally associated with bacterial infections and, therefore, adjuvants used with the invention generally include substances that mimic a bacterial substance. Th1 adjuvants can be modulators and / or agonists of Toll-like receptors (TLR). For example, they can be agonists of one or more of the TLR1, TLR2, TLR3, TLR4, TLR7, TLR8 and / or TLR9 proteins. Preferred agents are agonists of TLR7 (for example, imidazoquinolines) and / or TLR9 (for example, CpG oligonucleotides). These agents are useful for activating innate immunity pathways.

Immunostimulatory oligonucleotides

Immunostimulatory oligonucleotides may include nucleotide modifications / analogs such as phosphorothioate modifications and may be double stranded or (with the exception of the single stranded cRNA). References 111, 112 and 113 disclose possible analog substitutions, for example, guanosine replacements with 2'-deoxy-7-desazaguanosine. The adjuvant effect of CpG oligonucleotides is further analyzed in references 114-119. The CpG sequence can be directed to TLR9, such as the GTCGTT or TTCGTT motif [120]. The CpG sequence may be specific to induce a Th1 immune response, such as a CpG-A OD (oligodeoxynucleotide), or it may be more specific to induce a B lymphocyte response, such as a CpG-B ODN. In references 121-123 ODN CpG-A and CpG-B are analyzed. Preferably, the CpG is a CpG-A ODN. Preferably, the CpG oligonucleotide is constructed so that the 5 ′ end is accessible for receptor recognition. Optionally, two CpG oligonucleotide sequences can be joined at their 3 'ends to form "immunomers." See, for example, references 120 and 124-126. A useful CpG adjuvant is CpG7909, also known as ProMune ™ (Coley Pharmaceutical Group, Inc.).

As an alternative or in addition to the use of CpG sequences, TpG sequences [127] can be used. These oligonucleotides may lack unmethylated CpG motifs.

The immunostimulatory oligonucleotide can be rich in pyrimidine. For example, it may comprise more than one consecutive thymidine nucleotide (for example TTTT, as disclosed in reference 127), and / or it may have a nucleotide composition with> 25% thymidine (for example> 35%,> 40 %,> 50%,> 60%,> 80%, etc.). For example, it may comprise more than one consecutive cytosine nucleotide (for example, CCCC, as disclosed in ref. 127), and / or it may have a nucleotide composition with> 25% cytosine (for example> 35%, > 40%,> 50%,> 60%,> 80%, etc.). These oligonucleotides may lack unmethylated CpG motifs.

Immunostimulatory oligonucleotides will typically comprise at least 20 nucleotides. They can comprise less than 100 nucleotides.

3dMPL

3dMPL (also known as monophosphoryl lipid A or 3-des-O-acylated or 3-O-deacyl-4'-monophosphoryl lipid A) is an adjuvant in which position 3 of the terminal glycosamine reducer of lipid monophosphoryl A has deaciled. 3dMPL has been prepared from a Salmonella minnesota liver-free mutant, and is chemically similar to lipid A but lacks an acid labile phosphoryl group and a base labile acyl group. It activates cells of the monocyte / macrophage lineage and stimulates the release of several cytokines, including IL-1, IL-12, TNF-α and GM-CSF (see also ref. 103). In reference 129 the preparation of 3dMPL was originally described.

The 3dMPL can take the form of a mixture of related molecules, which vary by acylation (that is, they have 3, 4, 5 or 6 acyl chains, which can be of different lengths). The two glucosamine monosaccharides (also known as 2-deoxy-2-aminoglucose) are N-acylated on their carbons at position 2 (i.e. at positions 2 and 2 ') and there is also an O-acylation at position 3'. The group bound to carbon 2 has the formula NH-CO-CH2-CR1R1 ’. The group attached to carbon 2 ’has the formula -NH-CO-CH2-CR2R2’. The group attached to carbon 3 ’has the formula -O-CO-CH2-CR3R3’. A representative structure is:

image 1

5

10

fifteen

twenty

25

30

35

The groups R1, R2 and R3 are each, independently, - (CH2) n-CH3. The value of n is preferably between 8 and 16, more preferably between 9 and 12, and even more preferably it is 10.

The R1 ’, R2’ and R3 ’groups can be, independently: (a) -H; (b) -OH; or (c) -O-CO-R4, in which R4 is -H or (CH2) m-CH3, in which the value of m is preferably between 8 and 16, and more preferably is 10, 12 or 14 In position 2, m is preferably 14. In position 2 ', m is preferably 10. In position 3', m is preferably 12. The groups R1, R2 'and R3' are, therefore, preferably groups -O-acyl of dodecanoic acid, tetradecanoic acid or hexadecanoic acid.

When all of R1 ’, R2’ and R3 ’are -H, then the 3dMPL has only 3 acyl chains (one in each of positions 2, 2’ and 3 ’). When only two of R1 ’, R2’ and R3 ’are -H, then the 3dMPL can have 4 acyl chains. When only one of R1 ’, R2’ and R3 ’is -H, then the 3dMPL can have 5 acyl chains. When none of R1 ’, R2’ and R3 ’is -H, then the 3dMPL can have 6 acyl chains. The 3dMPL adjuvant used in accordance with the invention may be a mixture of these forms, with 3 to 6 acyl chains, but it is preferred to include 3dMPL with 6 acyl chains in the mixture, and in particular to ensure that the hexaacyl chain constitutes at least one 10% by weight of the total 3dMPL, for example, ≥20%, ≥30%, ≥40%, ≥50% or more. It has been found that 3dMPL with 6 acyl chains is the most adjuvant active form.

Thus, the most preferred form of 3dMPL for inclusion in the compositions of the invention is in Figure 2.

When 3dMPL is used in the form of a mixture, then references to amounts or concentrations of 3dMPL in the compositions of the invention refer to the 3dMPL species combined in the mixture.

Under aqueous conditions, 3dMPL can form micellar aggregates or particles of different sizes, for example, with a diameter <150 nm or> 500 nm. Either or both of them can be used with the invention, and the best particles can be selected by a routine test. Smaller particles (for example, small enough to provide a transparent aqueous suspension of 3dMPL) are preferred for use according to the invention due to their superior activity [130]. Preferred particles have an average diameter of less than 220 nm, more preferably less than 200 nm or less than 150 nm or less than 120 nm, and may even have an average diameter of less than 100 nm. However, in most cases, the average diameter will not be less than 50 nm. These particles are small enough to be suitable for sterilization by filtration. The particle diameter can be evaluated by the routine dynamic light scattering technique, which reveals an average particle diameter. When a particle is said to have a diameter of x nm, it will generally be a distribution of particles around this mean, but at least 50% in number (for example, ≥60%, ≥70%, ≥80%, ≥ 90%, or more) of the particles will have a diameter within the range x ± 25%.

3dMPL can be used advantageously in combination with an oil-in-water emulsion. Substantially all the 3dMPL can be located in the aqueous phase of the emulsion.

A typical amount of 3dMPL in a vaccine is 10-100 µg / dose, for example, about 25 µg or about 50 µg.

Vitamin E compounds

Reference 135 reports that a vitamin E supplement increases Th1 type responses. The improvement of humoral and cell-mediated immunity by a vitamin E supplement is also reported in reference 136, but it is indicated that administration as a vaccine adjuvant has a much greater effect. In addition, reference 104 reports that vitamin E has a significant impact on the expression of genes involved in the Th1 / Th2 balance. For example, vitamin E stimulation of immune cells can directly lead to increased production of IL-2 (i.e., a Th1 response).

Natural vitamin E exists in eight different forms or isomers: four tocopherols and four tocotrienols. All isomers have a chromanol ring, with a hydroxyl group that can donate a hydrogen atom to reduce free radicals and a hydrophobic side chain that allows penetration into biological membranes. There is an α, β, γ and δ form of both tocopherols and tocotrienols, determined by the number of methyl groups in the chromanol ring. Each form has its own biological activity, the measure of potency or functional use in the body.

Vitamin E compounds for inclusion in the compositions of the invention are tocopherols, and any of the tocopherols α, β, γ, δ, ε or ξ can be used. Α-tocopherols are preferred. Preferred tocopherols have antioxidant properties that can help stabilize the compositions, particularly emulsions [137].

Tocopherols can take various forms, for example, different salts and / or isomers. Salts include organic salts such as succinate, acetate, nicotinate, etc. Both D-α-tocopherol and DL-α-tocopherol can be used. A preferred α-tocopherol is DL-α-tocopherol, and the preferred salt of this tocopherol is succinate. Advantageously, it is known that α-tocopherol succinate is compatible with influenza vaccines and that it is a useful preservative as an alternative to mercurial compounds [11].

Since vitamin E compounds are normally oils, they can conveniently be included as a component in an oil-in-water emulsion, since such emulsions are known to be compatible with influenza vaccines (see below), and in reference 138 It is reported that oil-in-water emulsions containing tocopherols are Th1 induction adjuvants.

Oil-in-water emulsion adjuvants

It is known that oil-in-water emulsions are suitable as adjuvants in influenza virus vaccines, for example, the FLUAD ™ product contains an MF59 emulsion adjuvant. These emulsions typically include at least one oil and at least one surfactant, the oil or oils and the biodegradable (metabolizable) and biocompatible surfactants or surfactants being biocompatible. The components of the emulsion influence the Th1 / Th2 equilibrium and, therefore, not all emulsions are suitable for use with the invention. For example, the MF59 adjuvant preferably deflects the immune response towards a Th2 type response and, therefore, the invention does not use the MF59 adjuvant alone (although it can use MF59 in combination with an immunopotentiator, such as an immunostimulatory oligonucleotide). In contrast, the tocopherol-containing emulsions disclosed in reference 138 induce a Th1 response, and therefore can be used with the invention. The Th1 / Th2 equilibrium of a particular emulsion can be assessed by conventional tests, for example, using the double-color ELISPOT assays of refs. 139 and 140, the microsphere-based multiple test of ref. 141 or the rapid flow cytometry test of ref. 142.

Oil drops of a suitable emulsion are generally less than 5 µm in diameter, and can even have a submicron diameter, these small sizes being achieved with a microfluidizer to provide stable emulsions. Drops smaller than 220 nm are preferred, since they can be sterilized by filtration.

Emulsions may include oils such as those from an animal (such as a fish) or vegetable source. Sources of vegetable oils include nuts, seeds and cereals. Peanut oil, soybean oil, coconut oil and olive oil, which are the most commonly available, serve as examples of nut oils. Jojoba oil can be used, for example, obtained from jojoba. Seed oils include safflower oil, cottonseed oil, sunflower oil, sesame oil and the like. In the cereal group, corn oil is the most available, but oil from other cereals such as wheat, oats, rye, rice, teff, triticale and the like can also be used. Fatty acid esters of 610 carbons of glycerol and 1,2-propanediol can be prepared, although they do not occur naturally in seed oils, by hydrolysis, separation and esterification of the appropriate materials from nut and seed oils. Fats and oils from mammalian milk are metabolizable and, therefore, can be used in the practice of the present invention. The procedures for separation, purification, saponification and other means necessary to obtain pure oils from animal sources are well known in the art. Most fish contain metabolizable oils that can be easily recovered. For example, cod liver oil, shark liver oils and whale oil, such as whale sperm, serve as examples of several of the fish oils that can be used herein. Several branched chain oils are biochemically synthesized in 5-carbon isoprene units and are generally termed terpenoids. Shark liver oil contains branched unsaturated terpenoids known as squalene, 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexane, which is used herein. Fish oils, including squalene, can be easily purchased from commercial sources or can be obtained by procedures known in the art. As mentioned above, other preferred oils are tocopherols (see below). Mixtures of oils can be used.

Surfactants can be classified by their "EHL" (hydrophilic / lipophilic balance). Preferred surfactants of the invention have an EHL of at least 10, preferably at least 15 and more preferably at least 16. The invention can be used with surfactants that include, but are not limited to: the surfactants of polyoxyethylene sorbitan esters (commonly referred to as Tweens) , especially polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (OE), propylene oxide (OP), and / or butylene oxide (OB), sold under the trade name DOWFAX ™, such as linear OE / OP block copolymers; octoxinoles, which may vary in the number of repeated ethoxy (oxy-1,2-ethanediyl) groups, of particular interest being octoxynol 9 (Triton X-100, or toctylphenoxypolyethoxyethanol); (octylphenoxy) polyethoxyethanol (IGEPAL CA-630 / NP-40); phospholipids such as phosphatidylcholine (lecithin); noniphenol ethoxylates, such as the Tergitol ™ NP series; polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as monolauryl ether of triethylene glycol (Brij 30); and sorbitan esters (commonly known as SPAN), such as sorbitan trioleate (Span 85) and sorbitan monolaurate. Nonionic surfactants are preferred. Preferred surfactants are to include Tween 80 (polyoxyethylene sorbitan monooleate), Span 85 (sorbitan trioleate), lecithin and Triton X-100 in the emulsion.

Surfactant mixtures may be used, for example, Tween 80 / Span 85 mixtures. A combination of a polyoxyethylene sorbitan ester such as polyoxyethylene sorbitan monolaurate (Tween 80) and an octoxynol such as t-octylphenoxypolyethoxyethanol (Triton X-) is also suitable. 100). Another useful combination comprises laureth 9 plus a polyoxyethylene sorbitan ester and / or an octoxynol.

Preferred amounts of surfactants (% by weight) are: from 0.01 to 1% polyoxyethylene sorbitan esters (such as Tween 80), in particular about 0.1%; from 0.001 to 0.1% octyl- or nonylphenoxy polyoxyethanes (such as Triton X-100 or other detergents of the Triton series), in particular from 0.005 to 0.02%; from 0.1 to 20% of polyoxyethylene ethers (such as laureth 9), preferably from 0.1 to 10% and, in particular, from 0.1 to 1%, or about 0, 5%.

Two specific oil-in-water emulsions used as adjuvants with the invention are:

•

An emulsion of squalene, a tocopherol and Tween 80. The emulsion may include phosphate buffered saline. It can also include Span 85 (for example 1%) and / or lecithin. These emulsions may have 2 to 10% squalene, 2 to 10% tocopherol and 0.3 to 3% Tween 80, and the weight ratio between squalene and tocopherol is preferably ≤ 1 , since this provides a more stable emulsion. Squalane and Tween 80 may be present in a volume ratio of approximately 5: 2. This emulsion can be obtained by dissolving Tween 80 in PBS to give a 2% solution, and then mixing 90 ml of this solution with a mixture of (5 g of DL-α-tocopherol and 5 ml of squalene), and then subjecting the mixture to microfluidization. The resulting emulsion may have submicron oil droplets, for example, with an average diameter between 100 and 250 nm, preferably about 180 nm.

•

A submicron emulsion of squalene, Tween 80 and Span 85 [143-145], which also includes an immunostimulatory oligonucleotide. The composition of the emulsion by volume can be about 5% squalene, about 0.5% polysorbate 80 and about 0.5% Span 85. In terms of weight, these ratios become 4.3 % squalene, 0.5% polysorbate 80 and 0.48% Span 85, as described in more detail in Chapter 10 of ref. 146 and chapter 12 of ref.

147. The emulsion advantageously includes citrate ions, for example, 10 mM sodium citrate buffer.

These emulsions can be supplemented with 3dMPL and / or with a saponin, as described in reference 138.

The emulsions can be mixed with antigen before distribution, or they can be mixed with antigen extemporaneously, at the time of administration. In this way, the adjuvant and the antigen can be kept separated in a packaged or distributed vaccine, prepared for the final formulation at the time of use. The antigen will generally be in an aqueous form, so that the vaccine is finally prepared by mixing two liquids. The volume ratio of the two liquids for mixing may vary (for example, between 5: 1 and 1: 5), but is generally about 1: 1. After the antigen and adjuvant have been mixed, the hemagglutinin antigen will generally remain in the aqueous solution, but may be distributed in the oil / water interface. In general, a small amount of hemagglutinin will enter the emulsion oil phase.

Pharmaceutical compositions

The compositions of the invention are pharmaceutically acceptable. They may include components in addition to the fractionated antigen and the adjuvant, for example, typically include one or more pharmaceutical carriers and / or excipients. In ref. 148 a thorough analysis of these components is available.

The compositions will generally be in aqueous form. In fractionated antigen and adjuvant will typically be mixed.

The composition may include preservatives such as thiomersal or 2-phenoxyethanol. However, it is preferred that the vaccines are substantially lacking (ie, having less than 5 µg / ml) of mercurial material, for example, lacking thiomersal [11,149]. Vaccines that do not contain mercury are more preferred, and this can be conveniently achieved using an adjuvant containing tocopherol following ref. 11. Vaccines without preservatives are particularly preferred.

To control tonicity, it is preferred to include a physiological salt such as a sodium salt. Sodium chloride (NaCl), which can be present in amounts between 1 and 20 mg / ml, is preferred. Other salts that may be present include potassium chloride, diacid potassium phosphate, disodium phosphate dihydrate, magnesium chloride, calcium chloride, etc.

The compositions will generally have an osmolality between 200 mOsm / kg and 400 mOsm / kg, preferably between 240-360 mOsm / kg, and more preferably will be within the range of 290-310 mOsm / kg. It has previously been reported that osmolality does not have an impact on pain caused by vaccination [150], however it is preferred to maintain osmolality at this interval.

The compositions may include one or more buffers. Typical buffers include: a phosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer (particularly with an aluminum hydroxide adjuvant); or a citrate buffer. Buffers will typically be included in the 5-20 mM range. Conveniently an emulsion formed in phosphate buffered saline can be used.

The pH of a composition will generally be between 5.0 and 8.1, and more typically between 6.0 and 8.0, for example, between 6.5 and 7.5 or between 7.0 and 7.8. Therefore, a process of the invention may include a step of adjusting the pH of the vaccine in bulk before packaging.

The composition is preferably sterile. The composition is preferably non-pyrogenic, that is, it contains <1 EU (endotoxin unit, a conventional measure) per dose, and preferably <0.1 EU per dose. The composition preferably lacks gluten.

The composition may include material for a single immunization, or it may include material for multiple immunizations (ie, a "multidose" kit). In multidose arrangements the inclusion of a preservative is preferred. As an alternative (or in addition) to the inclusion of a preservative in multidose compositions, the compositions may be contained in a container having an aseptic adapter to extract the material.

Flu vaccines are typically administered in a dosage volume of approximately 0.5 ml, although half of a dose (i.e. approximately 0.25 ml) can be administered, for example, to children.

The compositions and kits are preferably stored at a temperature between 2 ° C and 8 ° C. They should not be frozen. Ideally, they should be protected from direct light.

Kits of the invention

The compositions of the invention can be prepared extemporaneously, at the time of administration. In this way, the invention provides kits that include the various components ready for mixing. The kit allows the adjuvant and antigen to be kept separate until the time of use, which can be useful when using an oil-in-water emulsion adjuvant.

The components are physically separated from each other within a kit, and this separation can be achieved in various ways. For example, the two components may be in two separate containers, such as vials. The contents of the two vials can then be mixed, for example, by removing the contents of one vial and adding it to the other vial, or by removing the contents of the two vials separately and mixing them in a third container.

In a preferred arrangement, one of the kit components is in a syringe and the other is in a container such as a vial. The syringe can be used (for example, with a needle) to insert its contents into the second container for mixing, and the mixture can then be extracted into the syringe. The mixed contents of the syringe can then be administered to a patient, typically through a new sterile needle. Packaging of a component in a syringe eliminates the need to use a separate syringe for administration to the patient.

In another preferred arrangement, the two kit components are maintained, but separately, in the same syringe, for example, in a double chamber syringe such as that disclosed in references 151-158 etc. When the syringe is operated (for example, during administration to a patient), then the contents of the two chambers are mixed. This arrangement avoids the need for a separate mixing stage at the time of use.

The kit components will generally be in aqueous form. In some arrangements, one component (typically the antigen component instead of the adjuvant component) is in dry form (for example, in a lyophilized form), the other component being in an aqueous form. The two components can be mixed to reactivate the dry component and provide an aqueous composition for administration to a patient. A lyophilized component will typically be located inside a vial instead of in a syringe. Dry components may include stabilizers such as lactose, sucrose or mannitol, as well as mixtures thereof, for example, lactose / sucrose mixtures, sucrose / mannitol mixtures etc. One possible arrangement uses an aqueous adjuvant component in a pre-filled syringe and a lyophilized antigen component in a vial.

Packaging of kit compositions or components

Suitable containers for the compositions of the invention (or kit components) include vials, syringes (eg, disposable syringes), nasal sprays, etc. These containers must be sterile.

When a composition / component is placed in a vial, the vial can be made of a plastic or glass material. It can be sterilized before the composition / component is added. To avoid problems with latex-sensitive patients, the vials can be sealed with a latex-free cap, and the absence of latex in all packaging materials is preferred. The vial may include a single dose of vaccine, or it may include more than one dose (a "multidose" vial), for example, 10 doses. Preferred vials are made of colorless glass.

A vial can have a cap (for example, a Luer-type closure) adapted so that a pre-filled syringe can be inserted into the cap, the contents of the syringe can be ejected into the vial (for example, to reconstitute the lyophilized material into inside) and the contents of the vial can be removed back into the syringe. After removing the syringe from the vial, a needle can be attached and the composition can be administered to the patient. The cover preferably if placed inside a seal or cover, such that the seal or cover has to be removed before the lid can be accessed. A vial may have a cap that allows aseptic removal of its contents, particularly in the case of multidose vials.

When a component is packaged in a syringe, the syringe can have a needle attached to it. If a needle is not attached, a separate needle can be supplied with the syringe for assembly and use. Said syringe may be covered. Safety needles are preferred. Typical are the 25.4 mm needles of 23 gauge, 25.4 mm 25 gauge and 15.87 mm gauge 25. The syringes can be provided with removable labels on which the lot number, the station can be printed of flu and the expiration date of the content, to facilitate record keeping. The syringe plunger preferably has a stop to prevent the plunger from being accidentally removed during aspiration. The syringes may have a rubber latex cap and / or plunger. Disposable syringes contain a single dose of vaccine. The syringe will generally have a cap on the tip to seal the tip before coupling a needle, and the tip cap is preferably made of a butyl rubber. If the syringe and needle are packaged separately, then the needle is preferably equipped with a butyl rubber cover. Preferred syringes are those sold under the trade name "Tip-Lok" ™.

The containers may be marketed to show a volume of half a dose, for example, to facilitate administration in children. For example, a syringe containing a dose of 0.5 ml may have a mark that shows a volume of 0.25 ml.

When a glass container (for example, a syringe or vial) is used, it is preferred to use a container made of a borosilicate glass instead of a sodium-calcium glass.

A kit or composition can be packaged (for example, in the same box) with a booklet that includes details of the vaccine, for example, instructions for administration, details of the antigens within the vaccine, etc. The instructions may also contain warnings, for example, that an adrenaline solution is available if an anaphylactic reaction occurs after vaccination, etc.

Vaccine treatment and administration procedures

The compositions of the invention are suitable for administration to human patients in a method for inducing an immune response in a patient, comprising the step of administering a composition of the invention to the patient.

The immune response induced by these procedures will generally include an antibody response, preferably a protective antibody response. Methods for evaluating antibody responses, neutralizing capacity and protection after influenza virus vaccination are well known in the art. Human studies have shown that antibody titers against human influenza virus hemagglutinin are correlated with protection (a serum hemagglutination inhibition titer of approximately 30-40 provides approximately 50% protection against homologous virus infection) [159]. Antibody responses are typically measured by hemagglutination inhibition, by microneutralization, by simple radial immunodiffusion (SRID) and / or by simple radial hemolysis (SRH). These test techniques are well known in this field.

The compositions of the invention can be administered in various ways. The most preferred route of immunization is by intramuscular injection (for example, in the arm or leg), but other available routes include subcutaneous, intranasal [160-162], oral [163], intradermal [164,165], transcutaneous injection. transdermal [166], etc.

The compositions of the invention can be used to treat both children and adults. Flu vaccines are currently recommended for use in pediatric and adult immunization, from the age of 6 months. In this way, the patient can be less than 1 year old, 1-5 years old, 5-15 years old, 15-55 years old or at least 55 years old. Preferred patients to receive vaccines are the elderly (for example, ≥50 years, ≥60 years, preferably ≥65 years), young children (for example, ≤5 years), hospitalized patients, health workers, armed service and personnel military, pregnant women, chronically ill immunodeficient patients, patients who have taken an antiviral compound (for example, an oseltamivir or zanamivir compound; see below) within 7 days prior to receiving the vaccine, people with egg allergies and people traveling abroad. However, vaccines are not only suitable for these groups and can be used more generally in a population. For pandemic strains, administration is preferred to all age groups.

Preferred compositions of the invention satisfy 1, 2 or 3 of the CPMP criteria for efficacy. In adults (18-60 years), these criteria are: (1) ≥70% seroprotection; (2) ≥40% seroconversion; and / or (3) a GMT increase of ≥2.5 times. In the elderly (> 60 years), these criteria are: (1) ≥60% seroprotection; (2) ≥30% seroconversion; and / or (3) a GMT increase of ≥2 times. These criteria are based on studies of open label with at least 50 patients.

The treatment can be performed by a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunization schedule and / or in a booster immunization schedule. In a multiple dose program, the different doses can be administered by the same route or by different routes, for example, parenteral stimulation and mucous reinforcement, mucous stimulation and parenteral reinforcement, etc. The administration of more than one dose (typically two doses) is particularly useful in immunologically virgin patients, for example, for people who have never received a flu vaccine before,

or for vaccination against a new subtype of HA (as in a pandemic outbreak). Multiple doses will typically be administered at an interval of at least 1 week (eg, about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.).

Vaccines of the invention can be administered to patients substantially at the same time (for example, during the same medical consultation or visit to the healthcare professional or vaccination center) as other vaccines, for example, substantially at the same time as a measles vaccine. , a mumps vaccine, a rubella vaccine, an MMR vaccine, a chickenpox vaccine, an MMRV vaccine, a diphtheria vaccine, a tetanus vaccine, a pertussis vaccine, a DTP vaccine, a vaccine of H. influenzae type b conjugate, an inactivated poliovirus vaccine, a hepatitis B virus vaccine, a meningococcal conjugate vaccine (such as a tetravalent AC-W135-Y vaccine), a respiratory syncytial virus vaccine, a pneumococcal vaccine conjugated, etc. Administration substantially at the same time as a pneumococcal vaccine and / or a meningococcal vaccine is particularly useful in elderly patients.

Similarly, the vaccines of the invention can be administered to patients substantially at the same time (for example, during the same medical consultation or visit to a healthcare professional) as an antiviral compound, and in particular an active antiviral compound against the virus of the influenza (for example, oseltamivir and / or zanamivir). These antivirals include neuraminidase inhibitors, such as (3R, 4R, 5S) -4-acetylamino-5-amino-3 (1-ethylpropoxy) -1-cyclohexane-1-carboxylic acid or 5- (acetylamino) -4 - [( aminoiminomethyl) -amino] -2,6-anhydro-3,4,5trideoxy-D-glycerol-D-galactonon-2-enonic, including esters thereof (for example, ethyl esters) and salts thereof (for example phosphate salts). A preferred antiviral is (3R, 4R, SS) -4-acetylamino-5-amino-3 (1-ethylpropoxy) -1-cyclohexene-1-carboxylic acid, ethyl ester, phosphate (1: 1), also known as phosphate of oseltamivir (TAMIFLU ™).

general

The term "comprising" encompasses "including" as well as "consisting", for example, a composition "comprising" X may consist exclusively of X or may include something additional, for example X + Y.

The term "substantially" does not "completely" exclude, for example, a composition that "substantially lacks" Y may completely lack Y. When necessary, the term "substantially" may be omitted from the definition of the invention.

The term "approximately" in relation to a numerical value x means, for example, x ± 10%.

Unless specifically indicated, a process comprising a mixing step of two or more components does not require any specific mixing order. In this way, the components can be mixed in any order. When there are three components, then two components can be combined with each other, and the combination can then be combined with the third component, etc.

When animal materials (and in particular cattle) are used in cell culture, they must be obtained from sources that are free of transmissible spongiform encephalopathies (TSEs) and, in particular, free of encephalopathy

5

10

fifteen

twenty

25

30

bovine spongiform (BSE). In general, it is preferred to grow cells in total absence of materials of animal origin. When a compound is administered to the body as part of a composition, then that compound can alternatively be replaced by a suitable prodrug.

When a cellular substrate is used for rearrangement or inverse genetic procedures, it is preferably one that has been approved for use in the production of human vaccines, for example, in general chapter 5.2.3 of the Ph Eur.

Brief description of the drawings

Figure 1 shows the number of cytokine positive cells, as a% of total CD4 + cells. The responses of two individual mice are shown. Mice were immunized with "A" or "B" fractional vaccines, without adjuvant or (i) alum or (ii) MF59 with a CpG oligonucleotide as adjuvants.

Figure 2 shows the formula of the most preferred form of 3dMPL for use with the invention.

Modes for carrying out the invention

Oil-in-water emulsion adjuvant that favors the response to Th1

Two trivalent influenza vaccines of commercially available fractionated virion without adjuvant ("SPLIT (A)" and SPLIT (B) ")) were obtained and used to immunize mice at a dose of 0.2 µg HA. were used without adjuvant or using adjuvant (i) aluminum hydroxide or (ii) a mixture of MF59 emulsion and 10 µg of an immunostimulatory CpG oligonucleotide Groups of 8 female Balb / C mice were immunized intramuscularly with the vaccines 8 weeks of age, with doses of 50 µl on days 0 and 28. Sera were obtained on days 14 and 42 and analyzed for anti-HA (IgG) titer, HI titer and T cells.

The serum IgG antibody titres (ELISA) on day 42 were as follows, considering each virus separately.

Without adjuvant

Alum MF59 + CpG

Anti-H1N1

SPLIT (A)

749 1329 8808

SPLIT (B)

1175 1991 6754

Anti-H3N2

SPLIT (A)

412 977 6032

SPLIT (B)

1111 1465 5308

Anti-B

SPLIT (A)

707 2534 11211

SPLIT (B)

1585 2520 10837

Antibody titers in HI serum on day 42 were as follows:

Without adjuvant

Alum MF59 + CpG

Anti-H1N1

SPLIT (A)

140 280 1387

SPLIT (B)

285 330 1371

Anti-H3N2

SPLIT (A)

290 780 800

SPLIT (B)

380 440 1371

Anti-B

SPLIT (A)

280 780 800

SPLIT (B)

550 440 1371

As shown in Figure 1, non-adjuvant vaccines induced very low levels of antigen-specific T cells. Alum as an adjuvant increased levels, in a deviated manner to Th2: in a mouse, alum produced a large increase in T-lymphocytes IL-5 + IFN-γ-TNF-α- (i.e., Th2 type), but not IL-5-IFNγ + TNF-α + cells (ie, Th1 type) were seen. On the contrary, a combination of MF59 and a CpG oligonucleotide produced both a large increase in the number of antigen-specific T lymphocytes and a shift towards a Th1 response.

Therefore, it is possible to use an oil-in-water emulsion adjuvant to raise the number of antigen-specific T lymphocytes induced by a fractional flu vaccine, and also to shift the immune response to a Th1 type response. In contrast, an alum adjuvant induces low levels of T cells with a Th2 response.

Human Trials [reference 167]

As described in reference 167, fractionated influenza vaccines were added as an adjuvant an oil-in-water emulsion having an organic phase consisting of two oils (α-tocopherol and squalene) and a

5 aqueous phase of phosphate buffered saline (PBS) containing Tween 80 as an emulsifying agent. It has a final concentration of 2.5% squalene (v / v), 2.5% α-tocopherol (v / v) and 0.9% polyoxyethylene sorbitan monooleate (v / v) (Tween 80). Initially it was prepared as a concentrate twice to mix with flu antigens. It is reported that this emulsion is a Th1 induction aid.

The emulsion was prepared by dissolving Tween 80 in (PBS) to give a 2% solution. To provide 100 ml of

10 a concentrate twice, 5 g of D, L-α-tocopherol and 5 ml of squalene were vortexed to mix thoroughly. 90 ml of the PBS / Tween solution was added to the oil mixture and mixed thoroughly. The resulting emulsion was then passed through a syringe and finally microfluidized. The resulting oil drops have a size of approximately 120-180 nm (mean Z).

In control experiments, the emulsion was not included.

15 Trivalent vaccines were administered to elderly human patients. The humoral and cell-mediated immune responses in the patients were measured as described in reference 167. Seroprotection and seroconversion were determined.

The percentages of seroprotection and seroconversion were as follows (left = control; right = with adjuvant):

A / New Caledonia

To Panama B / Shangdong

Seroprotection

98.0 98.0 91.8 100.0 95.9 100.0

Seroconversion

69.4 69.4 65.3 55.1 69.4 73.5

The anti-HA antibody responses were as follows:

Day

A / New Caledonia To Panama B / Shangdong

Control: no adjuvant

0

26.3 40.9 26.0

twenty-one

358.5 296.0 270.0

Emulsion as adjuvant

0

25.6 52.3 27.5

twenty-one

317.7 366.1 317.7

To compare Th1 and Th2 responses between the vaccine without control adjuvant and the emulsion vaccine as an adjuvant, cytokine responses were tested. CD4 + T lymphocytes taken from patients were re-stimulated

25 immunized with a fractionated antigen and evaluated with respect to the number secreting (i) at least interferon-γ (indicative of a Th1 response) and another of IL-2, TNFα or CD40L; or (ii) at least IL-2 (again, indicative of a Th1 response) and another of IFN-γ, TNFα or CD40L. The results, expressed as a difference before and after immunization, were as follows:

Without adjuvant

With adjuvant

IFN- image2

IL-2  IFN- image2   IL-2

CD4

1149 1738 2712 3465

30 Vaccine with adjuvant shows more Th1 response than control without adjuvant. In this way, the adjuvant containing tocopherol can modulate the Th1 / Th2 balance of a fractional flu vaccine.

It will be understood that the invention has been described by way of example only and that modifications may be made as long as it is kept within the scope of the invention.

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Claims (17)

one.

An immunogenic composition comprising a fractionated influenza virus antigen and a Th1 adjuvant, in which the antigen is prepared from a virus developed in a cell culture and does not include any egg protein and the Th1 adjuvant is in the form of (i) an oil-in-water emulsion that includes squalene, a tocopherol and polysorbate 80, or (ii) a submicrometric emulsion of squalene, polysorbate 80, sorbitan trioleate and an immunostimulatory oligonucleotide.

2.

The composition of claim 1, wherein the influenza virus antigen is a subtype of influenza A H1, H2, H3, H5, H7 or H9.

3.

The composition of claim 1 or claim 2, wherein the composition lacks ovalbumin, ovomucoid and chicken DNA.

Four.

The composition of any preceding claim, wherein the composition contains between 0 and 20 µg of hemagglutinin per viral strain.

5.

The composition of claim 4, wherein the composition contains approximately 15 µg of HA per strain

or contains approximately 3.8 µg of HA per strain.

6.

The composition of any preceding claim, wherein the tocopherol is DL-α-tocopherol.

7.

The composition of any preceding claim, wherein the emulsion has drops with a submicron diameter.

8.

The composition of any preceding claim, wherein the influenza virus antigen is prepared from a influenza virus having one or more RNA segments from a influenza virus A / PR / 8/34.

9.

The composition of any preceding claim, wherein the adjuvant comprises a monophosphoryl lipid A 3O-deacylated (3dMPL).

10.

The composition of claim 16, wherein at least 10% by weight of the 3dMPL is the hexaacyl chain form.

eleven.

The composition of any preceding claim, which substantially lacks mercurial material.

12.

The composition of any preceding claim, which includes one or more buffers.

13.

The composition of claim 12, wherein the buffer or buffers include: a phosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer; or a citrate buffer.

14.

The composition of any preceding claim, which has a pH between 5.0 and 8.1.

fifteen.

The composition of any preceding claim, wherein the composition includes two strains of influenza A and one strain of influenza B.

16.

The composition of any one of claims 1 to 14, wherein the composition is a monovalent vaccine against a strain of the pandemic influenza virus.

17.

A kit for preparing the composition of any preceding claim, comprising (a) a first component of the kit comprising a fractionated influenza virus antigen prepared from a virus developed in cell culture and which does not include any egg protein, and (b) a second kit component comprising a Th1 adjuvant in the form of (i) an oil-in-water emulsion that includes squalene, a tocopherol and polysorbate 80, or (ii) a submicron emulsion of squalene, polysorbate 80, trioleate of sorbitan and an immunostimulatory oligonucleotide; wherein the first and second components are in separate containers.