SI9400085A - Vaccine compositions comprising antigenic polypeptide and 3d-mpl, useful in preventing infection with influenza - Google Patents

Abstract

The present invention provides vaccine compositions capable of enhancing a protective response to a selected influenza antigen, said composition containing at least the antigen and 3D-MPL, and methods of enhancing an immune response to influenza using these compositions.

Description

(57) The present invention provides vaccine compositions capable of enhancing a protective response to a selected influenza antigen, the composition comprising at least an antigenic polypeptide and 3D-MPL, and methods for enhancing the immune response to influenza using these compositions.

1. SmithKline Beecham Corporation

2. SmithKline Beecham Biologicals (s.a.)

Vaccine compositions containing antigenic polypeptide and 3D-MPL useful in preventing influenza infection

This is a partial continuation of U.S. Patent Application Serial Number 021,535, filed Feb. 19, 1993, which is pending at the same time.

The present invention relates to vaccines useful in preventing influenza infection in humans.

Infection with influenza virus causes acute respiratory diseases in humans, horses and poultry, which are sometimes of a pandemic dimension. Influenza viruses belong to the orthomyxovirus family of RNA viruses and have wrapped virions of 80 to 120 nanometers in diameter with two external glycoprotein tips, hemagglutinin (HA) and neuraminidase (NA) and five internal proteins, a nucleoprotein, a matrix protein and three polymerases. Influenza viral RNA also encodes for two non-structural proteins (NS1 and NS2) that are produced in infected cells but are not incorporated into infectious virions.

Three types of influenza viruses, type A, type B and type C, infect humans. Type A viruses are responsible for most human epidemics in modern times, although there are also sporadic outbreaks of type B infections. Known swine, equine and poultry viruses are mostly type A, although type C viruses have also been isolated from pigs.

In the virus, genetic variation in the HA and NA surface proteins results in three important subtypes designated H1N1, H2N2 and H3N2. In type A, subtypes HI (swine influenza), H2 (Asian influenza) and H3 (Hong Kong influenza) are dominant in human infections.

of surface glycoproteins causing antigenic variation. It is most pronounced in type A virus, where the major genetic changes in HA or NA proteins have already expired (antigenic shifts). The emergence of these new viral subtypes results in infection of a pandemic dimension, resulting in considerable mortality and morbidity. E.g. H1N1 viruses, pre-1957 dominant, were replaced by H2N2 subtype viruses, which prevailed until 1968, and were then replaced by H3N2 subtype. In general, H3N2 strains are still circulating, but H1N1 viruses have re-emerged since 1977. HAs in a given subtype are also exposed to minor genetic changes (point mutations), every year or two (antigen flooding). Notably, they are limited to the antigenic determinants collected around sialic acid binding sites in HA1 and result in the formation of new viral strains. Although this antigen flooding does not cause serious mortality and morbidity of such magnitude as antigenic drift, it is responsible for annual influenza outbreaks.

Influenza vaccines are classified into three types, complete virion, cleft, and subunit. Vaccines are complete virions, based on intact viral particles, and although generally more immunogenic, tend to be more reactogenic and are therefore replaced by cleaved vaccines or subunits prepared from purified viral components obtained after the destruction of the virus by treatment with various chemical means. The difference between vaccines that are split and those that are subunits is that the vaccine subunit contains almost exclusively hemagglutinin and neuraminidase, the surface antigens of the virus, while the split vaccines additionally contain variable amounts of internal components of the virus, such as. ribonucleoprotein and matrix protein.

The generally available marketable influenza vaccines are based on the principle that the antibody for HA or NA provides protection. They consist of non-adjuvanted, inactivated, complete or split virus products that harness the virus grown in fertilized chicken eggs. All influenza vaccines generally contain preparations of H1N1, H3N2, and type B viral strains. Due to the annual antigenic variation, specific viral strains are harmonized on an annual basis according to WHO recommendations based on epidemiological surveillance of dominant circulating viral strains.

There is no universal vaccine for influenza virus, i.e. non-strain specific vaccine. Recently, attempts have been made to prepare such universal or semi-universal vaccines from reassortable viruses prepared by crossing different strains. More recently, such experiments have included recombinant DNA techniques focused primarily on the HA protein.

Influenza vaccines are less commonly used for reasons including doubts about efficacy, fear of side effects, the need for annual revaccination, and lack of interest among preparers. Existing vaccines have been shown to have about 60-80% efficacy against infection with influenza viruses, which are highly antigenically related to the viral strains used in the vaccine. This level of protective efficacy tends to decline when the HA antigen of an epidemic strain HA drifts out of the vaccine strain and would fall to zero if there were no shift in the subtype. In addition, protection is reduced in some immunocompromised groups, such as the elderly living in sanatoriums.

Therefore, the major disadvantages of widely available vaccines stem from the fact that, due to frequent antigen flooding, the component of viral strains needs to be changed annually and individuals need to be re-vaccinated.

Therefore, there is a need in the art for vaccine formulations and compositions capable of inducing protective responses in creatures for a wide variety of pathogens.

In one aspect, the present invention provides a vaccine composition capable of stimulating an increased immune and protective response in a vaccinated battle against influenza, the composition comprising a selected influenza antigen or antigenic polypeptide and an effective amount of 3-o-deacylated monophosphoryl lipid A (3DMPL).

In another aspect, the present invention provides a vaccine composition comprising a selected influenza antigen or antigenic polypeptide, an effective amount of 3D-MPL, and a liposomal preparation. The liposomal formulation is defined herein that, in addition to acting as a carrier, also acts as an adjuvant and offers considerable advantages in the manufacture and formulation.

In a further aspect, the present invention provides a method for enhancing a vaccine immune response for a selected influenza antigen. This process involves administering to the mammal, preferably a human, the vaccine composition described above.

Other aspects and advantages of the present invention are further described in the following detailed description of its preferred embodiments.

Figure 1 is a bar diagram showing the cross-protection for H1N1 and H2N2 subtype of influenza viruses in mice immunized with Flu D protein (SK&F 106160) in aluminum and 3D-MPL, as shown in Example 18.

Figure 2 is a bar diagram showing the splenic proliferative responses before challenge in mice vaccinated with Flu D formulations and controls. See example 20.

Figure 2B is a bar diagram showing the splenic proliferative responses after challenge in mice vaccinated with Flu D formulations and controls. See example 20.

Figure 3A is a bar diagram showing the proliferative responses of lymph nodes obtained on day 4 in mice vaccinated with 20 µg flu D in aluminum (blank columns) or aluminum and 3D-MPL (screwed columns). See example 21.

Figure 3B is a bar chart showing the proliferative responses of lymph nodes obtained on day 4 in mice vaccinated with 5 / w Flu D in aluminum (blank columns) or aluminum and 3D-MPL (screwed columns). See example 21.

Figure 3C is a bar diagram showing the proliferative responses of lymph nodes obtained on day 4 in mice vaccinated with 1 / w Flu D in aluminum (blank columns) or aluminum and 3D-MPL (screwed columns). See example 21.

Figure 4A is a bar graph showing the proliferation, on day 2, of lymph nodes, in mice immunized with 1 / wt of vaccine formulation of protein D. See example 21.

Figure 4B is a bar graph showing the proliferation of day 3 lymph nodes in mice immunized with 1 / wt of vaccine formulation of protein D. See example 21.

Figure 4C is a bar graph showing the proliferation of day 4 lymph nodes in mice immunized with 1 / wt of vaccine formulation of protein D. See example 21.

Figure 4D is a bar diagram showing IL-2 production, on day 2, of lymph nodes, in mice immunized with 1 / wt of vaccine formulation of protein D. See example 21.

Figure 4E is a bar chart showing IL-2 production, on day 3, of lymph nodes, in mice immunized with 1 / wt of vaccine formulation of protein D. See example 21.

Figure 4F is a bar diagram showing IL-2 production, on day 4, of lymph nodes, in mice immunized with 1 μ-g of vaccine formulation of protein D. See example 21.

Figure 5A is a diagram showing interferon levels in antigen-stimulated cultures in mice immunized as described in Example 24 below.

Figure 5B is a diagram showing IL-2 levels in antigen-stimulated cultures obtained from mice immunized as described in Example 24 below.

Figure 6A is a diagram showing viral titers determined in the nose by MDCK microtest on days 1, 3, 5, 7 and 9 after challenge (5 mice per group) for control containing aluminum and 3D-MPL (- squared- ), an influenza monovalent split vaccine containing A / PR / 8 strain without adjuvant (-triangle-), and strain A / PR / 8 adjuvanted with 3D-MPL (continuous line and circle). See example 28.

Figure 6B is a diagram showing viral titers determined in the nose by MDCK microtest on days 1, 3, 5, 7 and 9 after challenge (5 mice per group) for control containing aluminum and 3D-MPL (-square-) , an influenza monovalent split vaccine containing a Singaporean strain without adjuvant (-triangle-), and a Singaporean strain adjuvanted with 3DMPL (continuous line and circle). See example 28.

Figure 6C is a diagram showing viral titers determined in the trachea by MDCK microtest on days 1, 3.5, 7, and 9 after challenge (5 mice per group) for the same three vaccine formulations as in Figure 6A.

Figure 6D is a diagram showing the viral titers determined in the trachea by MDCK microtest on day 1,3, 5, 7 and 9 after challenge (5 mice per group) for the same three vaccine formulations as in Figure 6B.

Figure 6E is a diagram showing viral titers determined in lungs with MDCK microtest on days 1, 3, 5, 7 and 9 after challenge (5 mice per group) for the same three vaccine formulations as in Figure 6A.

Figure 6F is a diagram showing viral titers determined in lungs with MDCK microtest on days 1, 3, 5, 7 and 9 after challenge (5 mice per group) for the same three vaccine formulations as in Figure 6B.

The present invention provides vaccine compositions capable of eliciting an increased immune response in vaccinated hosts, including humans, as well as processes for the preparation and use of such vaccine compositions. The vaccine composition of the invention comprises an effective amount of the selected influenza antigen or antigenic polypeptide and 3-o-deacylated monophosphoryl lipid A (3D-MPL). In the present case, the liposome preparation may also be a component of the vaccine compositions of the invention.

The inventors have found that combinations of 3D-MPL and certain influenza antigens are effective in achieving protective influenza responses that are not achieved by the influenza antigen itself. E.g. with the antigenic polypeptide known as Flu D described below, this response is such that a lower amount of antigen is required to obtain the same results as with purified Flu D and complete Freund's adjuvant (CFA), a known potent animal adjuvant .

Furthermore, when the selected influenza antigen and 3D-MPL are incorporated into the liposome as described herein, a protective response is obtained that exceeds that obtained with the antigen and any other combination of adjuvant.

By the term enhanced immune response as used herein, it is meant that the vaccinated host produces a stronger cellular immune response (production of T protective lymphocytes) for the vaccine composition of the invention, such as or would be produced by the host in response to a selected antigen other than adjuvanted or adjuvanted with other conventional adjuvants suitable for internal administration. An increased antibody (cell B) response is also expected with this increased response.

By the term immunologically effective amount or effective amount as used herein is meant that amount of antigen that induces a protective immune response.

The term antigen, antigenic polypeptide or protein or immunogen as used herein is intended to mean a complete or inactivated pathogen, immunogenic protein, peptide or fragment from a pathogen, optionally fused to another peptide or protein that is homologous or heterologous sources. These terms also include the split virus described below. These terms may also include non-protein pathogens. Pathogens are preferably organisms that cause diseases that infect humans, although animal pathogens can also be used in these vaccines where desired for veterinary purposes. These terms refer to the ability of a complete pathogen, a cleaved virus, a peptide or fusion protein to elicit a protective immune response in a vaccinated host.

By the term monovalent vaccine is meant a vaccine containing antigens of a single type or subtype of influenza virus, e.g. H1N1, H2N2, H3N2 type A, type B and type C.

By the term multivalent vaccine is meant a vaccine containing antigens of more than one type or subtype of influenza virus, i.e. a trivalent vaccine may contain antigens from any of the three influenza types or subtypes, e.g. H1N1, H2N2, H3N2 type A, type B and type C.

The term split virus is intended to mean an influenza virus suspension obtained from fertilized chicken eggs inoculated with seed material that is partially purified and concentrated. The concentrated viral suspension is treated with a detergent such as e.g. sodium deoxycholate to break down the viral particles. Removal of viral phospholipids during this cleavage process produces an inactivated influenza antigen for which the reactogenicity potential is greatly reduced. The split virus suspension is completely inactivated by the combined effect of detergent and formaldehyde.

The following description of the compositions and methods of the invention describes in detail vaccine compositions for prophylactic use against influenza virus.

In one preferred embodiment of the invention, a vaccine composition capable of producing an enhanced immune response, protective against influenza virus infection, contains at least a selected influenza antigen polypeptide, such as e.g. NS1 ₁ ^ ₁ HA2 _{65 222} (referred to herein as Flu D, D protein, or Flu D protein) adjuvanted with 3D-MPL.

Generally, flu D protein is a preferred influenza antigen polypeptide for use in the vaccine compositions of the invention because it is most readily purified by influenza fusion proteins containing the entire carboxy terminal of the HA2 portion of the hemagglutinin region. The D protein contains the first 81 amino acids of NS1 fused to the amino acid of 65 truncated HA2 subunits (amino acids 65-222). In the present case, as regards the other NS1-HA2 fusion proteins shown herein, the linker sequence can be included between two fused sequences.

In the present preferred embodiment, the DNA coding sequence for the flu D protein is prepared as described in EP 0366238 by restriction of the HA2 coding sequence with PvuII and ligation of the C-terminal region of the Ncol site between amino acids 81 and 82 in the NS1 coding sequence by a synthetic oligonucleotide linker. This linker selection codes for glutamine-isoleucine-proline. The D protein, for which a 90-95% purity was obtained, requires the use of the purification procedures described herein to essentially remove host cell proteins (E. coli) and other contaminants.

Flu D protein and recombinant expression and purification thereof are detailed in U.S. Pat. 07 / 751,899, which is pending at the same time as its corresponding European patent application no. No. 366,238, published May 2, 1990, and U.S. Pat. no. No. 07 / 387,558 and European Patent Application no. No. 366,239, published May 2, 1990. These applications are incorporated by reference for the purpose of describing, expressing and purifying this protein.

In addition to Flu D, other suitable influenza antigen polypeptides can be used in the vaccine compositions of the invention, including those described in European patent application no. No. 366,238 and EP 366,239, both published May 2, 1990, and U.S. Pat. 07 / 751,898 07 / 751,896 and 07 / 837,773, which are simultaneously pending. Such proteins include Δ M, Δ M +, A, C, C13.03 short and Δ D. Others that are suitable include constructs D without Cys, HAZ ^ ₂₂₂ , and NS1H3HA2, such as e.g. those described in U.S. Patent Ser. no. 07 / 751,898, 07 / 751,896, 07 / 751,896 and 07 / 837,773 and WO 93/15763, which are contemplated simultaneously and are incorporated herein by reference. The H3HA2 constructs mentioned above are particularly desirable. Recent inventor studies show that mice immunized with a cocktail containing both NS1-H1HA2 and NS1-H3HA2 fusion proteins are protected against aviation challenge by both HI and H3 viral subtypes.

The coding sequences for HA2, NS1 and other influenza virus viral proteins can be prepared synthetically or can be derived from viral RNA using known techniques, or from accessible cDNA-containing plasmids, as described in published European applications cited above. E.g. in addition to the above references, the DNA coding sequence for HA from A / Japan / 305/57 strain is cloned and sequenced as cited by Gething et al, Nature, 287: 301-306 (1980); The HA coding sequence for strain A / NT / 60/68 is cloned as cited by Sleigh et al, and Both et al, both in Developments and Celi Biology, Elsevier Science Publishing Co., p. 69-79 and 81-89, (1980); The HA coding sequence for strain A / WSN / 33 is cloned as reported by Davis et al, Gene, 10: 205-218 (1980); and Hiti et al, Virology, 111: 113-124 (1981). The HA coding sequence for the poultry plaque virus is cloned as cited by Porter et al and Emtage et al, both in Developments and Celi Biology, (cited above), p. 39-49 and 157-168.

Systems for cloning and expression of vaccine polypeptides in a variety of microorganisms and cells, including e.g. E. coli, Bacillus, Streptomyces, Saccharomyces, mammalian and insect cells are known and available from private and public laboratories and warehouses and from commercial vendors.

The D protein can be purified by the method described in Example 13. Various conventional methods can be used to purify the protein of the compositions of the invention, although these processes are not necessary to obtain a highly purified pharmaceutical grade product. Such procedures may be used during, before or after the steps described above. One such optional step is diafiltration, a form of continuous dialysis that is extremely effective in achieving many buffer changes. Diafiltration is preferably carried out past the cellulose membrane or ultrafilter. Suitable membranes / filters are those with a molecular weight of about 1000, cut off for those with pore sizes up to 2.4 µm in diameter. Many different systems, adaptable to diafiltration, are commercially available, such as for example. 10 K Amicon Dual Spiral Cartridge. In the process of the invention, diafiltration using 20 mM tris buffer at about pH 8 can be effectively used to purify and subsequently concentrate the polypeptide used in the vaccine composition of the invention. In another preferred embodiment, the antigen used in the vaccine composition of the invention is a fully inactivated pathogen, such as e.g. the split virus detailed in Example 25. A monovalent split influenza vaccine containing a split virus or a multivalent (e.g. trivalent) split influenza vaccine containing more than one split virus can be adjuvanted with 3D-MPL. In one formulation, the vaccine contains a split virus prepared from an H1N1 strain, such as e.g. Singapore / 6/86 [Sachsisches Serumwerkl GmbH (SSW), Dresden, Germany and National Institute for Biological Standards and Control (NIBSC, London, England)], commonly used in conventional influenza vaccines. Alternatively, other H1N1 cleaved viruses can be prepared from A / PR / 8/34 (also referred to as A / PR / 8) described in T. Francis, Proc. soc. Exp. Biol. Med., 32: 1172 (1935) and available from American Type Culture Collection, Rockville, Maryland, USA under ATCC no. VR-95. The monovalent vaccine contains antigens from one strain of influenza virus. Alternatively, the vaccine may be multivalent, containing more than one influenza antigen, e.g. two or three split viruses to increase reactivity against more than one influenza virus. An example of a trivalent vaccine includes e.g. H1N1 Singapore / 6/86 strain, with H3N2 strain Beijing / 32/92 (SSW), Dresden, Germany] and type B strain, Panama / 45/90 [SSW, Dresden, Germany].

Influenza viruses that can be prepared as split viruses in a known manner as described in Example 25 include any strains, subtypes and types, especially those recommended by WH0, many of which are available from clinical specimens and from public storage, such as American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, 20852, USA (ATCC) and NIBSC. E.g. other suitable H3N2 viral strains include, without limitation, A / Victoria / H3 / 75, described in Fiers et al, Celi, 19: 683696 (1980); A / Udorn described in C. J. Lai et al, Proc. Natl. Acad. Sci., USA, 77: 210214 (1980); Palese and Schulman, virol., 57: 227-237 (1974); and A / HK / 8/68 described in WHO Weekly Epid. Record, Vol, 43: 401 (1968) and available from ATCC as no. VR544 as well as Beijing / 32/92. Other suitable Type B strains include, without limitation, those known as B / Lee / 40, described by Krystal et al, Away. Natl. Acad. Sci., USA 79: 4804-4900 (1982) and B / Taiwan, available from ATCC as no. VR-295, Panama / 45/90 and B / Yamaghta strains. H2N2 viruses can also be used in these vaccines.

It is understood that, in addition to other influenza viruses or inactivated viral preparations, other antigenic materials from other pathogens are provided for use in addition to the antigens illustrated, following the instructions given herein.

Another component of the vaccine composition of the invention is the 3D-MPL adjuvant. 3DMPL is described in detail in U.S. Pat. No. 4,912,094, which is incorporated herein by reference and is commercially available from RIBI Immunochem Research Inc., Hamilton, Montana. Briefly, 3D-MPL is an endotoxin derivative, monophosphoryl lipid A (MPL), a lipid A analog isolated from a non-heptose RE mutant of a gram-negative bacterium, such as e.g. Salmonella minnesota. MPL does not have a phosphate group in the C-1 position of glucosamine. Treatment of the MPL molecule to remove the acyl chain in position 3 of glucosamine yields 3D-MPL. 3D-MPL is non-toxic in contrast to other enterobacterial lipopolysaccharides but retains the antigenic activity of the original endotoxin. This molecule is useful in preventing gram-negative sepsis and endotoxemia.

3D-MPL can be dissolved in water to obtain solutions of vesicular aggregates likely composed of lipid bilayer membranes. Therefore, it is not possible to detect 3D-MPL as an individual molecule in the immune system because it prefers to interact with membrane-to-membrane contacts involving cell surfaces and vesicle surfaces.

In a preferred embodiment, the MPL particles are small and generally do not exceed 120 nm.

deacylated monophosphoryl lipid A with small particles generally not exceeding 120 nm can be made by the procedure described in GB 2 220 211 (or commercially available larger particulate MPL can be obtained from Ribi Immunochem.) and the product can then be sonicated until suspension is obtained. clear. The particle size can be determined using dynamic light scattering as described after.

Preferably, the particle size is in the range 60-120 nm.

Most preferably, the particle size is below 100 nm.

In accordance with the present invention, the inventors have determined that an influenza antigen polypeptide, e.g. flu D protein, When highly purified, it is neither immunogenic nor protective in the absence of an adjuvant. However, when adjuvanted with 3D-MPL as described herein and shown in Examples 14-24 below, the protein is capable of inducing protection against influenza infection. If the antigen cleaved virus is selected in a monovalent or trivalent composition, the composition of the invention has increased immunogenicity and cross-reactivity.

Furthermore, the inventors have found that if an influenza antigen polypeptide, e.g. flu D protein is adjuvanted with 3D-MPL, then a lower dose of flu D protein is required to achieve the same level of immune response as is obtained if the protein is adjuvanted with aluminum adjuvant only. If the selected antigen is a split influenza virus in a monovalent or trivalent composition, it is contemplated that the reactogenicity of the composition will be less if less antigen is used in the embodiment of the invention.

Additional adjuvants may also be included in the vaccine compositions of the invention. One suitable additional adjuvant is aluminum or aluminum hydroxide or aluminum phosphate. When adjuvanted with D aluminum and 3D-MPL, the flu D protein is equivalent to that detected with complete Freund's adjuvant (CFA), a classic T cell adjuvant that is not suitable for human administration.

For the preparation of a preferred vaccine composition of the invention containing an antigenic polypeptide, e.g. flu D protein and 3D-MPL, the required amount of flu D protein is mixed with an appropriate amount of 3D-MPL, as described in more detail below. In the present case, the lyophilized lipid A is mixed with a pre-liposomal gel detailed below before the antigen. The most preferred molar ratio of phosphatides to lipid A is 66: 1. However, the density of the asset can be varied to the desired level.

Other suitable agents for addition to the vaccine composition include, e.g. IL-2, QS21 [C. R. Kensil et al., J. Immunol., 146 (2): 431-437 (1991)] and muramyl dipeptides (MDP). In addition, other water-soluble or insoluble chemicals or drugs and / or adjuvants described above may be included in the vaccine compositions of the invention. For example, muramyl dipeptides can also be used in similar proportions as described above or as described above. necessary. Other drugs that may be part of such a vaccine may include any substance that, when contained in a vaccine, modifies one or more of its functions, e.g. as indicated in the official pharmacopoeia or substance used in the treatment or prevention of infections.

The vaccine compositions of the invention may further comprise suitable carriers well known to those skilled in the art of vaccines and easily selected. Examples of carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextrin, agar, pectin, peanut oil, olive oil, sesame oil, squalene and water. In a given case, the carrier or diluent may include temporal retention agents such as e.g. glyceryl monostearate or glyceryl distearate alone or with wax. Suitable chemical stabilizers may be used to improve the stability of the pharmaceutical composition. Suitable chemical stabilizers are well known in the art and include e.g. citric acid and other pH adjusters, chelating or sequestering agents and antioxidants. Alternatively, if the liposomal delivery system is part of the vaccine composition, then no carriers are required.

Another preferred vaccine composition of the invention comprises a selected antigen, e.g. flu D protein as described above and 3D-MPL, wherein the components are trapped or embedded in a liposomal preparation. Where appropriate, the liposome, flu D protein, and 3D-MPL containing the vaccine composition may also contain one or more additional influenza antigens or other antigens or suitable adjuvants and agents as described above. In a preferred formulation, the vaccine comprises an antigenic polypeptide flu D protein and 3D-MPL placed in a carrier liposome. In another formulation, the vaccine contains a mono- or multivalent influenza antigen, which is similarly contained in a carrier liposome.

The inventors have found that the liposomal preparations described herein are capable of acting not only as carriers but also as adjuvants and are particularly preferred because they do not require organic solvents or high shear areas, which is a significant advantage of the protein drug and All liposome ingredients are considered safe for internal administration. Due to the simplicity and flexibility of the preparative process, these liposome preparations are suitable for large-scale production.

The inventors have also found that 3D-MPL can be easily incorporated into the liposomal structures described herein in combination with influenza antigens to obtain results superior to those found when influenza antigens are combined with known conventional adjuvants. Typically, a vaccine composition containing D protein in 3D-MPL and a liposomal formulation has even greater efficacy than D protein in CFA. See, e.g. example 20 and 22 below.

Liposomal preparations useful in the vaccine compositions and methods of the invention are described in U.S. Pat. no. No. 07 / 714,984 (U.S. Pat. No. 5,230,899), which is contemplated by reference herein. In general, the word liposome has been proposed and accepted as a term for use in the scientific literature describing synthetic, oligolamel lipid vesicles. Such vesicles typically consist of one or more concentric natural or synthetic lipid bilayers surrounding the internal aqueous phase.

Specifically as defined herein and in accordance with the included reference, liposome preparations useful in the vaccine composition of the invention are prepared by dispersing in aqueous medium in a manner appropriate to liposome formation, the composition comprising a long-chain liposome-forming substance aliphatic or aromatic acid or amine; a hydrating agent having a charge opposite to that of the acid or amine present in a molar ratio between 1:20 and 1: 0.05 relative to the acid or amine; and water in an amount up to 300 mol relative to solids.

The preparation of liposomal adjuvant carriers useful according to the invention follows. Examples of liposome-forming substances include lipids that are administered and those that cannot be saponified, e.g. acyl glycerols, phosphoglycerides, sphingolipids, glycolipids, etc. Fatty acids include saturated or unsaturated alkyl (Cg-C ^) carboxylic acids, monoalkyl (C _g C ₂₇ ) esters of (C ₄ -C ₁₀ ) dicarboxylic acids, (e.g. cholesterol hemiatic acid and fatty acid derivatives of amino acids in which they are included also any N-acyl carboxylic acid (e.g. N-oleoyl threonine, Ndinoleoyl serine, etc.) Mono- or dialkyl (Cg-C ^) sulfonate esters and mono- or dialkyl (Cg-C ^) sulfonate esters and mono- or the dialkyl (Cg-C ^) phosphate esters may be substituted for fatty acids, and mono- or diacyl (Cg-C ^) glycerol derivatives of phosphoric acids and mono- or diacyl (Cg-C ^) glycerol derivatives of sulfuric acids may be used instead of fatty acids. .

In addition, fatty acids can also be replaced by amines (e.g., Cg-C ^ NHJ, Cg-C ^ fatty acid derivatives of amines (e.g., Cg-C ^ CONH-NH ₂ ), C _g -C ₂₄ fatty alcohol derivatives of amino acids ( e.g., Cg-C ^ OOC-NH ₂ ), and Cg-C ^ fatty acid esters of amines (e.g., Cg-C ^ COO-NH ₂ ).

Photopolymerizable lipids and / or fatty acids (or amines) (eg diacetylene fatty acids) may also be included that can provide closed liposomes with cross-linked membrane bilayers after photoinitiation of the polymerization.

Long-chain aliphatic and / or aromatic acids or amines include an acid or amine which has an open chain structure and consists of paraffin, olefin and acetylene hydrocarbons and their derivatives, e.g. saturated and unsaturated hydrocarbons or the backbone of such a chain contains an aromatic substituent. Such acids and amines may have more than one such function. The term long chain means that the framework of an aliphatic acid or amine chain contains 10 or more carbon atoms. If the chain contains an aromatic group, such as e.g. phenyl, the chain comprises a framework of at least five carbon atoms fused to an aromatic group. The chain of carbon atoms in the framework may be variously substituted by saturated or unsaturated aliphatic or aromatic functions.

The terms acid or amine mean commonly defined chemical functionalities. E.g. the acid function may be carboxylate or derived from phosphorus or sulfur, such as e.g. phosphate, phosphite or pyrophosphate or sulfate, sulfite, thiosulphate or similarly composed phosphorus or sulfur based acid. The amines must be sufficiently basic to have ionizable and / or hydrogen. capable of forming quaternary salts having such an ionization constant that they can form the necessary hydrate complex.

As used in the vaccine composition and depending on the amount of water used in the liposomal composition, the term solid refers to liposome forming agents and an acid or amine component, hydrating agents, 3D-MPL, aluminum and the selected antigenic substance to be encapsulated .

For the purposes of the invention, a hydrating agent means a compound with at least two ionizable groups, preferably of opposite charge, one being capable of dissociating an ionic salt, the salt being able to form a complex with the ionic functionality of an acid or amine in a substance which forms the liposome. The hydrating agent does not by itself form liposomes. Such an agent is also physiologically acceptable, i.e. does not have any adverse or adverse physiological effects on the host to which it is administered in connection with its use according to the invention.

Preferred hydrating agents are alpha amino acids with ionizable omega substitutions, such as e.g. carboxylate, amino and guanidine function and compounds represented by the formula:

x- (CH ₂ ) „- Y i where

XH ₂ NC (NH) -NH-, H ₂ N-, ZO ₃ S-, Z ₂ O ₃ P-, or ZO ₂ Where ZH is an inorganic or organic cation;

Y is -CHCNH ^ -CC ^ H, -NH ₂ -NH-C (NH) -NH ₂ -COOH, CH (NH ₂ ) SO ₃ Z or ZH (NH ₂ ) PO ₃ Z ₂ , where Z is defined above ; and n is an integer 1-10; or a pharmaceutically acceptable salt thereof.

Also included in the list of preferred compounds are Ν, Ν′-dialkyl substituted arginine compounds and similar compounds where the alkyl chain length varies. More preferred hydrating agents are omega substituted alpha amino acids, such as e.g. arginine, its N-acyl derivatives, homoarginine, gamma-aminobutyric acid, asparagine, lysine, ornithine, glutamic acid, aspartic acid or compounds represented by the following formulas:

H ₂ NC (NH) -NH- (CH ₂ ) _n -CH- (NH ₂ ) COOH II

H ₂ N- (CH ₂ ) _n -CH (NH ₂ ) COOH III

HzN-tCH ^ -CNHz) IV

H ₂ NC (NH) -NH- (CH ₂ ) _n -NH-CH (NH) - (NH ₂ ) V

HOOC- (CH ₂ ) _n -CH (NH ₂ ) COOH VI

HOOC- (CH ₂ ) _n -COOH VII

HO ₃ SC- (CH ₂ ) _n -CH (NH ₂ ) COOH VIII

H ₂ o ₃ s- (CH ₂ ) _n -CH (NH ₂ ) cooH IX

HO ₃ S- (CH ₂ ) _n -CH (NH ₂ ) SO ₃ HX

H ₂ O ₃ S- (CH ₂ ) _n -CH (NH ₂ ) PO ₃ H ₂ XI where n is 2-4.

The most preferred compounds are arginine, homoarginine, gamma-butyric acid, lysine, ornithine, glutamic acid or aspartic acid. The hydrating agents of the invention can be used alone or as a mixture. No restrictions are included or implied when using mixtures of these hydrating substances.

The hydrating agents of the invention are listed in the catalog of many chemical manufacturers, which are also commonly manufactured or can be made by methods known in the art. Arginine, homoarginine, lysine, glutamic acid, aspartic acid and other natural amino acids can be obtained by hydrolysis of the protein and separation of individual amino acids, or from bacterial sources.

The compounds of formula II can be made by the procedure of Eisele, K. et al, Justusliebigs, Ann. Chem., P. 2033 (1975). Further information on various representative examples of these compounds can be found in Chemical Abstracts Service, no. as follows: nonarginine, CAS # 14191-90-3; arginine, CAS # 74-79-3; and homoarginine CAS # 151-86-5. For representative examples of Formula III, see for 2,4-diaminobutanoic acid CAS # 305-62-4 and for lysine CAS # 56-87-1. Methods for making representative compounds of formula IV are available from Chemical Abstracts as follows: ethanediamine, CAS # 305-62-4; propane diamine-54618-94-9; and 1,4-diaminobutane, CAS # 52165-57-8. See specifically Johnson, T.B., J. am. Chem. Soc., 38: 1854 (1916).

Of the compounds of formula VI, glutamic acid is well known in the art and is available from many commercial sources. Descriptions for the manufacture of other representative compounds are contained in the literature, for example: 2-aminohexanoic acid - CAS # 62787-49-9 and

2- Aminoheptanedioic acid - CAS # 32224-51-0. Glutamic acid, a compound of formula VII wherein n is 2, is well known in the art and can be made by the process of Maryel and Tuley, Org. Syn., 5:69 (1925). Other representative compounds in this group may be made by the procedures referenced by the following CAS numbers: hexadioic acid, CAS # 123-04-9 and heptadioic acid, CAS # 111-16-0. Homocysteic acid is known in the art with reference CAS # 56892-03-6. Compound

3- Sulfovalin has been described in the literature with reference CAS # 23405-34-2.

Mixtures of hydrating agent, liposome-forming substance and discrete amounts of water form a gel-like mass. When in this form of gel, the hydrating agent and the acids or amines or amines, together with the liposome forming substances, are arranged in a hydrate complex, which is a highly ordered liquid crystal. Hydrate complex means a complex formed between a hydrating agent, an acid or an amine, in a liposome forming substance. Complexing in this connection means the formation of dissociative ion salts, where one functionality associates with the ionic functionality of the liposome-forming substance and the other functionality has hydrophilic properties that give the water solubility of the resulting complex. While the structure of the liquid crystal of the hydrate complex varies with the pH and amount of the hydrating agent, the structure of the liquid crystal is maintained. NMR spectroscopy confirms that this crystal structure consists of several lamellar lipid bilayers and hydrophilic layers that are alternately stacked together. The ³¹ P-NMR spectrum shows an anisotropic peak, confirming the existence of multilamellar bilayers.

Although the primary components of these liposomes are lipids, phospholipids and other fatty acids, various other components can also be added to modify liposomal permeability. We can add e.g. non-ionic lipid components, such as e.g. polyoxy alcoholic compounds, polyglyceride compounds or polyol esters, polyoxycholinolated alcohols; polyol esters and synthetic lipolipids such as cerebrosides. Other substances, such as long chain alcohols and diols, long chain amines and their quaternary ammonium derivatives; polyoxyethylene fatty amines, esters of long chain amino alcohols and their salts and quaternary ammonium derivatives; phosphorus esters of fatty alcohols, polypeptides and proteins.

The composition of a liposome can be made from more than one component of various types of lipids, fatty acids, alkyl amines, etc., and hydrating agents.

If the lipid component itself or the antigenic material or any other material to be added to the vaccine composition for encapsulation has the previously mentioned properties, then it is not necessary to include fatty acids (or amines) or hydrating agents in the lipid composition to form a pre-liposomal gel or liposomes. E.g. a mixture of dipalmitoylphosphatidylcholine (DPPC) and disteroyl phosphatidylethanolamine forms a pre-liposomal gel, respectively. liposomes with an aqueous solution of lutamic acid, a mixture of DPPC and oleic acid, form a pre-liposomal gel and liposomes with an aqueous epinephrine solution.

As an adjuvant substance for use in the vaccine composition, the liposome preparation preferably includes phospholipids, butyric acid (or phosphatidyl-ethanolamine) and arginine or lysine (or glutamic acid and / or aspartic acid).

About 1:20 the molar ratio of the hydrating agent to the liposome-forming substance provides beneficial effects of the invention, with an upward limitation of about 1: 0.05. The preferred concentration range for the hydrating agent is the molar ratio of the hydrating agent relative to the liposome forming substance between 1: 2 and 1: 0.5.

Preferably, therefore, if liposomes are prepared with a long-chain aliphatic or aromatic acid, hydration agents containing at least one ionizable nitrogen, such as e.g. arginine, homoarginine, lysine, ornithine and the like. Conversely, if amphiphatic substances are used to form liposomes containing long-chain aliphatic or aromatic amines, it is preferable to use a diacid such as e.g. glutamic acid, aspartic acid; any of the alkyl diacids, such as e.g. simple diacids such as e.g. valeric acid, caprylic, capric, caprine, etc .; or those diacids having two phosphate or sulfate functionalities; or those diacids having mixed -COOH / -SO ₃ H or -COOH / -PO ₃ H ₂ functions.

Certain aliphatic and aromatic acids and amines are preferred for use according to the invention. Such compounds may have multiple functions, with two or more acid or amine groups or combinations thereof. E.g. diacid, diamine or an acid and amine compound may be used. Preferred compounds are those having one or two acid or amine functions. More preferred are fat mono acids with 10 to 20 carbons, saturated and unsaturated. Most preferred are alkyl and alkenyl acids with 10 to 20 carbon atoms, especially oleic acid.

Mixtures of liposome-forming substances, long-chain aliphatic or aromatic acid or amine, one or more hydrating agents with up to 300 moles of water relative to the total amount of solid, with or without the selected amount of antigen, produce a gel from which liposomes are formed directly after the addition of aqueous solutions. This gel can be termed a pre-liposomal gel because its structural properties are essentially the same as those of the liposomes and it is capable of being converted to liposomes after dilution with aqueous solution. An aqueous solution in excess of about 300 mol equivalents causes the formation of liposomes to begin.

The structure of this gel is a highly ordered liquid crystal that forms an optically clear solution. X,

The Y and Z dimensions of the liquid crystal vary with the concentrations of the hydrating agent at constant pH as well as with the pH of the solution. By varying the concentration of the hydrating agent at constant pH or changing the pH while maintaining a constant percentage of hydrating agent, the size and number of lamellar structures of the lipid bilayer of later liposomal vesicles can be controlled.

The gel structure itself is adjusted to about 300 moles of water per mole of solids without disturbing its stability. The gel structure, as determined by proton nuclear magnetic resonance spectroscopy (NMR), comprises multilamellar lipid bilayers and hydrophilic layers alternately stacked together. The ³¹ P-NMR spectrum of this gel shows an anisotropic peak, which further confirms that the gel consists of several lamellar bilayers. This gel can be autoclaved, which is a convenient method of sterilization. In addition, no change in color is observed with the gel and remains clear at room temperature for at least one year after autoclaving. If desired and feasible, the gel may optionally be further sterilized by filtration through appropriate sterilization filters. After dispersing the gel in aqueous solution, liposomes are effective and spontaneously produced.

Pre-liposomal gel with or without 3D-MPL and the antigenic substance for encapsulation can also be dehydrated (eg, lyophilized) and the powder re-hydrated to form liposomes, spontaneously, even after a longer storage period. This ability provides vaccine compositions particularly useful for the administration of water-sensitive antigenic substances, where long-term pre-storage is required.

The pre-liposomal gel was prepared as follows. The semi-solid liquid crystalline gel referred to herein as the pre-liposomal gel is prepared by combining three basic ingredients: phospholipid, fatty acid and a hydrating agent in water. Depending on the lipid composition required, phospholipids or mixtures thereof belonging to the same species with respect to the transition temperature (Tm) of the gel-liquid can be used. The fatty acid can be selected according to the degree of saturation or chain length and is usually mixed with phospholipid in a thick paste. Cholesterol can be added to the lipid mixture to control the bilayer character. (In some cases, cholesterol increases the Tm of the membrane and thus affects its permeability for the agents involved).

Hydrating agent, preferably alpha amino acids, such as e.g. arginine, add the lipid mixture as a solution in water, slowly to obtain a homogeneous paste. For one of the desired formulations, egg phosphatidyl holimolic acid is used in a molar ratio of 1: 1.2. Arginine is added in water at about 1: 1.2 molar ratio of phospholipid to arginine.

The concentration of L-arginine in the component of the aqueous phase of the gel affects the diameter of the stable liposomal particles that eventually arise. The particle size depends on the route of administration and on the targeting of macrophage cells. E.g. for oral administration, the desired liposomal particle size is between about 1 and about 5 μτη. Particle size between about 200 and 500 nm is preferred for targeting lymphocytes. For the depot effect, the desired liposomal particle size is between about 5 and about 10 μτη. Different particle sizes are easily tested and selected by an expert.

The final pre-liposomal gel may contain from about 65 to about 70% by weight of water, has an ointment density and looks like a characteristic liquid crystal when viewed under a polarizing light microscope. The pre-liposomal gel can be stored under an inert atmosphere or lyophilized to a dry storage powder for an extended period of time.

In the formation of the vaccine compositions of the invention, the same stages of liposomal preparation are followed by the addition of an immunologically effective amount of the selected antigen, e.g. an antigenic polypeptide, in particular flu D protein and 3D-MPL, and optionally one or more additional immunogenic proteins, peptides or fragments from a selected pathogen mixed with the liposome preparation.

To prepare such vaccine compositions, the selected antigen is encapsulated or intercalated into a liposomal preparation by mixing it.

There are two methods by which 3D-MPL and the selected antigen can be incorporated into a liposomal composition to prepare a vaccine composition according to the present invention. One method involves the incorporation of the antigen into liposomes by hydration of the pre-liposome gel or the hydration of a lyophilized powder with an antigen solution in appropriate buffer. Another process involves physically mixing the pre-liposomal gel with a lyophilized antigen preparation. This mixture is then hydrated, resulting in the spontaneous formation of liposomes.

The method chosen is the one that results in the highest degree of association with the antigenliposome with the least loss of antigen as an unassociated fraction and depends on the physicochemical properties of the antigen in the presence of a lipid. In general, the ratio of components in such vaccine mixture is 20 mg of antigen protein: 2 mg of 3D-MPL: 300 mg of liposome gel. E.g. if the antigen is flu D, then the second method is used because the antigen has a rather hydrobial character and low water solubility and, as described below, to form a flu D composition of the invention 300 mg of pre-liposomal gel is mixed with 20 to 30 mg of flu D protein and 0.5 to 2 mg 3D-MPL.

It will be appreciated that one of skill in the art will readily determine the amount of antigenic substance to be used in the present invention in practice.

In this case, other agents coupled to the selected antigen may be added, e.g. flu D protein and 3D-MPL in liposomal composition. Suitable other means are described above.

After liposomes are formed in the presence of 3D-MPL, the selected antigen and any given case, additional components of any unassociated antigen can be removed in various ways. Typically, liposomes are centrifuged serially at about 100,000 g and the supernatants discarded. The final liposome pellet is reconstituted in acceptable liquid, 5% dextrose, normal saline or buffer solution for injection at the desired lipid or antigen concentration. Other methods known to those skilled in the art may be used to remove unassociated antigen. gel filtration or dialysis.

The description of the selected antigen, 3D-MPL, and liposomal formulation also includes the use of the split viruses described herein instead of or in addition to flu D.

The present invention also provides a method for enhancing an immune response, especially a T cell-mediated response in humans or other mammals, for the selected antigen in the vaccine composition. This process involves administering to a human or other mammal a vaccine composition of the invention comprising 3D-MPL and a selected antigen. In the present case, the liposome preparation may be part of the composition as described above. This procedure is not limited to any of the specific antigens illustrated herein. In a preferred embodiment, the method is useful for eliciting increased protection effective against influenza infection. In this embodiment, the vaccine composition comprises an effective amount of flu D and 3DMPL as described above. In another embodiment, the vaccine composition comprises an effective amount of one or more split influenza viruses and 3D-MPL as described above. Other vaccines can be prepared and administered in effective amounts in a similar manner.

As exemplified, in particular Examples 25-30, a vaccine composition that utilizes split viruses adjuvanted with 3D-MPL to result in superior viral clearance especially in the lung, stimulates higher neutralizing antibody titers; the occurrence of heterosubtypic (HjN ₂ ) cross-reactivity in the absence of neutralizing activity; and altered disease progression from early to lower respiratory tract (across the trachea).

The dosage and administration regulations can be optimized according to standard vaccination practice for these vaccine compositions. Typically, vaccines are administered intramuscularly, although other routes of administration are also useful such as e.g. subcutaneously, into the intestine, oral, pulmonary, intradermal, intraperitoneal or intravenous. The mode we choose may be determined by the type of immune response desired. E.g. subcutaneous mode may provide a classic depot effect or more prolonged stimulation than others. It is also possible that the chosen route of administration may generate stronger antibody responses than cellular responses or vice versa. E.g. the oral route of administration may allow the generation of an increased IgA response, which is useful for local immunity.

Based on what is known about other polypeptide vaccines, we expect a useful individual dosage for people of average age of the influenza antigen polypeptide vaccine, such as e.g. the vaccine of the invention containing the flu D protein in the range of from about 1 to about 1000 gg of D protein, preferably about 50 to about 500 gg and most preferably about 100 gg mixed with suitable amounts of 3DMPL adjuvant. When these doses of flu D protein are used, the amounts of 3D-MPL in the vaccine composition are between about 1 to about 500 gg of 3D-MPL / gg viral protein, more preferably about 10 to about 50 gg of 3D-MPL / gg of viral protein, and most preferably about 50 gg 3D-MPL.

When flu D and 3D-MPL are administered with a liposomal carrier as described herein, the preferred amount of D protein in the vaccine composition is between about 50 to about 500 gg and the amount of 3D-MPL is between about 10 and about 50 gg. Such 3D-MPL adjuvanted flu D vaccine composition contains from about 1 to about 10 mg and preferably about 3 mg of liposomal substance to about 0.2 mg of antigen protein.

If the flu D and 3D-MPL vaccine formulation optionally contains another adjuvant, such as e.g. aluminum or aluminum hydroxide, preferably a dosage of D protein is between about 10 to about 500 gg of protein; the preferred amount of aluminum or aluminum hydroxide is between about 10 to about 500 gg.

Similarly, given what is known about other split vaccines, it is expected that single dosing for middle-aged, influenza monovalent or multivalent split vaccines, such as e.g. those described herein, about 15 μ% hemagglutinin (HA) per strain, with the total protein in the range of about 80-300 μg / ml, in admixture with suitable amounts of 3D-MPL adjuvant. When these doses of the split virus are used, the amount of 3D-MPL in the vaccine composition is preferably 50 μ§ 3D-MPL per dose. Of course, experts can vary this amount of viral protein and 3D-MPL.

When administered with one or more split viruses and 3D-MPL with a liposomal carrier as described herein, the preferred amount of each split virus in the vaccine composition may be less than about 15 / xg Ha / strain and the amount of 3D-MPL is between about 10 and about 50 μ%. Such a vaccine composition of the influenza cleavage virus adjuvanted with 3D-MPL contains about 1 to about 10 mg and preferably about 3 mg of liposomal substance to about 0.2 mg of viral protein.

When the vaccine formulation of the split virus and 3D-MPL optionally contains another adjuvant, such as e.g. aluminum or aluminum hydroxide, preferably the downstream dosage of each split virus in the vaccine composition. The preferred amount of aluminum or aluminum hydroxide is similar to that described above for the antigenic polypeptide.

Any vaccine described by the present invention can be administered (preferably in 0.5 ml dosage units) at the beginning of late summer or early fall and may be re-administered 2 to 6 weeks later if necessary or periodically when immunity declines, e.g. . every two to five years.

The following examples illustrate preferred methods for preparing the vaccine compositions of the invention. These examples are for illustrative purposes only and do not limit the scope of the invention.

Example ί - gel preparation

Weigh 3.0 g of dipalmitoylphosphatidylcholine into a 50 ml beaker. Add 1.2 g of butyric acid and mix together to form a uniform paste.

0.72 g of arginine in 30 ml of distilled deionized water was added to the dipalmitoylphosphatidylcholinic acid trap and heated to 45 ° C. By mixing manually, the mixture forms a clear stable gel. The gel is stored and subsequently formed liposomes by diluting the gel with phosphate buffered saline.

Example 2 - Preparation of liposomes

120 mg of dipalmitoylphosphatidylcholine and 24 mg of butyric acid are combined and mixed thoroughly to form a white homogeneous paste.

Then 20 mg of arginine was dissolved in 60 ml of phosphate buffered saline (ionic strength = 0.15, pH = 7.4). Arginine - saline solution is added to the paste and heated at 40 ° C for half an hour or until a slightly cloudy solution is observed.

Example 3 - Large-scale gel and liposome preparation

i) Gel production: 20 g of N.F. oleic acid are added to 50 g of type 20 egg phosphatide powder (Asahi Chemicals). Stirring gives a white paste which is cooled to 4 ° C and ground into a fine powder. This powder was added to an aqueous solution containing 20 g of arginine and 500 g of distilled deionized water. The mixture was stirred with a spatula to warm the solution to about 35 ° C to aid in the hydration of the phospholipids. A homogeneous, slightly yellow gel is formed. This gel is then autoclaved and stored at 4 ° C, or it can be frozen and subsequently reconstituted.

ii) Production of liposomes: the gel prepared according to the procedure described in the previous paragraph is cold removed and warmed to room temperature. This was then mixed with 2 L of phosphate buffered saline, pH 7.4. A white cloudy liposomal solution is formed.

Example 4 - Gel Liposome Formation

A homogeneous paste is formed from 1.0 g of dipalmitoylphosphatidylcholine (DPPC) and 400 mg of butyric acid. Then, 300 mg of arginine is mixed in 10 ml of phosphate buffered saline, warmed to 45 ° C and added to DPPC / oleic acid traps to form liposomes.

Example 5 - Pre-liposomal gel g of dipalmitoylphosphatidylcholine (DPPL) was mixed with 400 mg of butyric acid to form a homogeneous paste. Mix 300 mg of arginine with 2 ml of water at 45 ° C until dissolved. The arginine solution was mixed with DPPC / oleic acid paste at about 45 ° C to give a thick gel. Liposomes are formed when this gel is diluted with phosphate buffered saline.

Example 6 - Different Liposomal Formulations

A. Liposomes containing cholesterol mg cholesterol are mixed with 100 mg dipalmitoylphosphatidylcholine (DPPC) to form a homogeneous powder. Then, 23 mg of butyric acid was added to the powder and stirred thoroughly to form a homogeneous paste. To make liposomes, 30 mg of arginine is added to 10 ml of phosphate buffered saline, warmed to 40 ° C and added to DPPC / cholesterol / oleic acid paste. The combination was stirred at 40 ° C to obtain liposomes.

B. Liposomes containing palmitic acid

250 mg of dipalmitoylphosphatidylcholine (DPPC) is mixed with 25 mg of palm tartaric acid to form a uniform powder. Then 80 mg of oleic acid is mixed with this powder and heated at 45 ° C with constant stirring until a uniform paste is formed. Dissolve 100 mg of arginine in 25 ml of distilled deionized water and warm to 45 ° C. The arginine solution was added to the paste at 45 ° C and stirred until a uniform homogeneous gel was formed. The gel is diluted with 10 times the amount of phosphate buffered saline to form liposomes.

C. Liposomes containing isostearic acid

Mix 100 mg dipalmitoylphosphatidylcholine with 50 mg isostearic acid to form a uniformly homogeneous paste. Make an arginine solution of 50 mg arginine in 2.0 mg distilled deionized water and add to the isostearic acid trap and heat at 45 ° C. The mixture was stirred until a clear gel was formed. The liposomes of us26 are thawed after dilution with phosphate buffered saline.

D. Liposomes containing oleoyl threonine

125 mg of dipalmitoylphosphatidylcholine and 75 mg of oleoyl threonine are put together and heated at 40 ° C to form a paste. Then, while stirring constantly at 40 ° C, 2 ml of distilled deionized water. A clear gel is formed which can be diluted with phosphate buffered saline at pH 5 to form liposomes.

E. Liposomes containing myristyl amine

192 mg of dipalmitoylphosphatidylcholine is added to 72 mg of myristyl amine and heated under constant stirring to form a uniform paste. 65 mg of glutamic acid are added to the paste in 5 ml of distilled deionized water and heated until a gel is formed. Phosphate buffered saline was added to the gel to form liposomes.

F. Liposomes containing DLPC mg dilavrylphosphatidylcholine (DLPC) are mixed with 20 mg oily acid to form a homogeneous paste. 20 mg of arginine was added to 10 ml of phosphate buffered saline, added to the paste and manually stirred until a cloudy liposomal solution was formed.

G. Phosphatidylethanolamine-glutamic acid liposomes mg dissolve L-glutamic acid in 2.0 ml of distilled deionized water and adjust the pH to 5.2 with 1.0 N sodium hydroxide. This solution was heated to 60 ° C and 100 mg of phosphatidylethanolamine was added. The solution was maintained at 60 ° C with constant stirring until a uniformly viscous gel was observed.

The phosphatidylethanolamine-glutamic acid gel is diluted with 1/10 phosphate buffered saline. Vesicle-like structures are observed under a phase contrast light microscope.

Example 7 - Effect of arginine concentration on liposome size

To 502 mg dipalmitoylphosphatidylcholine (DPPC) was added 10 μl (2-palmitoyl-1C ₁₄ ) (0.1 mCi / ml) dipalmitoylphosphatidylcholine. Chloroform is added to cause complete mixing of the radioactivity and then evaporated. 195 mg of oleic acid (OA) is then mixed into the lipid to form a paste. Add 5 ml of distilled water containing 119 mg arginine and stir at 45 ° C to form a clear gel.

weigh the gel into four different vials and add arginine as shown in the following Table 1.

Table 1

Composition of the sample

Sample ID DPPC: OA: Arg vial 1 + 1 ml water (1 ^; 1: 1) vial 2 + 1 ml mg / ml Arg in H ₂ O (1: 1: 3) vial 3 + 1 ml mg / ml Arg v H ₂ O (1: 1: 5) 4 + 1 ml vial

192 mg / ml Arg in H ₂ O (1: 1: 10)

Dilute half a gram of each solution to 50 ml of phosphate buffered saline at pH 7.8.

Estimated average diameters are obtained by chromatographic analysis on a Sephracyl S-1000 column using a ₁₄ C-isotope-labeled DPPC (ie diameters are determined based on scattering intensity using photon correlation spectroscopy (PCS)). The effects are listed in the following Table 2.

Table 2

Effects of arginine concentration on vesicle size

System The ratio PH Estimated diameter (nm) DPPC: OA: Arg 1: 1: 1 7.8 -220 DPPC: OA: Arg 1: 1: 3 7.8 -140 ΔΠΠΧ: ΟΑ: Αργ 1: 1: 3 7.8 -90 DPPC: OA: Arg 1: 1: 10 7.8 -20

Example 8 - effect of pH on vesicle size

In addition, the size of the vesicle can be varied by varying the pH of the aqueous buffer solution.

To the 100 mg dipalmitoylphosphatidylcholine (DPPC) was added 25 μΐ (2-palmitoyl-1C ₁₄ ) (0.1 mCi / ml) dipalmitoylphosphatidylcholine. Chloroform is added to cause complete mixing of the radioactivity and then evaporated. 40.1 mg of oleic acid (OA) is then mixed into the lipid to form a paste. 1 ml of a solution containing 24 mg / ml arginine in water was added to the lipid mixture and stirred at 45 ° C to form a clear gel.

Dilute two 100 mg aliquots of this gel in 10 ml of phosphate buffer at pH 9.0 and. 7.4.

Estimated average diameters were obtained by chromatographic analysis on a Sephracyl S-1000 column using dipalmitoylphosphatidylcholine, neglected with the ₁₄ C-isotope. The results are listed in the following Table 3.

Table 3

Effects of pH on vesicle size

System The ratio PH Estimated diameter (nm) DPPC: OA: Arg 1: 1: 1 7.4 - <300 DPPC: OA: Arg 1: 1: 1 7.8 -220 DPPC: OA: Arg 1: 1: 1 9.0 -25.4

Therefore, the desired sizes of liposomal vesicles can be prepared by varying the arginine concentration or pH of the aqueous buffer solution.

Example 9 - Liposomal Stability

Sterile liposomes can be prepared from thermally sterilized pre-liposomal gels. Alternatively, the liposome gel or liposomes can be sterile filtered through a suitable sterilization filter.

Liposomes prepared from DPPC: OA: Arg (1: 1: 2) at pH 8.0 are thermally sterilized and stored at room temperature for about 1 year without the addition of antimicrobials and antioxidants. No bacterial growth, discoloration or discoloration were observed. A negative color electron microscopic examination of one-year-old liposomes reveals that liposomal vesicles are stable.

Example 10 - sucrose latency

Encapsulated sucrose latency was measured using _C14 -saharoze encapsulated with the DPPC: OA: Arg (1: 1: 1) liposome system in aqueous phosphate buffer solution at pH 7.8. The results are presented in the following Table 4.

Table 4

Days% sucrose latency 100

97.4

93.4

91.4

The proposed liposomal system has excellent latency for drug administration.

Example 11 - Lyophilized Liposomes

30.0 g of butyric acid and 7.5 g of U.S.P cholesterol. we mix. Then 75.0 g of the type 20 phosphatide powder (Asahi Chemical Co.) was mixed with the oil / cholesterol mixture until a homogeneous paste was obtained.

Then 15.0 arginine (free base) was dissolved in 183 g of distilled, deionized water. This arginine solution was slowly mixed with the lipid paste to give a homogeneous gel. The gel was adjusted to pH 7.4 using 5.0 N HCl.

Transfer 10.0 g aliquots of this pre-liposomal gel into a 10 ml vial and lyophilize it. The resulting powder forms liposomes when diluted with 5 ml of phosphate buffered saline.

Example 12 - Construction of Flu D Expression Plasmids

Plasmid PaPR701 is a pBR322 derived cloning vector carrying coding regions for

Ml and M2 influenza viral proteins (A / PR / 8/34). This is described by Young et al, in The

Origin of Pandemic Influenza Viruses, 1983, ed. W. G. Laver, Elsevier Science Publishing Co.

Plasmid pAPR801 is derived from pBR322 from a clone vector carrying the NS1 coding region (A / PR / 8/34). This is described by Young et al, cited above.

Plasmid pASl is an expression vector derived from pBR322 containing a P _L promoter, an N usable site (to release transcriptional polarity effects in the presence of an N protein), and a cll ribosomal binding site, including a cll translation initiation codon, followed immediately by a BamHI site. This is described by Rosenberg et al, Methods Enzymol., 101: 123-138 (1983).

The plasmid pASldeltaEH was prepared by deletion of the non-essential EcoRI-HindIII region pBR322 onset from pAS1. A 1236 base pair of the BamHI fragment of pAPR801 containing the NS1 coding region in the 861 base pairs of viral onset and 375 base pairs of pBR322 onset is included in the BamHI site of pASldeltaEH. The resulting plasmid pASldeltaEH / 801 expresses authentic NS1 (230 amino acids). This plasmid has the Ncol site between the codons for amino acids 81 and 82 and the Nrul site 3 'for the NS sequences. The BamHI site between amino acids 1 and 2 is conserved.

The plasmid pASlAEH / 801 was cut off with Bglll, finally supplemented with DNA polymerase I (DNApolI; Klenow) and ligated closed to eliminate the Bglll site. The resulting plasmid pBgl 'was digested with Ncol, finally supplemented with DNA poly (Klenow) and ligated to a Bglll linker. The resulting plasmid pB4 contains a Bglll site in the NS1 coding region. Plasmid pB4 was digested with Bglll and ligated to the synthetic DNA linker as described in Example 4 EP 0366238.

The resulting plasmid pB4 + allows insertion of DNA fragments into the linker by coding region for the first 81 amino acids of NS1, followed by termination codons in all three reading frames. Plasmid pB4 + is digested with Xmal (cut in linker), finally replenished (Klenow) and ligated to a 520 base PvuII / Hindlll pair, finally replenished from the HA2 coding region. The resulting plasmid pD encodes for a protein comprising the first 81 amino acids of NS1, three amino acids derived from a synthetic DNA linker (Gln-Ile-Pro), followed by amino acids 65-222 HA2, as shown in FIG. 2 of European patent application no. 366,238.

To facilitate plasmid selection in fermentation production, a BamHI fragment derived from a pD expression plasmid encoding a recombinant flu D protein is ligated into the BamHI site of a pAS1 plasmid derivative containing the kanamycin resistant gene from Tn903 for selection [Berg et al, Microbiology, ed. D. Schlessinger, p. 13-15, American Society for Microbiology (Washington, DC 1978); Nomura et al, The Single-Stranded DNA Phages, ed. D. Denhardt et al, p. 467-472, Cold Spring Harbor Laboratory (New York 1978); Castellazzi et al, Molecul. Gen. Genet., 117: 211218 (1982)]. This results in the vector pC ₁₃ (H ₆₅ _ ₂₂₂ ) Kn.

As described by Shatzman and Rosenberg, Meth. Enzymol., 152: 661-673 (1987), pOTS207 is a clone vector derived from pAS carrying a kanamycin resistant gene from Tn903 [Berg, cited above; Nomura, cited above; Castellazzi, cited above]. This was constructed by degradation of the pUC8 plasmid [Yanisch-Perron et al, Gene, 33: 103-119 (1985)], by BamHI, and by ligation to a Bell fragment containing the kanamycin gene from Tn903. The resulting plasmid pUC8-Kan is digested with Ecorl and Pstl, and a fragment containing the kanamycin gene is inserted between the EcoRI and Pstl sites of pOTSV [Shatzman and Rosenberg, cited above]. The resulting plasmid is pOTS207.

A 520 bp fragment encoding the HA2 coding sequence was isolated from pJZ102 [a cloning vector carrying a coding region carrying the entire HA protein (A / PR / 8/34) derived from pBR322 (described by Young et al, The Origin of Pandemic Influenza Viruses, ed by WG Laver, Elsevier Science Publishing Co. (1983)] and incorporated into pB4 +, which was cut off by Xmal and finally replenished. The resulting plasmid pC ₁₃ (H ₆₅ _ ₂₂₂ ) Kn is degraded by BamHI and the flu D encoding fragment the protein isolated from this fragment was then ligated into the BamHI site of pOTS207 to produce plasmid pC ₁₃ (H ₆₅ _ ₂₂₂ ) Kn [SmithKline Beecham].

The sequence of the kanamycin resistant C ₁₃ (H _{65 222} ) Knplasmid is derived as follows: nucleotide positions 1-31, 3136-3964, 4021-4343, 4533-7166 are derived from pBR322 [Young et al, cited above]; nucleotide positions 32-1879, 4344-4532 derived from λ phage, nucleotide positions 1880-2122, 2682-3135 derived from NS1 gene, nucleotide positions 2123-2132, 2660-2681 derived from synthetic linker, nucleotide positions 2133-2659 derived from HA2 genes, nucleotide positions 3965-4020 are derived from pCV _] polylinker, and nucleotide positions 7167-8601 are derived from pUCKanl2 (Tn903: KN _r ). The DNA sequence of the coding region for the flu D protein derivative is confirmed by a dideoxy chain termination process for DNA sequencing [Sanger et al, Proc. Natl. Acad. Sci. U.S.A. 74: 5463-5467 (1977)].

This C ₁₃ (H _{65 222} ) Kn plasmid is essentially the same as the plasmids described in U.S. Pat. 07 / 387,200, which is pending at the same time, its corresponding published EPA no. No. 366,238 and U.S. Pat. No. 07 / 387,558, which is pending at the same time, its corresponding published EPA no. 366,239 except that the / 3-lactam marker is removed and replaced with the kanamycin marker. The above applications are incorporated by reference for their description of other relevant vectors.

pC ₁₃ (H _{65 222} ) Kn plasmid transformed into E. coli expression strain AR58 [SmithKline Beecham]; and flu D protein production was confirmed by immunoprecipitation analysis [Towbin et al, Proc. Natl. Acad. Sci. U.S.A. 76: 4350 (1979)], which reveals major immunoreactive species with a predicted molar mass of 27.7 kD.

Example 13 - purification of D protein

NSi ₁ ^ ₁ -HA2 _{65 222} or D protein (mm 27.7 kD) is purified as described in detail in published European patent application no. No. 366,239, corresponding to U.S. patent application Ser.No. 07 / 387,558.

After induction of D protein synthesis with any E. coli host strain AR58 [SmithKline Beecham] using the pC ₁₃ (H ₆₅ _ ₂₂₂ ) Kn system described in Example 12, bacterial cells were harvested by centrifugation and frozen at -70 ° C until use. This pellet is thawed and resuspended in 50 mM Tris, 2 mM EDTA, 0.1 mM dithiothreitol (DTT), 5% glycerol at pH 8. The resulting suspension is treated with lysozyme (final concentration of at least about 0.2 mg / ml) for about 1 hour at room conditions.

The suspension was then lysed on a Manton Gaulin homogenizer [APV Gaulin, Inc., Everet, Massachusetts at 5.4x10 ¹⁰ Pa in two passes. The resulting suspension was treated with Triton Χ-100 (1% final concentration) and deoxycholate (DOC; 0.1% final concentration). The D protein-containing pellet was suspended in 50 mM GlyNaOH buffer containing 2 mM EDTA and 5% glycerol at pH 10.5 using a Turk homogenizer. The resulting suspension, after the addition of Triton Χ-100 (1% final concentration), was stirred for about 1 hour in a cold room at 4 ° C and then centrifuged. The D protein-containing pellet was dissolved in 8 M urea, 50 mM Tris at pH 8.0 overnight at 4 ° C. The supernatant contains D protein.

After the addition of DTT (up to 50 mM final concentration), this supernatant was stirred at room temperature for about 1 hour and then charged to the DEAE flask rapidly.

Sepharose [Pharmacia] column equilibrated with 8M urea and 50 mM Tris buffer at pH 8. D protein was eluted with a 0 to 0.3 M NaCl gradient (over five column volumes or more) in balanced buffer.

The fractions containing D protein were concentrated on a Minisette Tangential Flow Apparatus [Pharmacia], treated with 10 mg SDS per mg protein, DTT [Sigma] (up to a final concentration of 50 mM) and stirred at room temperature for about 1.5 hours.

The resulting solution was filled into a Superose-12 column [Pharmacia] balanced with 25 mM Tris-Glycin buffer at pH 8.6 containing 1% SDS. The D protein is eluted at its predicted monomeric molecular weight in an isocratic gradient of balanced buffer. Fractions containing D protein of appropriate purity were collected, concentrated, treated with 10 mg SDS per mg protein and DTT (up to 50 mM final concentration), then chromatographed on a Superozni-12 column under the same conditions as previously described.

Fractions containing D protein of the highest purity were collected, concentrated and placed in a G25 Sephadex [Pharmacia] purification column balanced with 50 mM Tris buffer at 8.0 containing 8 M urea. D protein is eluted isocratic by equilibration buffer and fractions containing D protein without SDS are collected and concentrated. This highly purified sample of D protein was then concentrated, dialyzed against 20 mM Tris, 1 mM EDTA, pH 8.0, and sterile filtered.

Example 14 - Evaluation of Flu D Vaccine Compositions

Detailed descriptions of D protein and the procedures used for in vitro T cell assays and protection studies are described in S. B. Dillon et al, Vaccine, 10: 309 (1992), incorporated herein by reference.

CTLs and proliferation assays are performed as described previously [S. B. Dillon et al, cited above]. IL-2 was measured on an IL-2 dependent CTL line (CTLL) and IFN-γ was measured with a commercial ELISA kit.

The vaccine compositions of the invention containing the Superoso purified D protein (Example 13) in aluminum hydroxide adjuvant, with or without 3D-MPL, are prepared as follows: 3D-MPL (RIBI Immunochemical, Hamilton, MT) is reconstituted in sterile water for injection at working concentration of 1 mg / ml. Sonic stock solution was sonicated for 30 minutes and subsequent dilutions made in 5% dextrose sonicated for an additional 10 minutes before addition of a mixture of D protein in aluminum hydroxide prepared as previously described [Dillon et al, Vaccine, 10 (5): 309- 318 (1992)].

Antigen doses were titrated from 0.01, 0.1,1,0,10, 20 and 100 µg injection; and 3D-MPL doses were titrated from 0.025, 0.25, 2.5, 25 and 50 / tg injection dose. The 3DMPL: antigen ratio was maintained at 2.5: 1 (w / w) for all antigen doses except 100 / tg dose as shown in Table 5 below. Aluminum hydroxide is 100 / tg per dose in all cases. The control vaccine composition contains 20 / tg of antigen mixed with CFA as described in Dillon et al, Vaccine, 10 (5): 309-318 (1992). The final injection volumes are 0.2 ml per mouse.

For these mouse protection studies (CB6Fp 11-2 ^), inject subcutaneously with the selected vaccine dose at weeks 0 and 3 and challenge intranasally (under methophan anesthesia) at week 7 with 2 to 5 LD _S0 doses A / Puerto Rico / 8/34 [A / PR / 8/34 (type A influenza, H1N1)] virus.

Detailed descriptions of the procedures used for protection studies are described in S. Dillon et al, Vaccine, 10 (5): 309-318 (1992), which are incorporated herein by reference. Survival percentages at day 21 after challenge were compared between groups with Fischer's exact likelihood test.

Table 5

Antigenic 3D-MPL % survival dose (Mg) dose (Mg) (day 21) 100 50 27 100 0 53+ 20 50 86 ^{+, ++} 20 0 40 ** 10 25 67+ 10 0 4θ ** 1.0 2.5 80 + - ++ 1.0 0 7 0.1 0.25 20 0.1 0 20 0.01 0.025 20 0.01 0 13 0.0 50 13 0.0 0 0

⁺ p <0.003 against adjuvant control ** p <0.01 against adjuvant control ** p <0.01 against aluminum formulation at the same antigenic dose

From the results of Table 5 shows that the minimum dose of flu D antigen required for significant protection against lethal izzvom reduced from 10 South (only aluminum) per 1 Mg & j ^e 3D-MPL is included in the vaccine composition. Likewise, the percentage of mice surviving the outcome increases when 20 Mg (p - 0.01), 10 Mg (p 0.05; NS) and 1 Mg (p - 0.01) antigen doses are administered with 3D-MPL as well as aluminum against aluminum alone in the vaccine composition. Survival in the CFA control group at 20 Mg antigen was 73% in this experiment.

Example 15 - Evaluation of Flu D Vaccine Compositions

This experiment is carried out essentially as described in Example 14 above, except that the 3D-MPL doses are titrated and administered either alone with the minimally effective dose of flu D antigen (1 μ%) and aluminum adjuvant (100 μ g) or with flu D antigen (10 μ%) and aluminum adjuvant (100 / xg). The results reported in Table 6 show that the minimum effective dose of 3D-MPL is 2 μ-g.

Table 6

Antigen dose (μ-g) 3D-MPL dose (p, g)% survival (day 21)

10 10 80 ⁺ 10 2.0 73 ⁺ 10 0.4 53 ⁺ 10 0.0 53 * 1 10 80 ^{+, ++} 1 2.0 87 + - ++ 1 0.4 33 * 1 0.0 33 0 10 0 0 2.0 0 0 0.4 0 0 0.0 7

⁺ p <0.001 against adjuvant control * p <0.02 against adjuvant control ⁺⁺ p <0.01 against aluminum formulation at the same antigenic dose.

Example 16 - Evaluation of flu D vaccine compositions

In this experiment, the performance of liposomes and 3D-MPL is compared to liposomes (lipo) without

3D-MPL (Fish Immunochem] and A1 (OH) ₃ or CFA by determining the level of protection seen after challenge with 2,10 or 50 LD ₅₀ virus doses The vaccines used are the same as in Table 9 below.

Mice were injected at Week 0 and Week 3 and challenged with A / PR / 8/34 at Week 7. Survival is shown in Table 7 below on Day 21.

Table 7% survival

Antigen Dose (Mg) Adjuvant challenge dose (LD ₅₀ ) 2 10 50 D protein 50 Lipo 53 53 ^b 7 0 Lipo 27 0 0 D protein 50 Lipo / 3D-MPL ^a 100 ^b 73 ^b - ^c 47 ^b - ^c 0 Lipo / 3D-MPL 13 0 0 D protein 50 Al ³⁺ 86 ^b 20 13 0 Al ³⁺ 7 13 0 D protein 50 CFA 86 ^b 80 ^{b, c} 13 0 CFA 33 0 0

^a The amount of 3D-MPL per liposome has not been determined.

^b p <0.003 against adjuvant control ^c p <0.003 against aluminum hydroxide formulation at the same antigenic dose.

The results show that significant protection against the 50 LD ₅₀ challenge is achieved only with the liposomal / 3D-MPL formulation and with no other adjuvant formulation tested (Table 7). Considerable protection is achieved against the 2 LD ₅₀ challenge with all formulations and all formulations except aluminum are protective against the 10 LD ₅₀ challenge dose. Therefore, the D protein in the 3D-MPL / liposomal formulation is a vaccine with even greater potency than the D protein in CFA, which is a classic adjuvant for stimulating T cell response but is not suitable for use in humans.

Example 17 - Evaluation of Flu D Vaccine Compositions

In this experiment, the vaccine composition of the invention comprising flu D protein, 3D-MPL and liposomes is evaluated in comparison to a flu D vaccine composition,

3D-MPL and aluminum adjuvant. We measure the actual amount of 3D-MPL embedded in the liposomes.

Flu D protein (purified on a Superoso column) in 25 mM Tris / 1 mM EDTA, pH 8, was dialyzed against a total protein concentration of 2.49 mg / ml against 20 mM ammonium bicarbonate at pH 8 and lyophilized to powder in 20 mg aliquots.

Egg phosphatides (Asahi type 5) and oleic acid (Sigma) are mixed at a molar ratio of approximately 1: 1.2. L-arginine (free base), 0.38 M in water was added (1: 1) molar ratio of phosphatide: arginine) to the phosphatide / fatty acid mixture and stirred to form a smooth and homogeneous gel. This gel is a pre-liposomal gel.

The lyophilized protein was combined with a pre-liposomal gel (0.06-0.08 protein / gel (w / w) ratio) and stirred at room temperature to form a homogeneous paste. Monophosphoryl lipid A (Pisces / Immunochem) was added at a ratio of 1 μ, πιοί lipid A to 66 μηιοί phosphatides. It is a transparent colorless system consisting of nanoparticle structures (submicron diameter) of aggregated 3D-MPL monomers. The resulting liposomal suspension was further diluted with 25 mM Tris / 1 mM EDTA, pH 8.0 buffer and homogenized. The liposome preparation was then serially centrifuged (3x) to remove the non-incorporated protein and 3D-MPL. Each pellet was resuspended with tris / EDTA buffer. The final liposomal suspension was analyzed and stored under nitrogen at 5 ° C until use.

Liposomes are then tested to determine protein content by a modified Lowery colorimetric assay, phospholipid concentration by Bartlett assay [Bartlett, GR, J. Biol.m Chem., 234: 466-468 (1959)], and liposome size by photon correlation spectroscopy [Malvem model 4700]. All tests are performed using standard techniques. Liposomes have not been analyzed for lipid A content, but all of lipid A appears to remain in the final liposomal fraction.

In the following Table 8, the D protein LA and the LA-I control are prepared by incorporation of solid lipid A into the lipid phase. The LA-II control was prepared by hydration with a lipid A solution for incorporation.

Table 8

Liposomal sample Phospholipid (μιηοΐ / ml) Protein (mg / ml) 3D-MPL (/ ig / ml) diameter ZAg average (nm) control 4.37 not specified not specified 200,8 control + 3D-MPL 7.87 0.02 not specified 201.3 Flu D 21.8 4.89 not detected 487,0 FluD + 3D-MPL 14,8 4.06 270 502,4

Mice were immunized subcutaneously with 0.2 ml of antigen / vehicle / 3D-MPL formulation at week 0 and 3 and challenged intranasally at week 7 with 5 LD ₅₀ A / PR / 8/34 virus. The carrier is either aluminum hydroxide (Al) or liposomes (Upo). Survival is pursued for 21 days. These results are shown in Table 9 below. Unless otherwise stated, p is a measure against 3D-MPL control.

Table 9

Antigenic dose The carrier Dose 3D-MPL % survival p value 1.0 M-g Al - 33 0,084 1.0 Mg 2,5 gg 80 0.000 / 0.001 ³ 1,0 gg Al - 33 0,084 0,1 gg 2,5 gg 93 0.000 / 0.001 ³ Ogg Al 2,5 gg 7 - 0,1 gg Lipo - 73 0.001 1,0 gg 0,05 gg 80 0.000 0,1 gg Lipo - 46 0,054 0,1 gg 0,005 gg 87 0.000 / 0.020 ³ Ogg Lipo 0,05 gg 13 -

³ p value against homologous dose without 3D-MPL.

From these data, it can be seen that by using liposomes as carriers, the required dose of 3D-MPL (0.005 - 0.05 gg) can be dramatically reduced compared to the amount required with aluminum (about 2 gg). (See also Table 6 above).

Example 18 - Evaluation of the vaccine composition

CB6F ₁ (12 per group) was injected subcutaneously at weeks 0 and 3 with a vaccine composition containing 100 gg of flu D in aluminum (100 gg) and 3D-MPL (10 gg). Week 7 mice were challenged with 3-5 LD ₅₀ doses of the viruses shown in FIG. 1. Survival is pursued 21 days after the challenge.

From the results in Figs. 1 shows that the antigen specificity of the vaccine is equivalent to that shown in the yarn with Al ⁺³ adjuvant [SB Dillon et al, cited above] (i.e.

cross-protective for both HI and H2 subtypes, but lack of efficacy for H3 or type B). Survival was also higher in the vaccine group elicited by the heterologous H1N1 virus, A / Tai / 86, although the results were not significant, p <0.09. (see Fig. 1).

Example 19 - Evaluation of Flu D Vaccine Composition

CB6F _X mice were injected subcutaneously (sc) with a vaccine composition containing 1 ptg of D protein in aluminum (100 μ-g) and 3D-MPL (2.5 μ-g) or challenged with aluminum only at week 0 and 3.18 mice challenged week 7 with 5 LD ₅₀ doses of A / PR / 8/34 virus. 5 mice were killed on day 7 following a challenge for pulmonary titers. (Deaths in controls at day 7 were 60% for A1 / 3D-MPL and 40% for CFA). Spleens were removed from two mice in the pre-challenge group and three mice in the group on day 6 after the challenge for proliferation and cytokine assays (Table 15 below). Table 10 shows the survival (n = 10 / group) and purification of the lung virus (n = 5 / group) after the 5 LD ₅₀ challenge. The lung viral titer recorded on day 7 is listed in the fourth column of Table 10.

The results in Table 10 show that the decrease in lung viral titers in mice due to 5 LD _{50 is a} greater challenge with 3D-MPL versus CFA (2.4 log _w vs 0.9 log ₁₀ ), although survival is equivalent in these groups .

Table 10

Antigen (μ-g) Adjuvant Survival log ₁₀ TCID _S0 / lung 1.0 A1 / 3D-MPL 86% * 4.41 ± 0.83 ⁺ 0 A1 / 3D-MPL 0% 6.80 ± 0.45 ι, θ Al 33% 6.39 ± 0.41 ⁺ 0 Al 0% 7.39 ± 0.41 1.0 CFA 86% ** 6.00 ± 0.50 ⁺⁺ 0 CFA 26% 6.90 ± 0.72

* p <0.001 against adjuvant control group ** p <0.01 against adjuvant control group ⁺ p <0.003 against adjuvant control group ⁺⁺ p <0.03 against adjuvant control group,

Example 20 - Evaluation of Flu D Vaccine Composition

To determine if spleen cell proliferation is T, yIFN production is correlated with protection, these responses are observed in the spleens of mice immunized with 1 µg g of D protein in Al against A1 / 3D-MPL (2.5 ng 3D-MPL) adjuvants ( from the study shown in Table 10) before and after the challenge. On week 7, two mice are killed 6 days after the viral challenge. The spleen was co-cultured with D protein and pulsed with ³ H-thymidine on day 3. The supernatant cultures were harvested 8 hours later.

Overall proliferative spleen responses are similar before and after challenge for groups vaccinated with either the D protein in the Al or A1 / 3D-MPL formulation (maximum cpm in unstimulated cultures is 2750 cpm) (Fig. 3). The range of proliferative responses is also similar for the adjuvant group with a maximum stimulation index equal to 3.0 or lower (Fig. 2). Pre-challenge gamma levels of interferon in cultures of antigen-stimulated supernatants are only modestly (approximately 2-fold) increased and the two adjuvant groups are equivalent, as shown in Table 11.

However, the production of interferon gamma is increased more than 7-fold in antigen-stimulated cultures of the A1 / 3D-MPL group after challenge; while levels did not rise after challenge in the Al- ³ vaccinated group alone (Table 11). It is evident from these results that the production of interferon gamma is correlated with reduced pulmonary titers and survival after challenge, and further it is evident from these results that T cell proliferation by itself does not reflect the difference between the two adjuvant groups.

Table 11 ng / ml IFN-gamma

antigen injection dose adjuvant pg / ml in vitro before a challenge PO to the challenge D 1 P9 Al ⁺³ / 3D-MPL 0 <0.9 <0.9 1 1.0 > 7.0 10 1.8 > 7.0 - - Al ⁺³ / 3D-MPL 0 <0.9 <0.9 1 <0.9 1.0 10 0.94 <0.9 D and pg Al 0 <0.9 <0.9 1 1.9 1.0 10 1.9 1.0 - - Al 0 <0.9 1,2 1 <0.9 1.7 10 1.1 1.0

Example 21 - Evaluation of Flu D Vaccine Composition

To further investigate the role of CD4 ⁺ cells, we compare antigen-specific proliferation and cytokine production in mice vaccinated with vaccine compositions containing flu D and an aluminum adjuvant (Al) against vaccine compositions containing flu D and A1 / 3D-MPL formulations.

Lymph nodes of mice given a single injection of 1.5 or 20 / xg D protein in A1 / 3DMPL (3D-MPL: antigen ratio is 2.5: 1 w / w) or Al ⁺ adjuvant (100 μ-g) for 7 days the yarn is cultured with 0-30 mg / ml of purified D protein.

The results are shown in FIG. 3 and 4. Proliferation is clearly increased in the groups receiving A1 / 3D-MPL adjuvant and the difference is greatest at the lowest in vivo antigen dose (1 μ-g). In FIG. 3 shows the maximum cpm in unstimulated cultures, which is the same

1368 cpm. In FIG. 4 shows the maximum cpm in unstimulated cultures, which is the same

1600 cpm; and maximum cpm CTLL (IL-2 dependent CTL line) of cells cultured with supernatants from unstimulated cultures equal to 195 cpm). The peak (48 h) of IL-2 activity was higher in the groups vaccinated with A1 / 3D-MPL formulations, although the levels were generally lower in all cultures (Fig. 4).

Table 12 shows the results of the analysis of interferon gamma in supernatants from identical antigen-stimulated cultures. The levels of interferon in supernatants from adjuvant control cultures are all <1 ng / ml. Interferon gamma was measured with a Gibco-BRL ELISA kit. This cytokine, the highest on day 4 of the culture, is up to 5-fold higher in the A1 / 3D-MPL group.

Table 12 μ% antigen IFN-gamma antigen (ng / ml)

(in vivo) adjuvant (Aig / ml) in vitro day 2 day 3 day 4 1.0 Al ⁺ 0 <1 <1 <1 1 <1 <1 <1 10 <1 <1 <1,1 1.0 A1 / 3D-MPL 0 <1 <1 <1 1 <1 <1 1.9 10 1.1 2.9 4.9

The results in Tables 11 and 12 fully support the role of interferon gamma in the mechanism of action of MPL adjuvant.

Example 22 - Evaluation of Flu D Vaccine Composition

As the examples above show that 3D-MPL improves vaccine efficacy, we have done a study to determine if booster injection is needed for protection. Mice were vaccinated with single sc injection of 50 μ-g D and challenged 7 weeks later with 2 LD ₅₀ A / PR / 8/34. Alternatively, the second group is given a booster injection with the same antigen dose at 3 weeks. The 3D-MPL dose is 125 μ%. Flu D is adjuvanted with either aluminum hydroxide, aluminum and 3D-MPL or CFA. We perform controls for each adjuvant.

The results in Table 13 below (shown in Example 23) show that the incorporation of 3D-MPL (125 Mg) into the vaccine formulation with 50 Mg of antigen significantly increased survival in mice given either one or two injections when compared with the same dose of vaccine protein adsorbed on aluminum. Again, survival in the Al / MPL group is comparable to that seen in CFA.

Example 23 - Cytotoxic T lymphocyte assays

Detailed descriptions of the procedures used for in vitro T cell assays are described in S. Dillon et al, cited above. CTL memory cell detection assays are performed after a second in vitro stimulation with the virus as previously described [see F. Ennis et al, Lancet, 11: 887-891 (1981); A. Yamada et al, J. Exp. Med., 162: 663-674 (1985); and K. Kuwono et al, J. Exp. Med., 169: 1361-1371 (1989)]. Briefly, spleen cells or lymph node cells from immune or control mice were cultured at a ratio of 6: 1 with syngeneic spleen cells infected with influenza virus for 5 days. The culture medium is RPMI 1640 supplemented with 10% fetal calf serum [Hyclone Laboratories, Logan, UT], 2 mM glutamine, 5 χ 10 ′ ⁵ M 2-ME, 10 mM HEPES, penicillin and streptomycin.

The spleens of mice given one or two sc injections of 50 Mg deprotein (weeks 0 and 3) were removed for seven weeks and restimulated in vitro as described above. One lytic unit (LU ₃₅ ) is defined as the number of effector cells in the chromium release assay required for 35% lysis (determined by regression analysis). Table 13 lists the results of CTL activities specific for H1N1 in these spleens of mice immunized with SK&F 106160 (D protein) in aluminum hydroxide and 3D-MPL.

Table 13

LU ₃₅ on culture

control A / PR / 8/34 A / Taiwan / 86 % survival # injections: goals 1 2 1 goals 2 1 goals 2 1 2 Formulation L / A1 ⁺³ * <1 19 53 56 51 69 7 33 D / A1 ⁺³ <1 <1 41 43 64 53 40 + - ++ 73 ^{+, ++} / 3D-MPL * D / CFA * <1 <1 129 88 98 131 53 ^{+, ++} 93 ^{+> ++} Al ⁺³ <1 <1 <1 <1 35 35 N.D. 7 Al ⁺³ / 3D-MPL 18 18 <1 <1 29 29 N.D. 0 CFA <1 <1 <1 <1 <1 <1 N.D. 0

* = antigen dose is 50 gg flu D protein.

⁺ = p <0.05 vs adjuvant control group * + = p <0.05 vs ΑΓ ³ adjuvant group.

In the experiments of this example, 3D-MPL does not affect class I memory-restored CD8 + CTLs relative to the response generated by aluminum hydroxide (shown as Al ⁺³ ), as indicated in Table 13. In addition, the results in Table 13 show that that spleen CTLs are comparable in mice given 1 to 2 sc injection, whereas protection is clearly improved in mice given a second injection.

For this reason, we believe that a mechanism other than CD8 + CTL should be responsible for the enhanced immunity obtained when 3D-MPL is included in a vaccine composition containing an antigenic peptide and an aluminum adjuvant if given to a booster injection in mice.

Example 24 - Depression studies

To further determine which subset of guild T is best correlated with activity s

3D-MPL adjuvant, we performed depletion studies with anti-CD-4 or anti-CD-8 monoclonal antibodies (Mabs). Initial studies were performed using the vaccination rule for depletion of the T cell subset as follows. Mice (15 / group) were immunized with two subcutaneous injections of 50 / tg flu D (SK&F 106160) in A1 / 3D-MPL on days 0 and 21 and challenged with 5 LD ₅₀ doses of A / R / 8/34 on day 42. Antibodies (300 / tg / mouse) were given ip after vaccination on days 32, 33, 34, 39, 40 and 41 before challenge and on days 43, 48 and 53 after challenge. In another study, mice were treated with Mabs for the depletion of the T cell subset before the first vaccine injection (pre-vaccination rule) on days -3, -2, -1. After vaccination on day 0, mice were further treated with Mabs on days 7, 14, 18, 19 and 20 and then booster injected with the vaccine on day 21. The mice were further treated with Mabs on days 28, 35, 39, 40 and 41 before viral challenge on day 42. Mice were further treated with Mabs after challenge on days 43, 49 and 54. The results of the T cell subset depletion experiments are shown in Table 14 below. The results show that the effectiveness of vaccination is reduced if mice depleted of either CD4 ⁺ or CD8 ⁺ T cell subsets, the effect is more pronounced if Mab treatment was initiated before vaccination. Therefore, it is clear from these results that the mechanism of action of the flu D vaccine in the Al / MPL formulation is mediated by T cells, and both T cell subsets are required for efficacy.

Because the production of interferon gamma is correlated with protection (Table 11), a study was conducted to determine the phenotype of the T cell responsible for the production of this cytokine in vaccinated mice. Mice were depleted of CD4 ⁺ or CD8 ⁺ T cell subsets by injecting anti-CD4 or anti-CD8 Mab ip daily for 3 days (300 gg / injection). Four days after the last Mab injection, mice were immunized with 20 gg of flu D protein in aluminum hydroxide (100 gg) and the 3D-MPL (20 gg) formulation and the lymph nodes were removed 7 days later. Lymph node cells were restimulated in vitro with 0.1 or 10 gg / ml D protein, and the supernatants were collected on days 1-4 for interferon and IL-2 assays. From the results in Figs. 5 shows that IL-2 production is completely eliminated by anti-CD4 treatment and is also partially reduced in anti-CD-8 treated mice (Fig. 5B). The peak of IFNγ production (day 4 supernatants) was reduced by about 50% in anti-CD4 or anti-CD8 treated mice (Fig. 5C). Thus, both CD4 ⁺ and CD8 ⁺ T cell subsets produce IL-2 and IFNγ.

Table 14

Antigen (Mg) 3D-MPL (Mg) Mab processing % survival (Mabs after vaccination) % survival (Mabs before vaccination) 50 10 none 70 * 80 * 50 10 anti-CD4 30 20 ⁺ 50 10 anti-CD8 50 ** 0 ⁺ 50 10 anti-CD4 ⁺ anti-CD8 30 10 ⁺ 0 10 none 0 0

* p <0.002 against adjuvant control (group 5) are <0.02 against adjuvant control (group 5) ⁺ p <0.01 versus untreated control (group 1).

The examples presented above show that a recombinant influenza H1N1 vaccine formulated with aluminum and 3D-MPL facilitates viral purification and survival and lowers the antigen dose required to significantly protect against the aviation challenge while achieving an efficacy level equivalent to one obtained with CFA. From the data collected to date, it is evident that the mechanism by which 3D-MPL acts to enhance the activity of this recombinant influenza vaccine is generated by the selective ability of CD4 ⁺ T cell responses and may be restriction to IL-2-producing TH1 cells and IFNy.

Example 25 - preparation of a split virus

Split viruses, such as those manufactured by Sachsisches Serumwerkl GmbH (SSW) (Dresden, Germany) can be prepared by known methods such as those listed in European Pharmacopoeia PA / PH / Exp 3 (1992); DAB 10 Vaccines Influenzae ex virorum fragmentis preaparatum and requirements for influenza vaccines (inactivated), revised by World Health Organization draft (1990).

Such split viruses are prepared using one or more influenza virus strains recommended by WHO and EEC, such as e.g. A / Singapore / 6/86, A / Beijing / 32/92 and B / Panama / 45/90. These strains can be changed depending on which ones are popular in a given year.

As described elsewhere, influenza viruses are obtained from fertilized chicken eggs inoculated with the seed material. These viral suspensions are partially purified and concentrated. The concentrated viral suspension is treated with detergent, sodium deoxycholate to break down (or split) the viral particles. Following removal of viral phospholipids during the vaccination process, the potential for reactogenicity is greatly reduced. The split virus suspension is completely inactivated by the combined effect of detergent and formaldehyde.

In more detail, the process for making a split virus of the invention is as follows:

A. Preparation of a monovalent complete viral inoculum

On the day of inoculation of the fertilized eggs, a fresh inoculum is prepared by mixing the influenza-inducing phosphate buffer containing gentamicin sulfate 0.5 mg / ml and hydrocortisone 25 ml / ml. The viral inoculum was maintained at 2-8 ° C.

Up to 11 days old fertilized eggs are used for viral replication. The eggs are incubated on the farms before they arrive at the production plant and arrive at the production premises after decontamination of the shells. Eggs are inoculated with 0.2 ml of viral inoculum on an automatic egg inoculation machine. The inoculum is injected at a pressure of ± 0.3 MPa. The inoculated eggs were incubated at the appropriate temperature (depending on the viral strain) for 50 to 96 hours. At the end of the incubation period, the embryos are killed by cooling the eggs and storing them at 2-8 ° C for 12-60 hours.

The allantoic fluid from the cooled fertilized eggs is collected with an appropriate egg harvesting device. Usually, 8 to 10 ml of raw allantoic fluid per egg can be collected. 0.100 mg / ml thiomersal was added to the crude monovalent viral mass.

The collected allantoic fluid is purified by flow through a centrifuge with a volume of 100200 1 / hour and a centrifugal force of 1-17000 g. This previously purified liquid can be further purified on a 6-8 μτη membrane filter.

To obtain a CaHPO ₄ gel in the purified viral pool, 0.5 M Na ₂ HPO ₄ and 0.5 M CaCl ₂ solution were added to achieve a final CaHPO ₄ concentration of 1.5 g to 3.5 g CaHPO ₄ / l, depending on the viral strain. After sedimentation for at least 10 hours, the supernatant is removed and sediment containing the influenza virus is resolubilized by the addition of 0.26 M EDTA solution. The EDTA concentration varies between 4.5 and 10 g / l of the original volume collected.

The resuspended sediment is filtered on a 6 to 8 μπι membrane filter.

The influenza virus was concentrated by isopycnic centrifugation in a linear sucrose gradient (0-55%) at 90000 g. The volume flow is from 8-12 1 / h. At the end of the centrifugation, the contents of the rotor are recovered with four different fractions (sucrose is measured in a refractometer):

- fraction 1 55-52% sucrose

- fraction 2 52-38% sucrose

- fraction 3 38-20% sucrose

- fraction 4 20-0% sucrose

Only fraction 2 and 3 are used to further prepare the vaccine. Dilute fraction 3 to reduce the sucrose content to about 6%. The influenza virus present in this diluted fraction was pelleted at 53000 g to remove soluble contaminants. The pellet is resuspended and thoroughly mixed to give a homogeneous suspension. Fraction 2 and the resuspended pellet of fraction 3 were combined into pooles and phosphate buffer was added to give a volume of 30 1. At this stage, the product was called a monovalent complete viral concentrate.

B. Monovalent mass of the split virus

The selected influenza virus, preferably the monovalent complete viral concentrate from the previous part A, is broken down by centrifugation at 70000 g through a Nadoc linear sucrose gradient of 0-55% containing a linear distribution of sodium deoxycholate of 1.2-1.5%. Tweena 80 is 0.1% in gradient. The viral concentrate is pumped at a rate of 5 l / h. At the end of the centrifugation, the contents of the rotor are collected in three different fractions:

- fraction: 1 55-40% sucrose

- fraction: 2 40-13% sucrose

- fraction: 3 13-0% sucrose

Hemagglutinin is concentrated in fraction 2. Phosphate buffer containing thiomersal 0.01% and Tween 80 0.01% added to the dilution fraction 4 times (± 51).

The diluted fraction 2 was filtered on the filter membranes, ending with a 0.2 gm membrane. At the end of the filtration, the filters are washed with phosphate buffer containing 0.01% thiomersal and 0.01% Tween 80. The result is a final volume of filtered fraction 2, which is 5 times the volume of the original fraction. Depending on the viral strain, a brief sonication of the split virus material can be introduced to facilitate sterile filtration.

The filtered monovalent cleavage material was incubated at 22 ± 2 ° C for at least 84 hours to inactivate the viruses and mycoplasma with sodium deoxycholate. After this incubation time, phosphate buffer containing 0.01% thiomersal and 0.01% Tween 80 was added to bring the total protein content to a maximum of 250 gg / ml. Formaldehyde is added in an amount of 50 gg / ml and the inactivation takes place at 4 ° C ± 2 ° C for at least 72 hours.

The inactivated material of the cleaved virus is ultrafiltered on membranes whose average pore size is 20000 daltons. During the ultrafiltration, the content of formaldehyde, NaDOC and sucrose is significantly reduced. Keep the volume constant during ultrafiltration (diafiltration) by adding phosphate buffer containing 0.01% thiomersal and 0.01% Tween 80.

The ultrafiltered split material is filtered on membranes, ending with a 0.2 gm membrane, and depending on the viral strain, the last filtration membrane may be 0.8 gm. At this stage, the product is called: monovalent end mass. The monovalent final mass is stored at 2-8 ° C for a maximum of 18 months.

Example 26 - Vaccine composition of split virus (a) Preparation of MPL with 60-120 nm particle size.

Water for injection was injected into vials containing lyophilized 3 deacylated monophosphoryl lipid A (MPL) from Pisces Immunochem, Montana, using injections to obtain a concentration of 1 to 2 mg per ml. The preliminary suspension is obtained by vortex stirring. The contents of the vials were then transferred to a 25 ml round bottom Corex tube (10 ml suspension per tube) and sonicated using a water bath sonifier. When the suspension becomes clear, the particle size is estimated using dynamic light scattering (Malvem Zetasizer 3). The treatment is continued until the MPL particle size is in the range of 60-120 nm and preferably below 100 nm.

Suspensions can in some cases be stored at 4 ° C without significant aggregation for up to 5 months. Isotonic NaCl (0.15M) or isotonic NaCl and 10 mM phosphate induces rapid aggregation (size> 3-5 / xm).

(b) The vaccine composition of the split virus of the invention is prepared as follows.

The final buffer was prepared by adding water for injection of concentrated salt solutions: NaCl 4 mg; At ₂ HPO ₄ 0.52 mg; KH ₂ PO ₄ 0.19 mg; KC1 0.1 mg; and MgCl ₂ 0.05 mg and thiomersal 50 / xg. The resulting solution was stirred for 15 minutes before further use.

The monovalent mass of the split virus (15 / ig HA) was then mixed with 3D-MPL 50 / xg and the resulting mixture stirred for about an hour at room temperature.

The buffer mixture and the mixture of viral mass are then mixed together. After stirring at room temperature for 30 minutes, the pH was adjusted to 7.15 ± 0.1. The resulting final vaccine is stored at + 2- + 8 ° C.

To prepare a multivalent vaccine for a split virus, such as trivalent vaccine, monovalent cleavage viral pools of selected strains prepared as described above are mixed to form the final multivalent vaccine. The resulting pooled material was stirred for 30 minutes at room temperature (pH 7.15 ± 0.1). The resulting vaccine can be stored at temperatures between about 2 ° C and about 8 ° C. The vaccine is a colorless, slightly opalescent aqueous suspension of purified split influenza virus.

For the purpose of comparison in the tests of the following examples, the vaccine preparation is prepared without adjuvant. For the control vaccine, the final monovalent buffer mass is prepared by adding concentrated sox solutions to the water for injection: NaCl 4 mg; At ₂ HPO ₄ 0.52 mg; KH ₂ PO ₄ 0.19 mg; KC10.1 mg; MgCl ₂ 0.05 mg and thiomersal 50 μ-g. The resulting solution was stirred for 15 minutes before further use. The mixture was stirred for 15 minutes at room temperature. A monovalent mass (15 / ig HA) was added followed by stirring at room temperature for 30 minutes (pH 7.15 ± 0.1). The resulting final vaccine is stored at temperatures between + 2 ° C and + 8 ° C.

Two monovalent vaccine compositions of the invention and a monovalent control vaccine were prepared for the following cases using H1N1 strains, A / PR / 8 and Singapore mixed with 3D-MPL 50 gg (particle size <100 nm as obtained from Example 26 (a ).

Example 27 - Flying challenge in mice

The immunogenicity of the monovalent vaccine formulation of the invention is evaluated and compared with the immunogenicity of the monovalent non-adjuvanted formula and the antigen-free 3D-MPL adjuvant in the avian influenza challenge in mice.

For each vaccine, CB6F ₁ mice (30 per group) were immunized subcutaneously with 2 injections, given at intervals of 3 weeks, with a preparation containing 5 gg of the indicated strain ± 5 gg 3D-MPL. Seven weeks after the first injection, mice were challenged intranasally under anesthesia with methophan with 5 LD ₅₀ A / PR / 8. Survival and clinical signs of disease are monitored for 15 mice up to 3 weeks after challenge. Pulmonary viral titers were determined by MDCK microtest [AL Frank et al, J. Ciin. Microbiol., 12: 426-432 (1980)] in other animals (5 mice per group).

The results are listed in Table 15 below.

Table 15

Flying mice challenge: Survival rate and lung viral titers

Viral titer *

Vaccine group Survival (%) day 3 day 5 day 7 Aluminum -1- 3D-MPL13 8.90 ± 0.10 8.44 ± 0.55 8.69 ± 0.32 Singapore 80 8.10 ± 0.17 7.15 ± 0.23 3.09 ± 0.10 Singapore + 3D-MPL 100 # <1 <1 <1 A / Pr / 8 100 # 8.01 ± 0.029 3.93 ± 0.70 <1 A / PR / 8 + 3D-MPL 100 # <1 <1 <1

* TCID / lung log (geometric mean ± S.E.) # no visible clinical symptoms

Two strains (A / PR / 8 and Singapore) induce a certain level of protection. The survival rate for both groups is significantly higher than that for the antigen-free control preparation (Alum + 3D-MPL), and lung clearance is faster. Not surprisingly, the A / PR / 8 vaccine group (homologous to the challenge) behaves better than the Singapore vaccinated (heterologous to the challenge) group. Furthermore, both strains supplemented with 3D-MPL induce 100% survival, with virtually no pulmonary viral titre and no clinical symptoms. Obviously, with the 3D-MPL, the adjuvanted vaccine of the invention induces superior protection against infection of the lower respiratory tract and, therefore, against serious clinical phenomena such as e.g. viral pneumonia. This experiment therefore demonstrates that 3DMPL adjuvanting improves the homosubtypic and heterosubtypic activity of the split vaccine and is therefore expected to provide broader protection against antigenic displacement and float than non-adjuvanted vaccine.

Example 28 - Non-lethal challenge in mice

The prescription is the same as in Example 25 above except that the mice are anesthetized before the intranasal challenge. Viral titration in the respiratory tract, viral neutralization tests, and tracheal electron microscopy are performed.

Viral neutralization titrations against A / PR / 8 virus were performed on days 1, 5, and 9 after challenge (Table 16).

Table 16

Viral neutralization test

Neutralizing titer *

The vaccine group day 1 day 5 day 9 Aluminum + 3D-MPL <1 <1 <1 Singapore <1 <1 <1 Singapore <1 <1 <1 + 3D-MPL A / PR / 8 1.6 2.3 2.2 A / PR / 8 + 3D-MPL 3.5 3.6 3.3

* log ₁₀ average (5 mice)

Both A / PR / 8 groups produce neutralizing antibodies with a titer containing multiple 3D-MPL. By contrast, vaccination with Singapore does not induce neutralization of antibodies even with the addition of 3D-MPL. It follows that the heterosubtypic activity previously known with Singapore and 3D-MPL is not due to the antibody, but is strictly evident, to be responsible for cell-mediated immunity.

Linear electron microscopy (SEM) of the trachea was performed on samples collected on days 3 and 5. Tissues were evaluated for histopathological changes in the major and ciliated epithelial cells lining the trachea, especially for desquamation. These changes are indicative of the severity of the onset of influenza infection or recovery from infection (see Table 17 below).

Table 17

Non-lethal challenge in mice: SEM trachea average severity score *

The vaccine group day 1 day 5 the whole result Aluminum + 3D-MPL 1.7 1.0 0 Singapore 2.0 1.7 2+ Singapore + MPL 2.4 2.4 3 + A / PR / 8 2.0 2.0 2+ A / PR / 8 + 3D-MPL 2.0 2.4 3+

* semi-quantitative, made blind.

Regeneration: 1: Serious lesion

2: partial lesion 3: normal tissue ** overall score, from 0 (no protection) to 4+ (full protection)

The SEM results show that vaccination with either A / PR / 8 or Singapore strain alone provides protection; in both cases, this positive effect is enhanced by the addition of 3DMPL, showing that the adjuvanted split vaccine may alter the progression of influenza disease.

Viral titers were determined in the nose, trachea, and lungs by MDCK microtest on days 1, 3, 5, 7, and 9 after challenge (5 mice per group). The results are shown in FIG. 6A to 6F below. The nasal titers are higher than the tracheal or pulmonary ones, which is not surprising. However, not all treatments tested result in clear differences in nasal or tracheal titres. In contrast, pulmonary titers differ: A / PR / 8 + 3D-MPL and Singapore + 3D-MPL titers are always lower than or equal to the values of the antigen or 3D-MPL itself (Figs. 6A to 6F). This suggests that 3D-MPL adjuvanting offers better lung protection in mice.

Both sets of mouse experiments (lethal and non-lethal challenge) demonstrate that influenza vaccination with two subtypes of H1N1 strain (Singapore or A / PR / 8) and subsequent homotypic challenge improves the incorporation of 3D-MPL as an adjuvant in accordance with the present invention.

Example 29 - Immunogenicity of a vaccine

For each vaccine, CB6F ₁ mice (15 per group) were immunized subcutaneously with 2 injections given every 3 weeks, a preparation containing one tenth of a human dose of a conventional non-adjuvanted Singapore monovalent split vaccine or vaccine composition of the invention comprising a Singaporean strain of 3D- MPL. The human dose is defined as 0.5 ml injection containing 15 µg HA of each viral strain. The formulation used contains hemagglutinin (HA) against 3D-MPL in a ratio of 1.5 / ig HA per 5 / ig 3D-MPL to 5 / tg HA per 5 / tg 3D-MPL. The control group received only 3D-MPL (5 / ig). 3 weeks after the second injection, the blood was released to the mice and serum antibodies tested individually by inhibiting hemagglutination. The titers are calculated against a calibrated reference. Mice are considered responsive when antibody titers are greater than the cut off value.

The results are listed in Table 18. Both types of formulations induce anti-haemagglutination antibodies in all animals, so the sero-response rate is maximal. However, the immune response is significantly increased by the vaccine formulation of the invention containing 3D-MPL and as shown by geometric mean titers more than 5 times higher than that of the vaccine containing only the Singaporean strain.

Table 18

Vaccine Individual Titles * Geometric Sir group average average titer response 3D-MPL <25; <25; <25; <25; <25; (5 Mg) <25; <25; <25; <25; <25; <25; <25; <25; <25; <25; <25; 0/15 Singapore 100; 200; 200; 75; 200; (1.5 pg 100 25 800 300 50 150 15/15 HA) 200 200 600 200 50 Singapore 1200 600 400 400 1600 (1.5 pg 800 200 1600 1600 400 828 15/15 HA) + 3DMPL 5 pg 400 3200 400 3200 1200

* cut off value is less than 25.

This experiment demonstrates that the antibody response in mice is enhanced by the use of the 3D-MPL adjuvanted vaccine of the invention.

Example 30 - Guinea pig hypersensitivity study

A hypersensitivity study is performed in guinea pigs to determine if the addition of 3D-MPL (particle size <100 mm; Example 26) to a trivalent split vaccine modifies hypersensitivity. The trivalent vaccine, labeled trivalent Influsplit, was prepared as described in Example 25 and contained the H1N1 strain Singapore / 6/86, the H3N2 strain Beijing / 353/84 and the type B strain Β / Υamaghta / 16/88.

Sensitizing agent [allantoic fluid 0.5 or 2.5 mg ± 3D-MPL 50 μ-g; one human dose (0.5 ml injection containing 15 μξ HA for each of these influenza virus strains) of trivalent Influsplit ± 3D-MPL 50 μ-g] is given intraperitoneally with six injections daily 0, 3, 5, 7, 10 and 12. Guinea pigs are allowed to rest for 4 weeks and then challenged intravenously under anesthesia with a challenge agent (allantoic fluid 0.45 mg; single human dose of trivalent Influsplit ± 3D-MPL 50 μ%). We observe the animals 30 minutes after the challenge and again after 2 or 3 hours. Observed symptoms (scratching, breathing problems, cramps, death) are recorded. If no symptoms emerge after the first challenge, the animals are challenged again with allantoic fluid (1.3 mg) 24 hours later.

The results of this test are shown in Tables 19A and 19B below.

Table 19A - Hypersensitivity studies of Influsplit ± 3D-MPL

The challenge

The group Senzibi- lysing means An asset Death- nost Other find out howls 1 (negative control) none allantoic fluid 0/5 none 2 (negative control) none Influsplit 0/5 none 3 (negative control) none Influsplit 3D-MPL 0/5 none 4 (positive control) allantoic fluid allantoic fluid allantoic fluid 4/5 itching, dizziness, crunches 5 (vaccination split test) Influsplit allantoic fluid 0/5 none 6 (split vaccination test) allantoic fluid Influspl it 0/5 none 7 (positive control allant.tek./3D-MPL allantoic fluid 3D-MPL allantoic fluid 4/5 itching, dizziness, crunches 8 (split / 3D-MPL test) -Influsplit - 3D-MPL allantoic fluid 0/5 none 9 (split / 3D-MPL test) allantoic fluid Influsplit / 3D-MPL 0/5 none

Table 19B - Hypersensitivity studies of Influsplit ± 3D-MPL

; upina Sensitive means The challenge again An asset Death- nost Other find out howls (negative control) none (negative control) none (negative control) none (positive control) allantoic allantoic 1/5 itching, allantoic fluid fluid fluid dizziness, it cramps (vaccination split test) Influsplit allantoic 0/5 none fluid (vaccination split test) allantoic allantoic 5/5 itching, fluid fluid dizziness, it cramps (positive control allantoic allantoic 1/1 itching, alant.tek./3D-MPL fluid fluid dizziness, 3D-MPL it cramps (split split test./ -Influsplit allantoic 0/5 none 3D-MPL) - 3D-MPL fluid (split split test./ allantoic allantoic itching, 3D-MPL) fluid fluid 5/5 dizziness,

it cramps

The absence of effect for groups 1 to 3 (negative controls) indicates that hypersensitivity is required before intraperitoneal sensitization. The absence of hypersensitivity in group 5 indicates that Influsplit cannot sensitize to allantoic fluid. Similar conclusions can be drawn from group 6 (allantoic fluid sensitization and Influsplit challenge). Indeed, the results of groups 5 and 6 also show that the vaccine does not contain allantoic fluid protein residues. Addition of 3D-MPL to either the sensitizing agent (groups 7 and 8) or the challenge agent (group 9) does not change the response (comparison of groups 7 to 4; groups 8 to 5; groups 9 to 6).

The experiment shows that the trivalent Influsplit vaccine given as a sensitizing agent with or without 3D-MPL is not capable of inducing hypersensitivity. The trivalent Influsplit vaccine, administered as a challenge agent with or without 3D-MPL, is not capable of initiating any hypersensitivity reaction in animals previously hypersensitized with allantoic fluid; and under the experimental conditions 3D-MPL has no detectable effect on hypersensitivity reactions. Therefore, the vaccine compositions of the invention are safe for administration to mammals.

Many modifications and variants of the present invention are included in the description described above and are expected to be readily apparent to those skilled in the art. These modifications and alterations to the compositions and processes of the present invention are considered to be within the scope of the appended claims.

For

1. SmithKline Beecham Corporation

2. SmithKline Beecham Biologicals (s.a.):

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PATENT APPLICATIONS

Claims (24)

A vaccine composition characterized in that it is capable of eliciting an increased immune response to an influenza antigen containing an effective amount of influenza antigen and 3D-MPL. The vaccine composition of claim 1, wherein the influenza antigen is an antigenic polypeptide. A vaccine composition according to claim 1, characterized in that the antigenic polypeptide is selected from the group consisting of D protein NS1 ₁₈₁ HA2 ₆₅ _ ₂₂₂ , NS1 _{X 81} HA2 _{X 222} , HA2 _{66 222} , ΔΜ, ΔΜ +, A, C, 03, 03 short, Δϋ, cis-free D, HA2 _{66 222,} and NS1H3HA2 constructs. Vaccine composition according to claim 1, characterized in that the composition contains at least one split influenza virus. A vaccine composition according to claim 4, characterized in that it contains three split viruses with reactivity against at least three influenza virus strains. Vaccine composition according to claim 4, characterized in that the strains are selected from H1N1, H3N2, H2N2 and B type influenza strains. Vaccine composition according to claim 6, characterized in that the split virus is derived from a group of influenza strains consisting of A / PR / 8, A / Singapore, A / Udorn, A / Victoria, A / Texas, A / Beijing, A / Puerto Rico, B / Panama, B / Yamaghta, B / Lee / 40 and B / Taiwan. The vaccine composition of claim 1, further comprising an adjuvant containing aluminum. A vaccine composition according to claim 8, characterized in that the adjuvant is aluminum hydroxide or aluminum phosphate. A vaccine composition according to claim 9, characterized in that the influenza antigen is NS1 _Igl HA2 ₆₅ _ ₂₂₂ , D protein. A vaccine composition according to claim 10, characterized in that it consists of about 1 gg to about 1000 gg of D protein and between about 1 gg to about 50 gg of 3D-MPL. A vaccine composition according to claim 11, characterized in that the amount of D protein is about 2 gg and the amount of 3D-MPL is about 20 gg. A vaccine composition according to claim 3, characterized in that the antigen is a D protein, said composition comprising between about 50 gg to about 500 gg of D protein, between about 10 gg to about 50 gg of 3D-MPL and between about 100 gg up to about 500 gg of aluminum adjuvant. A vaccine composition according to any one of claims 1 to 13, further comprising a liposome preparation, wherein the liposome preparation comprises a liposome-forming substance comprising a long-chain aliphatic or aromatic acid or amine; a hydrating agent having a charge opposite that of the acid or amine, wherein the agent is present in a molar ratio between 1:20 and 1: 0.05 relative to the acid or amine; and water, in an amount of up to 300 moles, based on the solids present in the composition. The vaccine composition of claim 14, comprising between about 50 gg to about 500 gg of D protein, between about 10 gg to about 50 gg of 3D-MPL and between about 1 mg to about 10 mg of the lipomic preparation. A vaccine composition according to claim 14, characterized in that the flu D antigen consisting of between about 50 gg to about 500 gg of D protein is between about 10 gg to about 50 gg of 3D-MPL, from about 1 mg to about 10 mg of liposome preparation and between about 100 gg to about 500 gg aluminum adjuvant. A vaccine composition according to any one of claims 14 to 16, characterized in that the hydrating agent is an alpha amino acid with omega substitution, which is a carboxylate, amino ah guanidino function or a pharmaceutically acceptable salt or compound thereof represented by the formula: K-CCH.VV I where XH ^ - ^ NHj-NH-, H ^ -, ZO ₃ S-, Z ₂ O ₃ P-, or ZO ₂ Where ZH is an inorganic or organic cation; Y is -CHCNH ^ -CC ^ H, -NH ₂ , -NH-qNH) -NH ₂ -COOH, CH (NH ₂ ) SO ₃ Z, or ZH (NH ₂ ) PO ₃ Z ₂ wherein Z is defined above; and n is an integer 1-10; or a pharmaceutically acceptable salt thereof and are an acid or amine alkyl or alkenyl acid or an amine of 10 to 20 carbon atoms. A vaccine composition according to claim 17, characterized in that the hydrating agent is arginine, homoarginine, their N-acyl derivatives, gamma-aminobutyric acid, asparagine, lysine, ornithine, glutamic acid, aspartic acid or a compound of the following formula: H ₂ NC (NH) -NH- (CH ₂ ) _n -CH- (NH ₂ ) COOH II HN- (CH.) -CH (NH _o ) COOH III ^H 2 ^N < ^CH 2V ^NH 2) ^IV H ₂ NC (NH) -NH- (CH ₂ ) _n -NH-CH (NH) - (NH ₂ ) V HOOC- (CH ₂ ) _n -CH (NH ₂ ) COOH VI HOOC- (CH ₂ ) _n -COOH VII HO ₃ SC- (CH ₂ ) _n -CH (NH ₂ ) COOH VIII H ₂ O ₃ S- (CH ₂ ) _n -CH (NH ₂ ) COOH IX HO ₃ S- (CH ₂ ) _n -CH (NH ₂ ) SO ₃ HX H ₂ O ₃ S- (CH ₂ ) _n -CH (NH ₂ ) PO ₃ H ₂ XI or wherein n is 2-4, or a pharmaceutically acceptable salt thereof. A vaccine composition according to any of the preceding claims, characterized in that the particle size of the 3D-MPL does not exceed 120 nm. A vaccine composition according to claim 19, characterized in that the particle size is in the range 60-120 nm. A vaccine composition according to claim 19 or 20, characterized in that the particle size is below 100 nm. A vaccine composition according to claim 1, characterized in that it enhances the immune response to the influenza antigen. Use of a vaccine composition according to claim 1 in the manufacture of a medicament for enhancing the immune response to an influenza antigen. Process for the preparation of a vaccine composition according to claim 1, characterized in that it mixes said antigen and 3D-MPL. For 1. SmithKline Beecham Corporation 2. SmithKline Beecham Biologicals (s.a.): 1/9 Cross-protection in mice immunized with SK & F106 160 in A1 + 3D-MPL (H1N1) (H1N1) (H2N2) (H3N2) Regulation-SKF106160 10jjg / AI + 3D-MPL 10> ig SC (0)> SC (3)> challenge (7) -p <0.05 I I SK & F105160 / AI + 3D-MPL FIG. 1 PATI iUaJ L · * ki,. 1 'j 2/9 CD ο ο ο Ο ο σ ο σ σ σ ο σ ο σ σ σ ο ο ιη σ m ο LT) η CM Csl r ^- Wd3 3/9 20 / jg 4/9