AP515A - """Vaccine compositions""." - Google Patents

"""Vaccine compositions""." Download PDF

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Publication number
AP515A
AP515A APAP/P/1994/000629A AP9400629A AP515A AP 515 A AP515 A AP 515A AP 9400629 A AP9400629 A AP 9400629A AP 515 A AP515 A AP 515A
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mpl
vaccine composition
antigen
vaccine
hbsag
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APAP/P/1994/000629A
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AP9400629A0 (en
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Pierre Hauser
Pierre Voet
Moncef Slaqui
Nathalie Marie-Josephe Claude Garcon-Johnson
Pierre Desmons
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Smithkline Beecham Biolog
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7024Esters of saccharides
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/39Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/555Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505Inorganic adjuvants
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/555Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511Organic adjuvants
    • A61K2039/55555Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/555Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511Organic adjuvants
    • A61K2039/55566Emulsions, e.g. Freund's adjuvant, MF59
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/555Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511Organic adjuvants
    • A61K2039/55572Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection
    • Y02A50/30Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
    • Y02A50/38Medical treatment of vector-borne diseases characterised by the agent
    • Y02A50/398Medical treatment of vector-borne diseases characterised by the agent the vector-borne disease being caused by a bacteria
    • Y02A50/399Medical treatment of vector-borne diseases characterised by the agent the vector-borne disease being caused by a bacteria of the genus Borrellia
    • Y02A50/401Medical treatment of vector-borne diseases characterised by the agent the vector-borne disease being caused by a bacteria of the genus Borrellia the bacteria being Borrelia burgdorferi, i.e. Lyme disease or Lyme borreliosis
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection
    • Y02A50/30Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
    • Y02A50/46Medical treatment of waterborne diseases characterized by the agent
    • Y02A50/462The waterborne disease being caused by a virus
    • Y02A50/465The waterborne disease being caused by a virus the virus being the poliovirus, i.e. Poliomyelitis or Polio
    • Y02A50/466The waterborne disease being caused by a virus the virus being the poliovirus, i.e. Poliomyelitis or Polio the medicinal preparation containing antigens or antibodies, e.g. vaccines, antisera

Abstract

Novel vaccine compositions comprising small particles of 3-0-decylated monophosphoryl lipid A are provided. In Particular the particle size is below 120 nm. Such vaccine compositions have superior immunological properties.

Description

B45042 AP. Ο Ο 5 1 5

Vaccine Compositions

The present invention relates to novel vaccine formulations, methods forpreparing them and to their use in therapy. 5 3-O-deacylated monophosphoryl lipid A (or 3 De-O-acylated monophosphoryl lipid A) has formerly been termed 3D-MPL or d3-MPL to indicatethat position 3 of the reducing end glucosamine is de-O-acylated. For preparation,see GB 2 220 211 A. Chemically it is a mixture of 3-deacylated monophosphoryllipid A with 4,5 or 6 acylated chains. Herein the term 3D-MPL (or d3-MPL) is 10 abbreviated to MPL since 'MPL' is a Registered Trademark of Ribi

Immunochem.,Montana which is used by Ribi to denote unambiguously their 3-O-deacylated monophosphoryl lipid A product. GB 2 220 211A mentions that the endotoxicity of the previously usedenterobacterial lipopolysacharides (LPS) is reduced while the immunogenic 15 properties are conserved. However GB 2 220 211 cited these findings merely inconnection with bacterial (Gram negative) systems. No mention of the particle sizeof the MPL was made. In fact the particle size of the known 3-O-deacylatedmonophosphoryl lipid A has particles in excess of 500nm.

In WO 92/16231 a vaccine formulation comprising a Herpes Simplex Virus 20 glycoprotein gD or immunological fragments thereof in conjunction with 3 deacylated monophosphoryl lipid A was disclosed. Again, no mention of the particlesize of the 3 deacylated monophosphoryl lipid A was made.

In WO 92/06113 a vaccine formulation comprising HIV gp 160 or aderivative thereof such as gp 120 in conjunction with 3 deacylated monophosphoryl 25 lipid A was disclosed. No mention of particle size of the MPL was made.

The present invention provides a vaccine composition comprising an antigen in conjunction with 3-O-deacylated monophosphoryl lipid A (abbreviated herein toMPL) and a suitable carrier wherein the particle size of the MPL is 'small' and ingeneral do not exceed 120nm when prepared. 30 Such a formulation is suitable for a broad range of monovalent or polyvalent vaccines.

Surprisingly, it has been found that vaccine compositions according to theinvention have particularly advantageous properties as described herein. Inparticular such formulations are highly immunogenic. Additionally sterility of the 35 adjuvant formulation can be assured as the product is susceptible to sterile filtration. A further advantage of 'small' MPL arises when formulated with alum, as the MPLinteracts with the aluminium hydroxide and the antigen to form a single entity.

In an embodiment of the invention, the antigen is a viral antigen, for examplean antigen against hepatitis infection (Hepatitis A, B, C, D, or E) or herpes (HSV-1 or - 1 - B45042 HSV-2) infection as described hereinbelow. A review on modem hepatitis vaccines,including a number of key references, may be found in the Lancet, May 12th 1990 atpage 1142 ff (Prof A.L.W.F. Eddleston). See also 'Viral Hepatitis and Liver Disease'(Vyas, B.N., Dienstag, J.L., and Hoofnagle, J.H., eds, Grune and Stratton, Inc. (1984) 5 and Viral Hepatitis and Liver Disease' (Proceedings of the 1990 InternationalSymposium, eds F.B. Hollinger, S.M. Lemon and H. Margolis, published byWilliams and Wilkins). References to HSV-1 and HSV-2 may be found in WO92/16231.

Infection with hepatitis A virus (HAV) is a widespread problem but vaccines 10 which can be used for mass immunisation are available, for example the product*Havrix* (SmithKline Beecham Biologicals) which is a killed attenuated vaccineobtained from the HM-175 strain of HAV [see Inactivated Candidate Vaccines forHepatitis A' by F.E. Andre, A Hepburn and E.D'Hondt, Prog Med. Virol. Vol 37,pages 72-95 (1990) and the product monograph Havrix’ published by SmithKline 15 Beecham Biologicals (1991)].

Flehmig et al (loc cit., pages 56-71) have reviewed the clinical aspects, virology, immunology and epidemiology of Hepatitis A and discussed approaches tothe development of vaccines against this common viral infection.

As used herein the expression HAV antigen' refers to any antigen capable of 20 stimulating neutralising antibody to HAV in humans. The HAV antigen may comprise live attenuated virus particles or inactivated attenuated virus particles ormay be, for example an HAV capsid or HAV viral protein, which may convenientlybe obtained by recombinant DNA technology.

Infection with hepatitis B virus (HBV) is a widespread problem but vaccines 25 which can be used for mass immunisation are now available, for example the product*Engerix-B' (SmithKline Beecham pic) which is obtained by genetic engineeringtechniques.

The preparation of Hepatitis B surface antigen (HBsAg) is well documented.See. for example, Harford et al in Develop. Biol. Standard 54, page 125 (1983), 30 Gregg et al in Biotechnology, 5, page 479 (1987), EP-A- 0 226 846, EP-A-0 299 108and references therein.

As used herein the expression Hepatitis B surface antigen' or HBsAg'includes any HBsAg antigen or fragment thereof displaying the antigenicity of HBVsurface antigen. It will be understood that in addition to the 226 amino acid sequence 35 of the HBsAg S antigen (see Tiollais et al, Nature, 317,489 (1985) and references therein) HBsAg as herein described may, if desired, contain all or part of a pre-Ssequence as described in the above references and in EP-A- 0 278 940. In particularthe HBsAg may comprise a polypeptide comprising an amino acid sequencecomprising residues 12-52 followed by residues 133-145 followed by residues 175- -2- B45042 AP. Ο Ο 5 1 5

400 of the L-protein of HBsAg relative to the open reading frame on a Hepatitis Bvirus of ad serotype (this polypeptide is referred to as L*; see EP 0 414 374). HBsAgwithin the scope of the invention may also include the preSl-preS2 -S polypeptidedescribed in EP 0 198 474 (Endotronics) or analogues thereof such as those described 5 in EP 0 304 578 (Me Cormick and Jones). HBsAg as herein described can also referto mutants, for example the 'escape mutant' described in WO 91/14703 or EuropeanPatent Application Publication Number 0 511 855 Al, especially HBsAg wherein theamino acid substitution at position 145 is to arginine from glycine.

Normally the HBsAg will be in particle fonn. The particles may comprise for 10 example S protein alone or may be composite particles, for example (L*,S) where L*is as defined above and S denotes the S-protein of HBsAg. The said particle isadvantageously in the form in which it is expressed in yeast.

Herpes Simplex virus Glycoprotein D is located on the viral envelope, and isalso found in the cytoplasm of infected cells (Eisenberg R.J. et al J. of Virol. 1980 35 15 428-435). It comprises 393 amino acids including a signal peptide and has a molecular weight of approximately 60kD. Of all the HSV envelope glycoproteinsthis is probably the best characterized (Cohen et al J. Virology 60 157-166). in vivoit is known to play a central role in viral attachment to cell membranes. Moreover,glycoprotein D has been shown to be able to elict neutralizing antibodies in vivo 20 (Eing et al* J. Med Virology 127: 59-65). However, latent HSV2 virus can still bereactivated and induce recurrence of the disease despite the presence of highneutralizing antibodies titre in the patients sera. It is therefore apparent that theability to induce neutralizing antibody alone is insufficient to adequately control thedisease. 25 The mature recombinant truncated glycoprotein D (rgD2t) or equivalent proteins secreted from mammalian cells, is preferably used in the vaccineformulations of the present invention. Equivalent proteins include glycoprotein gDfrom HSV-1.

In a preferred aspect the rgD2t is HS V-2 glycoprotein D of 308 amino acids 30 which comprises amino acids 1 though 306 of the naturally occurring glycoproteinwith the addition of Asparagine and Glutamine at the C terminal end of the truncatedprotein. This form of the protein includes the signal peptide which is cleaved to yielda mature 283 amino acid protein. The production of such a protein in ChineseHamster ovary cells has been described in Genentech's European patent EP-B-139 35 417 and Science 222 p524, and Biotechnology p527 June 1984. Such a vaccine when formulated with small MPL according to the present invention has a superiortherapeutic potential as compared to known rgLty formulations.

Whilst certain experimental and commercially available vaccines affordexcellent results it is an accepted fact that an optimal vaccine needs to stimulate, not -3- B45042

15 20 Ο 30 only neutralising antibody but also needs to stimulate as effectively as possiblecellular immunity mediated through T-cells. A particular advantage is that the vaccine formulations of the invention are very effective in inducing protective immunity, even with very low doses of antigen. cr<d recurrent ' ,

They provide excellent protection against primaryjnfection and stimulate /advantageously both specific humoral (neutralising antibodies) and also effector cellmediated (DTH) immune responses.

To make 3 deacylated monophosphoryl lipid A with a small particle size, ingeneral not exceeding 120nm the procedure described in GB 2 220 211 may be to obtain tnown 3B - followedA(or commercial MPL of larger panicle size may be purchased from RibiImmunochem.) and the product may then be sonicated until the suspension is clear.

The size of the particles may be estimated using dynamic light scattering as describedhereinbelow. In order to maintain the MPL size to the 100 nm range after beingformulated with aluminium hydroxide, the antigen and the buffer, tween 80 orsorbitol can be added. Under these conditions, it has been established that MPL doesnot aggregate in the presence of phosphate buffer, as it may happen duringformulation without them. By doing so, the final formulation is further defined andcharacterized. It has also been established that under those conditions, MPL stillinteracts with aluminium hydroxide and the antigen forming one single entity. A clear solution of MPL forms an aspect of the invention. This solution maybe sterilised by passing through a filter.

Preferably the size of the particles is in the range 60-120nm.

Most advantageously the particle size is below lOOnm.

The MPL as defined above will normally be present in the range of 10-100gg,preferably 25-50gg per dose wherein the antigen will be typically present in a range2-50gg per dose. The vaccine formulation of the present invention may additionallycontain further immunostimulants, in a preferred embodiment the vaccines of thepresent invention may include QS21 (sometimes referred to as QA21). This is anHPLC fraction of a saponin extract derived from the bark of a tree, QuillajaSaponaria Molina and a method for its production is disclosed in US Patent5,057,540.

The carrier may optionally be an oil in water emulsion, a lipid vehicle, oralum (aluminium salt).

Non-toxic oil in water emulsions preferably contain a non-toxic oil, e.g.squalene and an emulsifier such as Tween 80, in an aqueous carrier. The aqueouscarrier may be, for example, phosphate buffered saline.

Preferably the vaccine formulations will contain an antigen or antigeniccomposition capable of eliciting an immune response against a human or animalpathogen, which antigen or antigenic composition is derived from HIV-1, (such as -4- 35 B45042 AP·Ο Ο 5 1 5 gpl 20 or gpl60; see WO 92/06113 and references therein), herpes virus such as gDor derivatives thereof or Immediate Early protein such as ICP27 from HSV-1 orHS V-2, gB (or derivatives thereof) from Human cytomegalovirus, or gpl, Π or ΙΠfrom Varicella Zoster Virus, or from a hepatitis virus such as hepatitis B virus or 5 from other viral pathogens, such as Respiratory Syncytial virus, human papillomavirus or Influenza virus, or against bacterial pathogens such as Salmonella, Neisseria,Borrelia (for example OspA or OspB or derivatives thereof), or Chlamydia, orBordetella for example P.69, PT and FHA, or against parasites such as plasmodium orToxoplasma. The vaccine formulations of the present invention may contain a 10 tumour antigen, and be useful as an anticancer vaccine.

One embodiment of the invention is HAV antigen (for example as in Havrix) in admixture with MPL and aluminium hydroxide as described hereinbelow. A further embodiment of the invention is HB Virus Surface (HBsAg) antigen (for example as in Engerix-B) in admixture with MPL and aluminium hydroxide as15 described hereinbelow. A further specific embodiment of the invention is HBsAg antigen as (L*,S)particles, defined hereinabove, in admixture with MPL and aluminium hydroxide.

Hepatitis A plus Hepatitis B combination vaccines may also be prepared inaccordance with the invention. 20 A further embodiment is a formulation according to the invention comrising mature truncated glycoprotein D (rgD2t) or equivalent proteins as describedhereinabove. Yet a further embodiment is a formulation of the invention comprisingan OspA antigen or derivative thereof from Borrelia burgdorferi. For example,antigens, particularly OSpA antigens from the ZS7 or B31 strains may be used. Yet a 25 further embodiment is a formulation of the invention comprising a flu antigen. Thisprovides an improved influenza vaccine, especially if a 'split' virus is used.

The formulation may also be useful for utilising with herpetic light particlessuch as described in International Patent Application No. PCT/GB92/00824 and,International Patent Application No. PCT/GB92/0O179. 30 Advantagously the vaccine composition of the invention contains other antigens so that it is effective in the treatment or prophylaxis of one or more otherbacterial, viral or fungal infections.

For example, hepatitis vaccine formulations according to the inventionpreferably contain at least one other component selected from non-hepatitis antigens 35 which are known in the art to afford protection against one or more of the following:diphtheria, tetanus, pertussis, Haemophilus influenzae b (Hib), and polio.

Preferably the vaccine according to the invention includes HBsAg ashereinabove defined. -5- B45042

Particular combination vaccines within the scope of the invention include aDTP (diphtheria-tetanus-pertussis) -hepatitis B combination vaccine formulation, anHib-Hepatitis B vaccine formulation, a DTP-Hib-Hepatitis B vaccine formulation andan IPV (inactivated polio vaccine) -DTP-Hib-Hepatitis B vaccine formulation. 5 The above combinations may advantageously include a component which is protective against Hepatitis A, especially the killed attenuated strain derived from theHM-175 strain as is present in Havrix.

Suitable components for use in such vaccines are already commerciallyavailable and details may be obtained from the World Health Organisation. For 10 example the IPV component may be the Salk inactivated polio vaccine. The pertussisvaccine may comprise whole cell or acellular product.

Advantageously the hepatitis or combination vaccine according to theinvention is a paediatric vaccine.

The invention in a further aspect provides a vaccine composition according to15 the invention for use in medical therapy, particularly for use in the treatment or prophylaxis of infections include viral and bacterial infections or for immunotherapeutic treatment of cancer. In a preferred aspect the vaccine according to theinvention is a therapeutic vaccine useful for the treatment of ongoing infections, forexample hepatitis B or herpetic infections in humans suffering therefrom. 20 Vaccine preparation is generally described in New Trends and Developments

in Vaccines, edited by Voller et al, University Park Press, Baltimore, MarylandU.S.A. 1978. Encapsulation within liposomes is described, for example, byFullerton, US Patent 4,235,877. Conjugation of proteins to macromoloecules isdisclosed, for example, by Likhite, US Patent 4,372,945 and by Armor et al, US 25 Patent 4,474,757.

The amount of antigen in each vaccine dose is selected as an amount whichinduces an immunoprotective response without significant, adverse side effects intypical vaccinees. Such amount will vary depending on which specific immunogensare employed. Generally it is expected that each dose will comprise l-1000pg of total 30 immunogen, preferably 2-100pg, most preferably 4-40pg. An optimal amount for aparticular vaccine can be ascertained by standard studies involving observation ofantibody titres and other responses in subjects. Following an initial vaccination,subjects may receive a boost in about 4 weeks.

In a further aspect of the present invention there is provided a method of 35 manufacture of a vaccine effective in preventing or treating infection, wherein themethod comprises mixing the antigen with a carrier and MPL wherein the particlesize of the MPL is no greater than 120nm, normally 60-120nm, preferably about orless than lOOnm.

The following examples illustrate the invention and its advantages. -6- B45042 AP. Ο Ο 5 1 5

Example 1: Preparation of MPL with a particle size of 60 -120 nm

Water for injection is injected in vials containing lyophilised 3-de-O-acylatedmonophosphoryl lipid A (MPL) from Ribi Immunochem, Montana using a syringe to 5 reach a concentration of 1 to 2 mg per ml. A preliminary suspension is obtained bymixing using a vortex. The content of the vials is then transferred into 25 ml Corextubes with round bottoms (10 ml suspension per tube) and the suspension is sonicatedusing a water bath sonicator. When the suspension has become clear, the size of theparticles is estimated using dynamic light scattering (Malvem Zetasizer 3). The 10 treatment is continued until the size of the MPL particles is in the range 60 -120 nm.

Suspensions can in some cases be stored at 4 degrees C without significant aggregation up to 5 months. Isotonic NaCl (0.15M) or isotonic NaCl plus lOmMphosphate induces a rapid aggregation (size >3-5pm). 15 Example 2: Production of large scale sterile soluble MPL with a particle size ofbelow lOOnm.

Lyophilised 3 de-O-acylated monophosphoryl lipid A was obtained from RibiImmunochem, and suspended in water for injection (WFI). The suspension wascontinuously pumped through an ultrasound flow cell. The flow cell is typically

20 made of glass or stainless steel with PTFE seals so as to comply with GMP constraints. The Ultrasound is generated utilising an Ultrasound generator and atitanium sonic horn (sonotrode) acquired from Undatim Ultrasonics (Louvain-La-Neuve, Belgium). A heat exchanger is incorporated into the loop to avoiddegradation of the product by heat. The temperature of the MPL between the inlet 25 and the outlet of the flow cell is kept between + 4°C and 30°C, and the difference ofthe temperature between the inlet and outlet is kept below 20°C. It will beappreciated that heat is also removed as the material passes through the apparatus.

The apparatus used is schematically depicted in figure 1. 2.1. Sonication 30 The MPL powder (50 to 500 mg) is suspensed in WFI at concentration between 1 and 2 mg/ml.

The MPL suspension (under stirring conditions) is continuously pumpedthrough the sonication loop (see figure 1) at a flow rate between 50 and 100 ml perminute in order to reach the equilibrium temperature of the system which is between 35 + 4and + 15°C.

The own frequency spectrum of the sonic horn in the configuration of thesystem (Power, Flow Cell, Liquid Flow rate, T°.) is set according to suppliersinstructions for the equipment Pre-established limits are fixed between 19000 Hertzand 21000 Hertz for the the 20,000 Hertz transducer. -7- B45042

The generator allows the control of the optimal efficiency of the sonication(more transmission of energy with less heat) at a given time interval.

The temperature during the process is maintained below 30°C to avoid MPLdegradation. 5 The process is complete when the particle size is reduced below lOOnm and the solution is clear by visual inspection. During the sonication process samples aretaken for particle size evaluation utilising (Malvern Zetasizer type 3) photocorrelationspectroscopy (dynamic light scattering) in the same manner as example 1. The totalliquid residence time in the flow cell is between 2.5 and 3.5 minutes (see table 1). 10 The total residence time corresponds to the additive time interval of presence of theMPL suspension into the flow cell along the cycles. This is normally less than 10individual exposures of 25 seconds in a 20ml flow cell at 50ml/min recirculation flowrate. 2.2. Sterilization Process 15 The resulting "solubilized" MPL is sterilized by dead end filtration on a hydrophilic PVDF 0.22mcm membrane. The observed pressure is currently below 1bar. At least 25 mg of "solubilized" MPL are easily processed per square centimetrewith a recovery above 85%. 2.3. Storage/Stability 20 Sterile "solubilized" MPL is stored at +2° to 8°C. Stability data (Malvern) did not show any significant difference of particle size after 6 months storage. (See Table2).

Example 3: Hepatitis B vaccine formulation 25 MPL (particle size less than 100 nm) was obtained as described in Example 1.

Aluminium hydroxide was obtained from Superfos (Alhydrogel).

The MPL was resuspended in water for injection at a concentration varyingfrom 0.2 to 1 mg/ml by sonication in a water bath until the particles reach a size ofbetween 80 and 500 nm as measured by photo correlation light scattering. 30 1 to 20gg of HBsAg (S- antigen as in Engerix B) in phosphate buffer solution at 1 mg/ml) is adsorbed on 30 to lOOgg of aluminium hydroxide (solution at 10.38AP+ mg/ml) for one hour at room temperature under agitation. To the solution isthen added 30 to 50gg of MPL (solution 1 mg/ml). Volume and osmolarity areadjusted to 600μ1 with water for injection and phosphate buffer 5x concentrated. 35 Solution is incubated at room temperature for 1 hour and kept at 4°C until use.Maturation of the formulation occurs during storage. This represents 10 injectingdoses for testing in mice. -8- B45042 AP . Ο Ο 5 1 5

Example 4: Hepatitis A vaccine formulation MPL (particle size less than lOOnm) was obtained as described in Example 1.

Aluminium hydroxide was obtained from Superfos (Alhydrogel). HAV (360 to 22 EU per dose) is preadsorbed on 10% of the aluminium 5 hydroxide final concentration (0.5mg/ml). The MPL (12.5 to HX^g per dose) isadded to the solution.

The remaining aluminium hydroxide is added to the solution and left for onehour at room temperature. Volumes are adjusted with phosphate buffer (phosphatelOmM, NaCl 150mM) and the final formulation is then stored at 4°C until use. 10

Example 5 - Comparison Of Adjuvant Efficacy Of A Recombinant HerpesSimplex Glycoprotein D Subunit Vaccine 5.1 In this study, the ability of various A1(OH)3 MPL formulations to improve theprotective immunity of a truncated glycoprotein D from Herpes Simplex virus type 2 15 (HSV2) (rgD2t) was evaluated in the prophylactic and therapeutic guinea pig models.Immunogenicity studies were also performed in primates. The aim of theseexperiments was to investigate the impact of the size of 3-de-O-acylatedmonophosphoryl lipid A (MPL) particles on the immunogenicity and protectiveefficacy of rgD2t Al(0H)3 MPL formulations in rodents and in primates. Three 20 different Al(0H)3 MPL formulations using different strategies to have small size MPL were tested:

Al(0H)3 MPL lOOnm (as described previously) A1(OH)3 MPL lOOnm sorbitolA1(OH)3 MPL lOOnm tween. 25 J 5,2 Antigen-adjuvant preparations and immunisation schedules © 5.2.1. Antigen formulations

Aluminium hydroxide (A1(OH)3) was obtained from Superfos (Alhydrogel 30 Superfos, Denmark). MPL was obtained from Ribi Immunochem Research Inc.

5.2.1.1. rgD2t (AI(OH)3)/MPL TEA MPL was resuspended by sonication in a water bath, giving sizes comprisedbetween 200 and 600nm. Formulation was prepared according to patent application 35 WO92/16231. Formulation was stored at 4°C until use. A dose contained 5pg rgD2t, 0.5 mg A1(OH)3 and 50gg MPL. -9- B45042 5.2.1.2. rgD2t/Al(OH)3ZMPL lOOnm MPL (particle size less than lOOnm) was obtained as described in Example 1.rgD2t was adsorbed on alum and incubated lh room temperature. MPL was added tothe solution at the required concentration and incubated further for 1 hour at room 5 temperature.

Formulation was completed with PBS to reach a final concentration of lOmMPO4,150mM NaCl. Formulation was further incubated for 30 minutes at roomtemperature and stored at 4°C until use. A dose contained 5pg rgD2t, 0.5 mg A1(OH)3 and 50pg MPL. 10 5.2.1.3. rgD2t/AI(OH)3/MPL lOOnm sorbitol MPL was prepared as described in Example 1. rgD2t was adsorbed on alumand incubated 1 h at room temperature. A 50% sorbitol solution was then added toreach a final concentration of 5%. Tris lOmM solution was then added to make up 15 the final desired volume, and the solution was incubated lh at room temperatureunder agitation.

Formulation was stored at 4°C until use. A dose contained 5gg rgD2t, 0.5 mg A1(OH)3 and 50gg MPL. 20 5.2.1.4. rgD2t/Al(OH)3/MPL lOOnm tween MPL was prepared as described in Example 1. In order to maintain the MPLsize to 100 nm, tween 80 was added to the solution at a concentration such that it willbe equal to 0.01% in the final formulation. The formulation is then prepared asdescribed in formulation 5.2.1.3 above. 25 A dose contained 5gg rgD2t, 0.5 mg A1(OH)3 and 50pg MPL. c θ 5.3. Guinea pig prophylactic experiment

In these experiments, groups of guinea pigs were vaccinated at days 0 and 28with 5gg rgD2t in two different Alum MPL formulations. Immunisations were given 30 subcutaneously in an 0.5ml dose. One month after the second vaccination, guineapigs were challenged intravaginally with 10^ pfu of HS V2 strain MS. They weremonitored daily for the development of primary and recurrent HSV2 disease (days 4to 39 post challenge). 35 5.3.1. Guinea pig therapeutic experiments

In these experiments, guinea pigs were challenged at day 0 with 10^ pfu HSV2 strain MS. After recovery from primary infection, they were evaluated dailyfor recurrent herpetic disease (days 13 to 21). Guinea pigs were vaccinated at days 21and 42 with rgD2t A1(OH)3 MPL vaccine. Vaccines were administrated - 10- B45042 AP. Ο Ο 5 1 5 subcutaneously in a 0.5 ml dose. Animals were monitored daily for herpetic lesionsuntil day ± 60 or ± 84. 5.3.2. Primate Immunogenicity studies 5 The immunogenicity of rgD2t A1(OH)3 combined with MPL in sorbitol was evaluated in African Green Monkeys. Groups of monkeys were vaccinated at days 0and 28 with 20pg rgD2t and 0.5mg A1(OH)3 combined with 50,20 or 5gg MPL insorbitol. Specific humoral (ELISA and neutralising titers) and effector cell mediated(delayed type hypersensitivity response :DTH) immune responses were evaluated. 10 Each monkey group contained 5 animals. The formulations were administeredintramuscularly in a 1 ml dose. Preparation of the formulations was done asdescribed above. Animals were bled ± every two weeks for antibody determination. DTH response was tested 14 days after the second vaccination. A descriptionof the skin test is given below. - 15 5.4. Read-outs

Assays were set up to evaluate the specific antibody response induced byvaccination with rgD2t A1(OH)3 MPL formulations (determination anti-rgD2t ELISAtiters and anti-HSV2 neutralising titers). The protective efficacy of these gD2 20 formulations was assessed in the prophylactic and therapeutical guinea pig models.Immunogenicity studies were also conducted in monkeys. Specific humoral andDTH responses were evaluated. 5.4.1. ELISA and neutralising titers 25 Anti-rgD2t antibody titers and anti-HS V2 neutralising activity were determined according the methods described in patent application No. WO92/16231 o 5.4.2. Delayed type hypersensitivity (DTH)

The rgD2t formulations were also tested for their ability to induce a T cell 30 specific immune response in monkeys as measured by the induction of delayed -typehypersensitivity (DTH) responses.

African Green monkeys were vaccinated at days 0 and 28 with 20μg gD2vaccine formulation administered intramuscularly. They were skin tested 14 daysafter the second vaccination by intradermal injection on the belly of 15 or 5gg of 35 rgD2t in saline. They were also skin tested with saline as control. The site of injection was inspected 24 and 48 hours later for erythema and induration and the sizeof these local reactions was measured. -11- B45042 5.4.3. Guinea pig intravaginal challenge model

The guinea pig model for HSV genital infection has been described by L.

Stanberry et al (J. of Infectious Diseases 1982.146: 397-403; Intervirology 1985,24:226-231). Briefly, in prophylactic experiments, the guinea pigs were challenged 5 intravaginally with 10^ pfu of HSV2 strain MS, one month after the last vaccination.The clinical course of primary disease was monitored by daily observation of theincidence and severity of genital skin lesions during the 4-12 day post challengeperiod. Animals were then examined daily for evidence of recurrent herpetic lesionsfrom days 13 to 39. In therapeutic experiments, guinea pigs were challenged at day 0 10 with 10^ pfu HS V2 strain MS. After recovery from primary infection, they wereevaluated daily for recurrent herpetic disease (days 13 to 21) and were thenrandomized according to their primary and recurrent scores (providing an equivalentdistribution of animals with mild to severe infection in each group) to receive eitherno treatment or vaccination. Vaccines were administered on days 20 and 41 after 15 challenge. The pattern of recurrent disease was generally observed until ±70 postchallenge.

The herpetic lesions were quantitated by using a lesion score scale rangingfrom 0 to 32. 20 Scoring system

Lesion type Score None 0 Vaginal lesions - - Bleeding 0.5 - Redness for one or two days without bleeding 0.5 - Redness and bleeding for a day 1 - Redness without bleeding for at least 3 days 1 External herpetic vesicles - < 4 small vesicles 2 - 2:4 small vesicles or only one big vesicle 4 - £ 4 large lesions 8 - Fusing large lesions 16 - Fusing large lesions on all external genital area 32 -12- B45042 AP .00515

Clinical read-outs

Primary infection - Lesion severity = sum of the daily scores for the days 4 to 12 post infection. 5 The lesion severity is expressed as arithmetic mean ± SD as well as median (more appropriate for non-parametric test). - Primary infection incidences = % of animals having experienced a maximumlesion score of 0,0.5,1, 2,4, 8 or 16 (rarely 32).

Primary infection index - Σί (max.score i) x (incidence %) with i = 0,0.5,2,10 4, 8 or 16.

Recurrent disease - Recurrence day number = number of recurrence days for the days 13 to 39post-infection. One recurrence is preceded and followed by a day without lesion and 15 characterized by at least two days with erythema or one day with vesicle(s).Recurrence day numbers are expressed as arithmetic means ± SD and medians. - Recurrence severity = sum of the daily scores for the days 13 to 39 post-infection. Results are expressed as arithmetic means ± SD and medians. 20 5.5. Results

The protective efficacy of different rgD2t A1(OH)3 MPL formulations wascompared in prophylactic and therapeutic experiments in guinea pigs.Immunogenicity studies were also conducted in primates. The aim of theseexperiments was to compare the immunogenicity and protective efficacy of rgDjt 25 A1(OH)3 combined with different MPL particles size. 5.5.1. Prophylactic experiments

Two experiments were performed to evaluate the potential of different rgD2tA1(OH)3 MPL vaccines to provide protection against primary and recurrent HSV2 30 disease, when administered to guinea pigs prior to intravaginal viral inoculation.

Experiment 1: Comparison of MPL 100 nm sorbitol versus MPL TEA

Group of female hartley guinea pigs (200-250g) were immunized at days 0and 28 with 5gg rgD2t Al(0H)3 combined with small size MPL particles (lOOnm; 35 MPL in sorbitol), or with larger MPL particles (MPL in TEA). Control animals wereinjected according to the same protocol with adjuvant alone or were untreated. Theanimals were bled 14 and 28 days after the second vaccination for antibodydeterminations by ELISA and neutralization assays. They were challenged 29 daysafter the second vaccination with 10$ pfu of HSV2 strain MS intravaginally. After -13- B45042 challenge, the guinea pigs were monitored daily for clinical signs of acute infection(days 4 to 12 post challenge) and for evidence of recurrent herpetic disease (days 13to 39 post challenge). a) Induction of humoral immunity 5 As shown in Table 3, higher ELISA and neutralizing titers were induced when the small size MPL particles were used in the rgD2t A1(OH)3 formulation. b) Effect of vaccination on primary HSV2 infection (Table 3)

As compared to the control group that became infected and experienced acuteprimary infection, both vaccinated groups showed significantly reduced lesion 10 severity (p < 0.00005). A significantly lower skin lesion incidence was observed inthe rgD2t A1(OH)3 MPL 100 nm vaccinated group (p<0.06). c) Effect of vaccination on recurrent HSV2 disease

Results are given in Table 4. As compared to the controls, both vaccines wereable to alter the development of recurrent herpetic disease, as measured by reduction 15 in the number of recurrent episodes (p<0.02 for rgD2t A1(OH)3 MPL 100 nm) d) Conclusions

Both formulations were able to provide significant protection against primaryinfection and to reduce recurrent disease. These results show that the rgD2t A1(OH)3formulation containing small size MPL particles has a very potent prophylactic 20 efficacy.

Experiment 2: Efficacy of AI(OH)3 MPL 100 nm

Hartley guinea pigs (200-250g) were immunized at days 0 and 28 with 5gg gD2 formulated in Al(0H)3 MPL 100 nm. Immunizations were given 25 subcutaneously in a 0.5 ml dose. A dose of 50pg MPL was used in the A1(OH)3MPL formulation. Control animals were injected according the same protocol withadjuvant alone or were untreated. The animals were bled 14 and 28 days after thesecond vaccination for antibody determination by ELISA and neutralization assays.The guinea pigs were challenged 29 days after the last immunization with 1(P pfu of 30 HSV2 strain MS intravaginally. a) Induction of humoral immunity

As shown in Table 3, the vaccinated group produced good ELISA andneutralizing titers. Control group did not develop any detectable antibody response. b) Effect of vaccination on primary HSV2 infection (Table 3) 35 As compared to the control group that became infected and experienced acute primary infection, the vaccinated group showed significant reduced lesion severity (p< 0.00005) and incidence (p<0.002). There was no evidence of external skin lesionsin any of the vaccinated guinea pigs. -14- B45042 AP.00515 c) Effect of vaccination on recurrent HSV2 disease (Table 4)

As compared to the controls, the rgD2t A1(OH)3 MPL vaccine was able toalter the development of recurrent herpetic disease, as measured by significantreduction in the severity of recurrent episodes (p<0.00005), and in the incidence of 5 recurrent episodes (p<0.01). d) Conclusions rgD2t A1(OH)3 combined with small size MPL particles is very potent inproviding protection against primary and recurrent HSV2 infection in guinea pigs.

From the experiments described above one could conclude that small size10 MPL A1(OH)3 formulations obtained through two different strategies induce at least as potent prophylactic response as does large size MPL A1(OH)3 formulation. Inaddition small size MPL has the advantage of being sterilised easily before use.

5.5.2. Therapeutic experiments 15 The aim of these experiments was to compare the therapeutical potential of different rgD2t A1(OH)3 MPL formulations on the course of the recurrent herpeticdisease in guinea pigs with established HSV2 infection.

Guinea pigs were inoculated intravaginally at day 0 with 10^ pfu HSV2 strainMS. They were monitored daily for clinical signs of acute infection (days 4 to 12) as 20 well as for evidence of recurrent herpetic disease (days 13 to 20). Animals wererandomized into different experimental groups according to their primary andrecurrent scores, providing an equivalent distribution of animals with mild to severeinfection in each group. Guinea pigs without evidence of clinical signs of infectionwere not enrolled in the protocol. Vaccines were administrated subcutaneously at 25 days 21 and 42 after challenge.

The therapeutic efficacy of rgD2t Al(0H)3 MPL formulations was evaluatedin three different experiments.

Experiment 1: Efficacy of rgD2t A1(OH)3 combined with large size MPL 30 particles (MPL in TEA)

Guinea pigs experiencing recurrent disease were randomized to receive either20pg rgD2t A1(OH)3 combined with large size MPL particles (MPL TEA) oradjuvant alone. Vaccines were administered at days 21 and 42 post challenge. Thepattern of recurrent disease was observed until day 84. 35 As shown in Table 3, the rgD2t Al(0H)3 MPL TEA formulation was not effective in reducing the ongoing recurrent disease.

Experiment 2: Efficacy of rgDjt AI(OH)3 MPL lOOnm

Two groups of guinea pigs were either vaccinated with 20gg rgD2t Al(0H)3combined with small size MPL particles (MPL lOOnm) or untreated. -15- B45042 t;

Vaccinations were given at days 20 and 41 post challenge. The recurrentdisease was followed until day 69.

As shown in Table 5 in contrast with data in Experiment 1 where large sizeMPL was used, vaccination with the rgEty A1(OH)3 MPL lOOnm vaccine altered the 5 recurrences of established HSV2 disease, as compared to the control group, by significantly decreasing the recurrence severity (-39%, p<0.05) and the recurrenceday number (-28%, p<0.1).

Experiment 3: Comparative efficacy of A1(OH)3 combined with small size MPL10 particles

In this experiment, a third strategy for obtaining small size MPL was used:addition of tween, eg Tween 80.

The experimental groups were as follows:

Group 1 (n=15): 20 gg rgD2t/Al(OH)3 MPL lOOnm tween 15 Group 2 (n=l5): 20 gg rgD2t/Al(OH)3 MPL lOOnm sorbitol

Group 3 (n=16): controls

Controls were either untreated or vaccinated with A1(OH)3 MPL alone.

Vaccines were administered at days 21 and 42 post challenge. The pattern ofrecurrent disease was observed until day 60 post challenge. 20 The results are shown in Table 5. A clear significant therapeutical effect was observed in animals vaccinated with the two rgD2t/Al(OH)3 MPL formulations.

Both formulations significantly reduced the recurrence severity, the recurrence daynumber and the number of recurrent episodes. 25 Conclusions A very potent therapeutic effect against established recurrent HSV2 genitaldisease was observed with rgD2t A1(OH)3 MPL formulations containing small sizeMPL particles (ca. lOOnm). In contrast, this therapeutic effect was not observedwhen large MPL size particles (MPL in TEA) were added to the rgD2t A1(OH)3 30 vaccine.

In conclusion, results obtained in guinea pigs clearly demonstrate theprophylactic efficacy of rgD2t Al(0H)3 MPL formulations prepared with small sizeMPL particles. These formulations have an improved therapeutic potential ascompared to rgD2t A1(OH)3 combined with large size MPL particles. 35 5.5.3 Immunogenicity studies of rgD2t AI(OH)3 combined with small size MPLparticles in primates

The immunogenicity of rglty A1(OH)3 combined with small size MPLparticles (MPL lOOnm sorbitol) was evaluated in primates (African Green Monkeys). -16- B45042 AP .00515

Doses of 50, 20 or 5gg of MPL lOOnm were combined with 20gg of rgD2t andA1(OH)3 (0.5mg). Two vaccinations were given at 0 and 1 months. Specifichumoral (ELISA and neutralizing titers) and effector cell mediated (DTH) immuneresponses were measured. 5 a) Experimental procedure

Three groups of 5 African Green Monkeys were vaccinated at days 0 and 28 with 20pg of gD2t Alum formulation containing 50,20 or 5gg of MPL.Immunisations were given intramuscularly in a 1ml dose. Animals were bled everytwo weeks for antibody determination by ELISA (anti-gD2 titers) and neutralization 10 assays. The three vaccine formulations were also compared for their ability to inducea T cell mediated immunity in vivo, as measured by the induction of a specificdelayed-type hypersensitivity (DTH) response. Three monkeys in each group wereskin tested 14 days after the second vaccination with 15 or 5gg of gD2t, given in -s saline on the belly. They were also skin tested with saline alone as control. Erythema 15 and induration at the site of the intradermal inoculation were monitored 24 hours and48 hours later. b) Results

Serological and DTH responses are shown in Table 6. Groups of monkeysvaccinated with the gD2t A1(OH)3 formulation containing either 50 or 20gg of MPL 20 produced significantly more neutralizing antibodies than the group receiving the 5ggMPL dose (p<0.003 and p<0.008, respectively). There was no significant differencein the ELISA or neutralizing titers measured in the 50 or 20gg MPL groups. Acorrelation between the MPL dose and the effect on the effector cell mediatedimmune response was observed. A strong DTH response was detected in the majority 25 of the monkeys (3/4) vaccinated with the 50 or 20gg MPL formulation. In contrast, only one monkey from the 5pg MPL group developed a skin test response. o c) Conclusions

The data described above demonstrate that the adjuvant effect of A1(OH)3 30 combined with small size MPL particles is also effective in primates and is notrestricted to small animal species. A correlation between the MPL dose and theimmunogenicity of the rgD2t Alum MPL formulation could be observed in monkeys,with 20 and 50pg giving the best serological and DTH responses. - 17- B45042

Example 6: CLINICAL STUDIES with Lyme and Hepatitis B vaccines andsmall MPL 6.1. I^me disease vaccine comprising a fusion protein of NS1(1-81) frominfluenza virus and OspA derived from B.burgdorferi ZS7. 5 Formulations preparations 6.1.1. NSl-OspA/alum NS 1-OspA prepared according to the procedures described in WO 93/04175was adsorbed on alum and incubated 1 hr at room temperature. Final volume was 10 adjusted with phosphate buffer (PO4 10 mM, NaCl 150 mM). Formulation wasstored at 4°C until use. A dose contains 10pg NSl-OspA/500gg alum

6.1.2. NSl-OspA/alum/MPL 15 NS 1-OspA was adsorbed on alum and incubated 1 hr at room temperature. MPL prepared as described previously was then added to the formulation andincubated again 1 hr at room temperature. The formulation was then adjusted to thefinal volume with phosphate buffer (lOmM PO4,150 mM NaCl). Formulation wasstored at 4°C until use.

20 A dose contains 10pg OspA/500gg Al(OH)3/50pg MPL 6.3. Immunization schedule

Human volunteers were injected three times intramuscularly with 1ml of agiven formulation at days 0, 31 and 62. Sera were taken 30 days post I, Π and ΙΠ. 25 They were then analysed by ELISA for total IgG anti OspA and for LA-2 like antibody response in an inhibition test (LA-2 Mab was shown to be protectiveantibody against infection in mice). 6.4. HBsAg/MPL formulations in humans 30 6.4.1. Preparation of Formulations HBsAg 20pg/Alum 500pg HBsAg was adsorbed on the total final amount of alum and final volume wasadjusted with phosphate buffer saline (PO4 10 mM, NaCl 150 mM) at 1ml per dose.Formulation wa stored at 4°C until use. 35 6.4.2. HBsAg 20pg/Alum 100pg HBsAg was formulated as described previously but adsorbed only on lOOpgof A1(OH)3. The final volume was maintained constant (lml) -18- B45042 AP .00515 10 15 20 25 30 35 6.4.3. HBsAg 20gg/Alum lOOgg/MPL 50gg HBsAg was adsorbed on lOOgg alum and incubated lh at room temperature.MPL was then added at the necessary concentration and incubated lh at roomtemperature. The formulation was then adjusted to the final volume (1ml per dose)with appropriate buffer (as above) and stored at 4°C until use. 6.5. Immunization schedule

Human volunteers (20 per group) were injected IM- with 1ml of one of thegiven formulations. Sera were collected on months 0,1,3 and 6. They wereanalysed for neutralizing antibodies with the commercially available Abbot test.

6.6. RESULTS

Table 9 shows that MPL used in combination with alum and NS 1-OspA in aform of particles of lOOnm is efficient at producing higher antibody titers ofinhibitory nature than the antigen on alum and that the kinetics of seroconversion arefaster.

This established that for a soluble antigen, like NS 1-OspA, in humans, MPLformulated as small particle keeps the adjuvant properties that it already exhibited inanimals with other soluble antigens.

Table 7 shows that the adjuvant effect lost by reducing the amount of alum present in the formulation can be recovered by adding MPL in the form described inalso i/njyewes this patent the mpl^ the seroconversion rate.

Example 7: Combination vaccine formulation·Hepatitis B +Hepatitis A HBsAg is adsorbed on 90% of the final amount of aluminium hydroxide(0.5mg/ml) and incubated overnight at room temperature. The pH is adjusted to 6.2and the preparation is left 14 days at room temperature for maturation.

Hepatitis A antigen at 360 to 22EU per dose, in the form of an inactivatedderivative of the HM-175 strain (as in Havrix) is preadsorbed on 10% of thealuminium hydroxide final concentration (0.5mg/ml). The remaining aluminiumhydroxide is then added to the solution and left for one hour at room temperatureunder agitation.

The HAV adsorbed on aluminium hydroxide is then added to the HBsAgformulation. MPL (particle size less than 100 nm) is added to the HAV/HBsAg solution ata final concentration of 12.5 to lOOug per 1 ml dose, the volume is adjusted to thefinal dose volume, and the formulation is stored at 4°C until used. -19-

I f B45042

Example 8: Combination vaccines containing additional antigens

Combination vaccines may be prepared by adding one or more of the desired antigens to the formulations described in Example 2 or Example 3 or Example 4above. 5

Example 9: Increase Of Humoral Immunity And Induction Of Cell MediatedImmunity By Immunization Of Mice With HBsAg formulated With AluminiumHydroxide And MPL 10 9.1. Effect Of AI(OH)3 + Mpl On Induction Of Anti-HBs Antibodies

Balb/c mice were immunized by the subcutaneous route or by the intradermal

route with recombinant HBsAg adsorbed on Al(0H)3 with MPL as adjuvant. Micewere immunized twice with HBsAg/Al/MPL formulations and the antibody responsewas measured after the first and the second doses. Total Ig were measured by ELISA 15 or AUS AB kit (Abbott Lab, Π1.) and a particular attention was given to the induction of antibodies of the IgG2a isotype since this isotype is mainly induced by secretion ofg-Interferon. The induction of this isotype thus indirectly reflects the activation ofcell mediated immunity, namely the activation of Thl.

The ratio HBsAg/MPL has been investigated as well as the size of MPL 20 particles. 9.1.1. EXPERIMENT I - Effect of MPL (> 500 nm) dose on immunogenicity ofrec.HBsAg adsorbed on A1(OH)3

Groups of 10 female Balb/c mice were injected by the subcutaneous route 25 with 2.5 meg of recHBsAg adsorbed on 50 meg of A1+++ (as A1(OH)3) and increasing amounts of MPL (3.1 to 50 meg) with a particle size of > 500 nm. Themice were injected twice in a volume of 100 mcl and at 2 weeks interval. They werebled 2 weeks after the first injection (partial bleeding) and one week after the booster.Total anti-HBs IgG and specific IgG2a were measured by ELISA using recHBsAg as 30 capture antigen. The titers were expressed as the reciprocal of the dilution corresponding to 50% of the maximal value (mid-point dilution). The results indicatean increase of both specific IgG and IgG2a with increasing doses of MPL,particularly for doses of 12.5 to 50 meg. The effect is seen for both primary andsecondary responses and is particularly obvious for IgG2a (up to 20 fold increase) 35 indirectly indicating a secretion of g-interferon induced by the immunization withMPL. -20- B45042 AP- Ο Ο 5 1 5 9.1.2. EXPERIMENT Π - Comparison of clinical lots of adsorbed recHBsAgcontaining or not containing MPL (> 500 nm) 3 clinical lots of recHBsAg adsorbed on A1(OH)3 were prepared: lotDSAH16 contained no MPL and served as control. Lots DSAR501 and 502 were 5 prepared in a similar way (20 meg of recHBsAg adsorbed on 0.5 mg A1+++ asA1(OH)3) but contained 50 meg of MPL (> 500 nm).

The 3 lots were injected subcutaneously to groups of 10 mice (200 mclcontaining 2.5 meg HBsAg, 100 meg A1+++ and 6.25 meg MPL), twice at 2 weeksinterval. The mice were bled at day 14 and 1 week after the booster. Anti-HBs 10 antibodies were measured using AUSAB kit or an in-house ELISA for either IgG orIgG2a. The results are given in table 2. They indicate that, 2 weeks after the firstinjection, the 2 lots containing MPL induce a very significant anti-HBs response (12.4and 41.9 mlU/ml) while the lot which does not contain MPL only induces a marginalresponse (0.75 mlU/ml). The number of responders is also higher with MPL (9/10 15 and 9/10 versus 1/10 in absence of MPL). The effect of MPL is confirmed after thebooster since the titers obtained for lots DSAR501 and 502 are about 6 fold higherthan that observed without MPL.

This indicates that, at least in mice, MPL (> 500 nm) can improve both thekinetics of the anti-HBs response and the level of the anti-HBs response. 20 These results were confirmed when specific IgG and IgG2a are measured after immunization with lots DSAH16 (without MPL) and DSAR502 (with MPL): the anti-HBs IgG titer is 5 (primary response) and 3 (secondary response) times higher whenMPL is present.

For the IgG2a response, the effect of MPL is even more striking, at least after 25 the second dose, indicating a preferential induction of IgG2a. This indirectly reflectsactivation of cell-mediated immunity (secretion of gamma-interferon) by thepreparation containing MPL. 9.1.3. Experiment ΠΙ: Effect of MPL (< 100 nm) dose on immunogenicity of 30 recombinant HBsAg adsorbed on Al(0H)3

Groups of 10 mice (Balb/c, female, 7 weeks old) were injected subcutaneously with 1 meg of recombinant HBsAg adsorbed on 50 meg of A1+++ (as A1(OH)3) andin presence of increasing amounts (3.1 to 25 meg) of MPL(< 100 nm). The micewere injected twice at 2 weeks interval with a volume of 200 mcl. They were bled 2 35 weeks after the first injection and 1 week after the booster. The anti-HBs response was evaluated by ELISA (total Ig, IgG, IgG2a) on pooled sera. The titers wereexpressed as mid-point dilutions (reciprocal of the dilution giving 50 % of the highestvalues). The results indicate that as few as 3.1 meg of MPL induce a strong increaseof the antibody response both for primary and secondary responses. The response -21- B45042 culminates for 6.25 meg and decreases afterwards to become similar to that foundwithout MPL when high doses of MPL (25 meg) are used. The pattern of theantibody response is similar for IgG, IgG2a and total Ig. It contrasts with resultsobtained for MPL of higher size (> 500 nm) and shows that small size (<100 nm) 5 particles of MPL are more effective than larger size (> 500 nm) particles (at least forhumoral immunity), since less MPL is needed to obtain the maximal effect. Thehighest activity of small size MPL was confirmed in several experiments.

As shown for larger size MPL (> 500 nm), the adjuvant effect of MPL ishigher for IgG2a than for total IgG or Ig. At the maximal effect of the secondary 10 response (6.25 meg of MPL), there is a 25 fold increase for IgG2a while theincrease for IgG or total Ig was 7.6 and 4.3 respectively.

9.2. Induction of Cell-Mediated Immunity by recHBsAg adsorbed onAI(0H)3 - effect of MPL 15 If humoral immunity is sufficient to protect against Hepatitis B, the induction of cell-mediated immunity (CTL, Thl) could be of particular importance for thetreatment of the disease.

New formulations are required however for therapeutic vaccines sinceA1(OH)3 is capable of improving humoral immunity but not cell mediated immunity. 20 We have investigated the effect of MPL on the induction of Th 1 cells capable of secreting IL-2 and g-(i.e. gamma) interferon in Balb/c mice immunized withrecHBsAg adsorbed on Al(0H)3. 9.2.1. EXPERIMENT I - Effect of MPL (> 500 nm) on induction of Thl cells 25 after immunization of Balb/c mice with A1(OH)3 adsorbed HBsAg A group of 10 Balb/c mice (female, 5 weeks old) were immunized by injection in each footpad of 30 mcl containing 10 meg of HBsAg, 15 meg of A1+++(as A1(OH)3) and 15 meg of MPL. Control mice were injected similarly with thesame amount of recHBsAg either mixed with FCA (positive control) or adsorbed on 30 Al(0H)3 without MPL (negative control).

Six days after the immunization, the mice were killed and the popliteal lymph nodes were removed. The lymph node cells (LNC 2.105/ml) were cultivated fordifferent periods of time (24 hrs to 74 hrs) in RPMI medium supplemented with 1 %of negative mouse serum and containing 5 mcg/ml of recHBsAg. After termination 35 of the culture, the amount of IL-2, INF-g and IL-4 secreted in the medium was measured. IL-2 was estimated by its ability to stimulate the proliferation (evaluatedby incorporation of 3H-Thymidine) of an IL-2-dependent CTL line (VDA2 cells) andthe titer was expressed as stimulation index (SI = amount of 3H-Thymidineincorporated in stimulated cells/amount of 3H-Thymidine incorporated in non -22- B45042 AP. Ο Ο 5 1 5 € stimulated cells). The amount of IL-4 and INF-g was measured using commercialELISA kits (Holland Biotechnology for EFN-g and Endogen for IL-4). The titerswere expressed in pg of IFN-g/ml.

The results indicate that no significant amount of IL-2, DL-4 or INF-g is5 secreted by LNC from mice immunized with HBsAg adsorbed on Al(0H)3. On the contrary, high levels of IL-2 (I.S. = 38 at 48 hrs) and a significant amount of INF-gare secreted by LNC from mice immunized with HBsAg adsorbed on Al(0H)3 +MPL. This secretion is similar (INF-g) or higher (IL-2) to that observed for miceimmunized with HBsAg + FCA and the in vitro secretion occurs earlier. 10 No IL-4 was detected after immunization with HBsAg adsorbed on A1(OH)3 even in presence of MPL.

This secretion profile indicates that specific Thl cells (IL-2, INF-g) have beeninduced by immunization with adsorbed HBsAg in presence of MPL but not in o absence of MPL. However, no Th2 (IL-4) were detected in these conditions of 15 immunization. 9.2.2. EXPERIMENT Π * Effect of the dose of MPL (< 100 nm) on theinduction of Thl cells after immunization of Balb/c mice with A1(OH)3 adsorbedrecHBsAg 20 Groups of 5 Balb/c mice were immunized in each of the 2 footpads with30 mcl containing 10 meg of recHBsAg adsorbed on 15 meg of A1+++ (as A1(OH)3) andwith increasing amounts of MPL (100 nm, 0 to 15 meg).

Six days after the injection, the mice were killed and the popliteal lymph nodecells (LNC) were cultivated at 2.106 cells/ml in RPMI supplemented with 1 % 25 negative mouse serum for different periods of time (24 hrs to 96/25) in presence of 5mcg/ml of recHBsAg.

The secretion of IL-2 was measured by stimulation of the proliferation ofVDA2 cells and concentration of IL-2 is expressed as Stimulation Index (SI); thesecretion of INF-g was measured using a commercial kit and expressed in pg/ml. 30 It was found that the secretion of IL-2 is dramatically increased by the lower dose of MPL (7.5 meg) and a maximal effect is obtained for 15 meg of MPL.

The secretion of IL-2 is generally more important at 24 hrs than at 48 or 72hrs.

The secretion of INF-g is absent when HBsAg is adsorbed on A1(OH)3 in35 absence of MPL. A small dose (7.5 meg) of MPL induces a secretion of INF-g and again, the maximal effect is obtained for 15 meg of MPL. Contrary to what isobserved for IL-2, the secretion of INF-g is delayed in the culture and increases withtime up to 96 hours. -23- B45042

Taken together these data indicate that MPL (less than 100 nm) is a potentinducer of Thl when combined with HBsAg adsorbed on A1(OH)3.

The effect of a formulation containing HBsAg adsorbed on A1(OH)3 and MPL on theinduction of both humoral and cell-mediate immunity in Balb/c mice has 5 been investigated. The results indicate that MPL clearly improves the kinetics of theanti-HBs response since much more anti-HBs antibodies are found after both theprimary and secondary immunizations. The quality of the anti-HBs is also modifiedand a preferential induction of IgG2a has been observed, reflecting indirectlysecretion of INF-g and thus induction of a cell-mediated immunity. 10 Direct evaluation of the induction of Thl cells by formulations containing HBsAg, Al(0H)3 and MPL clearly indicates that MPL is a potent inducer of Thlcells secreting both IL-2 and INF-g. This kind of formulation is thus important in thedevelopment of therapeutic vaccines.

Best results were obtained using MPL of less than lOOnm particle size. 15 For Tables showing the results of experiments described above, see Tables 9- 14 below. 13. Conclusions

It is now possible to produce sterile vaccine formulations comprising small

20 MPL

The data presented herein show that the use of small MPL (around lOOnm)gives an adjuvant effect at least as good as the previous form (big MPL) and in somecases better (therapeutic gD and therapeutic Hepatitis B).

Furthermore, in both clinical studies, addition of small size MPL to25 aluminium hydroxide resulted in a significant increase in antibody titers, as compared to aluminium hydroxide only formulations. ¢2) Overall the data suggests that small MPL is an improved immunostimulant in primates including man, over large size MPL A1(OH)3 formulations in which nosignificant increase in antibody titers was observed over the A1(OH)3 formulations 30 for herpes gD2t trial, and hepatitis B, (Engerix B) MPL trial. This combined with theability to make large scale sterile lots renders small MPL as a suitableimmunostimulant for a range of human or animal health vaccines. -24- B45042 AP.00515

Table 1: MPL particle and filtration recovery using different sonication parameters

Trial n° Concentration (mg/ml) Total residence time in the flow cell (min) Particle size before filtration (nm) Recovery afterfiltration (%) 16 1 2,5 92 104 17 1 3 79 78,5 18 1 3,5 95 86,4 19 2 2,8 77 N.A. 20 1 2,8 98 N.A.

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B45042 TABLE 6

IMMUNOGENICITY OF gD2t ALUM MPL lOOnm (sorbitol)IN PRIMATES

SEROLOGICAL AND DTH RESULTS

VACCINE MONKEY No. ANTIBODY RESPONSE* ** DTH RESPONSE ♦♦ (Induration) ELISA TITER NEUTRA TITER PBS gD2 5gg gD2 I5gg KQ 100 18630 800 - + ++ 20gg gD2t KQ 101 5554 1600 - - - Alum KQ 102 14870 800 - ++ +++ 50gg MPL KQ 103 5486 1600 - ++ +++ KQ104 GMT 16270 10655 1600 1213 ND ND ND KQ 105 16170 800 - + ++ 20gg gD2 KQ 106 4389 800 - - - Alum KQ 107 20440 1600 - ++ +++ 20gg MPL KQ 108 5613 800 - + + KQ 109 GMT 6765 8876 1600 1056 ND ND ND KQ 110 2486 200 - - - 20gg gD2t KQ 111 9918 800 - ++ +++ Alum KQ 112 2526 400 - - - 5pgMPL KQ113 7137 400 - - - • KQ114 GMT 8396 5181 400 400 ND ND ND * Measured 14 days post II/GMT = geometric mean titer ELISA titer= midpoint titer NEUTRA titer = reciprocal of the highest serum dilution giving 100% protectionagainst the cytopathogen effect **skin test on day 14 post IIInduration 24 hrs reading + = 1mm ++ = l-5mm+++=>5mm -30-

B45O42 t"

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B45042

Table 8

Immunogenicity of clinical batches of OspA In HumansAnti-OspA in the LA-2 inhibition assay(ng equiv LA-2/ml) (GMT)

Vaccine Pre Do Post I 30D28 Post Π 30 D56 Post III 30 D84 NS 1-OspAon Alum 118 233 409 768 SC (%) 2.6 77.2 86.5 100 NSl-OspA+MPLon Alum 134 269 865 2424 SC (%) 2.6 88.6 97.2 100 C’ N=8010gg/doseIm route -32- AP .0 0 5 1 5 δ «η

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Claims (32)

B45042/A i* ,-r i -i »15» id U. ·*» drc iM/c :;·· .1.ih*t * (i ·’ I LL.ul· i· Claims
1. A vaccine composition comprising an antigen in conjunction with 3-O-deacylated5 monophoshoryl lipid A (MPL) and a suitable carrier wherein the particle size of the MPL does not exceed 120nm.
2. A vaccine composition as claimed in Claim 1 in which the particle size of theMPL is in the range 60-120nm.
3. A vaccine composition as claimed in Claim 1 or Claim 2 in which the particle sizeof the MPL is less than lOOnm.
4. A vaccine composition as claimed in any one of Claims 1 to 3 in which the carrierIS is alum.
5. A vaccine composition as claimed in any one of Claims 1 to 3 in which the carrieris an oil in water emulsion or other lipid based vehicle. 20
6. A vaccine composition as claimed in any one of claims 1 to S in which the antigen is a viral antigen.
7. A vaccine composition as claimed in any preceding claim wherein the antigen isan antigen against Hepatitis A. o 25
8. A vaccine composition as claimed in Claim 7 wherein the Hepatitis A antigen is an Q inactivated whole cell composition derived from the HM-175 strain.
9. A vaccine formulation as claimed in any one of Claims 1 to 6 wherein the antigen30 is an antigen against hepatitis B.
10. A vaccine composition as claimed in Claim 9 wherein the antigen comprisesHepatitis B surface antigen (HBsAg) or a variant thereof. 35
11. A vaccine composition as claimed in Claim 10 wherein the HBsAg comprises the S antigen of HBsAg (226 amino acids).
12. A vaccine composition as claimed in Claim 11 wherein the HBsAg additionallycomprises a pre-S sequence. - 1- B45042/A AP. Ο Ο 5 1 5
13. A vaccine composition as claimed in Claim 11 or Claim 12 wherein the HBsAgis the composite particle of the formula (L*,S) wherein L* denotes a modified Lprotein of hepatitis B virus having an amino acid sequence comprising residues 12-52 5 followed by residues 133-145 followed by residues 175-400 of the L protein and Sdenotes the S-protein of HBsAg.
14. A vaccine composition according to any one of Claims 9 to 13 additionallycomprising a Hepatitis A antigen. 10
15. A vaccine composition as claimed in any preceding claim comprising one ormore hepatitis antigens and at least one other component selected from a non-hepatitisantigen which affords protection against one or more of the following: diphtheria,tetanus, pertussis, Haemophilus influenzae b (Hib), and polio. 15
16. A vaccine composition according to Claim 15 selected from a DTP (diphtheria-tetanus-pertussis) HBsAg combination, an Hib-HBsAg combination, a DTP-Hib-HBsAg combination and an IPV (inactivated polio vaccine) -DTP-Hib-HBsAgcombination. 20
17. A vaccine composition according to claim 16 additionally comprising a hepatitisA antigen.
18. A vaccine composition as claimed in any one of claims 1 to 6 comprising an25 HSV glycoprotein D or an immunological fragment thereof.
19. A vaccine composition as claimed in Claim 18 wherein the glycoprotein D is atruncated protein. 30
20. A vaccine composition as claimed in Claim 19 wherein the truncated protein is HSVgD2 and is devoid of the C terminal anchor region.
21. A vaccine composition as claimed in any one of claims 1 to 6 comprising HTVgpl60 or a derivative thereof. 35
22. A vaccine composition as claimed in Claim 21 wherein the derivative of gp 160is gp 120. -2- B45042/A
23. A vaccine composition as claimed in any preceding claim wherein the 3-0-deacylated monophosphoryl lipid A is present in the range 10pg-100pg per dose.
24. A vaccine composition as claimed herein, additionally comprising either Tween 5 80 or Sorbitol.
25. A vaccine composition as claimed herein for use in medicine.
26. 3-0-deacylated monophosphoryl lipid A wherein the particle size is less than 10 120nm.
27. A clear sterile solution of 3-O-deacylated monophosphoryl lipid A.
28. A method for preparing a clear sterile solution of 3-O-deacylated 15 monophosphoryl lipid A comprising suspending 3-O-deacylated monophosphoryllipid A in water and subjecting the resulting suspension to sonication.
29. A method for preparing a vaccine as claimed herein comprising admixing theproduct of claim 27 with an antigen. 20
30. Use of an antigen in conjunction with 3-O-deacylated monophosphoryl lipid Ahaving a particle size of no greater than 120nm in the manufacture of a medicamentfor the prophylaxis or treatment of infections. 25
31. A method of treating a human subject suffering from or susceptible to infection comprising administering an effective amount of a vaccine according to any of ofclaims 1 to 23.
32. 3-O-deacylated monophosphoryl lipid A having a particle size of less than 30 120nm for use in medicine. -3-
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