Composition of vaccines containing 3-0-deacylated monophosphoryl lipoid a

The subject of the invention is a new suspension of particles of 3-0-deacylated monophosphoryl lipid A and its preparation and a vaccine composition comprising an antigen in combination with 3-0-deacylated monophosphoryl lipid A and a method for its preparation.

The 3-0-deacylated monophosphoryl lipid A (or 3-de-O-acylated monophosphoryl lipid A) was formerly called 3D-MPL or d3-MPL to indicate that the 3-position of the reducing end of glucosamine is de-O-acylated. The preparation is given in British Patent No. 2220211A. Chemically, it is a mixture of 3-deacylated monophosphoryl lipid A with 4.5 or 6 acylated chains. In the description, the term 3D-MPL (or d3-MPL) is shortened to MPL because "MPL" is the registered name Ribi Immunochem. Montana, used by Ribi to uniquely name its 3-0-deacylated monophosphoryl lipid A.

British Patent No. 2220211A mentions that the endotoxicity of previously used enterobacterial lipopolysaccharides (LPS) is reduced while maintaining immunogenic properties. However, the description gives this information simply in connection with bacterial (gram-negative) systems. MPL particle sizes are not mentioned. In fact, the particle sizes of 3-0-deacylated monophosphoryl lipid A exceed 500 nm.

Publication WO / 92/16231 describes a vaccine containing the herpes virus gD glycoprotein or immunological fragments thereof in combination with 3-0-deacylated monophosphoryl lipid A. Also not mentioned is the particle size of 3-0-deacylated monophosphoryl lipid A.

Publication WO / 92/06113 describes a vaccine containing HIV glycoprotein 160 or immunological fragments thereof in combination with 3-0-deacylated monophosphoryl lipid A. No particle size MPL is mentioned.

The present invention relates to a suspension of 3-0-deacylated monophosphoryl lipid particles

A (MPL), characterized in that it is visually clear and sterilizable by blind filtration of a 0.22 pm PVDF on a hydrophilic membrane, preferably the suspension particles have a size generally smaller than 120 nm.

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The invention also relates to a process for preparing a suspension of 3-0-deacylated monophosphoryl lipid A particles, characterized in that it consists of suspending 3-0-deacylated monophosphoryl lipid A in water and subjecting the resulting suspension to ultra-sounds to produce a suspension of visually clear and sterilizable particles by blind filtration of a 0.22 pm PVDF nahydrophilic membrane, preferably with a suspension particle size generally less than 120 nm.

The invention also provides a vaccine composition comprising an antigen in combination with a suspension of 3-0-deacylated monophosphoryl lipid A (MPL) and a suitable carrier, characterized in that it contains MPL in the form of a suspension of particles visually clear and sterilizable by blind filtration on a hydrophilic PVDF membrane 0.22 pm, preferably with a suspension particle size generally less than 120 nm, at 10-W0 pg per dose and antigen.

Preferably the composition comprises MPL with a particle size of 60-120 nm, and particularly preferably with a particle size less than 100 nm.

In another preferred embodiment, the vaccine composition comprises aluminum hydroxide as the carrier.

Another preferred carrier is an oil-in-water emulsion or other liquid lipid carrier.

Preferably, the vaccine of the invention antigen comprises a viral antigen. Particularly preferred antigens are: Hepatitis A antigen, especially a deactivated composition of whole cells of the HM-175 strain; Hepatitis B antigen, especially the Hepatitis B surface antigen (HBsAg) or its variant, the HBsAg antigen being the S antigen HBsAg (226 amino acids), the HBsAg antigen additionally containing the pre-S sequence, the HBsAg antigen containing the complex particle of formula (L *, S), wherein L * is a modified Hepatitis B virus L protein having an amino acid sequence of residues 12-52, followed by 133-145 and 175-400 L protein, and S is HBsAg S protein; Hepatitis A antigen; one or more Hepatitis antigens and at least one component selected from compounds different from the Hepatitis antigen, which protects against one or more diseases selected from diphtheria, tetanus, whooping cough, Haemofilis influenzae b (Hib) and polio.

Particularly preferred are compositions comprising the DTP (diphtheria-tetanus-whooping cough) -HBsAg combination, the Hib-HBsAg combination, the DTP-Hib-HBsAg combination and the IPV (inactive polio vaccine) -DTP-Hib-HBsAg combination, and optionally additionally the Hepatitis A antigen.

In another embodiment, the vaccine composition comprises HSV glycoprotein D or an immunological fragment thereof, in particular a truncated sequence D glycoprotein, preferably HSVgD₂ without anchor end C.

The vaccine composition preferably contains HIV gp 160 or an immunological fragment thereof, and in particular contains a gp 120 derivative as a gp 160 derivative.

The invention also relates to a method for producing a vaccine composition comprising an antigen in combination with an MPL suspension comprising mixing the MPL suspension with the carrier and the antigen in a pharmaceutically acceptable excipient.

A preferred subject of the invention is a vaccine composition comprising an antigen in combination with 3-0-deacylated monophosphoryl lipid A (abbreviated to MPL) and a suitable carrier in which the MPL particle size is "small" not exceeding 120 nm.

Such preparations are suitable for many monovalent or multivalent vaccines.

It has surprisingly been found that the vaccine compositions of the invention have the particularly advantageous properties described below. In particular, such compositions are highly immunogenic. In addition, the sterility of the adjuvant composition can be ensured because the product is suitable for sterilizing filtration. Another benefit of "small" MPL particles arises with aluminum hydroxide compositions because MPL interacts with aluminum hydroxide and the antigen to form an isolated unit.

In the compositions of the invention, the antigen is a viral antigen, e.g., antigen to jaundice infection (Hepatitis A, B, C, D or E) or herpes (HSV-1 or HSV-2)

178 578 as described below. Description of modern anti-jaundice vaccines with a number of references^- can be found in the Lancet of May 12, 1990 on page 1142 ff (prof.

ALWF Eddleston). See also "Viral Hepatitis and Liver Disease" (ed. Vyas, BN, Dienstag, JL iHoofnagle, JH, Grune and Stratton, Inc. (1984)) and "Viral Hepatitis and Liver Disease" (Proceedings of the 1990 International Symposium, ed. FB Hollinger, SM Lemon and H. Margolis, publ. Williams and Wilkins). References for HSV-1 and HSV-2 can be found in WO92 / 16321.

Hepatitis A virus infection (HAV) is a widespread problem, but vaccines suitable for mass vaccination are available, e.g. Havrix (SmithKline Beecham Biologicals) which is a still-vaccinated vaccine from the HM-175 HAV strain [see 'Inactivated Candidate Vaccines for Hepatitis A', FE Andre , A. Hepburn and E. D'Hondt, Prog Med. Virol., Vol. 37, pp. 72-75 (1990) and the monograph of the product "Havrix" published by SmithKline Beecham Biologicals (1991)].

Flehming et al. (loc. cit., pp. 56-71) reviewed the clinical aspects, virology, immunology and epidemiology of Hepatitis A and discussed the approach to developing vaccines against this common viral infection.

As used herein, the expression "HAV antigen" refers to an antigen capable of neutralizing the HAV antibody in humans. The HAV antigen may contain live attenuated virus particles or inactivated attenuated virus particles, or may be a HAV capsid or viral protein, convenient for production by genetic engineering.

Hepatitis B virus (HBV) infection is a widespread problem, but vaccines suitable for mass vaccination are available, e.g. Engerix-B (SmithKline Beecham plc) obtained by genetic engineering.

The production of the Hepatitis B surface antigen (HBsAg) is well documented. See, e.g., Hartford et al., Develop. Biol. Standard 54, p. 125 (1983), Gregg et al., Biotechnology, 5, p. 479 (1987), European patents EP-A-0226846, EP-A-0299108 and references therein.

As used herein, the term "Hepatitis B surface antigen" or "HBsAg" includes any HBsAg antigen or fragment thereof exhibiting the antigenicity of the hBv surface antigen. It should be understood that in addition to the 226 amino acids of the HBsAg S antigen sequence (see Tiollais et al., Nature, 317,489 (1985) and references therein), HBsAg described herein may, if desired, contain all or part of the pre-S sequence described in the above references and in European EP-A-0278940. In particular, HBsAg may be a polypeptide containing an amino acid sequence containing residues 12-52, followed by 133-145 and 175-400 of the HBsAg L protein relative to the open reading frame of the B hepatitis ad serotype virus (this polypeptide is referred to as L *, see European Patent EP 0414374). HBsAg within the scope of the invention may also comprise the preS1-preS2-s polypeptide described in EP 0198474 (Endotronics) or analogs thereof as described in EP 0304578 (McCormick and Jones). HBsAg described herein may also refer to mutants, e.g., the "escape mutant" described in WO 91/14703 or European Patent Application No. 0511855A1, and particularly to HBsAg in which glycine was substituted on position 145 with arginine.

Usually HBsAg is present in the form of particles. The particles may contain, e.g., the S protein alone or in the form of composite particles, e.g. (L *, S), where L * is as defined above and S is the HBsAg S protein. Said particle is preferably in a form in which it is a product of expression in yeast.

Herpex Simplex virus D glycoprotein is housed in a virus envelope, and can also be found in the cytoplasm of infected cells (Eisenberg RJ et al., J. of Virol. 1980, 35, 428-435). It consists of 393 amino acids, including a signal peptide, and has a molecular weight of about 60 kD. Of all glycoproteins, this is probably the best studied (Cohen et al. Virology 60,157-166). It is known that in vivo it plays a major role in the binding of the virus to the cell membrane. In addition, glycoprotein D secretes neutralizing antibodies in vivo (Eing et al. J. Med. Virology 127, 59-65).

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However, the latent HSV2 virus may still come alive and induce relapse despite high levels of neutralizing antibodies in the patient's plasma. It is therefore obvious that the ability to induce only neutralizing antibodies is not sufficient to properly combat the disease.

The vaccine compositions of the invention preferably use mature recombinant truncated D glycoprotein (rgD2t) or equivalent proteins. Equivalent proteins include HSV gD glycoprotein.

In a preferred aspect, rgD₂is glycoprotein D with 308 amino acids, containing amino acids 1 to 306 from natural glycoprotein with the addition of asparagine or glutamine at the C-terminus of the truncated protein. This form of protein includes a signal peptide that is cleaved to give a mature protein with 238 amino acids. The production of such a protein in Chinese hamster ovary cells is described in the European patent specification Genetech EP-B-139417 and Science 222, p. 524, as well as Biotechnology, June 1984, p. 527. Such a vaccine, composed with the small MPL of the invention, has much greater therapeutic potential than known rgD compositions₂Although some experimental and commercially available vaccines give excellent results, it is well known that an optimal vaccine must not only stimulate a neutralizing antibody, but also should stimulate T cell-mediated cell resistance as effectively as possible.

It is particularly preferred that the vaccine compositions of the invention effectively induce protective immunity, even at very low antigen doses.

They provide excellent protection against primary and recurrent infection, and also favorably stimulate both specific extracellular (neutralizing antibodies), as well as effector cell-mediated (DTH) immune responses.

To produce 3-deacylated monophosphoryl lipid A with small particle sizes, usually not exceeding 120 nm, the procedure described in British Patent GB 2200211 can be used to obtain known 3D-MPL (or commercial MPL with larger particle sizes can be purchased from Ribi Immunochem) and then the product can be sonicated until a clear solution is obtained. Particle sizes can be estimated using dynamic light scattering as described below. In order to maintain MPL sizes in the 100 nm range after combining with aluminum hydroxide, antigen and buffer, Tween 80 or sorbitol may be added. Under these conditions, it was determined that MPL does not aggregate in the presence of phosphate buffer as is possible in its absence. By doing so, the final composition is more accurately determined. MPL still interacts with aluminum hydroxide and antigen to form an isolated unit. The clear MPL suspension is also an aspect of the invention. The suspension can be sterilized by passing it through a filter.

Preferably, the particle sizes in the suspension are 60-120 nm.

Most preferably the suspended particle sizes are below 100 nm.

mPl as defined above is present in an amount of 10-200 pg, preferably 25-50 pg per dose in which the antigen is present in an amount of 2-50 pg or more. The vaccine composition of the invention may further comprise immunostimulants, and in a preferred embodiment QS21 (sometimes called QA21). This is the HPLC fraction of the saponin extract derived from the bark of the Quillaja Saponaria Molina tree, and the method of its production is given in US Patent No. 5,057,540.

The carrier may optionally be an oil-in-water emulsion, a lipid carrier or aluminum hydroxide (aluminum hydroxide 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 aqueous carrier may e.g. be phosphate buffered saline.

Preferably, the vaccine compositions will contain an antigen or antigenic composition that can elicit an immune response to human or animal pathogens, wherein the antigen or antigenic composition is from HIV-1 (such as gp120 or gp160, see WO 92/06113 and references therein), virus herpes, such as gD or

178 578 its derivative, or Immediate Early protein such as ICP27 from HSV-1 or HSV-2, gB (or its derivatives) from human cytomegalovirus, or gpl, II or III from Varicella zoster virus, or from hepatitis virus such as hepatitis B virus or against other viral pathogens such as Respiratory Syncytial Virus, human papilloma or influenza virus, or bacterial pathogens such as Salmonella, Neisseria, Borrilia (e.g. OspA or OspB or derivatives thereof), or Chlamydia or Bordetella, e.g. P.69, PT or FHA, and parasites such as plasmodium or Toxoplasma. The vaccine compositions of the invention may contain a tumor antigen, and be suitable for cancer vaccine.

One embodiment of the invention is a composition comprising the HAV antigen (e.g., as in Havrix) in a mixture with MPL and aluminum hydroxide as described below.

Another embodiment of the invention is a composition comprising the HB surface antigen (HBsAg) (e.g., as in Engerix) in a mixture with MPL and aluminum hydroxide as described below.

Another embodiment of the invention is a composition comprising the HBsAg antigen as particles (L8, S) in a mixture with MPL and aluminum hydroxide.

Hepatitis A and Hepatitis B combined vaccines can be prepared according to the method of the invention.

Another embodiment of the invention is a vaccine composition containing mature truncated D glycoprotein (rgD2) or equivalent proteins as described above. So another one. a variant of the invention is a composition according to the invention comprising the OspA antigen or a derivative thereof from Borelia btagdorferi. For example, antigens may be used, particularly OsaA antigens from ZS7 'or B31 strains. Thus, another embodiment of the invention is a vaccine composition comprising an influenza antigen. As a result, an improved influenza vaccine is obtained, especially when using a split virus.

The composition can also be used with light herpes particles, such as those described in International Patent Application No. PCT / GB92 / 00824 and International Patent Application No. PCT / GB / 00179.

Preferably, the vaccine compositions of the invention contain other antigens for the effective treatment or prevention of one or more bacterial, viral or fungal infections.

For example, the jaundice vaccine compositions of the invention preferably containing at least one component such as non-jaundice antigen known to confer resistance to one or more of the above infections: defectite, tetanus, whooping cough, Haemofilis influenzae b (Hib) and polio.

Preferably the vaccine according to the invention comprises HBsAg as defined above. The particular vaccine combination according to the invention includes the DTP vaccine (diphtheria-tetanus-pertussis ^ hepatitis B vaccine, Hib-hepatitis B vaccine combination, the DTP-Hib-hepatitis B vaccine combination, and the IPV vaccine (inactive Aoli vaccine) -DTP-Hib-hepatitis B vaccine .

The above combinations may advantageously contain a protective component against Hepatitis A, a particularly dead attenuated strain derived from the HM-175 strain present in Havrix.

Suitable components for such vaccines are commercially available, and details can be obtained from the World Health Organization. For example, the IPV component may be a deactivated Salk polio vaccine. Pertussis vaccine may contain whole cells or cell products.

Preferably the hepatitis vaccine or combination is a pediatric vaccine.

The vaccine compositions of the invention are used in medical therapy, particularly for the treatment or prevention of infections, including viral and bacterial, or for the immunotherapeutic treatment of cancer. In a preferred aspect, the vaccine of the invention is a therapeutic vaccine for treating existing infections, e.g. hepatitis B or herpes in humans.

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Vaccine preparations are described generally in New Trends and Developments in Vaccines, ed. Voller et al., University Park Press, Baltimore, Maryland, USA, 1978. Liposome encapsulation is described, e.g., by Fullerton, U.S. Patent No. 4,372,945 and Armor et al., U.S. Patent No. 4,474,757.

The amount of antigen in each vaccine dose is selected so as to induce an immunoprotective response without significant adverse side effects of conventional vaccines. This amount will vary depending on the type of immunogens used. Each of them will usually be expected to contain 1-1000 pg of complete immunogen, preferably 2-100 pg, most preferably 4-40 pg. The optimal amount for a particular vaccine can be determined in a standard manner, including observation of antibody titers or other responses in subjects. After initial vaccination, patients may receive a secondary vaccination after approximately 4 weeks.

In a further aspect, the invention provides a method for producing a vaccine effective in preventing or treating infection, which method comprises mixing the antigen with the carrier and the MPL suspension, the MPL particle size preferably not exceeding 120 nm, usually 60-120 nm, more preferably about 100 nm or less.

The following examples illustrate the invention and show its benefits.

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

Water for injection is introduced into vials containing 3-de-O-acylated monophosphoryl lipid A (MPL) from Ribi Immunochem, Montana, using a syringe to a concentration of 1 to 2 mg / ml. The initial suspension was obtained using vortex mixing. The contents of the vials were transferred to 25 ml Corex round bottom tubes (10 ml suspension per tube) and the suspension was sonicated in an ultrasonic bath. When the suspension became clear, the particle size was assessed by dynamic light scattering (Malvern Zetasizer 3). The processing was continued until MPL particles 60-120 nm in size.

Suspensions can in some cases be stored at 4 ° C without significant aggregation for up to 5 months. Isotonic NaCl (0.15 M) or isotonic NaCl with 40 mM phosphate induces rapid aggregation (size> 3-5 pm).

Example 2: Large-scale production of sterile soluble MPL with particle sizes below 100 nm.

The lyophilized 3-de-O-acylated monophosphoryl lipid A (MPL) from Ribi Immunochem was suspended in water for injection (WFI). The suspension was pumped continuously through an ultrasonic flow chamber. This chamber is made of glass or stainless steel with a PTFE seal to meet the requirements of general medical practice. Ultrasound is generated with a suitable generator and sonotrode from Undati Ultrasonics (Louvain-La-Neuve, Belgium). The heat exchanger attaches to the loop to avoid heat degradation of the product. The MPL temperature between the inlet and outlet of the chamber is from + 4 ° C to 30 ° C, and the difference in these temperatures does not exceed 20 ° C. It should be understood that heat is removed as the material passes through the apparatus.

This apparatus is schematically shown in Figure 1.

2.1. Ultrasounds

MPL powder (5 Odo 500 mg) is suspended in WFI at a concentration of 1 to 2 mg / ml.

The MPL suspension (under agitation) is continuously pumped through an ultra-cU ^^^ vectic loop (Fig. 1) at a rate of 50 to 100 ml per minute to achieve a system equilibrium temperature of +4 to + 15 ° C .

The spectrum of the sonotrode's own frequency in the system (power, flow chamber, liquid flow rate, temperature) is set in accordance with the manufacturer's instructions. The pre-set limits range from 19000 to 21000 Hz for the 20,000 Hz transducer.

The generator allows you to control the optimal performance of ultrasonic treatment (more energy and less heat) at a given time.

The temperature during the process is below 30 ° C to avoid degradation of MPL.

The process is complete when the particles have sizes below 100 nm and the solution is clearly transparent. Samples are taken during ultrasonic treatment to determine particle sizes

178 578 by photocorrective spectroscopy (dynamic light scattering) using Malvern Zetasizer 3 as in Example 1. The total residence time of the liquid in the chamber is calculated to be from 2.5 to 3.5 minutes (see Table 1) at a 20 ml chamber and a recirculation rate of 50 ml per minute. This gives an average residence time of 25 s per cycle, and usually less than 10 cycles are needed to achieve the desired effect - small MPL particles.

2.2. Sterilization process

The resulting "dissolved" MPL is sterilized by blind filtration on a 0.22 pm PVDF hydrophyte membrane. The pressure observed is below 1 bar. At least 25 mg of "dissolved" MPL is easily processed into 1 cm² with over 85% recovery.

2.3. Storage / Stability

Sterile "dissolved" MPL is stored at +2 to 8 ° C. Stability data (Malvern) do not show significant differences in particle size after 6 months storage (see Table 2).

Example 3: Hepatitis B vaccine composition

MPL with particle sizes below 100 nm were obtained as in Example 1. Aluminum hydroxide was obtained from Superfos (Alhydrogel).

MPL was suspended in water for injection at concentrations from 0.2 to 1 mg.ml by sonication in a water bath until a particle size of 80 to 500 nm was reached, as measured by photocorrective light scattering.

1 to 20 pg HBsAg (S-antigen as in Engerix B) in phosphate buffer solution (1 mg / ml) adsorbed 30 to 100 pg aluminum hydroxide (10.3 8 mg / ml Al solution³⁺) for 1 hour at room temperature with stirring. 30 to 50 pg MPL (1 mg / ml solution) was then added to the solution. The volume and osmotic activity was set at 600 [mu] l with water for injection and concentrated phosphate buffer 5 times. The solution was incubated at room temperature for 1 hour and kept at 4 ° C until use. The composition matures during storage. The total is 10 doses injected during tests on mice.

Example 4: vaccine composition against Hepatitis A.

MPL with particle sizes below 100 nm were obtained as in Example 1. Aluminum hydroxide was obtained from Superfos (Alhydrogel).

HAV (360 to 22 EU (ELISA units) per dose) was pre-adsorbed to a 10% final concentration of aluminum hydroxide (0.5 mg / ml). MPL (12.5 to 100 pg per dose) was added to the solution.

The remaining aluminum hydroxide was added to the solution and left for one hour at room temperature. The volumes were supplemented with phosphate buffer and the final composition was kept at 4 ° C until use.

Example 5: Comparison of the effectiveness of adjuvants in a recombinant vaccine D glycoprotein Herpes Simplex subunit.

5.1. This study determines the ability of the Al (OH) composition₃ with MPL to improve the protective immunity of truncated D glycoprotein Herpes Simplex type 2 virus. Immunogenic stages were performed on primates. The purpose of the experiments was to investigate the effect of 3-de-O-acylated monophosphoryl lipid A (MPL) particle sizes on the immunogenicity and efficacy of the rgD2t, Al (OH) composition₃ and MPL in rodents and primates. Three different Al (OH) compositions were tested₃ from MPL with small particle sizes:

Al (OH) 3 with MPL 100 nm (as previously described)

Al (OH) 3 with MPL and sorbitol

Al (OH) 3 from MPL and Tween

5.2. Antigenic compositions

Aluminum hydroxide (Al (OH) 3) was obtained from Superfos (Alhydrogel Superfod, Denmark). MPL was obtained from Ribi Immunochem Research Inc.

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5.2.1.1. RGD₂tz Al (OH)₃/ MPL and TEA (triethyl aluminum)

MPL was placed by ultrasonic treatment in a water bath to obtain particle sizes from 200 to 600 nm. The formulations were prepared in accordance with patent application No. WO 92/16231 and stored at 4 ° C before use.

The dose contained 5 pg fgD₂t, 0.5 mg Al (OH)₃ and 50 pg MPL.

5.2.1.2. rgD2t with Al (OH)₃/ MPL 100 nm

MPL (particle size below 100 nm) was obtained as in example 1. rgD2t was adsorbed onto aluminum hydroxide and incubated for an additional hour at room temperature.

The preparation was completed by adding PBS buffer with a final concentration of 10 nM PO₄ and 150 mM NaCl. The final composition was further incubated for 30 minutes at room temperature and stored at 4 ° C before use.

The dose contained 5 pg fgD2t, 0.5 mg Al (OH) 3 and 50 pg MPL.

5.2.1.3. RGD₂tz Al (OH) 3 / MPL 100 nm and sorbitol

MPL was prepared as in example 1. rgD2t was adsorbed onto aluminum hydroxide and incubated for an additional hour at room temperature. A 50% sorbitol solution was then added to a final concentration of 5%. A 10 mM Tris solution was then added to the final volume and incubated for an additional hour at room temperature with stirring.

The composition was stored at 4 ° C before use.

The dose contained 5 pg fgD2t, 0.5 mg Al (OH) 3 and 50 pg MPL.

5.2.1.4. RGD₂t with Al (OH) 3 / MPL 100 nm and Tween

MPL was prepared as in Example 1. To maintain particle size, MPL 100 nm was added to the solution with Tween 80 at a concentration such that it gave a concentration of 0.01% in the final composition. The composition was further prepared as above in 5.2.1.3.

The dose contained 5 pg fgD2t, 0.5 mg Al (OH) 3 and 50 pg MPL.

5.3. Preventive experiment on guinea pigs

In experiments, a group of guinea pigs was vaccinated on days 0 and 28 with 5 pg rgD₂in two different compositions of MPL with aluminum hydroxide. Vaccinated subcutaneously with a 0.5 ml dose. One month after the first vaccination, mumps were administered vaginally 10⁵ HSV2 units of the MS strain. They were observed daily for primary and recurrent HSV2 disease (days 4 to 39 after infection).

5.3.1. Therapeutic experiments on guinea pigs

A group of guinea pigs was infected in the experiments 10⁵HSV2 units of the MS strain. After recovery from the primary infection, they were observed daily for recurrent herpes disease (days 13 to 21). Mumps were vaccinated on day 21 and 42 with the rgDfoA / OH /./ MPL vaccine. Vaccines were administered subcutaneously at a dose of 0.5 ml. Animals were observed daily for the presence of herpes lesions until about day 60 to 84.

5.3.2. Studies on immunogenicity in primates

RgD immunogenicity₂t / Al (OH) 3 / MPL in combination with MPL in sorbitol was performed on African green monkeys. Groups of monkeys were vaccinated on days 0 and 28 with 20 pg rgD2t and 0.5 mg Al (OH) 3 of 50.20 or 5 pg MPL in sorbitol. Specific extracellular (ELISA and neutralizing titer) and effector cells (delayed-type DTH) hypersensitivity responses were tested. The compositions were administered intramuscularly in a 1 ml dose. Composition preparations were prepared as described above. The blood of the animals was tested every two weeks for the presence of antibodies.

The DTH response was tested 14 days after the second vaccination. A description of the skin test is given below.

5.4. Information readings

Tests were performed to determine the specific antibody response induced by rgD compositions₂t / Al (OH) .3 / MPL (determination of anti-rgD2t ELISA titers and anti-HSV2 neutralizing titers). The protective efficacy of the gD2 composition was evaluated in prophylactic and therapeutic models in guinea pigs. Immunogenicity studies were also conducted in monkeys. Extracellular and DTH specific responses were determined.

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5.4.1. ELISA and neutralizing titers

Anti-rgD antibody titers were determined₂and anti-HSV2 neutralizing activity according to the methods given in patent application WO 92/16231.

5.4.2. Delayed type hypersensitivity (DTH)

The rgD2t compositions were tested for their ability to induce T cell-specific immune responses measured by induction of delayed-type hypersensitivity (DTH) responses.

African green monkeys were vaccinated on days 0 and 28 with 20 pg of intramuscularly administered gD2 vaccine composition. Skin tests were performed on them 14 days after the second vaccination by intradermal injection on the abdomen 15 or 5 pg rgD₂in brine. Skin tests with brine as a control fluid were performed on them. The injection site was examined 24 and 48 hours later for erythema and sclerosis. Local response sizes were measured.

5.4.3. Vaginal model of guinea pig infection

The guinea pig model of HSV genital infection was described by L. Stanberry et al. (J. of Infectious Diseases 1982, 146: 397-403); Intervirology 1985, 24: 226-231). Briefly, in preventive experiments, guinea pigs were infected vaginally 10⁵HSV2 units of the MS strain one month after the last vaccination. The clinical course of the primary disease and the severity of genital skin damage were observed daily 4-12 days after infection. The animals were tested daily for the presence of recurrent optic lesions on days 13 to 39. In therapeutic experiments, guinea pigs were infected on day 10⁵HSV2 units of the MS strain. After recovery from the primary infection, they were examined daily for recurrent herpes (days 13 to 21), and then divided into groups randomly according to the effects of the primary and secondary disease (equivalent distribution of animals with mild and acute infection in each group) for vaccination or failure to vaccinate. The vaccine was administered on days 20 and 41 after infection. Distribution of the presence of relapse was observed normally about 70 days after infection.

Herpetic lesions were quantified using a scale of 0 to 32.

Grading scale

<td>Damage Type</td><td>Rating</td>

<td>Lack</td><td> 0</td>

<td>Vaginal damage</td><td></td>

<td>bleeding</td><td> 0,5</td>

<td>- redness for 1 to 2 days without bleeding</td><td> 0,5</td>

<td>- redness and bleeding for a day</td><td> 1</td>

<td>- redness without bleeding for at least 3 days</td><td> 1</td>

<td>External herpes vesicles</td><td></td>

<td>- &lt;4 small bubbles</td><td> 2</td>

<td>-&gt; 4 small bubbles or 1 large</td><td> 4</td>

<td>-&gt; 4 major damage</td><td> 8</td>

<td>- large merging damage</td><td> 16</td>

<td>- merging large lesions all over the genitals</td><td> 32</td>

Clinical Readings

Primary infection

- Cautious focus - for large readings from 12 to 12 days after infection.

The severity of damage is expressed as the arithmetic mean ± standard deviation as well as the median (better for a parameterless test)

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- Prevalence of primary infection =% of animals having maximum grades of 0.0,: 5,12,4, or 16 (less often 32).

Primary infection index = Ą (max. Rating i) x (% of occurrences) zi = 0.0,5,2,4,8 or 16.

Relapse

- Number of relapse days = number of days with relapse for days 13 to 39 after infection. One relapse must be preceded and followed by one day without damage, and within it must be two days with erythema or one with blisters. The number of recurrence days is expressed as the arithmetic mean ± standard deviation and the medians.

- Relapse severity = the sum of daily evaluations for days 13 to 39 after infection. The results are given as arithmetic means ± standard deviations and medians.

5.5. Results

Protective efficacy of various rgD compositions₂t / Al (OH)₃/ MPL was compared in preventive experiments on guinea pigs. Primary immunogenicity studies were also conducted. The purpose of these experiments was to compare immunogenicity and protective efficacy ^^ D₂t^fromAl (OH)₃ in combination with MPL with different particle sizes.

5.5.1. Preventive experiments

Two experiments were carried out to determine the potential of various rgD2t / Al (OH) vaccines₃/ MPL in protection against primary and return HSV2 disease when administered to guinea pigs against vaginal infection.

Experiment 1: Comparison of MPL 100 nm with sorbitol from MPL with TEA

A group of female Hartley guinea pigs (200-250 g) were vaccinated on days 0 and 28 with 5 pg rgD2t / Al (OH)₃ in combination with MPL with small particle sizes (100 nm, MPL in sorbitol) or larger (MPL in TEA). Control animals were vaccinated according to the same adjuvant protocol or not vaccinated at all. Blood was collected on days 14 and 28 after the second vaccination to determine the antibodies in the ELISA or neutralization test. Animals were infected 29 days after the second vaccination 10⁵HSV2 units of the MS vaginally. After infection, they were observed daily for signs of acute infection (days 4 to 12 after infection) and evidence of recurrent herpes disease (days 13 to 39 after infection).

a) induction of extracellular immunity

As shown in Table 3, higher ELISA and neutralizing titers were obtained when MPL with small particle sizes were used in the composition ng.₂t / Al (OH)₃.

b) Effect of vaccination on primary HSV2 infection (Table 3)

Compared with the control group infected and undergoing acute primary infection, both vaccinated groups showed significantly lower levels of damage (p &lt;0.00005). Less frequent skin damage was observed in the rgD vaccine group₂t / Al (OH) 3 / MPL 100 nm (p &lt;0.06).

c) Impact of vaccination on the re-infection of HSV2

The results are given in Table 4. Compared with the control group, both vaccines were able to stop the development of recurrent herpes disease, as indicated by a lower number of relapse cases (p &lt;0.02 for rgD₂t / Al (OH)₃MPL 100 nm).

d) Conclusions

Both compositions are able to provide known protection against primary infection and reduce the extent of disease recurrence. The results show that the rgD2t / Al (OH) 3 / MPL composition with MPL particles of small size has very high prophylactic effectiveness.

Experiment 2: Efficiency Al (OH) 3 / MPL 100 nm

A group of Hartley guinea pigs (200-250 g) were inoculated on days 0 and 28 with 5 pg gD2 in Al (OH) 3 with MPL 100 nm. Vaccination was performed subcutaneously at a dose of 0.5 ml. Control animals were vaccinated according to the same adjuvant protocol or not vaccinated at all. Blood was collected on days 14 and 28 after the second vaccination to determine the antibodies in the ELISA or neutralization test. Animals were infected 29 days after the second vaccination 10⁵ HSV2 units of the MS vaginally.

a) induction of extracellular immunity

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As shown in Table 3, the vaccinated group showed good ELISA and neutralizing titers. The control group did not show a detectable antibody response.

b) Effect of vaccination on primary HSV2 infection (Table 3)

Compared with the control group infected and undergoing acute primary infection, the vaccinated group showed significantly lower levels of damage (p &lt;0.00005) and incidence (p &lt;0.002). No external skin damage was observed in vaccinated guinea pigs.

c) Effect of vaccination on recurrent HSV2 infection (Table 4)

Compared with the control group, rg'D vaccine₂t^/Al (OH)₃/ MPL was able to change the development of relapse of herpes disease, as indicated by a significant reduction in the severity of relapse (p &lt;0.00005) and the frequency of relapses (p &lt;0.01)

d) Conclusions

RgD composition₂t / Al (OH)₃ with MPL particles of small size has very high efficiency in providing protection against primary and secondary HSV2 infection in guinea pigs.

It can be concluded from the experiments described above that the Al (OH) compositions₃ from MPL of small size obtained in two different ways inducing the least strong prophylactic response as Al (OH) 3 compositions from MPL of large size. In addition, small sized MPLs are preferably easily sterilized prior to use.

5.5.2. Therapeutic experiments

The purpose of these experiments is to compare the therapeutic potential of different rgD2t / Al (OH) compositions₃/ MPL during relapse of herpes in guinea pigs with established infection with HSV2.

Guinea pigs were vaginally vaccinated on day 10-10⁵HSV2 units of the MS strain. They were observed daily for primary infection (days 4 to 12) and recurrent herpes infection (days 13 to 20). The animals were divided into groups randomly according to the effects of primary and secondary disease (equivalent distribution of animals with mild and acute infection in each group). Guinea pigs without clinical signs of infection were not entered in the protocol. The vaccine was administered subcutaneously on days 21 and 42 after infection.

Therapeutic rgD infection₂t / Al (OH)₃/ MPL was evaluated in three different experiments.

Experiment 1: Comparison of rgD₂t / Al (OH) 3 with MPL with large particle sizes (MPL in TEA)

Relapsed guinea pigs were divided into groups, giving either 20 pg rgD2t / Al (OH) 3 of MPL with large particle size (MPL in TEA), or adjuvant alone. Vaccines were administered subcutaneously on days 21 and 42 after infection. Distribution of disease relapses was observed up to day 84.

As table 3 shows, the rgD2t / Al (OH) 3 / MPL composition with TEA was not effective in attenuating persistent recurrence of infection;

Experiment 2: Efficacy of rgD2t / Al (OH) 3 with MPL 100 nm

Two groups of guinea pigs were inoculated with 20 pg rgD2t / Al (OH) 3 from MPL with small particle size (MPL 100 nm), or vaccination was discontinued.

Vaccines were administered on days 21 and 42 after infection.

Distribution of relapses was observed up to day 69.

As Table 5 shows, compared to data from Experiment 1, where MPL with large particle sizes were used, rgD vaccination₂t / Al (OH) 3 / MPL 100 nm changed the incidence of HSV2 disease compared to the control group, reducing relapse severity (-39%, p &lt;0.05) and the number of days with relapse (-28%, p &lt;0.1 ).

Experiment 3: Comparison of the effectiveness of Al (OH) 3 in combination with MPL with small particle sizes

A third strategy for obtaining MPL with small particle sizes was used in the experiment: addition of Tween, e.g. Tween 80.

The experimental groups were as follows: Group 1 (n = 15): 20 pg rgD2t / Al (OH)₃ from MPL

100 nm from Tween

Group 2 (n = 15): 20 pg rgD2t / Al (OH)₃ with MPL 100 nm with sorbitol

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Group 3 (n = 16): control

Control groups were not vaccinated or only Al (OH) was vaccinated_:, from MPL. Vaccines were administered on days 21 and 42 after infection. Distribution of disease relapses was observed up to day 60.

The results are shown in Table 5. Significant, pronounced therapeutic effect was observed in animals vaccinated with the two compositions rgD2t / Al (OH) 3 from MPL. Both compositions significantly reduced the severity of relapses, the number of days they lasted and the number of relapses.

Conclusions

A very strong therapeutic effect was observed against steady-state recurrence of HSV2 genital disease with two rgD2t / Al compositions (OH&gt; 3 with MPL of small particle size (about 100 nm). In contrast, no therapeutic effect was observed for MPL with large particle size ( MPL in TEA) added to the rgD vaccine₂t / Al (OH) 3.

The results obtained for guinea pigs clearly show the prophylactic efficacy of the rgD2t / Al (OH) 3 composition with MPL of small particle size. They have higher therapeutic potential than rgD2t / Al (OH) 3 with large particle size MPL.

5.5.3. RgD immunogenicity studies₂t / Al (OH) 3 in combination with MPL with small primate sizes

Doses of 50.20 or 5 pg MPL 100 nm were combined with 20 pg rgD2t and A / OH) (0.5 mg). Two vaccinations were carried out on day 0 and after one month. Specific extracellular (ELISA and neutralizing titer) and effector cell (DTH) immune responses were measured.

a) experimental procedure

Three groups of green African monkeys were vaccinated on days 0 and 28 with 20 pg gD₂t with Al (OH) 3 containing 50.20 or 5 pg MPL. Animal blood was tested every two weeks to determine the presence of antibodies in ELISA (anti-gD2 titers) and neutralization tests. The three vaccine compositions were compared for their ability to induce T cell-specific immune responses as measured by induction of delayed-type hypersensitivity (DTH) responses. On three monkeys from each group, skin tests were performed 14 days after the second vaccination by abdominal injection of 15 or 5 pg gD2t in brine. Skin tests with brine as a control fluid were also performed. The injection site was examined 24 and 48 hours later for erythema and sclerosis.

b) Results

The serological and DTH responses are shown in Table 6. Groups of monkeys vaccinated with the rgD composition₂ti Al (OH) 3 containing 50 or 20 pg MPL produced significantly more neutralizing antibodies than the 5 pg MPL dose group (p &lt;0.003 and p &lt;0.008, respectively). There was no clear difference in ELISA and neutralizing titers measured in the 50 or 20 pg MPL groups. A correlation was observed between the dose of MPL and the effect on the immune response of effector cells. A strong DTH response was detected in most monkeys (3/4) vaccinated with compositions with 50 or 20 pg MPL. In contrast, only one monkey in the 5 pg MPL group showed a skin test response.

c) Conclusions

The data given above show that acting as an Al (OH) 3 adjuvant in combination with MPL with small particle sizes is also effective in primates, not just small animal species. In monkeys, a correlation between the dose of MPL and the immunogenicity of the rgD2t / Al (OH) 3 / MPL composition can be observed, with 50 and 20 pg giving the best serological response and DTH.

Example 6: CLINICAL STUDIES of Lyme and Hepatitis B vaccines and MPL small particles

6.1. Lyme disease vaccine containing the NS1 (1-81) fusion protein from group i virus

OspA from B. burgdorferi ZS7.

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Composition making

6.1.1. NS 1 -OspA / aluminum hydroxide

NSl-OspA prepared according to the procedure of patent application WO 93/04175 was adsorbed on aluminum hydroxide and incubated at room temperature for 1 hour. The final volume was reached by adding phosphate buffer (PO₄ 10 mM, NaCl 150 mM). The composition was stored at 4 ° C until use.

The dose contains 10 pg NS1-OspA / 500 pg aluminum hydroxide.

6.1.2. NS1-OspA / w Aluminum hydroxide / MPL

NS1-OspA was adsorbed onto aluminum hydroxide and incubated at room temperature for 1 hour. MPL, prepared as previously described, was added to the composition and again incubated at room temperature for 1 hour. The final volume was achieved by adding phosphate buffer (PO₄ 10mM, NaCl 150 mM). The composition was stored at 4 ° C until use.

The dose contains 10 pg NSl-OspA / 500 pg aluminum hydroxide / 50 pg MPL.

6.1.3. The course of vaccination

Volunteers received three ml intramuscular injections of the composition on days 0, 31 and 62. Plasma was collected on day 30 after I, II and III vaccination. They were subjected to ELISA analysis for total IgG anti-OspA content and an LA-2-like anti-antibody response in the inhibition assay (LA-2 showed the protective effect against infection in mice as an antibody).

6.2. Κοιτφί ^ ο ^ ε HHsAg / 'MPL for. luuzi

6.2.1. Composition making

HBsAg 20 pg / aluminum hydroxide 500 pg

HBsAg was adsorbed on the final amount of aluminum hydroxide and the final volume was achieved by adding phosphate buffer with brine (PO4 10 mM, NaCl 150 mM) for a dose of 1 ml. The composition was stored at 4 ° C until use.

6.2.2. HBsAg 20 pg / aluminum hydroxide 100 pg

HBsAg was composed as above by adsorbing onto 100 pg aluminum hydroxide. The final volume was 1 ml per dose.

6.2.3. HBsAg 20 pg / aluminum hydroxide 100 pg / MPL 50 pg

HBsAg was adsorbed onto 100 pg aluminum hydroxide. The correct concentration of MPL was added and incubated at room temperature for 1 hour. The final volume was reached by adding the appropriate buffer (as above) and stored at 4 ° C until use.

6.2.4. The course of vaccination

Volunteers (20_;in the group) received 1 ml intramuscular injections of one of the given compositions. Plasma was collected at months 0, 1, 3 and 6. They were analyzed for protective antibodies using the commercially available Abbot test.

6.3. WWNIKJ

Table 8 shows that MPL, used in combination with aluminum hydroxide and NS1-OspA in the form of 100 nm particles effectively allows the formation of inhibitory antibody titers higher than that of aluminum hydroxide antigen, and the kinetics of seroconversion are faster.

It follows that for soluble antigens, such as NS 1 -OspA, in humans MPL composed in the form of small particles has the adjuvant properties already demonstrated in animals for other soluble antigens.

Table 7 shows that the support effect lost as a result of the reduction in the amount of aluminum hydroxide in the compositions for Hepatitis B can be recovered by adding MPL as described herein. MPL also improves the speed of seroconversion.

Example 7: Combined vaccine composition - Hepatitis B + Hepatitis A

HBsAg is adsorbed onto 90% of the final amount of aluminum hydroxide (0.5 mg / ml) and incubated at room temperature for 1 hour. The pH is adjusted to 6.2 and the preparation is allowed to ripen for 14 days at room temperature.

Hepatitis A antigen in an amount of 360 to 22 UE per dose, in the form of a deactivated derivative of the HM-175 strain (as in Havrix) is subjected to preliminary adsorption on aluminum hydroxide

178 578 at 10% final concentration (0.5 mg / ml). The rest of the aluminum hydroxide is then added to the solution and left for one hour at room temperature with stirring.

HAV adsorbed on aluminum hydroxide is then added to the composition and HBsAg.

MPL (particles smaller than 100 nm) is added to the HAV / HBsAg solution to a final concentration of 12.5 to 100 pg per 1 ml dose, made up to the final volume and the composition stored at 4 ° C before use.

Example 8: Combined vaccines containing additional antigens

Combined vaccines may be prepared by adding one or more of the desired antigens to the compositions described in Example 2, 3 or 4 above.

Example 9: Increase in extracellular immunity and induction of cellular immunity by immunization with HBsAg mice composed of aluminum hydroxide and MPL.

9.1. Impact of Al (OH)₃ with MPL for induction of anti-HBs antibodies

Balb / c mice were immunized subcutaneously or transdermally with recombinant HBsAg adsorbed to Al (OH)₃ with MPL as an adjuvant. Mice were vaccinated twice with HBsAg (Al (MPL and first and second dose antibody responses. The total amount of Ig was measured by ELISA or AUSAB (Abbott Lab, 111.), paying particular attention to the induction of IgG2a isotype because it is mainly induced by secretion g-interferon Induction of this isotype indirectly reflects the activation of cellular immunity, specifically Th1 activation.

The HBsAg / MPL ratio and MPL particle sizes were tested.

9.1.1. Experiment 1: Effect of MPL dose (&gt; 500 nm) on immunogenicity of rec HBsAg adsorbed on Al (OH)₃

A group of 10 female Balb / c mice were immunized subcutaneously with 2.5 pg recHBsAg adsorbed to 50 pg Al^LT + (as Al (OH&gt; 3 containing increasing amounts of MPL (3.1 to 50 pg) with particle sizes&gt; 500 nm. Mice were vaccinated twice with 100 pl every two weeks. Blood samples were taken 2 weeks after the first injection (partial) and after week from the second 2. Total anti-HBs IgG and specific IgG2a ELISA were measured using recHBsAg as capture antigen. Titers were expressed as the inverse of the dilution corresponding to 50% of the maximum value (middle dilution). Results indicating an increase in specific IgG and IgG2a with an increase in MPL doses, especially at a dose of 12.5 to 50 pg. The effect is visible for the primary and secondary responses, and is particularly pronounced for IgG2a (almost a 20-fold increase), indirectly indicating the secretion of g-interferon induced by MPL immunization.

9.1.2. Experiment II - Comparison of clinical sets of adsorbed recHBsAg with or without MPL (&gt; 500 nm)

Three clinical sets of recHBsAg adsorbed on Al2 OHty were prepared. DSAH16 set did not contain MPL and was a control set. DSAR501 and 502 sets were prepared in a similar manner (20 pg recHBsAg was adsorbed onto 0.5 mg Al⁺⁺⁺ as Al (OH ^) but contained 50 pg MPL (&gt; 500 nm).

Three sets were injected into groups of 10 mice (200 µl contains 2.5 pg HBsAg, 100 pg Al⁺++ and 6.25 pg MPL), twice every two weeks. Blood samples were taken 14 days after the first injection and one week after the second. Anti-HBs antibody levels were measured with the AUSAB kit or IgG or IgG2a home ELISA. The results are given in Table 2. The results indicate that two weeks after the first injection, both sets containing MPL inducing a very pronounced anti-HBs response (12.4 and 41.9 mIU / ml), and the set without MPl only a minimal response (0.75 mIU / ml). The number of responses is also higher when MPL is present (9/10 and 9/10 versus 1/10 without MPL). The effect of MPL also underlines the condition after the second injection, because the obtained titers for the DSAR501 and DSAR502 kits are about 6 times higher than in the absence of MPL.

This indicates that at least MPL (&gt; 500 nm) can improve both the kinetics of the anti-HBs response and the level of this response.

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The results were confirmed by the level of specific IgG and IgG2a after immunization with DSAH 16 (without MPL) and DSAR502 (with MPL): anti-HBs IgG titers are 5 (primary response) and 3 (secondary response) times more in the presence of MPL.

9.1.3. Experiment III: Effect of MPL dose (&lt;100 nm) on immunogenicity of recombinant Albs (HBsAg) adsorbed onto Al (OH)₃

A group of 10 mice (Balb / c, females, 7 weeks old) were immunized subcutaneously with 1 pg of recombinant HBsAg adsorbed to 50 pg A +++ (as Al (OH)₃) containing increasing amounts of MPL (3.1 to 25 pg) with particle sizes &lt;100 nm. Mice were vaccinated twice with 200 pl every two weeks. Blood samples were taken 2 weeks after the first injection and a week after the second. The anti-HBs response (total Ig, IgG, IgG2a) on measured plasma was measured by ELISA. Titers are expressed as the middle dilution (the inverse of the dilution corresponding to 50% of the maximum value). The results indicate that even a low dose of 3.1 pg MPL induces a strong increase in the antigen response, both primary and secondary. The response is maximum for 6.25 pg and decreases to values ​​in the absence of MPL at high doses of MPL; (25 pg). The response pattern is similar for IgG, IgG2a and total Ig. The results are opposite to those for MPL of large size (&gt; 500 nm) and indicate that MPL particles of small size (&gt; 100 nm) are more effective (at least for extracellular immunity) because less MPL is needed for maximum effect. Higher MPL activity with small particle sizes was confirmed by several experiments.

As seen for MPL with large particle sizes (&gt; 500 nm), the MPL support effect is higher for IgG2a than total IgG or Ig. With a maximum secondary response effect (6.25 pg MPL) there is a 25-fold increase in IgG2a content, and 7.6 and

4.3 for IgG and total Ig.

9.2. Induction of cellular immunity by recHBsAg adsorbed to Al (OH)₃ - impact

MPL

If eosocellular immunity is sufficient to protect against Hheαtjtjtd B virus, induction of cellular immunity (CTH, Th1) should be of great importance in the treatment of the disease.

New compositions are needed for therapeutic vaccines because Al (OH) 3 is able to improve extracellular but not cellular immunity.

The effect of MPL on the induction of Th1 cells capable of secreting IL-2 and g- (i.e. gamma) - interferon in Balb / c mice immunized with Al-adsorbed recHBsAg (OHty) was investigated

9.2.1. Experiment I - Effect of MPL (&gt; 500 nm) on the induction of Th1eo cells by the immersion of Balb / c HBsAg adsorbed on Al (OH) 3

A group of 10 Balb / c mice (female, 5 weeks old) jmmunizowm with foot injection 30 pl of a solution containing 10 pg HBsAg 15 pg Al⁺⁺⁺ (as Al (OHty) and 15 pg MPL. Control mice were injected with the same amount of recHBsAg mixed with FCA (positive control) or adsorbed to Al (OH) 3 (negative control).

Six days after immunization, the mice were killed and the excavated lymph nodes removed. Node cells (LNC 2,105 / ml) were cultured for various periods (24 to 74 hours) on RPMI medium supplemented with 1% negative mouse plasma and 5 pg / ml recHBsAg. At the end of the culture, the amount of IL-2, INF-g and IL-4 secreted into the medium was measured. IL-2 was assessed by its ability to stimulate proliferation (its measure was the attachment of 3H-thymidine) of IL-2 dependent CTL lines (VDA2 cells), and the titer was expressed as the stimulation index (SI = the amount of 3H-thymidine incorporated into the stimulated cells / the amount of 3H) thymidine to multimusive cells). The amount of IL-4 and INF-g was measured using commercial ELISA kits (Holland Biotechnology for IFN-g and Endogen for IL-4). Titers were expressed in pg IFN-g / ml.

The results indicate LNC from mice immunized with Al (OH) 3 adsorbed HBsAg does not secrete significant amounts of IL-2, IL-4 or INF-g. In contrast, large amounts of IL-2 (SI = 38 after 48 hours) and significant amounts of INF-g secrete LNC from mice immunized with HBsAg adsorbed onto Al (OH) 3 from MPL. This secretion is similar (INF-g) or higher (IL-2) compared to that observed for HBsAg immunized with FCA mice, and occurs earlier in vitro.

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No IL-4 detected after immunization with HBsAg absorbed on Al (OH) 3 even in the presence of MPL.

This secretion profile indicates that specific Th1 cells (IL-2, INF-g) were induced by immunization with adsorbed HBsAg in the presence of MPL, but not in its absence. However, Th2 (IL-4) was not detected under these immunization conditions.

9.2.2. Experiment I - Effect of MPL dose (&lt;100 nm) on induction of Th1 cells after immunization with Alb OH 3 adsorbed Balb / c HBsAg mice.

A group of 5 Balb / c mice were immunized by injection into two foot pads of 30 µl solution containing 10 pg HBsAg adsorbed to 15 pg A +++ (as Al (OH ^) with increasing amounts of MPL (100 nm, 0 to 15 pg).

Six days after injection, mice were killed and popliteal node cells (LNC, 2,106 cells / ml)) were cultured in RPMI medium supplemented with 1% negative mouse plasma for various periods (24 hours to 96/25) in the presence of 5 pg / ml rccHBsAg.

IL-2 secretion was measured by stimulating VDA2 cell proliferation and IL-2 concentration was expressed as stimulation index (SI). INF-g secretion was measured using commercial kits⁷ and expressed in pg / ml.

IL-2 secretion has been found to increase significantly at a low MPL dose (7.5 pg) and the maximum effect occurs at 15 pg MPL.

IL-2 secretion is significantly more important after 24 hours than after 48 or 72 hours.

The secretion of INF-g does not occur in the case of HBsAg adsorbed to Al (OH) 3 in the absence of MPL. A low dose (7.5 pg) of MPL induces the secretion of INF-g, and again the maximum effect occurs at 15 pg MPL. In contrast to IL-2, INF-g secretion is delayed in culture and increases with time up to 96 hours.

Taken together, these data indicate that MPL (below 100 nm) is a strong Th1 inducer in combination with HBsAg adsorbed to Al (OH) 3. The effect of compositions containing HBsAg adsorbed on Al (OH) 3 and MPL on the induction of extracellular and cellular immunity in Balb / c mice was investigated. The results indicate that MPL clearly improves the kinetics of the anti-HBs response because much more anti-HBs antibodies were found after primary and re-vaccination. The anti-HBs quality has also been modified and the preferred IgG2a induction is observed, which indirectly indicates the secretion of INF-g, and thus the induction of cellular immunity.

Direct determination of Th1 cell induction by compositions containing HBsAg, Al (OH) 3 and MPL clearly indicates that MPL very strongly induces IL-2 and INF-g secretion by Th1 cells. Such compositions make an important contribution to the development of therapeutic vaccines.

The best results were obtained with MPL with particle sizes smaller than 100 nm.

The results of the experiments described here are given in Tables 9-14.

13. Conclusions

Cumulative data suggest that MPL with small particle sizes is an improved immunostimulant in primates, including humans, compared to MPL with large particle sizes. This feature combined with the possibility of large-scale sterilized preparation makes ML with small particle sizes a suitable immunostimulator for many human and animal vaccines.

Table 1

MPL particles and filtration recovery at various ultrasonic performance parameters

<td>Attempt No.</td><td>Concentration mg / ml</td><td>Total residence time in the chamber (min.)</td><td>Particle size before filtration (nm)</td><td>Recovery after filtration (%)</td>

<td> 16</td><td> 1</td><td> 2,5</td><td> 92</td><td> 104</td>

<td> 17</td><td> 1</td><td> 3</td><td> 79</td><td> 78,5</td>

<td> 18</td><td> 1</td><td> 3,5</td><td> 95</td><td> 86,4</td>

<td> 19</td><td> 2</td><td> 2,8</td><td> 77</td><td>Lack</td>

<td> 20</td><td> 1</td><td> 2,8</td><td> 98</td><td>Lack</td>

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Table 2

Particle size stability of the sterile MPL solution determined by light correlation spectroscopy (Malvern), 1 mg / ml

<td>Test No.</td><td>Particle size after filtration (nm)</td><td colspan="4">Particle size after stabilization at 4 ° C (nm)</td>

<td></td><td></td><td>8 days</td><td>1 month</td><td>3 months</td><td>6 months</td>

<td> 9</td><td> 94</td><td> 81</td><td> 74</td><td> 88</td><td> 82</td>

Table 3

Prophylactic efficacy of rgD compositions₂t / A1 (OH) 3 / MPL in guinea pigs Extracellular response and impact of vaccination on primary HSV2 disease

<td>Group</td><td>Composition</td><td colspan="2">Titers (GTM)</td><td colspan="10">Primary infection</td>

<td></td><td></td><td colspan="2">28 days after III</td><td colspan="2">Sharpness</td><td colspan="7">Occurrence of damage ratings **%</td><td>in-</td>

<td></td><td></td><td></td><td></td><td colspan="2">damage *</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>deks</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>PI ***</td>

<td></td><td></td><td>ELISA</td><td>NEUTRA</td><td>Śr.arytm.</td><td>Median</td><td> 0</td><td> 0,5</td><td> 1</td><td> 2</td><td> 4</td><td> 8</td><td> 16</td><td></td>

<td></td><td></td><td></td><td></td><td>± SD</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>Experiment 1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>ln = 12</td><td>rgD2t 5 pg Al (OH) 3 / MPL (Sorbitol)</td><td> 10439</td><td> 673</td><td> 2,2±311</td><td> 0,5</td><td> 50</td><td> 17</td><td> 0</td><td> 33</td><td> 0</td><td> 0</td><td> 0</td><td> 75</td>

<td>2n = 12</td><td>rgD2t 5 pg Al (OH) and / MPL TEA</td><td> 5454</td><td> 378</td><td> 4,6±6,3</td><td> 1,5</td><td> 42</td><td> 8</td><td> 8</td><td> 25</td><td> 17</td><td> 0</td><td> 0</td><td> 130</td>

<td>3n = 11</td><td>control</td><td> &lt;100</td><td> &lt;50</td><td> 55,3±51,8</td><td> 55</td><td> 18</td><td> 0</td><td> 0</td><td> 0</td><td> 27</td><td> 0</td><td> 55</td><td> 988</td>

<td></td><td>Experiment 2</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>1n = 10</td><td>rgD2t Al (OH)₃/ MPL</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>100 nm</td><td> 21039</td><td> 696</td><td> 0,5±0,7</td><td> 0</td><td> 60</td><td> 30</td><td> 10</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 25</td>

<td>2n = 10</td><td>control</td><td> &lt;100</td><td> &lt;50</td><td> 28,5±29,1</td><td> 31,5</td><td> 30</td><td> 0</td><td> 0</td><td> 0</td><td> 10</td><td> 40</td><td> 20</td><td> 680</td>

* Sum of service assessments from 4 to 12 o'clock and inferiority ** Damage ratings: none (0), vaginal (0.5 or 1), external skin bubbles (2, 4, 8 or 16 *** Index of primary infection = Σί (max. Rating i) x (% of occurrences); i = 0, 0.5, 1, 2, 4, 8 or 16

Table 4

Prophylactic efficacy of rgD compositions₂t / Al (OH) 3 / MPL in guinea pigs Effect of vaccination on relapse of HSV2 disease

<td rowspan="3">Group</td><td rowspan="3">Composition</td><td colspan="10">Relapses</td>

<td colspan="2">Relapse sharpness *</td><td colspan="2">Number of recurrence days **</td><td colspan="6">Number of relapses (%)</td>

<td>Śr.arytm. ± SD</td><td>Median</td><td>Śr.arytm. ± SD</td><td>Median</td><td> 0</td><td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td>

<td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td><td> 7</td><td> 8</td><td> 9</td><td> 10</td><td> 11</td><td> 12</td>

<td></td><td>Experiment 1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>1n = 12</td><td>rgD2t 5 pg</td><td> 5,44±6,2</td><td> 3,5</td><td> 4±5</td><td> 2,5</td><td> 33</td><td> 42</td><td> 8</td><td> 8</td><td> 8</td><td> 0</td>

<td></td><td>Al (OH) 3 / MPL</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>(Sorbitol)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>2n = 12</td><td>rgD2t 5 pg</td><td> 6,5±5,9</td><td> 6,5</td><td> 4,3±3,9</td><td> 3</td><td> 27</td><td> 27</td><td> 9</td><td> 27</td><td> 9</td><td> 0</td>

<td></td><td>Al (OH) j / MPL</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>TEA</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>3n = 11</td><td>control</td><td> 8±5,4</td><td> 9</td><td> 5,1±3,1</td><td> 6</td><td> 18</td><td> 0</td><td> 18</td><td> 64</td><td> 0</td><td> 0</td>

178 578

Table 4 - continued

<td> 1</td><td> 2</td><td> 9</td><td> 4</td><td> 5</td><td> 6</td><td> 7</td><td> 8</td><td> 9</td><td> 10</td><td> 11</td><td> 12</td>

<td>1n = 10</td><td>Experiment 2 rgD2t</td><td> 1,6±9,9</td><td> 0</td><td> 0,5±1,1</td><td> 0</td><td> 80</td><td> 20</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td>2n = 10</td><td>Al (OH) 3 / MPL 100 nm kcnti-clna</td><td> 6,1 ±6</td><td> 6,75</td><td> 4,9±4,9</td><td> 4,5</td><td> 40</td><td> 0</td><td> 20</td><td> 20</td><td> 0</td><td> 20</td>

* Total occn damagedoddee from ddia 1 3dd3 9infections ** Number of recurrence days for days cd 19 dc 99 pc infection.

One recurrence is thinned and it takes a day without damage, and it means at least two days with erythema or one day with blisters.

T abe1 a 5

Therapeutic efficacy of Ocmpczczenie rgD2t / Al (OH)₃/ MPL

<td rowspan="3">Group</td><td rowspan="3">Kdmodzyzja</td><td colspan="6">Therapeutic effect</td>

<td colspan="2">Sharpness*</td><td colspan="2">Number of recurrence days **</td><td colspan="2">Number of conversions ***</td>

<td>Śr.arytm. ± UD</td><td>Median (% kcnt.)</td><td>Śr.arytm. ± UD</td><td>Median (% kcnt)</td><td>Śr.arytm ± UD</td><td>Median (% kcnt.)</td>

<td></td><td>Experiment 1</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>1n = 18</td><td>rgD2t 20 pg Al (OH) 9 / MPL TEA</td><td>lack</td><td>lack</td><td>lack</td><td> 11</td><td>lack</td><td> 7</td>

<td>2n = 18</td><td>kcnti-lamb</td><td>lack</td><td>lack</td><td>lack</td><td></td><td>lack</td><td> 5</td>

<td></td><td>Experiment 2</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>1n = 14</td><td>rgD2t Al (OH) 9 / MPL 1-10 nm</td><td>11.1 ± 8.7 (-99%) p &lt;0.05</td><td>10.25 (-41%) P &lt;0.1</td><td>± 8.4 (-28%) P &lt;0.1</td><td>8.5 (-29%) P &lt;0.31</td><td>β 2 ± 2</td><td> 4</td>

<td>2n = 19</td><td>kdftrdlfa</td><td> 18,9±10,9</td><td> 17,5</td><td> 11,7±6,8</td><td> 11</td><td> 4,4±2,1</td><td> 4</td>

<td></td><td>Experiment 9</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>1n = 15</td><td>rgD2t Al (OH) 9 / MPL 100 nm, Tween</td><td> 10,9±10,1</td><td>6 (-54%) p &lt;0.07</td><td> 6,9±5,8</td><td>4 (-49%) P &lt;0.1</td><td>2.7 ± 2 p &lt;0.1</td><td>9 (-25%) p &lt;0.1</td>

<td>2n = 15</td><td>rgD2t Al (OH) 9 / MPL 100 nm, scrbitcl</td><td> 8,9±6,7</td><td>6.5 (-50%) p &lt;0.09</td><td> 5,4±4,4</td><td>4 (-49%) p &lt;0.1</td><td> 2,7±1,5</td><td>9 (-25%) P &lt;0.1</td>

<td>9n = 16</td><td>Odntrcina</td><td> 12,5±8,1</td><td> 19</td><td> 8,5±4,5</td><td> 7</td><td> 9,6±1,6</td><td> 4</td>

* Total price of injuries cd 21 dc 60 pc infection ** Total number of relapse days in animals for cd 21 dc 60 pc infection days * * * I ^ iizzn ρι ^ Γ ^ ΙΟ ^ n returns for 2 2 to 66 pp&gt; infenoii. Jenen nfrottent and nfls Sepo mm a day without bruises, and means cn at least two days with erythema (spear = 0.5) or one day with external vesicles (price ^ 2). Immundterapie: intradermal injections on day 21 and 42 of the pc of infection, statistical analysis: WilccKcna test with rank relative to essential adjuvants (found for p&gt; 0.1).

178 578

Table 6

Immunogenicity of gD2t, aluminum hydroxide, MPL 100 nm compositions in primates Serological results and DTH

<td rowspan="2">Vaccine</td><td rowspan="2">number monkeys</td><td colspan="2">Antibody response *</td><td colspan="3">DTH (hardening) response *</td>

<td>Titre ELISA</td><td>Titre NEUTRA</td><td>PBS</td><td>GD2 5 Pg</td><td>gd₂15 pg</td>

<td>20 μ gD2t</td><td>KQ 101</td><td> 5554</td><td> 1600</td><td> -</td><td> -</td><td> -</td>

<td>aluminum hydroxide</td><td>KQ 102</td><td> 14870</td><td> 800</td><td> -</td><td> ++</td><td> +++</td>

<td>50 pgMPL</td><td>KQ 103</td><td> 5846</td><td> 1600</td><td> -</td><td> ++</td><td> +++</td>

<td></td><td>KQ 104 GTM</td><td> 16270 10665</td><td> 1600 1213</td><td>lack</td><td>lack</td><td>lack</td>

<td></td><td>KQ 105</td><td> 16170</td><td> 800</td><td> -</td><td> +</td><td> ++</td>

<td>20 pg gD2t</td><td>KQ 106</td><td> 4389</td><td> 800</td><td> -</td><td> -</td><td> -</td>

<td>aluminum hydroxide</td><td>KQ 107</td><td> 20440</td><td> 1600</td><td> -</td><td> ++</td><td> +++</td>

<td>20 pg MPL</td><td>KQ 108</td><td> 5613</td><td> 800</td><td> -</td><td> +</td><td> +</td>

<td></td><td>KQ 109 GTM</td><td> 6755 8876</td><td> 1600 1056</td><td>lack</td><td>lack</td><td>lack</td>

<td></td><td>KQ 110</td><td> 2486</td><td> 200</td><td> -</td><td> -</td><td> -</td>

<td></td><td>KQ 111</td><td> 9918</td><td> 800</td><td> -</td><td> ++</td><td> +++</td>

<td>20 pg gD2t</td><td>KQ 112</td><td> 2526</td><td> 400</td><td> -</td><td> -</td><td> -</td>

<td>aluminum hydroxide</td><td>KQ 113</td><td> 7137</td><td> 400</td><td> -</td><td> -</td><td> -</td>

<td>5 pgMPL</td><td>KQ 114 GTM</td><td> 8396 5181</td><td> 400 400</td><td>lack</td><td>lack</td><td>lack</td>

* measured in 6 cdii after IIIGTM = geometry measure titer ELISA titer = midpoint titer NEUTRA titer titer = inverse of the highest dilution giving 100% protection against cytopathogenicity ** skin test 14 day after II multiple sclerosis - 24 hours reading + = 1 mm ++ = 1-5 mm +++ =&gt; 5 mm

Table 7

<td>Time</td><td>N</td><td>seroconversion</td><td> %</td><td>GMT</td><td>Min. titre</td><td>Max. titre</td>

<td>aluminum hydroxide (500 pg), HBsAg before</td><td> 20</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td>PI (month 1)</td><td> 20</td><td> 10</td><td> 50</td><td> 6</td><td> 1</td><td> 58</td>

<td>PII (month 3)</td><td> 20</td><td> 19</td><td> 95</td><td> 80</td><td> 7</td><td> 565</td>

<td>aluminum hydroxide (100 pg), HBsAg before</td><td> 20</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td>PI (month 1)</td><td> 18</td><td> 7</td><td> 36,8</td><td> 4</td><td> 1</td><td> 56</td>

<td>PII (month 3)</td><td> 19</td><td> 18</td><td> 94,7</td><td> 24</td><td> 2</td><td> 320</td>

<td>aluminum hydroxide (100</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>pg), HBsAg before</td><td> 20</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td>PI (month 1)</td><td> 20</td><td> 12</td><td> 60</td><td> 10</td><td> 1</td><td> 66</td>

<td>PII (month 3)</td><td> 20</td><td> 20</td><td> 100</td><td> 73</td><td> 6</td><td> 605</td>

178 578

Table 8

Immunogenicity of clinical OspA strains in humans Anti-OspA in the LA-2 inhibition test (ng LA-2 equivalents / ml) (GTM)

<td>Vaccine</td><td>Before day 0</td><td>After I 30 day 28</td><td>After II 30 day 56</td><td>After III 30 day 84</td>

<td>NS1-OspA on aluminum hydroxide</td><td> 118</td><td> 233</td><td> 409</td><td> 768</td>

<td>SC (%)</td><td> 2,6</td><td> 77,2</td><td> 86,5</td><td> 100</td>

<td>NS1-OspA + MPL on aluminum hydroxide</td><td> 134</td><td> 269</td><td> 865</td><td> 2424</td>

<td>SC (%)</td><td> 2,6</td><td> 88,6</td><td> 97,2</td><td> 100</td>

N = 80 pg / dose

intramuscular

Table 9

Effect of increasing dose of MPL (&gt; 500 nm) on the immunogenicity of recHBsAg adsorbed on Al (OH) 3

<td rowspan="3">Amount of MPL (pg / dose)</td><td colspan="4">Anti-HBs response</td>

<td colspan="2">IgG fusion</td><td colspan="2">IgG2a</td>

<td>day 14</td><td>day 21</td><td>day 14</td><td>day 21</td>

<td> 0*</td><td> 69</td><td> 743</td><td> 3.2</td><td> 11</td>

<td> 3.13</td><td> 122</td><td> 541</td><td> 3.8</td><td> 20</td>

<td> 6.25</td><td> 296</td><td> 882</td><td> 6.4</td><td> 24</td>

<td> 12.5</td><td> 371</td><td> 1359</td><td> 10</td><td> 48</td>

<td> 25</td><td> 456</td><td> 1493</td><td> 18</td><td> 138</td>

<td> 50</td><td> 403</td><td> 1776</td><td> 33</td><td> 242</td>

* HBsAg on Al.

Table 10

Comparison of 3 clinical sets with and without MPL. AUSAB reply

<td>Set</td><td>HBsAg dose for Al (OH)₃ (PG)</td><td>MPL dose (pg)</td><td colspan="2">GMT anti-HBs (mlU / ml)</td>

<td>DSAH16</td><td> 2,5</td><td> 0</td><td> 0,75</td><td> 15,1</td>

<td>DSAR501</td><td> 2,5</td><td> 6,25</td><td> 12,4</td><td> 96,7</td>

<td>DSAR502</td><td> 2,5</td><td> 6,25</td><td> 41,9</td><td> 89,2</td>

178 578

Table 11

Comparison of 2 clinical sets with and without MPL (&gt; 500 nm). Anti-HBs IgG and IgG2a response

<td rowspan="3">Set</td><td rowspan="3">HBsAg dose for Al (OH)₃(PG)</td><td rowspan="3">MPL dose (pg)</td><td colspan="4">Anti-HBs response</td>

<td colspan="2">IgG</td><td colspan="2">IgG2a</td>

<td>d15</td><td>d21</td><td>d15</td><td>d21</td>

<td>DSAH16</td><td> 2,5</td><td> 0</td><td> 20</td><td> 178</td><td> &lt;5</td><td> 5</td>

<td>DSAR502</td><td> 2,5</td><td> 6,25</td><td> 113</td><td> 641</td><td> &lt;5</td><td> 28</td>

Table 12

Effect of MPL dose (&lt;100 nm) on the immunogenicity of recHBsAg adsorbed on Al (OH) 3

<td rowspan="3">Dose HBsAg adsorb, Al (OH)₃(PG)</td><td rowspan="3">MPL dose &lt;100 nm (pg)</td><td colspan="6">Anti-HBs response</td>

<td colspan="2">Total IG</td><td colspan="2">IgG</td><td colspan="2">IgG2a</td>

<td>d15</td><td>d21</td><td>d15</td><td>d21</td><td>d15</td><td>d21</td>

<td> 1</td><td> 0</td><td> 30</td><td> 637</td><td> 67</td><td> 516</td><td> 15</td><td> 99</td>

<td> 1</td><td> 3,12</td><td> 312</td><td> 2302</td><td> 335</td><td> 3532</td><td> 167</td><td> 1752</td>

<td> 1</td><td> 6,25</td><td> 538</td><td> 2719</td><td> 856</td><td> 3932</td><td> 261</td><td> 2521</td>

<td> 1</td><td> 12,5</td><td> 396</td><td> 2104</td><td> 485</td><td> 3625</td><td> 125</td><td> 139</td>

<td> 1</td><td> 25,0</td><td> 38</td><td> 446</td><td> 141</td><td> 638</td><td> 28</td><td> 233</td>

Table 13

Effect of MPL (&gt; 500 nm) on HBsAg-specific Th1 cell induction in Balb / c mice

<td rowspan="3">Dose HBsAg (Pg / mouse)</td><td rowspan="3">Composition</td><td colspan="9">In vitro secretion</td>

<td colspan="3">IL-2 (SI)</td><td colspan="3">INF-γ (pg / ml)</td><td colspan="3">IL-4 (pg / ml)</td>

<td>24</td><td>48h</td><td>72h</td><td>24</td><td>48h</td><td>72h</td><td>24</td><td>48h</td><td>72h</td>

<td> 20</td><td>FCA</td><td> 1,3</td><td> 2,0</td><td> 8,0</td><td> &lt;125</td><td> &lt;125</td><td> 385</td><td>NT</td><td>NT</td><td>NT</td>

<td> -</td><td>FCA</td><td> 0,7</td><td> 1,8</td><td> 0,7</td><td> &lt;125</td><td> &lt;125</td><td> &lt;125</td><td>NT</td><td>NT</td><td>NT</td>

<td> 20</td><td>Al (OH)₃</td><td> 1,0</td><td> 1,4</td><td> 1,2</td><td> &lt;125</td><td> &lt;125</td><td> &lt;125</td><td> &lt;40</td><td> &lt;40</td><td> &lt;40</td>

<td> 20</td><td>Al (OH) + MPL (30 pg)</td><td> 2</td><td> 38</td><td> 10</td><td> &lt;125</td><td> 280</td><td> 280</td><td> &lt;40</td><td> &lt;40</td><td> &lt;40</td>

Following the one described in the text, lymph node cells were cultured from monocytes, cultured with 5 pg recHBsAg for the indicated period and IL-2, INF-γ and IL-4 secretion were measured using a VDA2 T cell line and two commercial ELISA kits

178 578

Table 14

Effect of different doses of MPL (&lt;100 nm) on the induction of HBsAg-specific Thi cells

<td rowspan="2">HBsAg dose (p / mouse)</td><td rowspan="2">Dose MPL</td><td colspan="7">In vitro secretion</td>

<td colspan="3">IL-2 (SI)</td><td colspan="4">INF-γ (pg / ml)</td>

<td></td><td></td><td>24 h</td><td>48 h</td><td>72 h</td><td>24 h</td><td>48 h</td><td>72 h</td><td>96 h</td>

<td> 20</td><td> 0</td><td> 2,6</td><td> 28</td><td> 21,8</td><td> &lt;67</td><td> &lt;67</td><td> &lt;67</td><td> &lt;67</td>

<td> 20</td><td> 7,5</td><td> 207</td><td> 173</td><td> 58</td><td> &lt; 67</td><td> 207</td><td> 522</td><td> 698</td>

<td> 20</td><td> 15</td><td> 270</td><td> 71</td><td> 36</td><td> 275</td><td> 878</td><td> 1249</td><td> 1582</td>

<td> 20</td><td> 30</td><td> 41</td><td> 59</td><td> 36</td><td> &lt;67</td><td> &lt;67</td><td> &lt;67</td><td> 207</td>

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