MXPA97009732A - Vaccine comprising a conjugate of antigen depolisacarido protein carrier and protein carrier li - Google Patents

Abstract

The present invention relates to combination vaccines comprising a conjugated polysaccharide antigen linked to a carrier protein, and wherein the carrier protein is also present as a free antigen in the composition of the cell, characterized in that the ratio of the invention relates to a multivalent vaccine, which is a vaccine for the improvement or treatment of more than one state of infirmity. The present invention also relates to the production and use of such vaccine in medicine.

Description

VACCINE COMPRISING A CONJUGATE OF POLYACARIDE ANTIGEN - CARRIER PROTEIN AND FREE CARRIER PROTEIN The present invention relates to the combination of vaccines comprising a polysaccharide antigen conjugated to a carrier protein, and wherein the carrier protein is also present as a free antigen in the composition of the vaccine. In particular, the composition of the vaccine of the present invention refers to a multivalent vaccine, that is, a vaccine for the improvement or treatment of more than one state of disease. The present invention also relates to the production and use of such a vaccine in medicine. Vaccines that use polysaccharides are known in the art. For example, a vaccine for the prevention of Haemophilus influenzae b (Hib) infections is based on capsular polysaccharide (PRP) conjugated to a carrier protein. Normally the porterant protein is a tetanus toxoid or diphtheria. It has also been suggested to produce a vaccine for the prevention of Streptococcus pneumonia, which is based on a capsule polysaccharide conjugated to a carrier protein, such as tetanus toxoid or diphtheria toxoid. Examples of such conjugated vaccine antigens are described in US 4 365 1 70 US 4 673 574 EP 208 375 EP 477508 and EP 1 61 1 88. It is desirable to administer such conjugate vaccines with other antigens or vaccines at the same time and this may involve multiple injections. Problems associated with multiple injections include a more complicated administration procedure and a large total injection volume. This is a particularly acute problem when the vaccine for infants is intended. Consequently, it has been proposed to produce combination vaccines. A well-known combination vaccine provides protection against diphtheria, tetanus and B. pertussis infections. This vaccine comprises a component (either B. Pertussis dead cell or pertussis "accellular", which typically consists of up to three antigens - PT, FHA and 69kDa) and diphtheria toxoid and tetanus toxin. Such vaccines are frequently referred to as DTPw (whole cell) or DTPa ("accellular"). It would be desirable to add vaccines of polysaccharide conjugate to such a combination. However, we have found that the simple mixing of the components results in a reduction of titres for the polysaccharide component and that the persistence of protective levels of such antibody titres is reduced. Thus, it has been observed that the immunogenicity of conjugated PRP Hib polysaccharide, for the detoxified Tetanus toxoid (TT) decreases in infants immunized with the TT-PRP conjugate combined with DTPa as compared with infants immunized with TT-PRP conjugate and DTaP concomitantly, but in separate sites. In the monovalent situation, that is, when a vaccine comprises only a simple polysaccharide (PS) conjugated with a carrier-borne protein, the higher the content of the conjugate, the higher the antibody response to the conjugate. polysaccharide. This is thought to be due to the fact that the induction of a T cell-dependent polysaccharide-specific antibody response requires the covalent coupling of a protein carrier to the polysaccharide. The protein carrier needs to be present at concentrations (ie, ratio of PS to protein) so that a good TH cell response is induced to the carrier, and that this TH response can recognize peptides expressing PS-specific B cell clones. derivatives of the carrier protein covalently coupled. Typically, the monovalent vaccines of the prior art have a polysaccharide: protein carrier ratio of 1: 3 (weight: weight). Surprisingly and in complete contrast to the position in the monovalent vaccine, the present inventors have discovered that when the conjugated component of polysaccharide is part of a multivalent vaccine comprising a free form of the carrier protein, the immunogenicity of the polysaccharide component is improved by reducing the protein content of the conjugated antigen. Accordingly, the present invention provides a combined vaccine containing a polysaccharide antigen conjugated to a carrier protein, and wherein the carrier protein is also present as a free antigen characterized in that the ratio of polysaccharide to carrier protein is about 1: 0.3 to 1: 2. A preferred ratio is 1:05 to 1: 1.5, typically in the order of 1: 1. The polysaccharide conjugate can be prepared by any known coupling technique via a thioether linkage. This method of conjugation depends on the activation of the polysaccharide with 1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAp) to form a cyanate ester. The activated polysaccharide can thus be coupled directly or via a spacer group to an amino group on the carrier protein. Preferably, the cyanate ester is coupled with hexane diamine and the amino-derivatized polysaccharide is conjugated to the carrier protein using heteroligation chemistry that involves the formation of the thioether linkage. Such conjugates are described in the published PCT application 2093/15760 Uniformed Services University. The conjugates can also be prepared by direct reducing amination methods as described in US 4365170 (Jennings) and US 4673574 (Anderson). Other methods are described in EP-0-161-188, EP-208375 and EP-0-477508. A further method involves the coupling of an activated polysaccharide of cyanogenbromide derivative with a hydrazide adipic acid (ADH) to the protein carrier by carbodiimide condensation (Chu et al., Infect. Immunity, 1983245256). Preferably, the carrier protein is an antigen derived from a Tetanus or Diphtheria preferably tetanus toxoid or diphtheria toxoid. The carrier can also be derived from Bordetella. Typically the antigen is a non-toxic derivative of a tetanus or diphtheria toxin. Preferably, the compositions of the invention contain a suitable auxiliary. A preferred auxiliary is MPL (3-deacylated monophosphoryl lipid A, also known as 3D-MPL). 3D-MPL is known from GB 2 220 211 (Ribi) as a mixture of 3 De-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains and is manufactured by Ribi Immunochem, Montana. A preferred form of MPL is described in International Patent Application 92/116556.

Another auxiliary, which can be used in the present invention is known as QS21. QS21 is a purified non-toxic fraction Hplc of a saponin from the bark of the South American tree Quillaya saponaria molina and its production method is described (as QA21) in US Pat. No. 5,057,540. The combinations of QS21 and 3 D-MPL are known to produce a synergistically effective vaccine formulation and are described in the international patent application WO 94/00153. Frequently, the Hib component vaccines of the invention will not require any specific carrier, and will be formulated in an aqueous or any other pharmaceutically acceptable buffer. In some cases it may be that the entire vaccine of the present invention is presented in an oil-in-water emulsion, or other suitable vehicle, such as for example, liposomes, microspheres or encapsulated antigen particles. The vaccine formulations may contain additional antigens. Antigenic compositions or antigens known in the art can be used in the compositions of the invention, including antigens or antigenic compositions derived from HIV-1 (such as gp120 or gp160), animal or human herpes viruses, such as gD or derivatives of the same or Immediate Early proteins such as ICP27 from HSV1 or HSV2, cytomegalovirus (especially human) (such as gB or derivatives thereof). Varicella Zoster Virus (such as gpl, II or III), or a hepatitis virus such as hepatitis B virus, for example Hepatitis B surface antigen or a derivative thereof, hepatitis A virus, hepatitis C virus and hepatitis E virus, or other viral pathogens, such as respiratory syncytial virus, human papillomavirus or influenza virus, or poliovirus such as inactivated poliovirus (I PV) or derived from bacterial pathogens, such as Diphtheria (D), Tetanus (T), Salmonella, Neisseria, particularly Neisseria Meningitis A, B or C. Borrelia (for example OspA or OspB or derivatives thereof), or Chlamydia or Bordetella by example P.69, PT and FHA of B. Pertussi (P), or derived from parasites such as Plasmodium or Toxoplasma. Particularly preferred vaccines of the invention include, DTPa Hib; and DTPa Hib Hep B; and DTPa Hib Hep B I PV. It will be appreciated that in the combination vaccine of the invention, the Hib component can be formulated with the other antigens just prior to administration. Thus, a freeze-dried Hib component can be reconstituted before use, by mixing it with the aqueous formulation of other antigens. The above combinations may optionally include a component, which is protective against hepatitis A. Suitable components for use in such vaccines are already commercially available and details may be obtained from the World Health Organization. For example, the IPV component can be the Salk inactivated polio vaccine. The diphtheria, tetanus and pertussis vaccines may comprise an "accellu lar" product, such as Infanrix DTPa (SmithKine Beecham Biologicals). The component that provides protection against Hepatitis A is preferably the product known as "H avris" (Smith Kine Beecham Biologicals) with a killed attenuated vaccine derived from the H M-1 75 species of HAV [see "I nactivated Candidate" Vaccines for Hepatitis A "by FE Andre, A Hepburn and E: D'Hondt, Proa Med. Virol. Vol 37, pages 72-95 (1990) and the monograph of the product "Havrix" published by SmithKine Beecham biologicals (1991)]. The Hepatitis B component may comprise the "S" antigen as in "Engerix-B". Advantageously, the Haemophilus Influenzae B vaccine or the combination vaccine according to the invention is a pediatric vaccine. The preparation of the vaccine is described generally in Vaccine Design - the subunit and Adjuvant approach, edited by Michael F Powell and Mark Newman, Plenum Press. The encapsulation within liposomes is described, for example, by Fullerton, US Patent 4,235,877. The conjugation of proteins with macromolecules is described, for example, by Likhite, U.S. Patents 4,372,945 and by Armor et al., U.S. Patent 4,474,757. The amount of antigen in each vaccine dose is selected as an amount, which induces an immunoprotective response without significant adverse side effects in typical vaccinates. Such amount will vary depending on which specific immunogenic substances are being used. Generally, it is expected that each dose will comprise 1-100ug of the total immunogenic substance, preferably 2-100ug, most preferably 4-40ug. An optimal amount for a particular vaccine can be ascertained by standard studies involving the observation of antibody titers and other responses in subjects. Following an initial vaccination, subjects can receive one or two booster injections at approximately 4 week intervals. The following examples will illustrate the invention.

Examples Example 1 a) Vaccine comprising DTPa - Hib PRP-TT - and HbsAg. Production of conjugate Hib TT Materials and Methods Materials Hib PRP is extracted and purified from inactivated cells. The purified material meets the WHO and US specifications in terms of residual protein, nucleic acid, endotoxin, structural sugars and molecular size distribution. The tetanus toxoid produced by Behringwerke also meets WHO specifications. The material is additionally pu rified by acid pH precipitation and gel filtration chromatography to isolate the monomeric form (1 50 kDa) from TT.

Activation and coupling chemistry 20 mg of Hib PRP were dissolved in 4 ml of NaCl 2 M 1 50 mcl of CDAP (1-cyano-4-dimethylamino-pyridine io tetraflu oroborate) was added to the polysaccharide solution (from one solution 1000 mg / ml support in acetonitrile). One minute later, 300 mcl of 0.2 M triethylamine was added. Activation of the polysaccharide was carried out for 2 minutes at 0 ° C to pH 9.5- 1 0. mg or 40 mg of tetanus toxoid (initial ratio of PRP / Protein 1/1 or Vi) at a concentration of 15 mg / ml was added, and the coupling reaction was performed at 25 ° C for 1 hour. The reaction was then quenched for 1 hour at 25 ° C with 3 ml of a 1 M glycine solution of pH 5.0, and then overnight at 4 ° C. The conjugate is purified by gel filtration chromatography using a Sephacryl H R 500 gel filtration column equilibrated with 0.2 M NaCI. The protein and carbohydrate content of the levigated fractions was determined. The conjugate was combined and displayed in a sterile manner. The PRP / protein ratio and the free PS content in the conjugate preparation were determined.

Example 1 b) Formulation of DTP combination vaccine Hib PRP TT HbsAg The formulation of DTPa (comprising diphtheria toxoid, tetanus toxoid, tosin erin toxoid, pertactin and FHA) with or without Hepatitis B vaccines are known in the art. technique. For example, such a vaccine is described in International Patent Application No. WO 93/241 48. The Conjugate as described in Example 1 was added to the vaccine and mixed prior to injection for studies of immunogenicity. In the present studies, the following steps were undertaken: • Adjustment of the content of f-enoxi-ethanol (bacteriostatic) to 5 mg / ml • Adjustment of pH to 6.1 + 0.1 • Adjustment of PRP concentration to 1 00 mcg / ml • Addition of DTPaH BV (0.5 ml) to 0.1 ml of PRP-CDAP-TT solution.

The final composition of the vaccine was for a dose: PRP'S 10 mcg D 25 Lf T 10 Lf PT 25 mcg FHA 25 mcg 69K 8 mcg HepB 10 mcg A1 (OH) 3 Behring 0.35 mg A1 (OH) 3 Superpos 0.15 mg A1 PO4 Superpos 0.20 mg NaCl 150 mM 2fEt-OH 3 g (? -5 mg / ml) PH 6.1 + 0.1 Vol 0.6 ml Example 2 Immunogenicity The immunogenicity of the proportions of polysaccharide derived from haemophilus Influenzae type B: protein (weight: weight) (1: 3, 1: 2 and 1: 1), using two different coupling chemistries, is compared after a first and second inoculation in young OFA rats. The data obtained at 28 days after the first inoculation of the vaccine clearly shows an improved anti-PS antibody titer that correlates with a decreased protein content in the conjugate vaccine, when the conjugated antigen is combined with DTPa Hepatitis B, but not when the conjugate is injected alone. This is in clear contrast to the situation observed when the conjugate vaccine is provided alone, where the PS-minor-TT ratio (ie more carrier protein) results in a better antibody response to the polysaccharide (see day 42 anti-PS group title). 1 to 3 going from 25 to 18 to 5 μg / ml with a decreasing amount of TT, while ranging from .3, 4 to 8 to 19 μg / ml for the same contents of TT, but when given in combination with the DTPa He B vaccine Example 3 Effect of the TT / PS ratio on the compatibility of Hib with DTPa Groups of 10 rats (OFA, 5 weeks of age, female) were immunized 2 times at 24-day intervals by the subcutaneous route with the different TT.PRP vaccines. provided in 2 doses: 0.5? PS / rat (table 2) or 0.05? PS / rat (table 3). The Hib001 vaccine was reconstituted with a saline solution or 1 dose of DTPa (lot 119) to have a concentration of: 10? PS / ml. The liquid vaccines were diluted with saline or 1 human dose of DTPa to have: 2.5? PS / ml or: 0.25? PS / ml. The rats were injected with 200 μl of each vaccine within one hour after dilution. The animals were bled on day 24 and day 38, and the anti-PRP IgG Abs was measured by ELIS in individual sera and expressed in? / Ml. The GMT was calculated for each group.

Example 4 Comparison of Immunogenicity of DTPa Vaccine, and DTPa Hepatitis B mixed with a low or "normal" ratio PRP: TT Groups of 1 0 rats (OFA, 1 week old) were immunized three times (SC) at two week intervals with 0.5 μg of PS combined 1 hour before injection with 1 / 20- of a human dose of DTaP or DTaP HepB. The animals were bled 2 weeks after the third dose and the antibodies against pertussis toxoid (PT), FHA, pertactin (69k), diphtheria toxoid (D), tetanus toxoid (T) and Hepatitis B surface antigen. (H BS), were measured by Elisa. The titers, expressed as a dilution of average amount using a reference, are shown in Table 4. The results of this experiment together with the summary of Example 3 demonstrated that the Hib response is intensified in a combination vaccine, if the amount of protein in the conjugate is reduced. Moreover, there is no significant interference to the immune response of any other component of the vaccine.

TABLE 1 TABLE 2 TABLE 3 TABLE 4

Claims (1)

1. REIVI NDICATIONS A combination vaccine comprising: (a) a polysaccharide antigen conjugated to a carrier protein; and (b) a free carrier protein; characterized in that the ratio of polysaccharide to carrier protein in the conjugate is from 1: 0.3 to 1: 2 (w / w). A combination vaccine as claimed in claim 1, wherein the carrier protein is a diphtheria or tetanus antigen. A combination vaccine as claimed in claims 1 or 2, wherein the ratio of polysaccharide: carrier protein in the conjugate is from 1: 05 to 1: 1.5 (w / w). A combination vaccine as claimed in claim 1, wherein the polysaccharide component is derived from Haemophilus Influenzae type B, or Streptococcus Pneu monia or Neisseria meningitis A, or B or C. A combination vaccine as claimed in claim 4, which also includes antigens capable of inducing protection against diphtheria, tetanus and pertussis infections. A combination vaccine as claimed in claim 4, or claim 5, further comprising an antigen derived from the hepatitis B virus. A combination vaccine as claimed in any of claims 4 to 6, further comprising an antigen derived from the vi rus. of hepatitis A. A combination vaccine as claimed in any of claims 4 to 7, further comprising an antigen capable of inducing immunity against polio. A combined vaccine as claimed in any of claims 4 to 7, further comprising an antigen capable of inducing immunity to Streptococcus pneumoniae and Haemophilus I nflu enzae. A combination vaccine according to any of claims 4 to 9, further comprising a suitable auxiliary. A vaccine as claimed in any of the previous claims, to be used in medicine. A method of treating a human susceptible to or suffering from an infectious disease comprises administering an effective amount of a vaccine as claimed in claims 1 to 10.