MXPA97010521A - A composition of vaccines including an antigen conjugated of polysaccharid adsorbided in alumi phosphate - Google Patents

Abstract

The present invention relates to a vaccine formulation for the prevention of infections of Haemophilus influenzae Type B (Hib) and where the antigen is absorbed in aluminum phosphate. The invention also relates to a multivalent vaccine, which is a vaccine for the relief or treatment of more than one disease state. The present invention also relates to the production and use of such vaccines in medicine.

Description

A VACCINATION COMPOSITION UNDERSTANDING A CONJUGATED POLYACARIDE ANTIGEN ADSORBIDO IN ALUMINUM PHOSPHATE The present invention relates to new vaccine formulations, comprising a conjugated polysaccharide antigen linked to a carrier protein. In particular, the invention relates to a vaccine formulation for the prevention of Haemophilus Influenzae Type B (Hib) infections and where the antigen is adsorbed to an aluminum phosphate. The invention also relates to a multivalent vaccine, which is a vaccine for the relief or treatment of more than one disease state The present invention also relates to the production and use of such vaccines in medicine. Vaccines using pohsacápdos are known in the art. For example, a vaccine for the prevention of Haemophilus infections. Influenzae b (Hib) are based on the conjugate of capsular polysaccharide (PRP) with a carrier protein The polysaccharide is a polymer of ribose, pbitol and phosphate These vaccines are normally present as simple formulations (ie without auxiliaries) Although in one case, (Pedvax Hib produced by Merck) a diluent containing aluminum hydroxide is used to reconstitute the lyophilized conjugate Typically the carrier protein is a tetanus or diphtheria toxoid or an outer membrane protein of N menigitidis Examples of such conjugate vaccine antigens are described in US 4 365 1 70, US 4 673 574, EP 208 375, EP 477508 and EP 161 188 It is desirable to administer such vaccines conjugated 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 is administered. intended for infants 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 whole cell or an acellular pertussis component that typically consists of two or three antigens - (detoxified PT, FHA and frequent, but not exclusively 69kDa) although under certain circumstances other B pertussis antigens may also be present and toxoid diphtheria and tetanus toxins Such vaccines are frequently referred to as DTPw or DTPa. Other antigens would be desirably added to such a combination vaccine for the prevention of diseases such as hepatitis B or polio. It would be desirable to add polysaccharide conjugate vaccines to such a combination. However, it has been found that mixing simple of the components results in a reduction of antibody titers for the polysaccharide component. The present inventors have discovered that this reduction can be inhibited if the conjugated antigen is adsorbed on aluminum phosphate. In contrast, if the antigen is adsorbed on aluminum hydroxide , there is a complete reduction of antibody titers for the polysaccharide component. Accordingly, the present invention provides a vaccine composition comprising a polysaccharide conjugated antigen adsorbed on aluminum phosphate. Preferably the antigen is a capsular polysaccharide (PRP) of Hib conjugated with a carrier protein. Preferably, the carrier protein is either tetanus toxoid or diphtheria, Diphtheria Crm197 protein or an outer membrane protein of a bacterium such as N menigitidis. The polysaccharide conjugate can be prepared by any known coupling technique. For example, the polysaccharide can be coupled via a thioether bond This method of conjugation depends on the activation of the pohsacápdo with 1-c? ano-4-d? met? lamino pipdinio tetrafluoroborato (CDAP) to form a cyanate ester In this way, the activated polysaccharide can be coupled directly or via a spacer group to an ammo group on the carrier protein Preferably, the cyanate ester is coupled with hexane diamine and the amino-derivative polysaccharide is conjugated to the carrier protein using heteroligation chemistry involving the formation of thioether linkage Such conjugates are described in the published PCT application WO93 / 15760 from Uniformed Services Umversity The conjugates can also be prepared by direct reductive 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 activated polysaccharide of cyanogen bromide derivative with adipic acid hydrazide (ADH) to the protein carrier by carbodiimide condensation. Such conjugation is described in Chu C et al Infect Immunity, 193 245 256. In a preferred embodiment of the invention the ratio of polysaccharide PRP to the carrier protein is reduced from a typical 1: 3 to 1: 03 to 1: 2. Such low-ratio conjugates are convenient, since even in a non-auxiliary state, they do not suffer from interference problems. In a preferred embodiment of the invention, the formulation preferably contains at least one other selected component of antigens, which provide protection against one or more of the following: Hepatitis A virus (HAV), diphtheria, tetanus, pertussis, Hepatitis B and polio. Particular combination vaccines within the scope of the invention include a combination vaccine formulation DTPa (diphtheria-tetanus-acellular pertussis) -Hib, a Hib-Hepatitis B vaccine formulation, a DTPa-Hib-Hepatitis B vaccine formulation, and an IPV (inactivated polio vaccine) -DTPa-Hib-Hepatitis B vaccine formulation. 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 the details They can be obtained from the World Health Organization. For example, the IPV component can be the Salk inactivated polio vaccine. The Diphtheria, Tetanus and Pertussis vaccine may comprise an acellular product such as Infanrix DTPa (SmithKine Beecham Biologicals). The component that provides protection against Hepatitis A is preferably the product known as "Havrix" (SmithKine Beecham Biologicals) which is a killed attenuated vaccine derived from the HM-175 species of HAV [see "Inactivated Candidate Vaccines for Hepatitis A" by FAITH Andre, A Hepburn and E. D'Hondt, Prog Med. Virol. Vol 37, pages 72-95 (1990) and the product monograph "Havrix" published by Smith KIine Beecham Biologicals (1991)] The Hepatitis B component may comprise the "S" antigen as in "Engerix-B". Advantageously, the Haemophilius Influenzae B or the combination vaccine according to the invention is a pediatric vaccine. The preparation of the vaccine is generally described in Vaccine Design - The Subunit and adjuvant approach Ed Powell and Newman; Pellum Press. Encapsulation within liposomes is described, for example, by Fullerton, US Patent 4,235,877. The conjugation of proteins to macromolecules is described, for example, by Likhite, U.S. Patent 4,372, 945 and by Armor et al. , U.S. Patent 4,474, 757. The amount of conjugated antigen in each vaccine dose is selected as an amount which induces a immunoprotective response without significant adverse side effects in typical vaccinates. Such amount will vary depending on which specific immunogens are employed. Generally, it is expected that each dose will comprise 1 -1000ug of total immunogen, preferably 2-100ug, more 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. In a further aspect according to the invention, there is provided a method for producing the vaccine comprising adsorbing the antigen of the conjugate to an aluminum phosphate. The adsorption is preferably made at a pH between 5 and 6, preferably to about 5.4. in one embodiment, the vaccine is dehydrated by freezing after being maintained for more than 24 hours. Alternatively, the vaccine of the invention can be combined with other antigens in a liquid form. The invention further provides the first medical use of such a vaccine. In a further embodiment, the invention provides a method for preventing or alleviating infections of Haemophilus Influenzae B, the method comprising administering an effective, non-toxic amount of the vaccine of the invention. The following examples illustrate the invention.

EXAMPLE 1 Vaccine formulation comprising polysaccharide HiB conjugated to Tetanus toxoid adsorbed on aluminum phosphate Synthesis of Tetanus toxoid conjugate (TT) capsular polysaccharide (PRP) of Haemophilus influenzae type B 1a Coupling of cyanogen bromide The covalent binding of PRP and TT is carried out by a coupling chemistry developed at the NIH (Chu C. et al. (1983), additional studies on the immunogenicity of pneumococcal polysaccharide protein conjugates type 6a and Haemophilus influenzae type B. Infection Immunity, 245-256). The PRP is activated under conditions controlled by cyanogen bromide and derivatized with an adipic hydrazide separator. After derivatization, the activated polysaccharide (PRP-AH) is purified by diafiltration. The coupling of the two purified components (PRP-AH and TT) is effected by carbodiimide condensation. The conjugate is then purified by ultrafiltration and gel filtration to remove the reagent and the TT and PR P unconjugated.

Synthesis of Conjugates PRP-TT 1 . b CDAP Coupling 30 mg of PR P Natural Hib were dissolved in 6 ml of 2M NaCl. 225 mcl of CDAP (1 c? ano-4-dimethylamino-pyridinium tetrafluoroborate) was added to the polysaccharide solution (100 mg / ml of stock solution in acetonitrile). 90 seconds later, 450 mcl of 0.2 M triethylamine were added. Activation was performed at pH 10.0 for 1 minute on ice and one minute at room temperature. 90 mg of tetanus toxoid (PS / initial portéin ratio of 1/3) were added to the activated polysaccharide and the coupling reaction was performed at room temperature for 1 hour. Then, the reaction was quenched with 3 ml of 1 M glycine solution, pH 5.0 for 30 minutes at room temperature and overnight at 4 ° C. The conjugate was purified by gel filtration on a HR 500 column of sephacryl equilibrated in NaCl 0.2M. The carbohydrate and protein content was determined in each fraction. The conjugate was combined and sterile filtered (Minisart membrane 0.222 μm).

Adsorption on aluminum phosphate 1 .c To 0.15 mg of aluminum phosphate were added 12.5 mcg of the polysaccharide conjugate of example 1 (a). This was stirred for two hours, the pH adjusted to 5.1. The mixture was allowed to stand for one day at room temperature and the adsorbed conjugate is then left for an additional 9 days at 2 to 8 ° C. To prepare a dehydrated product by freezing, the adsorbed product is diluted in lactose (1. 5.75 mg) to give a final composition of 25 mcg of polysaccharide / ml and 0.4 mg Al / ml and the resulting composition was filled into 0.5 ml vials and It was dehydrated by freezing. To prepare a liquid product, the adsorbed conjugate is diluted in water for injection with 150 mM NaCl and 5 mg / ml phenoxyethanol, to give a final composition of 20 mcg of polysaccharide / ml and 0.32 mg of Al / ml. 1 .d The formulation of a Diphtheria Tetanus and Pertussis (acellular) vaccine with and without hepatitis B was made according to the methods of WO 93/24148 (SmithKine Beecham Biologicals). 1 .e Preparation of a PRP-TT "low proportion" pre-adsorbed aluminum phosphate conjugate. The conjugate was prepared in a manner analogous to Example 1 a, but with reduced amount of Tetanus used (30mcg, 60mcg) to give a product with polysaccharide: Protein ratio of 1: 1 or 1: 2. The conjugate is then adsorbed to aluminum phosphate according to the method of example 1 c. The final preparation dehydrated by freezing contains 12.5 μg of conjugate, 0.15 mg of AIP04, 15.75 mg of lactose. This is reconstituted in 0.1 5 ml of water for injection before being used at a pH of 0.1 +/- 0.1.

Example 2: Immunogenicity of PRP-TT conjugate preadsorbed on aluminum phosphate and combined with DTPa or DTPa-HB The Hib conjugate of example 1 a), either simple or pre-adsorbed on AIPO4 (both vaccines were lyophilized) was mixed with DTPa or DTaP HB no more than 1 hour before injection and the combination was injected into baby rats (1 week old) by the subcutaneous route at a dose corresponding to? / 20ßs, human dose mo (0 5 μg PRP) Rats were boosted 2 weeks and 4 weeks later and serum was collected after each immunization to measure anti-PRP antibodies Controls included Hib vaccines (adsorbed or not in AIP04 ) reconstituted in saline Groups of 10 random baby rats (1-week OFA species) were immunized 3 times subcutaneously at 0-14-28 days with 1 / 20th of human dose of Hib vaccine, alone or combined with DTPa or DTaP HB (1 / 20th of human dose) The reconstitution of the lyophilized Hib vaccine with saline or combinations (DTPa or DTPa HB) was done less than 1 hour before the immunization. The rats were bled under anesthesia at 14- 28-42 and 56 days The anti-PRP antibodies were measured by ELISA in individual sera and the titers were expressed in? / Ml using a calibrated reference The GMT was calculated for each group and for each time point The confidence limits of 95 % were calculated for the titres obtained after the third immunization As shown in Table 1, the adsorption of the Hib conjugate in AIP04 does not modify its immunogenicity. Some anti-PS were produced after the second dose and a good reinforcing effect is shown after the third dose as seen in human babies The mixing of Hib vaccine with DTPa or DTPa HB reduces by 3 to 8 times the anti-PRP response and in the case of DTPa-HB, this decrease is important. In contrast, pre-adsorption of the Hib vaccine in AIPO4 restores the anti-PRP response to a level at least equivalent to that obtained with the single vaccine.

Conclusion: The formulation of Hib / aluminum phosphate has thus the potential to solve the compatibility problem encountered when mixing Hib with other pediatric combinations.

Table 1 Immunogenicity in a model of baby rats of PRP-TT conjugate pre adsorbed on AIPO4 and combined with DTPa or DTPa-HB

Claims (6)

2. ) A combination vaccine comprising a capsular polysaccharide of Haeomphilius influenzae B conjugated to a carrier protein and at least one other antigen, characterized in that the conjugate is adsorbed to aluminum phosphate. ) A combination vaccine comprising a capsular polysaccharide of Haemophilius influenzae B conjugated to a carrier protein, mixed with one or more antigens selected from the group: Hepatitis A virus, diphtheria, tetanus, pertussis, Hepatitis B and Polio; characterized in that the conjugate is adsorbed to aluminum phosphate. ) A combination vaccine as claimed in claim 1 or 2 comprising a capsular polysaccharide of Haemophilius influenzae B conjugated to a carrier protein; wherein the carrier protein is selected from the group comprising: Diphtheria toxoid, protein from
3. Diptheria CRm 197, Meningococcal outer membrane protein and Tetanus toxoid. ) A combination vaccine as claimed in any of claims 1 to 3, wherein the carrier protein conjugated to the capsular polysaccharide of Haemophilius influenzae B is tetanus toxoid. ) A combination vaccine as claimed in any of claims 1 to 4 wherein the proportion of the polysaccharide of Haemophilius influenzae B to carrier protein is from 1: 0.3 to 1: 2 (p: p).
4. ) A vaccine as claimed in any of claims 1 to
5. 5 wherein the adsorbed conjugate is dehydrated by freezing. ) A vaccine as claimed in any of claims 1 to
6. 6 wherein the adsorbed conjugate is suspended in water for injection. ) An assembly comprising a container of a dehydrated vaccine by freezing according to claim 6 and a second container with a vaccine against a second pathogen. A method for producing a vaccine according to claims 1 to 7 comprising conjugating a polysaccharide antigen to a protein carrier, mixing with one or more antigens selected from the group: Hepatitis A virus, diphtheria, tetanus, pertussis, Hepatitis B and Polio; and adsorbing said antigens in aluminum phosphate. 0) A vaccine composition as defined in any of claims 1 to 7 for use in medicine. 1) A method for treating a patient suffering from or susceptible to a Haemophilius influenzae B infection, comprising administering an effective and safe amount of a vaccine composition according to any of claims 1 to 7.