SA96170157B1 - composition vaccine antigen conjugated polsaccharide aluminum phosphate - Google Patents

Abstract

Abstract: This invention relates to a vaccine formulation for the prevention of infection with Haemophilus Influenzae Type (Hib) B, where the antigen is adsorbed on aluminum phosphate. It also relates to multivalent vaccines, ie a vaccine used to mitigate or treat more than one medical condition. This invention also relates to the production and use of these vaccines in medicine.

Description

b : Vaccine composition comprising a pi conjugated polysaccharide antigen Adsorbed on aluminum phosphate Full Description Background This invention relates to 303s vaccine formulations comprising Mga polysaccharide antigen Conjugated polysaccharide antigen associated with a carrier protein This invention is concerned with the formulation of a vaccine used to prevent infection with °Cus Haemophilus influenzae B (Hib) The antigen is adsorbed on aluminum phosphate It is also related to a multipurpose vaccine »; Any vaccine used to relieve or treat more than one condition. This invention also relates to the production and use of these vaccines in medicine. Vaccines containing polysaccharides have been known in this technique. For example, a vaccine against hemorrhagic influenza A virus (Hib) B is based on a capsular polysaccharide (PRP) encapsulated in a carrier protein. The polysaccharide consists of a ribose polymer, ribitol and catamoram phosphate. These vaccines are usually found in the form of simple formulations, that is, without adjuvants, and in one case a diluent containing aluminum hydroxide (Pedvax Hib) Vo 4230 (Merck Reconstitution) is used. Lyophilized conjugate: Lyophilised conjugate. The carrier protein is usually diphtheria or tetanus toxoid (3S 48) or outer membrane protein of N.Menigitidis 17. Examples of such conjugate antigens have been described in the US patent. 718, US Patent No. 5797708174, European Patent No. 7 018 _, European Patent No. 4,775078, European Patent No. ey

Y: Nal antigens It is desirable that such conjugate vaccines be administered with antigens or other vaccines at the same time and may involve multiple injections. Problems include increasing the volume of injection solution. This Tada associated with multiple injections is the method of giving the most serious problem, especially when administering the vaccine to an infant.

° Hence, it was proposed to produce combination vaccines. One of the well-known combination vaccines protects against diphtheria, tetanus, and whooping cough 5 B.pertussis. This vaccine includes a pertussis component of whole cells or acellular usually composed of two or three antigens (detoxified pertussis toxin PT and FHA) and often 64KDa, although

01 (to be limited to this) and under certain circumstances other antigens against pertussis 3, toxoided diphtheria and tetanus toxins may also be present. often; These vaccines are called DTPw or W01. It is desired to add other antigens to such a recombinant vaccine to prevent diseases such as hepatitis BB or polio. Polio: Vo. It is desirable to add polysaccharide conjugated vaccines. conjugate vaccines to a combination of this cdl, however, simple mixing of these components was found to lower mia levels of antibodies in the polysaccharide component. The inventors discovered that this reduction could be inhibited if the conjugated antigen was adsorbed on aluminum phosphate to convert it. In contrast, if antigen.9 is adsorbed on aluminum hydroxide, antibody levels in the polysaccharide component are completely reduced. $Y

GENERAL DESCRIPTION OF THE INVENTION Accordingly, the present invention presents a vaccine composition comprising a polysaccharide conjugated antigen 0 adsorbed on aluminum phosphate, preferably a polysaccharide (PRP) encapsulated from Hib protein conjugate pregnant. Preferably, the carrier protein is a diphtheria protein, a tetanus toxoid, a diphtheria protein, Crmjygy, or an outer membrane protein of the bacteria Jie meningitis N. meningitidis. The coupled polysaccharide may be prepared by any known conjugation technique. For example, a polysaccharide may be conjugated by means of a thioether bond, and this conjugation method depends on the activation of the dale saccharide with 1-cyano-;-di-aminopyridinium 1-cyano-4- dimethylamino pyridinium tetrafluoroborate (CDAP) to form a "Jud cyanate ester" and thus the activated polysaccharide may be conjugated directly or via a spacer group to an amino group on the carrier protein; It is preferable that the cyanate ester be coupled with a hexane diamine and the polysaccharide, Vo, derived from the amino derivatised polysaccharide, be coupled to the carrier protein according to the heteroligation chemistry methods that include the formation of a bond of ether thioether Conjugation of this kind was described in International Patent Application No. 97/157566 issued Gg to the Patent Cooperation Treaty at the Uniformed Services University The conjugate materials can also be prepared by direct reductive amination methods methods as described by Jennings US Patent No. 4,751970 and Anderson US Patent_No. 704. Other methods have been described in European Patent No. 1,151,144 and Malala No

Another method involves coupling a cyanogen bromide activated polysaccharide derivative with an adipic acid hydrazide (ADH) to a carrier protein by condensing it with a carbodiimide. Conjugation of this was described by Jul in the journal Imjmunity. 108 LC. C.Chu and collaborators; XOU-YE0 a VAY In a preferred embodiment of the present invention the ratio of the encapsulated polysaccharide (PRP) to the carrier protein is reduced from the normal ratio FY to 7:1-0,7:1. Conjugates at this lower ratio are preferred. Because even if they do not contain auxiliary additives, they do not face interference problems. In a preferred embodiment of the invention, it is preferable that the composition contain at least one other component at >0 chosen from antigens that protect against one or more diseases. These include: aS viral infections (HAV) A, diphtheria, tetanus, pertussis, hepatitis-3, and polio. Special recombinant vaccines within the scope of this invention include a vaccine composition comprising a combination of 27108 (antigens against diphtheria, tetanus and acellular pertussis) and Hib influenzae ve, a vaccine composition of antigen against influenza hemorrhagic influenza A0 and viral hepatitis (B) and a combination of Vaccine against diphtheria, tetanus and pertussis (DTPa), influenza A (Hib BA IL) and viral hepatitis 3 and a vaccine formulation including attenuated polio vaccine (TPV) (inactivated polio vaccine) and against diphtheria, tetanus and pertussis (DTPa), hemorrhagic influenza 8 and viral hepatitis 8 1. Optionally, the combinations mentioned in the previous paragraph include a component that prevents hepatitis A virus, and the appropriate components for use in these vaccines are already commercially available and details can be obtained from the World Health Organization (Word Health Organization). The attenuated polio vaccine component is SALK attenuated polio vaccine.” It could be a GY Laie diphtheria, tetanus and pertussis vaccine such as Cz

Infanrix against whooping cough, diphtheria and tetanus (DTPa) (from SmithKline Beecham Biologicals #DNA: 5). Preferably, the ingredient that prevents viral hepatitis A is known as “Havrix #20” (from Smithkline Beecham Biologicals), an attenuated killed vaccine from strain MHM178. (224-175) from HAV [see the paper “Inactivated Candidate Vaccines A for hepatitis A” by F.E.Andre, AHepburn, ED 53 E.DHondt “Prog Med.Virol” vol. YY pp. 95-77 in 490 2) and article “Havrix #1070” in one article published by Smithkline Beecham Biologicals in 1951. 1 M. The ye component of the vaccine against viral hepatitis 8 may include antigen S as in Engerix B 5. It is useful, according to this invention, to have the vaccine against hemorrhagic influenza 8 or against a group of recombinant diseases The subunit and adjuvant approach by Powell and Newman asl. The encapsulation of the vaccine in liposomes was described in US Patent No. 770/77 _ under the name of Fullerfon. AIS Likhite reported on the conjugation of proteins with macromolecules in a patent. US Patent No. 57977949; As disclosed by Armor et al. in US Patent No. 4497697817. Yes, the amount of conjugate antigen in each vaccine dose should be chosen to be an amount that induces a protective immune response without the appearance of harmful side effects in typical vaccines. This amount will vary depending on the particular immunogen used. In general, each dose is expected to contain an amount in the range from 1 to 1,000 micrograms of total immunostimulants, preferably in the range from 7 to 100 micrograms, and better than; to 0 Yo | μg. The optimal dose of each vaccine can be known by conducting standardized studies involving observation of antibody levels and other responses in treated individuals. After giving one or a booster injection, individuals can be given two booster injections (BY) vaccination, separated by a period of about; Weeks. In another aspect, the invention provides a method for producing a vaccine that includes the adsorption of the conjugated antigen. It is preferable that the adsorption occurs at the degree of aluminum phosphate. On aluminum phosphate, pH ranges from © to 1 and is best around 9.4. In another embodiment, the vaccine is freeze-dried for 1 hour; Alternatively, the vaccine of the present invention may be mixed with YE after it has settled for no less than other antigens in liquid form. The invention also provides the first medical use of such a vaccine. In another embodiment, the invention provides a method of prevention. From or reduce infections with influenza Veo This method includes giving an effective amount; Non-toxic from the vaccine of this invention to a patient who needs Al 8 Al 0 to describe the description The following examples illustrate this invention:

Aluminum Phosphate Tetanus Adsorbed on Aluminum Phosphate Against Influenza (TT) and Tetanus Toxoid (PRP) Encapsulated Polysaccharide Conjugate Gan 8 Hemorrhagic Cyanogen Bromide Cyanogen Bromide Conjugation A1 7 & 77 Valence covalent bonding according to a conjugation chemistry developed by the PRP Society. Other studies are conducted on 1988 NHS ChuC and collaborators (conducted by C. ChuC) and pneumococcus type 68 consisting of B conjugates. Immunity to Haemorrhagic Influenza E 100 pages Journal Infec.Immunity ¢protein and polysaccharide protein £Y

A

It is derived using cyanogen bromide under controlled conditions with cyanogen bromide PRP and the adipic hydrazide spacer activates a spacer of adipic hydrazide de gana

Liv Lodi 4aspd fi (PRP-AH) and after its derivation: the activated polysaccharide (17) is purified by condensation with carbo-diimide (PRP-AH) and the coupling of the two purified components is induced by diafiltration and ultrafiltration, then the conjugate material is purified by ultrafiltration Accuracy “carbodiimide °

TT 5 PRP for removal of reactant and unconjugated material from gel filtration and 77 PRP for synthesis of conjugates from 1-cyano-conjugate tetrafluoroborate; -Dimethylaminopyridinium B-1 1-cyano-4-dimethylamino-pyridinum tetrafluoroborate (molecular molarity 7) NaCl in + ml of natural Hib against PRP 0 mg of solvent ye 1 microliter of 1-cyano-;-dimethylaminopyridinium tetrafluoroborate YYO was added to a polysaccharide (CDAP) solution of 1-cyano-4-dimethylamino-pyridinum tetrafluoroborate mg/ml in acetonitrile 001 stock concentration solution (taken from a crude molecular polysaccharide solution) after 7 ( triethylamine from triethylamine Aly Sea £00) and (acetonitrile) was added while keeping it for one minute 01 seconds. The activation process was carried out at a pH of 0 on ice and for one minute at room temperature. 90 mg of tetanus toxoid was added (so that the ratio of the primary polysaccharide to the activated polysaccharide to the activated polysaccharide was carried out (¥: 1 protein at At room temperature for 1 hour; then water the reaction with © ml of glycine solution 1 min at room temperature To molecular at pH 0 for 1 (glycine - 0 at) and overnight at

Sephacryl © += J The conjugate was purified by gel filtration on a sphacryl H molecular column. The ratio of carbohydrates was determined 1:7 (NaCl, equilibrium was obtained in HR-500 solution and protein in each part; the conjugate material was mixed and filtered after sterilization by a carbohydrate gammameter). Minzart Cr) 7 5657 |

4 Adsorption on aluminum phosphate a sie am) Y.0 1 μg of conjugated polysaccharide tes of example 1-a was added to ve mg of aluminum phosphate phosphate and stirred the previous mixture for two hours, adjusting the pH at (0) and leaving the mixture to settle for one day at room temperature (0), then the adsorbent conjugate was left for an additional 9 days at a temperature ranging from “A” to (A) to prepare a dried product By lyophilization, the adsorbed product was diluted in 16.75 mg of lactose, resulting in a final composition product containing YO μg of polysaccharide/ml ty mg of aluminum ¢Jo/aluminium. The resulting composition was packaged in JS bottles. of which 0, + ml Ching by freezing. 0 To prepare a liquid gia, the conjugate adsorbed in water was diluted for injection with NaCl (mM) and 5 mg/ml of phenoxy ethanol resulting in a final composition To se micrograms ga polysaccharide per ml oe pile TY aluminum J/aluminium . : 1-D Prepare a vaccine formulation against diphtheria, tetanus and pertussis (acellular) in the presence or vo in the absence of hepatitis virus (b) Preparation of Gy by methods described in International Patent Application No. 97/741544 (SmithKline Beecham Biosciences Company). 1-e Preparation of a conjugate from PRP and 17 at a low percentage previously adsorbed on aluminum phosphate. The adjuvant was prepared in a manner similar to that described in the HY example, but using reduced Jia © from tetanus (i.e. 30 μg instead of 60 micrograms) yielded a product in which the ratio of polysaccharide to protein was 1:1 or .:1, then the conjugate was adsorbed on aluminum phosphate (Gy aluminum phosphate) according to the example method 1- Ag, and the final freeze-dried product contained 17.5 micrograms of conjugate and 6215 pile of pile 10,V0 5 AIPO, of lactose. This substance was reconstituted in 0.5 mL of water for injection prior to use at dan Yo acidity of cp Ee $y

: 1 example? and 77 previously adsorbed on aluminum phosphate PRP immunoprecipitation resulting from the conjugate of -HB-DTPa or DTPa mixed with ; aluminum phosphate, which was prepared in example 1-a, whether in the form of Hib. The conjugate of the two vaccines were mixed in (simple aluminum phosphate) or pre-adsorbed on aluminum phosphate 0 one hour before injection only, and rats were injected Age of DTPa-HB or DTPa (both cases) mixed with a dose of 1/70 each one week of the combination vaccine subcutaneously at a dose equal to 5 weeks after PRP (PRP). It is given to humans (0.0 μg of vaccine administration and serum collection after each vaccination dose to measure the levels of antibody to or not adsorbed) reconstituted in AIPO, (both adsorbed on Hib and controls contained JPRP Co vaccines .saline saline solution 1 week Lia rats age 10 each randomly vaccinated groups consisting of three times subcutaneously on day 0, day 14 and day (ago-OFA) (from the strain

Hib of the amount of the dose given to humans of the twenty 1/70 vaccine in a dose of (alll) of the dose given 1/70 (the dose amount of DTPa-HB or with DTPa alone or with - 1 or with DTPa lyophilized in saline (or in combinations with Hib for humans).Vaccine was reconstituted no later than 1 hour prior to vaccine administration. (DTPa-HB measured ON, Day 47 and Day YA Day VE and anesthetized rats bled on day (ELISA) by PRP antibody levels in individual serum and the levels were expressed in gamma/ml using a calibrated reference. At each point of time, the GMT confidence limits were calculated as the standard geometric mean of the levels produced after the third inoculation. Yao confidence limits on Hib phosphate does not modify the adsorption of the conjugate from (1) shown in Table WS Of its immunizing ability: Some antibodies were produced from aluminum phosphate aluminum polysaccharide after the second dose, and a good stimulant effect appeared after the third dose, as was observed in ve.

So human infants. The combination of Hib vaccine with DTPa or with DTPa-HB reduced the antibody response to PRP from three to A-fold. In the case of mixing the vaccine with DTPa-HB, this decrease was significant. In contrast, gb pre-vaccine adsorption of Hib on aluminum phosphate restores the antibody response to PRP to a level at least 0 equivalent to that obtained when using a simple vaccine.

Conclusion: Hib vaccine formulations adsorbed on aluminum phosphate can therefore solve the problem associated with mixing Hib vaccine with other childhood vaccine combinations.

i VY (1) Table previously adsorbed on aluminum phosphate TT PRP Immunoconjugate from a model of age rats DTPa-HB mixed with 7 or with aluminum phosphate every one week .: sree, | cw ona 67 Today YA Today VE asl Third Vaccine) | (after the third vaccine) aay | (SED (after first vaccine) | (after vaccine) + (Dhib-024) (119) DTPa+AIPO4 (16705) 1 term (veo ey (16705)DTPa-Hb+AIPO,

NY

Claims (1)

Vy protective elements: capsular polysaccharide containing an encapsulated polysaccharide combination vaccine Recombinant vaccine 1-1 carrier Haemophilus Influenzae B Antigenic influenzae Y Tetanus toxoid cdiphtheria toxoid 08m mixed With antigens against one or more diphtheria v toxoids Al antigens Al gay pertussis and tetanus toxoid ¢ Anti © Vga Hepatitis A virus A Choose from the following group: Hepatitis A virus ° And poliovirus Hepatitis B surface antigen B surface of hepatitis B virus 1 on the adsorbed conjugate material where the attenuated cInactivated Polio Virus is adsorbed 7 .aluminium phosphate aluminum phosphate A 1 7 - Recombinant Tg combination vaccine for protection element 1 » where the carrier protein Y is selected from the group consisting of: diphtheria toxoid protein 1 diphtheria 197 Diphtheria CRML 197 CRML and outer membrane protein for meningococci Meningococcal outer membrane protein ¢ -y 1 recombinant vaccine Gy combination vaccine for protective factor 1 where the carrier protein Y is conjugated to the capsular polysaccharide against influenza ¥ Haemophilus Influenzae B It is a tetanus toxoid Jl 3S 1 | ¢— a recombinant Gy combination vaccine for protectant 1 in which the ratio of polysaccharide Y against Haemophilus Influenzae B B to carrier protein G ,7:0 (carrier protein) to 0:1 (w/w). $Y " —o 1 a combination vaccine according to claim 1 where the conjugate is dried freeze-dried adsorbed conjugate Y zal 5 1 recombinant combination vaccine according to claim ©” in which conjugate 4 adsorbed, lyophilized, is suspended in water for injection . 1-1 method for producing a vaccine according to claim 1; includes conjugating a capsular polysaccharide 7 of an influenzae antigen Haemophilus Influenzae B ¥ with a carrier protein adsorption of the conjugate mentioned on aluminum phosphate and mix the product with diphtheria toxoid Tetanus toxoid and cpertussis antigen Another antigen 1 One or more choose from the group that consists of the virus Ada Sl For Hepatitis A virus, Hepatitis B surface B antigen A, and Inactivated Polio Virus. 8 A method for treating a patient suffering from or at risk of infection with the hemorrhagic influenza virus +0 vaccine, which includes administering an effective and safe dose of the “Haemophilus Influenzae Y. 1” vaccine composition according to compostion 7. kit pik - 1 includes a container: 7 contains lyophilized vaccine. The Y capsular polysaccharide mRNA consists of Haemophilus Influenzae Yoo antibody B conjugated to a carrier protein and adsorbed on ¢ aluminum phosphate and a second container containing antigens against diphtheria toxoid ° diphtheria toxoid tetanus toxoid and antigens against whooping cough 1 58 with one or more other antigens The elderly choose the group: $Y Vo Hepatitis A virus A surface antigen | Inactivated Polio Virus Hepatitis B surface antigen B A on coated polysaccharide Gly combination vaccine Recombinant vaccine 1 1 Haemophilus Influenzae B from antibody to influenza A capsular polysaccharide aluminum and adsorbent On aluminum phosphate tetanus toxoid and combined with tetanus toxoid “diphtheria toxoid” with the following antigens: diphtheria toxoid phosphate 1 acellular non-cellular pertussis antigens. tetanus toxoid Tetanus ° Hepatitis B surface B Hepatitis B surface antigen 6019518 5 hl Inactivated Polio Virus and attenuated poliovirus antigen a1 as on polysaccharide J dy combination vaccine #18 -11 1 Haemophilus Influenzae 8 4— 3 From an influenza antibody capsular polysaccharide Y alurninium adsorbed on aluminum phosphate tetanus toxoid conjugated with tetanus toxoid ¥ diphtheria toxoid with the following antigens: diphtheria toxoid $ phosphate “whole-cell pertussis antigens Cough Whole cell pertussis ctetanus toxoid Tetanus °. Hepatitis B surface antigen B and hepatitis B surface antigen B1 $Y