Immunogenic composition of haemophilus influenzae conjugated to protein d

Abstract

The present invention relates to a multi valent vaccine composition comprising antigens against Diphtheria toxoid (DT), Tetanus toxoid (TT), Whole-cell pertussis (wP) / Acellular Pertussis (aP), Polio / Inactivated Poliomyelitis (IPV), Hepatitis (Hep) and Haemophilus influenzae (Hib) wherein Haemophilus influenzae (Hib) conjugated to Protein D or protein D fragment as a carrier protein. This invention also relates to processes for their preparation and immunogenic compositions comprising them, wherein protein D induces protective responses against invasive infections caused by Haemophilus influenzae (Hib).

Description

[0001] IMMUNOGENIC COMPOSITION OF HAEMOPHILUS INFLUENZAE CONJUGATED TO PROTEIN D

[0002] Field of Invention:

[0003] The present invention relates to a multi valent vaccine composition comprising antigens against Diphtheria, Tetanus, Pertussis, Polio, Hepatitis B and Haemophilus influenzae-, wherein capsular polysaccharide antigen from Haemophilus influenza serotype b (Hib) is conjugated to Protein D or protein D fragment as a carrier protein. This invention also relates to processes for their preparation and immunogenic compositions comprising them, wherein protein D induces protective responses against invasive infections caused by Haemophilus influenzae.

[0004] Background of the Invention:

[0005] Haemophilus influenzae is responsible for significant morbidity and mortality worldwide in children younger than 5 years. In 1933, Fothergill and Wright demonstrated that the great majority of Hib meningitis cases occurred in children younger than 5 years.

[0006] The first-generation Hib vaccine contained the pure polysaccharide of the Hib capsule. The polysaccharide vaccine was introduced in the early 1980s but was poorly immunogenic in children younger than 18 months, the age group in which the Hib disease burden is highest. Hib conjugate vaccines were introduced in 1985.

[0007] Polysaccharides are T-cell independent antigens able to directly stimulate B cells to produce antibodies. Disease burden caused by polysaccharide-encapsulated bacteria is highest in the first year of life, where plain polysaccharides are not generally immunogenic, limiting their use as vaccines. This limitation has been overcome by covalent coupling carbohydrate antigens to proteins that provide T cell epitopes. Haemophilus influenzae is a significant human- specific pathogen colonizing in the mucosa of the upper respiratory tract. H. influenzae isolates can be divided into 2 major groups: the encapsulated strains, many of which cause invasive diseases and the non-encapsulated, non-typeable (NT) strains, which are responsible for the majority of mucosal H. influenza infections. Encapsulated forms can penetrate the epithelium of the nasopharynx and invade blood capillaries, with their polysaccharide capsule allowing them to resist phagocytosis and complement- mediated lysis in nonimmune hosts. Six serotypes (a— f) have been identified; H. influenza serotype b (Hib) is the leading cause of invasive infections in children.

[0008] In regions of the world where a conjugate vaccine is not introduced, sepsis, meningitis, and pneumonia caused by Hib are still a major burden of disease.

[0009] Non-typeable H. influenzae strains are rarely associated with invasive disease in healthy children and adults, but they are associated with respiratory tract infections in both populations. These strains are frequently (up to 40% of the time) the cause of acute otitis media (AOM) in children. In addition, NT H. influenzae strains are also commonly isolated in purulent secretions of patients with cystic fibrosis and chronic obstructive pulmonary disease. The burden of disease caused by Nontypeable H. influenza justifies immune prophylaxis preferentially by active vaccination. A number of NT H. influenzae proteins have been evaluated in preclinical studies for their potential as vaccine antigen. Identifying ideal NT H. influenzae vaccine candidates has not been easy, because NT H. influenzae demonstrates extensive sequence and antigenic variation among gene products interacting with the immune system, such as outer membrane proteins, adhesins, lipopolysaccharides, and secreted virulence factors.

[0010] No-ntypeable Haemophilus influenzae is a significant pathogen in children, causing otitis media, sinusitis, conjunctivitis, pneumonia, and occasionally invasive infections. H. influenzae type b conjugate vaccines have no effect on infections caused by non-typeable strains because non-typeable strains are non-encapsulated. Approximately, one-third of episodes of otitis media are caused by non-typeable H. influenzae and the bacterium is the most common cause of recurrent otitis media.

[0011] The development of vaccines against polysaccharide-encapsulated pathogens e.g. Haemophilus influenzae type b is challenging because polysaccharides do not elicit a strong and long-lasting immune response (i.e. T-cell independent). This can be overcome by conjugating the polysaccharide to a protein carrier (e.g. tetanus toxoid, cross -reacting material 197 [CRM]), which vastly improves the immune response and induces memory to the polysaccharide (T-cell dependent).

[0012] Here, is a list of Discontinued U.S. Vaccines which comprises Hib antigen conjugated with carrier protein Multicomponent combination vaccine compositions comprising Hib antigen and which have been developed until now are reviewed for conjugation properties of Haemophilus b polysaccharide antigen. It was observed from referred prior arts that only four carrier proteins have been used to develop conjugated Hib vaccines i.e. diphtheria toxoid (PRP-D), tetanus toxoid (PRP-T), a mutant diphtheria toxin (HbOC / CRM) and an outer membrane protein of meningococcus (PRP-OMP). PRP-T conjugates achieve protective antibody levels only after the administration of the 2nd dose of the vaccine.

[0013] None of the marketed vaccine composition comprising Hib antigen possesses Hib conjugated with protein D as a carrier protein.

[0014] W0200056360 also cited the disadvantages of using these commonly used protein carriers. Despite the common use of these carriers and their success in the induction of anti polysaccharide antibody responses they are associated with several disadvantages.

[0015] It is known that antigen specific immune responses may be suppressed (epitope suppression) by the presence of preexisting antibodies directed against the carrier, For e.g. In the population at large, a very high percentage of people will have preexisting immunity to both DT and TT as people are routinely vaccinated with these antigens.

[0016] In addition, for vaccines which require regular boosting, the use of highly immunogenic carriers such as TT and DT are likely to suppress the polysaccharide antibody response after several injections. These multiple vaccinations may also be accompanied by undesirable reactions such as delayed type hyper-responsiveness.

[0017] Therefore polysaccharide based vaccine requires a balance between the necessity to use a carrier working in all recipients, the induction of high levels of antipolysaccharide antibody responses, while selecting a carrier protein.

[0018] Protein D is an antigenically conserved, surface-localized outer membrane protein of all Haemophilus influenzae species, including non-typeable (NT) H. influenzae,

[0019] It is also reported in the prior art that the increased use of acellular pertussis combination vaccines in the UK from late 1999 onwards precipitated a large number of Hib vaccine failures. Therefore an object of the present invention is to develop a multicomponent combination vaccine composition, wherein Hib is conjugated with protein D as a carrier protein.

[0020] Summary of the Invention:

[0021] The main aspect of the present invention is to develop a multicomponent combination vaccine composition, wherein Hib is conjugated with protein D or protein D fragment as a carrier protein, wherein protein D induces protective responses against invasive infections caused by Haemophilus influenzae.

[0022] Present invention also comprises process of conjugation of Hib and Protein D or protein D fragment as a carrier protein

[0023] Present invention also relates to a combination vaccine composition containing structurally unrelated antigens, which comprises Hib antigen conjugated with protein D, wherein, in addition to the response against other antigens, protective responses is induced against protein D for invasive infections caused by non-type able Haemophilus influenzae.

[0024] Present invention also relates to a combination vaccine composition which comprises Hib antigen conjugated with protein D, wherein vaccine composition is devoid of issues like carrier induced epitope suppression, antigen competition, immune interference and epitopic load.

[0025] Brief Description of Figures:

[0026] Figure 1: Anti-PRP response against Hib polysaccharide.

[0027] Figure 2: Anti- Protein D response against carrier protein PrD.

[0028] Figure 3: Bactericidal activity of Vaccine containing Hib conjugated with protein PrD. Detailed Description of the Invention:

[0029] The objective of the present invention is to explore the dual role of the Protein D or protein D fragment as a conjugate carrier for polysaccharide antigens and as a protective antigen.

[0030] This invention relates to a combination vaccine containing a mixture of antigens to protect against diseases.

[0031] In particular the present invention relates to a combination vaccine composition comprising capsular polysaccharide antigen from Haemophilus influenza serotype b (Hib) conjugated to protein D.

[0032] Present invention also relates to a combination vaccine composition which comprises capsular polysaccharide antigen from Haemophilus influenza serotype b (Hib) conjugated with protein D, wherein protein D induces protective responses against invasive infections caused by Haemophilus influenzae.

[0033] Present invention comprises tetra and / or penta and / or hexa and / or multi valent vaccine composition comprising antigens against Diphtheria, Tetanus, Pertussis, Polio, Hepatitis B and Haemophilus influenzae-, wherein capsular polysaccharide antigen from Haemophilus influenza serotype b (Hib) is conjugated to Protein D or protein D fragment as a carrier protein.

[0034] Present invention also comprises multicomponent vaccine composition comprising antigens Diphtheria toxoid (DT), Tetanus toxoid (TT), Whole-cell pertussis (wP) / Acellular Pertussis (aP), Hepatitis (Hep) and Haemophilus influenzae (Hib) conjugated to Protein D or protein D fragment as a carrier protein.

[0035] Present invention further comprises multicomponent vaccine composition comprising antigens antigens Diphtheria toxoid (DT), Tetanus toxoid (TT), Wholecell pertussis (wP) / Acellular Pertussis (aP) and Haemophilus influenzae (Hib) conjugated to Protein D or protein D fragment as a carrier protein Present invention also relates to a immunogenic multicomponent vaccine composition comprising antigens Diphtheria toxoid (DT), Tetanus toxoid (TT), Whole-cell pertussis (wP) / Acellular Pertussis (aP), Polio, and Haemophilus influenzae (Hib) conjugated to Protein D or protein D fragment as a carrier protein

[0036] Present invention also comprises process of conjugation of Hib and Protein D as a carrier protein.

[0037] The invention also provides a kit for multicomponent combination vaccine comprising all the antigenic components in a single vial / prefilled syringe or a kit comprising two different containers, vials, prefilled syringes or dual chamber syringe.

[0038] The first aspect of the present invention is to provide a multivalent vaccine composition comprising Hib antigen conjugated with protein D as a carrier protein.

[0039] The invention also provides a combination vaccine comprising antigens for protecting a subject against at least diphtheria ('D'), tetanus ('T'), pertussis ('P') and H. influenzae type b ('Hib') antigen, wherein Hib antigen is conjugated with protein D or protein D fragment as a carrier protein.

[0040] In one embodiment, invention provides a combination vaccine comprising antigens for protecting a subject against at least diphtheria ('D'), tetanus (T), pertussis ('P'), Hepatitis B surface antigen and H. influenzae type b (Hib) antigen; wherein Hib antigen is conjugated with protein D or protein D fragment as a carrier protein.

[0041] In one embodiment, invention provides a combination vaccine comprising antigens for protecting a subject against at least diphtheria ('D'), tetanus (T), Hepatitis B surface antigen and H. influenzae type b (Hib) antigen; wherein Hib antigen is conjugated with protein D or protein D fragment as a carrier protein.

[0042] In one embodiment, invention provides a combination vaccine comprising antigens for protecting a subject against at least diphtheria ('D'), tetanus (T), pertussis ('P'), Hepatitis B surface antigen (HepB), Inactivated Poliomyelitis antigens (IPV) and H. influenzae type b (Hib); wherein Hib antigen is conjugated with protein D or protein D fragment as a carrier protein.

[0043] According to one other aspect of the invention, Inactivated Poliomyelitis antigens are one or more Salk strains which may be selected from the group of Mahoney type 1, MEF type 2 and Saukett type 3 or one or more Sabin strains selected from the group of Sabin types 1 , 2 and 3 and polio Virus-like particles molecules that mimic viruses but are not infectious.

[0044] Present invention provides tetravalent, pentavalent and hexavalent combination vaccine compositions; wherein Hib antigen is conjugated with protein D as a carrier protein. Vaccine composition may further comprises one or more antigens from Hepatitis (A, C, D, E, F and G strains), meningitis A, B or C, Influenza, Pneumococci, Streptococci, anthrax, dengue, malaria, measles, mumps, rubella, BCG, Respiratory Syncytial Virus (RSV), Japanese encephalitis, Rotavirus, smallpox, yellow fever, typhoid, Singles, Varicella virus, and others.

[0045] This Invention further provides a combination vaccine composition either in the completely liquid form or Hib antigen may be used extemporaneously by formulating the vaccine just prior to administration.

[0046] The pertussis antigen used according to the invention may be cellular (e.g. whole cell) or acellular.

[0047] One other aspect of the invention also relates to a process of conjugation of Hib and Protein D.

[0048] According to one embodiment of the invention, the Hib antigen, which is derived from the capsular polysaccharide, The Hib antigen preparation used in the vaccine of the invention comprises Hib antigen preferably conjugated to Protein D as a carrier protein. According to other embodiment of the invention, Protein D which is derived from the Escherichia coli (E. coli) strain or Haemophilus influenzae (Hib) or other recombinant means thereof.

[0049] The polysaccharide conjugate of Hib and Protein D may be prepared by any known coupling technique. For example the polysaccharide can be coupled via a thioether linkage. This conjugation method relies on activation of the polysaccharide with 1- cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) to form a cyanate ester. The activated polysaccharide may thus be coupled directly or via a spacer group to an amino group on the carrier protein. The conjugates can also be prepared by direct reductive animation methods. Another method involves the coupling of a cyanogen bromide (CNBr) activated polysaccharide derivatised with adipic acid hydrazide (ADH) to the protein carrier by carbodiimide condensation. Any other known method may be used to prepare the polysaccharide conjugate used in the vaccine of the invention.

[0050] Other Antigens of the vaccine composition of the invention

[0051] Diphtheria is caused by Corynebacterium diphtheriae, a Gram-positive non- sporing aerobic bacterium. This organism expresses a prophage-encoded ADP- ribosylating exotoxin ('diphtheria toxin'), which can be treated (e.g. using formaldehyde) to give a toxoid. This toxoid is no longer toxic but still remains antigenic and is able to stimulate the production of specific anti-toxin antibodies after injection. The Diphtheria antigen preparation used in the vaccine of the invention preferably comprises Diphtheria toxoid.

[0052] Tetanus is caused by Clostridium tetani, a Gram-positive, spore-forming bacillus. This organism expresses an endopeptidase ('tetanus toxin'), which can be treated to give a toxoid that is no longer toxic. However it still remains antigenic and is able to stimulate the production of specific anti-toxin antibodies after injection. The Tetanus antigen preparation used in the vaccine of the invention preferably comprises Tetanus toxoid. Pertussis or whooping cough is caused by Bordetella pertussis. The whole cell pertussis (wP) antigen may be prepared from the Bordetella pertussis strains that are likely to cause infections. The wP antigen of the invention may be inactivated by a number of ways such as use of chemical. However, inactivated B. pertussis (wP) can be used in the vaccine composition of the invention, preferably, is heat inactivated at 56 ±1°C, 30 minutes. The wP antigen preparation used in the vaccine of the invention is preferably made from Bordetella pertussis 134, 509 and 10536. The single Harvests of strains 10536, 509 and 134 are preferably mixed in the ratio of 1: 1: 1 ratio based upon their opacity.

[0053] The acellular pertussis (aP) antigens may be obtained from any of the known Bordetella pertussis strains. For the purpose of the invention the aP antigens may preferably be obtained from the B. pertussis Tohama strain. Any of the appropriate media may be used for the isolation, culturing, proliferation and fermentation of the culture. For the purpose of the invention the modified Stainer- Scholte may preferably be used. The acellular pertussis (aP) antigens used in the vaccine of the present invention comprise at least one or more antigens selected from the group of Pertussis toxoid (PT), Filamentous hemagglutinin (FHA), Pertactin (P69 or PRN) and FIM (fimbrial antigens- 1, 2 or 3). However, according to the preferred embodiment of the invention, the aP antigen preparation used in the vaccine of the invention comprises PT, FHA and PRN (P69) antigens.

[0054] Hepatitis is caused by various Hepatitis strains such as A, B, C, D, E, F or G. Hepatitis B virus (HBV) is one of the major agents which cause viral hepatitis. The HB V virion consists of an inner core surrounded by an outer protein coat or capsid. The major component of the capsid is a protein known as HBV surface antigen or, more commonly, 'HBsAg'. When this antigen is administered to a vaccinee it stimulates the production of anti-HBsAg antibodies which protect against HBV infection. According to one preferred aspect of the invention, the Hepatitis (Hep) antigen preparation used in the vaccine of the invention comprises Hep antigens derived from the surface antigen of Hepatitis B strain (HBsAg). For vaccine manufacture, HBsAg can be made either by purifying the antigen in particulate form from the plasma of chronic hepatitis B carriers, as large quantities of HBsAg are synthesized in the liver and released into the blood stream during an HBV infection or by expressing the protein by recombinant DNA methods. HBsAg for use in the vaccine of the invention may be prepared in either way.

[0055] Poliomyelitis is caused by the polio viruses. The vaccine of the invention may comprise Sabin (Sabin 1 and / or Sabin 2, and / or Sabin 2) or Salk strains of Poliovirus. According to one preferred embodiment of the invention, the vaccine of the invention comprises Salk strains. There are 3 types of Salk strains that can cause poliomyelitis. The three types are similar and cause identical symptoms, but they are antigenically very different and infection by one type does not protect against infection by others. Salk Poliovirus includes 3 strains- Type 1 (e.g. Mahoney strain), poliovirus Type 2 (e.g. MEF-I strain), and poliovirus Type 3 (e.g. Saukett strain). According to a preferred embodiment of the invention, the vaccine of the invention may comprise one or more of the said Salk strains.

[0056] Polioviruses may be grown in cell culture. Vero cell line, which is a continuous cell line derived from monkey kidney, may be used to grow the polioviruses. After growth, virions may be purified using already known techniques. The inactivation of the viruses may be done. Quantities of poliovirus are typically expressed in the 'DU' unit (the "D antigen unit"). The IPV antigen preparation used in the manufacturing of the vaccine of the invention is preferably prepared so as to comprise one or more strains that are used in the manufacturing of the vaccine. This bulk preparation is then used to formulate the vaccine of the invention.

[0057] Other antigens envisaged for the invention are VLP, Muti-epitope designer antigens, multimeric vaccine candidate, subunit, Split or purified proteins of Polioviruses, HPV, COVID, Hepatitis, Varicella, Shingles etc. Non antigenic components:

[0058] Along with the antigenic components the vaccine may comprise a number of non- antigenic components that are pharmaceutically acceptable excipients. These include but are not restricted to pH modifiers, buffers, adjuvants, preservative, carrier and tonicity modifying agents.

[0059] Aluminum based adjuvants are the most commonly used adjuvants. These adjuvants have also been approved by FDA for use in vaccines. Studies have shown that many aluminum-containing vaccines cause higher and more prolonged antibody responses than comparable vaccines without the adjuvant. The benefit of adjuvants has usually been observed during the initial immunization series rather than with booster doses. Preferred adjuvant used in the vaccine composition of the invention is Aluminum phosphate.

[0060] The vaccine may also contain, novel adjuvants selected from the group of Emulsion adjuvant, TLR 3, TLR 4, TLR 5, TLR 7, TLR 8, TLR 9 and TLR 10 agonist.

[0061] The vaccines are prone to contamination by bacteria. Thus, to avoid the potentially life threatening contamination with harmful microbes, that may be introduced in a vaccine incorporated during the event of accidental contamination, a preservative may be included in the composition of the vaccine while formulating it. The preservatives that have been used include Benzethonium chloride (Phemerol), thiomersal, Phenol and 2-phenoxyethanol (2-POE). Preferred preservative used in the vaccine composition of the invention is 2-Phenoxyethanol.

[0062] A composition according to the invention may be administered by any conventional route which is used in the field of vaccines, in particular by the systemic, i.e. parenteral route, e.g. by the subcutaneous, intramuscular, intradermal or intravenous route.

[0063] Modes for Carrying Out the Invention

[0064] Example 1: Tetra-valent vaccine composition of Hib antigen conjugated to Protein D as a carrier protein and method of their preparation. 340 Composition: The composition of Diphtheria toxoid, Tetanus toxoid, Hepatitis B (rDNA) and Haemophilus influenzae vaccine is presented in the table 1 below.

[0065] Table 1

[0066] NLT: Not Less Than, NMT: Not More Than

[0067] 345 Formulation of bulk vaccine:

[0068] The required quantity of individual drug substance and other excipients was calculated based on the batch size and the label claim.

[0069] Component 1

[0070] A calculated quantity of buffer solution was transferred to formulation vessel to 350 which the required quantity of adjuvant (75% of the required quantity) and phenoxyethanol (50% of required qty) was added. Afterwards the required quantity of Tetanus toxoid, Diphtheria toxoid and whole cell pertussis was added under constant stirring. The volume of resultant blend was made up to 60% of required batch size with buffer Saline. The pH of bulk was adjusted to 6.0+0.5 and it was 355 incubated for NLT 18 hrs at 25°±2°C with stirring at 150 rpm. Component 2

[0071] A calculated quantity of buffer solution was transferred to formulation vessel to which the required quantity of adjuvant (25% of the required quantity) and phenoxyethanol (50% of required qty) was added. Afterwards the required quantity Hepatitis B antigen was added under constant stirring. The volume of resultant blend was made up to 15 % of required batch size with buffer Saline. The pH of bulk was adjusted to 6.0+0.5 and it was incubated for NLT18 hrs at 25°±2°C with stirring at 150 rpm.

[0072] Mixing of different components

[0073] After completion of incubation, the component I and II were mixed and the resultant final bulk was again incubated for NLT 18 hrs at 25°±2°C with stirring at 150 rpm. If required the pH of bulk was adjusted to 6.5+0.5. After completion of incubation Hib components was added to above mixture and the resultant final bulk was again incubated for NLT 4 hrs at 25°+2°C with stirring at 150 rpm. If required the pH of bulk was adjusted to 6.5+0.5.

[0074] Post incubation the final bulk vaccine was stored at 2-8°C for pending filling. The final bulk vaccine was filled into USP type I, tubular glass vials, stoppered with bromobutyl rubber stopper and sealed with flip off aluminum seal.

[0075] Stored temperature: Store at 2-8°C.

[0076] Protein D used as a carrier in above cited example. However it can additionally induce an immune response against non-typeable Haemophilus influenzae. Experiments in mice revealed that vaccination with PD induced high serum IgG and IgA levels, as well as significant bactericidal activity against homologous and heterologou s strain s .

[0077] Example 2: Determination of Antibody (IgG) titer against Hib-PRP and Protein D.

[0078] Animal immunization:

[0079] Swiss mice were immunized with Heamophilus influenzae b PRP conjugated to tetanus toxoid or Protein D either individually or combined in pentavalent 385 composition. Group of 10 Swiss mice were immunized thrice on day 0, 14 and 28 via subcutaneous route with test vaccine. One group of 10 Swiss mice was kept as un-inoculated. Animals were bled after 10 days of third dose. The sera were separated by centrifuging at 4000 rpm for 15 minutes. The serum samples were processed by ELISA method to determine IgG antibodies against Poly

[0080] 390 ribosylribitolphosphate (PRP) and Protein D of Heamophilus influenzae.

[0081] Antibody Determination:

[0082] In-house ELISA method for the determination of IgG antibodies against PRP and Protein D to Haemophilus influenzae type b in mouse serum samples was used.

[0083] 395

[0084] Test sera samples diluted 100 fold was added to PRP or Protein D antigen coated wells. Specific Horseradish Peroxidase (HRP) conjugated secondary antibody was added followed by substrate that catalysis by the enzyme leads to a change in color indicating the presence of antibody. Results are presented in the table 2 and is

[0085] 400 reflected in the figure 1 and figure 2.

[0086] Table 2: Geo-mean of Antibody (IgG) titer against Hib-PRP and Protein D: This indicates that Anti-PRP response (i.e. against Hib polysaccharide) of plain Hib-TT and plain Hib-PrD is comparable in nature. Hib-TT and Hib-PrD based 05 pentavalent formulation is comparable in nature as indicated in figure 1.

[0087] Anti-Protein D response (i.e. against carrier protein PrD) of plain Hib-Prd and Hib- PrD based pentavalent formulation is comparable in nature as disclosed in figure 2. Thus, apart from Anti PRP response, Hib-PrD and its combination vaccine, are able 10 to induce anti-Protein D response also.

[0088] Example: 3: Hexavalent vaccine composition of Hib antigen conjugated to Protein D as a carrier protein and method of their preparation.

[0089] Composition: The composition of Diphtheria toxoid, Tetanus toxoid, Inactivated 15 whole cell pertussis, IPV (Sabin strain), Hepatitis B (rDNA) and Haemophilus influenzae vaccine is presented in table 3 below.

[0090] Table 3

[0091] Formulation of bulk vaccine 20 The required quantity of individual drug substance and other excipients was calculated based on the batch size and the label claim.

[0092] Component 1

[0093] A calculated quantity of buffer solution was transferred to formulation vessel to which the required quantity of adjuvant (75% of the required quantity) and 25 phenoxyethanol (50% of required qty) was added. Afterwards the required quantity of Tetanus toxoid, Diphtheria toxoid and whole cell pertussis was added under constant stirring. The volume of resultant blend was made up to 60% of required batch size with buffer Saline. The pH of bulk was adjusted to 6.0+0.5 and it was incubated for NLT18 hrs at 25°±2°C with stirring at 150 rpm. 30 Component 2

[0094] A calculated quantity of buffer solution was transferred to formulation vessel to which the required quantity of adjuvant (25% of the required quantity) and phenoxyethanol (50% of required qty) was added. Afterwards the required quantity Hepatitis B antigen was added under constant stirring. The volume of resultant 35 blend was made up to 15 % of required batch size with buffer Saline. The pH of bulk was adjusted to 6.0+0.5 and it was incubated for NLT18 hrs at 25°±2°C with stirring at 150 rpm.

[0095] Mixing of different components

[0096] After completion of incubation, the component I and II were mixed and the resultant 40 final bulk was again incubated for NLT 18 hrs at 25°±2°C with stirring at 150 rpm.

[0097] If required the pH of bulk was adjusted to 6.5+0.5. After completion of incubation, Hib and IPV components were added to above mixture and the resultant final bulk was again incubated for NLT 4 hrs at 25°±2°C with stirring at 150 rpm. If required the pH of bulk was adjusted to 6.5+0.5.

[0098] 445 Post incubation the final bulk vaccine was stored at 2-8°C for pending filling.

[0099] The final bulk vaccine was filled into USP type I, tubular glass vials, stoppered with bromobutyl rubber stopper and sealed with flip off aluminum seal.

[0100] Stored temperature: Store at 2-8°C.

[0101] 450 Examples 4: Hexavalent vaccine composition of Hib antigen conjugated to Protein D as a carrier protein and method of their preparation.

[0102] Composition: The composition of Diphtheria toxoid, Tetanus toxoid, inactivated whole cell pertussis, Hepatitis B (rDNA), Haemophilus influenzae and inactivated polio virus (Salk) is presented in table 4 below.

[0103] 455

[0104] Table 4

[0105] NLT: Not Less Than, NMT: Not More Than

[0106] Formulation of bulk vaccine

[0107] 460 The required quantity of individual drug substance and other excipients was calculated based on the batch size and the label claim.

[0108] Component 1

[0109] A calculated quantity of buffer solution was transferred to formulation vessel to which the required quantity of adjuvant (75% of the required quantity) and

[0110] 465 phenoxyethanol (50% of required qty) was added. Afterwards the required quantity of Tetanus toxoid, Diphtheria toxoid and whole cell pertussis was added under constant stirring. The volume of resultant blend was made up to 60% of required batch size with buffer Saline. The pH of bulk was adjusted to 6.0+0.5 and it was incubated for NLT 18 hrs at 25°±2°C with stirring at 150 rpm.

[0111] 470 Component 2

[0112] A calculated quantity of buffer solution was transferred to formulation vessel to which the required quantity of adjuvant (25% of the required quantity) and phenoxyethanol (50% of required qty) was added. Afterwards the required quantity Hepatitis B antigen was added under constant stirring. The volume of resultant

[0113] 475 blend was made up to 15 % of required batch size with buffer Saline. The pH of bulk was adjusted to 6.0+0.5 and it was incubated for NLT 18 hrs at 25°±2°C with stirring at 150 rpm.

[0114] Mixing of different components

[0115] After completion of incubation, the component I and II were mixed and the resultant

[0116] 480 final bulk was again incubated for NLT 18 hrs at 25°±2°C with stirring at 150 rpm. If required the pH of bulk was adjusted to 6.5+0.5. After completion of incubation, Hib and IPV components were added to above mixture and the resultant final bulk was again incubated for NLT 4 hrs at 25°+2°C with stirring at 150 rpm. If required the pH of bulk was adjusted to 6.5+0.5. Post incubation the final bulk vaccine was stored at 2-8°C for pending filling.

[0117] The final bulk vaccine was filled into USP type I, tubular glass vials, stoppered with bromobutyl rubber stopper and sealed with flip off aluminum seal.

[0118] Stored temperature: Store at 2-8°C. Examples 5: Hexavalent vaccine composition of Hib antigen conjugated to Protein D as a carrier protein and method of their preparation.

[0119] Composition: The composition of Diphtheria toxoid, Tetanus toxoid, inactivated whole cell pertussis, Hepatitis B (rDNA), Haemophilus influenzae and inactivated polio virus (Salk) is presented in table 5 below.

[0120] Table 5

[0121] NLT: Not Less Than & NMT: Not More Than Formulation of bulk vaccine

[0122] The required quantity of individual drug substance and other excipients was calculated based on the batch size and the label claim.

[0123] Component 1

[0124] A calculated quantity of buffer solution was transferred to formulation vessel to which the required quantity of adjuvant (75% of the required quantity) and phenoxyethanol (50% of required qty) was added. Afterwards the required quantity of Tetanus toxoid, Diphtheria toxoid and whole cell pertussis was added under constant stirring. The volume of resultant blend was made up to 60% of required batch size with buffer Saline. The pH of bulk was adjusted to 6.0+0.5 and it was incubated for NLT18 hours at 25°±2°C with stirring at 150 rpm.

[0125] Component 2

[0126] A calculated quantity of buffer solution was transferred to formulation vessel to which the required quantity of adjuvant (25% of the required quantity) and phenoxyethanol (50% of required qty) was added. Afterwards the required quantity Hepatitis B antigen was added under constant stirring. The volume of resultant blend was made up to 15 % of required batch size with buffer Saline. The pH of bulk was adjusted to 6.0+0.5 and it was incubated for NLT18 hours at 25°±2°C with stirring at 150 rpm.

[0127] Mixing of different components

[0128] After completion of incubation, the component I and II were mixed and the resultant final bulk was again incubated for NLT 18 hours at 25°±2°C with stirring at 150 rpm. If required the pH of bulk was adjusted to 6.5+0.5. After completion of incubation, Hib and VLP of IPV components were added to above mixture and the resultant final bulk was again incubated for NLT 4 hours at 25°+2°C with stirring at 150 rpm. If required the pH of bulk was adjusted to 6.5+0.5.

[0129] Post incubation the final bulk vaccine was stored at 2-8°C for pending filling. The final bulk vaccine was filled into USP type I, tubular glass vials, stoppered with bromobutyl rubber stopper and sealed with flip off aluminum seal.

[0130] Stored temperature: Store at 2-8°C.

[0131] Examples 6: Pentavalent vaccine composition of Hib antigen conjugated to Protein D as a carrier protein and method of their preparation.

[0132] Composition: The composition of Diphtheria toxoid, Tetanus toxoid, Acellular pertussis, Hepatitis B (rDNA) and Haemophilus influenzae vaccine is presented in table 6 below.

[0133] Table 6

[0134] NLT: Not Less Than & NMT: Not More Than

[0135] Formulation of bulk vaccine

[0136] The required quantity of individual drug substance and other excipients was calculated based on the batch size and the label claim.

[0137] Component 1

[0138] A calculated quantity of buffer solution was transferred to formulation vessel to which the calculated quantity of adjuvant (75% of the required quantity) and phenoxyethanol (50% of required qty) was added. Afterwards the required quantity of Tetanus toxoid, Diphtheria toxoid and acellular pertussis component (Pertussis toxin (PT), Filamentous hemagglutinin (FHA) and Pertactin (PRN)) was added under constant stirring. The volume of resultant blend was made up to 60% of required batch size with buffer Saline. The pH of bulk was adjusted to 6.0+0.5 and it was incubated for NLT18 hrs at 25°±2°C with stirring at 150 rpm

[0139] Component 2

[0140] A calculated quantity of buffer solution was transferred to formulation vessel to which the calculated quantity of adjuvant (25% of the required quantity) and phenoxyethanol (50% of required qty) was added. Afterwards the required quantity Hepatitis B antigen was added under constant stirring. The volume of resultant blend was made up to 15 % of required batch size with buffer Saline. The pH of bulk was adjusted to 6.0+0.5 and it was incubated for NLT18 hrs at 25°+2°C with stirring at 150 rpm.

[0141] Mixing of different components

[0142] After completion of incubation, the component I and II were mixed and the resultant final bulk was again incubated for NLT 18 hrs at 25°+2°C with stirring at 150 rpm. If required the pH of bulk was adjusted to 6.5+0.5. After completion of incubation Hib components was added to above mixture and the volume of resultant blend was made up to required batch size with buffer Saline. The resultant final bulk was again incubated for NLT 4 hrs at 25°+2°C with stirring at 150 rpm. If required the pH of bulk was adjusted to 6.5+0.5.

[0143] Post incubation the final bulk vaccine was stored at 2-8°C for pending filling.

[0144] The final bulk vaccine was filled into USP type I, tubular glass vials, stoppered with bromobutyl rubber stopper and sealed with flip off aluminum seal.

[0145] Stored temperature: Store at 2-8°C.

[0146] Examples 7: Hexavalent vaccine composition of Hib antigen conjugated to Protein D as a carrier protein and method of their preparation. Composition: The composition of Diphtheria toxoid, Tetanus toxoid, Acellular pertussis, Hepatitis B (rDNA), Haemophilus influenzae and Inactivated polio virus (Sabin) is presented in table 7 below.

[0147] Table 7

[0148] NLT: Not Less Than & NMT: Not More Than

[0149] Formulation of bulk vaccine

[0150] The required quantity of individual drug substance and other excipients was 85 calculated based on the batch size and the label claim. Component 1

[0151] A calculated quantity of buffer solution was transferred to formulation vessel to which the required quantity of Aluminum Phosphate adjuvant and phenoxyethanol (50% of required qty) was added. Afterwards the required quantity of Tetanus toxoid, Diphtheria toxoid and acellular pertussis component (Pertussis toxin (PT), Filamentous hemagglutinin (FHA) and Pertactin (PRN)) was added under constant stirring. The volume of resultant blend was made up to 60% of required batch size with buffer Saline. The pH of bulk was adjusted to 6.0+0.5 and it was incubated for NLT18 hrs at 25°±2°C with stirring at 150 rpm

[0152] Component 2

[0153] A calculated quantity of buffer solution was transferred to formulation vessel to which the required quantity of Aluminum hydroxide adjuvant and phenoxyethanol (50% of required qty) was added. Afterwards the required quantity Hepatitis B antigen was added under constant stirring. The volume of resultant blend was made up to 15 % of required batch size with buffer Saline. The pH of bulk was adjusted to 6.0+0.5 and it was incubated for NLT18 hrs at 25°+2°C with stirring at 150 rpm.

[0154] Mixing of different components

[0155] After completion of incubation, the component I and II were mixed and the resultant final bulk was again incubated for NLT 18 hrs at 25°+2°C with stirring at 150 rpm. If required the pH of bulk was adjusted to 6.5+0.5. After completion of incubation IPV and Hib components was added to above mixture and the volume of resultant blend was made up to required batch size with buffer Saline. The resultant final bulk was again incubated for NLT 4 hrs at 25°+2°C with stirring at 150 rpm. If required the pH of bulk was adjusted to 6.5+0.5.

[0156] Post incubation the final bulk vaccine was stored at 2-8°C for pending filling.

[0157] The final bulk vaccine was filled into USP type I, tubular glass vials, stoppered with bromobutyl rubber stopper and sealed with flip off aluminum seal.

[0158] Stored temperature: Store at 2-8°C. Examples 8: Hexavalent vaccine composition of Hib antigen conjugated to Protein D as a carrier protein and method of their preparation.

[0159] Composition: The composition of Diphtheria toxoid, Tetanus toxoid, Acellular pertussis, Hepatitis B (rDNA), Haemophilus influenzae and inactivated polio virus 0 (Salk) is presented in table 8 below.

[0160] Table 8

[0161] NLT: Not Less Than & NMT: Not More Than 5 Formulation of bulk vaccine

[0162] The required quantity of individual drug substance and other excipients was calculated based on the batch size and the label claim. Component 1

[0163] A calculated quantity of buffer solution was transferred to formulation vessel to which the calculated quantity of Aluminum adjuvant (75% of the required quantity) and phenoxyethanol (50% of required qty) was added. Afterwards the required quantity of Tetanus toxoid, Diphtheria toxoid and acellular pertussis component (Pertussis toxin (PT), Filamentous hemagglutinin (FHA) and Pertactin (PRN)) was added under constant stirring. The volume of resultant blend was made up to 60% of required batch size with buffer Saline. The pH of bulk was adjusted to 6.0+0.5 and it was incubated for NLT18 hrs at 25°±2°C with stirring at 150 rpm

[0164] Component 2

[0165] A calculated quantity of buffer solution was transferred to formulation vessel to which the required quantity of Aluminum adjuvant (25% of the required quantity) and phenoxyethanol (50% of required qty) was added. Afterwards the required quantity Hepatitis B antigen was added under constant stirring. The volume of resultant blend was made up to 15 % of required batch size with buffer Saline. The pH of bulk was adjusted to 6.0+0.5 and it was incubated for NLT18 hrs at 25°±2°C with stirring at 150 rpm.

[0166] Mixing of different components

[0167] After completion of incubation, the component I and II were mixed and the resultant final bulk was again incubated for NLT 18 hrs at 25°±2°C with stirring at 150 rpm. If required the pH of bulk was adjusted to 6.5+0.5. After completion of incubation IPV and Hib components was added to above mixture and the volume of resultant blend was made up to required batch size with buffer Saline. The resultant final bulk was again incubated for NLT 4 hrs at 25°+2°C with stirring at 150 rpm. If required the pH of bulk was adjusted to 6.5+0.5.

[0168] Post incubation the final bulk vaccine was stored at 2-8°C for pending filling.

[0169] The final bulk vaccine was filled into USP type I, tubular glass vials, stoppered with bromobutyl rubber stopper and sealed with flip off aluminum seal.

[0170] Stored temperature: Store at 2-8°C. Examples 9: Hexavalent vaccine composition of Hib antigen conjugated to Protein D as a carrier protein and method of their preparation.

[0171] Composition: The composition of Diphtheria toxoid, Tetanus toxoid, Acellular pertussis, Hepatitis B (rDNA), Haemophilus influenzae and Virus like particle of Poliovirus is presented in table 9 below.

[0172] Table 9

[0173] NLT: Not Less Than & NMT: Not More Than * VLP of polio type 1, 2, 3 at a dose based on protein content. The specific antigen content is determined based on D-Ag ELISA

[0174] Formulation of bulk vaccine

[0175] The required quantity of individual drug substance and other excipients was calculated based on the batch size and the label claim.

[0176] Component 1

[0177] A calculated quantity of buffer solution was transferred to formulation vessel to which the required quantity of Aluminum Phosphate adjuvant and phenoxyethanol (50% of required qty) was added. Afterwards the required quantity of Tetanus toxoid, Diphtheria toxoid and acellular pertussis component (Pertussis toxin (PT), Filamentous hemagglutinin (FHA) and Pertactin (PRN)) was added under constant stirring. The volume of resultant blend was made up to 60% of required batch size with buffer Saline. The pH of bulk was adjusted to 6.0+0.5 and it was incubated for NLT18 hrs at 25°±2°C with stirring at 150 rpm

[0178] Component 2

[0179] A calculated quantity of buffer solution was transferred to formulation vessel to which the required quantity of Aluminum hydroxide adjuvant and phenoxyethanol (50% of required qty) was added. Afterwards the required quantity Hepatitis B antigen was added under constant stirring. The volume of resultant blend was made up to 15 % of required batch size with buffer Saline. The pH of bulk was adjusted to 6.0+0.5 and it was incubated for 8 hrs at 25°±2°C with stirring at 150 rpm.

[0180] Mixing of different components

[0181] After completion of incubation, the component I and II were mixed and the resultant final bulk was again incubated for NLT 18 hrs at 25°±2°C with stirring at 150 rpm. If required the pH of bulk was adjusted to 6.5+0.5. After completion of incubation IPV and Hib components was added to above mixture and the volume of resultant blend was made up to required batch size with buffer Saline. The resultant final bulk was again incubated for NLT 4 hrs at 25°+2°C with stirring at 150 rpm. If required the pH of bulk was adjusted to 6.5+0.5.

[0182] Post incubation the final bulk vaccine was stored at 2-8°C for pending filling.

[0183] The final bulk vaccine was filled into USP type I, tubular glass vials, stoppered with bromobutyl rubber stopper and sealed with flip off aluminum seal.

[0184] Stored temperature: Store at 2-8°C.

[0185] Example 10: Determination of functional antibody against NTHi

[0186] The levels of bactericidal activity of immune sera were determined by Bactericidal assay. Serum Bactericidal Assay (SBA) is used as a measure of vaccine-induced antibody function in conjunction with complement to kill bacteria. The assay gives information on whether a particular serum sample has a level (titre) of bactericidal antibodies sufficient to reach a protective threshold.

[0187] To perform the assay Sera were incubated at 56°C for 30 min to remove complement activity. NTHi strain 3655 was grown to log phase in brain heart infusion broth and suspended at 5 x 104 CFU / ml in sterile PCM buffer (PBS with 0.15 mM CaC12 and ImM MgC12) containing 1% BSA.

[0188] Thereafter about 20 pl of bacteria, 20 pl of serially diluted antiserum, 20 pl of rabbit complement, and finally 40 pl of 1% BSA in PCM buffer were mixed. The initial bacterial concentration, was determined on chocolate agar plates. The samples were incubated thereafter at 37°C for 1 hr with gentle shaking and 10 pl of the sample was plated in duplicate. Sear and complement control were also test for ensure test validity.

[0189] The agar plates containing sample from each group were incubated at 37°C overnight and the bactericidal activities of serially diluted antisera were assessed by determining the numbers of CFU the following day. The dilutions manifesting 50% killing were noted for comparison of the activities of different sera. The mean values of serum dilutions yielding 50% killing were noted for comparison. Results are presented in the table 10 and is reflected in the figure 3.

[0190] Table 10

[0191] *The Bactericidal Titers are Expressed as the Reciprocal of the Greatest Serum Dilution Yielding 50% Bacterial Death, Compared to the Controls

[0192] The bactericidal activity of serum collected form vaccine samples wherein Hib is conjugated with protein D are significantly higher than vaccine samples containing Hib conjugated with Tetanus toxoid.

Claims

CLAIMS:

1. An immunogenic multicomponent vaccine composition comprising a polysaccharide antigens against Diphtheria or / and Tetanus or / and Pertussis or / and Polio or / and Hepatitis or / and Haemophilus influenzae b conjugated to protein D or a protein D fragment thereof.

2. The immunogenic multicomponent vaccine composition of claim 1 comprising a Haemophilus influenza b - protein D polysaccharide conjugate antigen.

3. The immunogenic multicomponent vaccine composition of claim 1 comprising conjugated capsular polysaccharides, Haemophilus influenza b, wherein the carrier protein for at least one of the polysaccharides is protein D.

4. The immunogenic multicomponent vaccine composition of claim 3 wherein Haemophilus influenza serotype b antigen preferably conjugated to Protein D as a carrier protein.

5. The immunogenic multicomponent vaccine composition as claimed in claim 1 wherein the polysaccharide - protein D conjugate antigen is adsorbed onto aluminum-based adjuvant.

6. The immunogenic multicomponent vaccine composition of claim 1 comprising tetra and / or penta and / or hexa and / or multi valent vaccine composition.

7. The immunogenic multicomponent vaccine composition of claim 6 comprising antigens Diphtheria toxoid (DT), Tetanus toxoid (TT), Wholecell pertussis (wP) / Acellular Pertussis (aP), Hepatitis (Hep) and Haemophilus influenzae (Hib) conjugated to Protein D as a carrier protein.

8. The immunogenic multicomponent vaccine composition of claim 6 comprising antigens Diphtheria toxoid (DT), Tetanus toxoid (TT), Wholecell pertussis (wP) / Acellular Pertussis (aP) and Haemophilus influenzae (Hib) conjugated to Protein D as a carrier protein.

9. The immunogenic multicomponent vaccine composition of claim 6 comprising antigens Diphtheria toxoid (DT), Tetanus toxoid (TT), Wholecell pertussis (wP) / Acellular Pertussis (aP), Inactivated Poliomyelitis antigens (IPV) and Haemophilus influenzae (Hib) conjugated to Protein D as a carrier protein.

10. The immunogenic multicomponent vaccine composition of claim 1 provides a either a fully liquid composition or kit comprising all the antigenic components in a single vial / prefilled syringe or a kit comprising two different containers, vials, prefilled syringes or dual chamber syringe.

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