WO2020165920A1

WO2020165920A1 - Multivalent vaccine composition

Info

Publication number: WO2020165920A1
Application number: PCT/IN2020/050141
Authority: WO
Inventors: Sivakumar Ayyaswamy PERNAMALLUR; Vikram Madhusadan Paradkar; Narender Dev MANTENA; Mahima DATLA; Martin Reers
Original assignee: Biological E Limited
Priority date: 2019-02-12
Filing date: 2020-02-12
Publication date: 2020-08-20

Abstract

The present invention pertains to a multivalent vaccine formulation comprising at least one inactivated poliovirus (IPV) S19 strain selected from S19 Type 1, S19 Type 2 and S19 Type 3 and at least one antigen selected from Diphtheria toxoid (DT), Tetanus toxoid (TT), whole-cell Bordetella pertussis (wP), acellular Pertussis (aP), Hepatitis B surface protein, Haemophilus influenzae Type b polysaccharide, Inactivated Japanese encephalitis (JE) virus and Inactivated Rotavirus 116E strain (IRV). The multivalent vaccine formulation of the present invention provide protection against several diseases, simplify vaccine administration and improve vaccine compliance.

Description

MULTIVALENT VACCINE COMPOSITION

FIELD OF INVENTION

The present invention relates to a multivalent combination vaccine comprising hyper- attenuated polio virus e.g. S 19 strains of inactivated poliomyelitis vims (IPV). More particularly, the present invention provides a vaccine composition comprising S 19 strains of inactivated poliomyelitis virus and one or more antigens selected from e.g. Diphtheria (D) toxoid, Tetanus (T) toxoid, whole cell Pertussis (wP) or acellular Pertussis (aP), Hepatitis B surface protein, Haemophilus influenzae type b capsular polysaccharide, Inactivated Japanese encephalitis (JE) virus and Inactivated Rotavirus (IRV).

BACKGROUND OF INVENTION

The development of combination vaccines for protection against multiple diseases began with the combination of individual Diphtheria, Tetanus, and Pertussis (DTP) vaccines into a single product; this combined vaccine was first used to vaccinate infants and children in 1948. It has become the cornerstone of pediatric and adult immunization programs, and over the years, the vaccination program has seen the addition of other vaccines to the combination and the replacement of components to improve its safety profile. An important advancement was the replacement of whole-cell pertussis antigens (wP) with less reactogenic acellular antigens (aP) in the early 1990s. This paved the way for the combination of Diphtheria, Tetanus, and acellular Pertussis antigens (DTaP) with other routine vaccines such as inactivated polio vaccine, Haemophilus influenzae vaccine (Hib), and Hepatitis B vaccine (HepB).

Combination vaccines, which provide protection against multiple pathogens are desirable in order to minimize the number of immunizations required to confer protection against multiple pathogens, to lower administration costs, and to increase acceptance and coverage rates.

Few examples of combination vaccines available in the market such as Pediarix^®, which is a combination of DTaP, Hep B, and IPV (polio); Kinrix^® - combination of DTaP and IPV (polio); Pentacel^® - combination of DTaP, IPV (polio), and Hib; Quinvaxem^® and ComBEfive^® fully liquid vaccine containing DTwP-HepB-Hib.

Despite the introduction of two live oral vaccines (Rotarix and RotaTeq™) in many countries, rotavirus (RV) is still the leading cause of severe gastroenteritis and diarrhea, resulting in deaths among children under the age of 5 years worldwide. The two vaccines have shown high efficacy in high and middle-income countries. However, concerns linger about rare but severe adverse events, such as intussusception and their efficacy against the full range of rotavirus serotypes. In order to overcome these risk factors inactivated rotavirus vaccine (IRV) as an alternative approach is being considered. Various studies have demonstrated the protective role of serum antibody in animals and children and the robust serum antibody response and protection against rotavirus infection in animal models following parenteral immunization with IRV.

WO 1998/000167A1; WO 2002/000249 A2; WO 2008/028957A2; WO 2010/046934A1 and US 2013/0280293A discloses various multivalent vaccine compositions containing two or more antigens such as Diphtheria (D) toxoid, Tetanus (T) toxoid, whole cell Pertussis (wP) or acellular Pertussis (aP), Hepatitis B surface protein, IPV and Haemophilus influenzae type b capsular polysaccharide.

The combination vaccines mentioned in the above references, as well as commercially available combination vaccines all contain IPV antigens from Salk or Sabin. Each 0.5 ml dose of commercially available Salk based IPV contains 40 D antigen units of type 1, Mahoney (MahPl), 8 D antigen units of type 2 MEF-1 (MEF2P1) and 32 D antigen units of type 3, Saukett (SktPl) of the polio vims.

However, the current production of IPV is more expensive and may even be prohibitively expensive for less and least developed countries, where a strong need for polio vaccines still exists. The culture systems for producing bulk poliovirus material contribute to a large extent to the relatively high costs, in addition to the high-level containment requirements and handling challenges in particular for Salk and Sabin polioviruses. Due to the risks associated with the handling of large quantities of poliovirus needed for IPV production, while global cessation of poliovirus transmission is occurring, a high-level containment (BSL- 3/GAPIII) for all manufacturing and quality control areas where Salk and Sabin live vims is handled must be implemented.

The present inventors have endeavoured to develop multivalent combination vaccines containing inactivated antigens that originated from live S 19 strains. It is assumed that multivalent combination vaccine based on IPV S 19 strains is at least as efficacious as compared to commercially available multivalent combination vaccine based on Salk and / or Sabin strains. However, the containment requirements for the manufacturing of S 19 strains IPV bulk antigens are less as compared to commercially available Salk and Sabin bulk inactivated antigens.

OBJECTIVE OF THE INVENTION

It is an objective of the invention to provide a safe and effective multivalent immunogenic composition comprising inactivated polio antigens that originated from S 19 strains. Yet another objective of the present invention is to provide a process for the preparation of a multivalent combination vaccine comprising inactivated polio antigens originated from S19 strains for immunization against poliomyelitis and various other diseases.

SUMMARY OF THE INVENTION

The present invention relates to an immunogenic composition comprising at least one inactivated poliomyelitis virus originated from S19 strains, and at least one other antigen selected from Diphtheria toxoid (DT), Tetanus toxoid (TT), killed whole-cell Bordetella pertussis (wP), or two or more acellular Pertussis components (aP) (e.g. pertussis toxoid (PT), filamentous haemagglutinin (FHA) and pertactin (PRN)).

The present invention also relates to a tetravalent vaccine, comprising inactivated poliomyelitis vims S19 strains, Diphtheria toxoid (DT), Tetanus toxoid (TT), killed whole cell Bordetella pertussis, or two or more acellular Pertussis components (aP (e.g. Pertussis toxoid (PT), filamentous haemagglutinin (FHA) and pertactin (PRN)).

The present invention also relates to a pentavalent vaccine, comprising inactivated poliomyelitis vims S19 strains, Diphtheria toxoid (DT), Tetanus toxoid (TT), killed whole cell Bordetella pertussis, or two or more acellular Pertussis components (aP) (e.g. Pertussis toxoid (PT), filamentous haemagglutinin (FHA) and pertactin (PRN)) and an antigen from Hepatitis B or a conjugate of the capsular polysaccharide of Haemophilus influenzae type B (Hib) and carrier protein.

The present invention also relates to a hexavalent vaccine, comprising inactivated poliomyelitis vims S19 strains, Diphtheria toxoid (DT), Tetanus toxoid (TT), killed whole cell Bordetella pertussis, or two or more acellular Pertussis components (aP) (e.g. Pertussis toxoid (PT), filamentous haemagglutinin (FHA) and pertactin (PRN)); an antigen from Hepatitis B, a conjugate of the capsular polysaccharide of Haemophilus influenzae type B (Hib) and carrier protein.

The present invention also relates to a heptavalent vaccine, comprising inactivated poliomyelitis vims S19 strains, Diphtheria toxoid (DT), Tetanus toxoid (TT), killed whole cell Bordetella pertussis, or two or more acellular Pertussis components (aP) (e.g. Pertussis toxoid (PT), filamentous haemagglutinin (FHA) and pertactin (PRN)); an antigen from Hepatitis B, a conjugate of the capsular polysaccharide of Haemophilus influenzae type B (Hib) and carrier protein; and inactivated rotavims antigen (IRV).

The present invention also relates to a multivalent vaccine, comprising inactivated poliomyelitis vims originated from S19 strains, Diphtheria toxoid (DT), Tetanus toxoid (TT), killed whole-cell Bordetella pertussis, or two or more acellular Pertussis components (aP) (e.g. Pertussis toxoid (PT), filamentous haemagglutinin (FHA) and pertactin (PRN)); and one or more other antigen selected from Hepatitis B, a conjugate of the capsular polysaccharide of Haemophilus influenzae type B (Hib) and carrier protein; inactivated rotavirus antigen (IRV) and Inactivated Japanese encephalitis (JE) virus.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods belong. Although any formulation, composition and method similar or equivalent to those described herein can also be used in the practice of the methods and compositions, representative illustrative methods and compositions are now described.

Where a range of values is provided, it is understood that each intervening value between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the methods and compositions of the present invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within by the methods and compositions, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the methods, compositions and delivery systems.

It is appreciated that certain components or features, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the compositions and methods, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. It is noted that, as used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

As used herein, the term "comprises" or "comprising" as used herein permits the presence of one or more additional components in the compositions.

The term “immunogenic composition” is optionally substitutable with the term “vaccine” and vice versa.

The term“D-antigen units” (DU also referred to as“international units” or IU): The D antigen units referred to herein are the measured total D-antigen units of each unadsorbed bulk IPV antigen prior to formulation of the final vaccine which are added in each human dose of formulated vaccine. The methods of measuring D-antigen units are well known in the art. The terms“S 19 type 1” refers to S19 serotype 1 , Mahoney (MahPl),“S 19 type 2” refers to S 19 serotype 2, MEF-1 (MEF2P1), and“S19 type 3” refers to S 19 serotype 3, Saukett (SktPl).

IPV S 19 strains is a chimeric recombinant poliovirus, comprising non-coding region based on serotype 3 of a Sabin strain and the coding region based on Sabin strain with the capsid protein from wild type / Salk strain serotypes Mahoney, MEF and Saukett. The IPV S 19 strains includes its mutants e.g. S 18 which may be obtained after a series of passages.

The term “immunogenic composition” is optionally substitutable with the term “vaccine” or“vaccine composition” and vice versa.

In an embodiment, the present invention provides a multivalent immunogenic composition comprising at least four antigens selected from inactivated poliomyelitis virus S 19 strains, Diphtheria toxoid (DT), Tetanus toxoid (TT), killed whole-cell Bordetella pertussis ( wP ) or two or more acellular Pertussis components (aP) (e.g. pertussis toxoid (PT), filamentous haemagglutinin (FHA) and pertactin (PRN)); an antigen from Hepatitis B, a conjugate of the capsular polysaccharide of Haemophilus influenzae type B (Hib) and carrier protein; and inactivated rotavirus (IRV) antigen, wherein the one or more these antigens may be adsorbed to an adjuvant or unadsorbed.

In a preferred embodiment, the present invention provides a hexavalent vaccine composition comprising:

(a) IPV S 19 Type 1 at a dose of 4 to 40 D-antigen units, IPV S 19 Type 2 at a dose of 0.8 to 8 D-antigen units and IPV S 19 Type 3 at a dose of 3.2 to 32 D-antigen units,

(b) Tetanus toxoid,

(c) Diphtheria toxoid,

(d) killed whole-cell Bordetella pertussis, or two or more acellular Pertussis components (aP) (e.g. Pertussis toxoid (PT), filamentous haemagglutinin (FHA) and pertactin (PRN)),

(e) a conjugate of the capsular polysaccharide of Haemophilus influenzae type b and carrier protein,

(f) an antigen from Hepatitis B and

(g) one or more pharmaceutically acceptable carrier.

In an embodiment, the present invention provides a method of preparing a multivalent immunogenic composition comprising at least four antigens selected from inactivated poliomyelitis virus S 19 strain, Diphtheria toxoid (D), Tetanus toxoid (T), killed whole - cell Bordetella pertussis ( wP ), or two or more acellular Pertussis components (aP) (e.g. Pertussis toxoid (PT), filamentous haemagglutinin (FHA) and pertactin (PRN)); an antigen from Hepatitis B, a conjugate of the capsular polysaccharide of Haemophilus influenzae type B (Hib) and a carrier protein; and inactivated rotavirus (IRV) antigen, wherein the one or more these antigens may be adsorbed to an adjuvant or unadsorbed.

The immunogenic composition further comprises one or more pharmaceutically acceptable excipient or carrier.

The multivalent vaccine composition of the present invention provides protection in a host against the infections caused by Bordetella pertussis, Clostridium tetani, Corynebacterium diphtheriae, poliovirus, Hepatitis B, rotavirus, Japanese encephalitis virus and/ or Haemophilus influenza.

In yet another embodiment, the IPV S 19 strain is a chimeric recombinant poliovirus, comprising non-coding region based on serotype 3 of a Sabin strain and the coding region based on Sabin strain with the capsid from wild type / Salk strain serotypes Mahoney, MEF and Saukett.

In an embodiment, S 19 strain Type 1 of the present invention is a chimeric recombinant poliovirus comprising non-coding region from serotype Sabin 3 and the coding region on Sabin strain with the capsid from poliovirus Type 1 Mahoney (MahPl) or Bmnhilde strain (Type-1).

In an embodiment, S 19 strain Type 2 of the present invention is a chimeric recombinant poliovirus comprising non-coding region from serotype Sabin 3 and the coding region on Sabin strain with the capsid from poliovirus Type 2 (MEF-1).

In an embodiment, S 19 strain Type 3 of the present invention is a chimeric recombinant poliovirus comprising non-coding region from serotype Sabin 3 and the coding region on Sabin strain with the capsid from poliovirus Type 3 (Saukett).

In yet another embodiment, the IPV S 19 strains selected from S 19 Type 1 is present at a dose of 4 to 60 D-antigen units, S 19 Type 2 at a dose of 0.8 to 12 D-antigen units and S19 Type 3 at a dose of 3.2 to 48 D-antigen units.

The vaccines of the invention comprise Diphtheria, Tetanus and/or Pertussis component(s). The Diphtheria antigen is typically a Diphtheria toxoid. The preparation of Diphtheria toxoids (DT) is well known in the art and any suitable Diphtheria toxoid may be used. For instance, DT may be produced by purification of the toxin from a culture of Corynebacterium diphtheriae followed by chemical detoxification, alternatively the Diphtheria toxoids (DT) is made by purification of a recombinant, or genetically detoxified analogue of the toxin. In one embodiment DT is present at an amount of 5 Ff to 35 Ff, preferably 10 to 30 Ff per dose. In one embodiment, the Diphtheria toxoid may be adsorbed onto an aluminium salt such as aluminium hydroxide or aluminium phosphate or a mixture of aluminium hydroxide and aluminium phosphate.

The Tetanus toxoid of the present invention may be produced by purification of the toxin from a culture of Clostridium tetani followed by chemical detoxification. Alternatively, the Tetanus toxoid may be made by purification of a recombinant or genetically detoxified analogue of the toxin. The TT may be present at an amount of 2.5-30 Lf, preferably 5 to 20 Lf per dose. In one embodiment, the Tetanus toxoid (TT) may be adsorbed onto an aluminium salt such as aluminium hydroxide or aluminium phosphate or a mixture of aluminium hydroxide and aluminium phosphate.

The Pertussis component of the invention may be either acellular (aP) where purified Pertussis antigens are used or whole-cell (wP) where killed whole cell Pertussis is used as the Pertussis component. wP may be inactivated by several known methods, including mercury- free methods. Those methods may include heating at 50-70°C, preferably at 55-60°C, for a period of 10-60 minutes, treatment with formaldehyde, glutaraldehyde and the like. The wP component of the present invention may be present in an amount of 5-50 IOU, preferably 10- 30 IOU per dose.

Acellular pertussis (aP) vaccine may comprise 2 or more antigens selected from a group comprising Pertussis toxoid (PT), filamentous haemagglutinin (FHA), pertactin (PRN), agglutinogens 2 & 3. In one embodiment, the aP comprises PT, FHA and PRN. Pertussis Toxoid (PT) may be present in an amount of 20 to 50 mg, Filamentous Haemagglutinin (FHA) may be present in an amount of 10 to 40 mg, Pertactin (PRN) in an amount of 1 to 5 mg and Fimbriae antigen Types 2 and 3 (FIM) may be present in an amount of 2 to 10 mg per dose of vaccine.

In one embodiment, the Pertussis component may be adsorbed onto an aluminium salt such as aluminium hydroxide or aluminium phosphate or a mixture of aluminium hydroxide and aluminium phosphate.

In another embodiment, one or more antigens of the invention may be adsorbed onto an aluminium salt such as aluminium hydroxide or aluminium phosphate or a mixture of both or may be unadsorbed. The aluminium content (Al⁺³) in the vaccine may in the range of 0. lmg to 2 mg, preferably, 0.1 to lmgper dose.

The Hepatitis B surface antigen (HBsAg) of the present invention may be prepared by the method known in the art. For example, it may be prepared by a method which involves purifying the antigen in particulate form from the plasma of chronic hepatitis B carriers. Another method involves expressing the protein by recombinant DNA methods. The HBsAg may be prepared by expression in the yeast-like Saccharomyces cerevisiae, Pichia pastoris and insect cells (e.g. Hi5) or mammalian cells. The Hepatitis B surface antigen may be adsorbed onto an aluminium salt such as aluminium hydroxide or aluminium phosphate or a mixture of aluminium hydroxide and aluminium phosphate. In one embodiment, HBsAg is present in an amount of 5-20 mg, preferably 5-15 mg per dose.

Antigens from Haemophilus influenzae type B may be prepared by methods known in the art. The antigen may be capsular polysaccharide (PRP) from Haemophilus influenzae type B (Hib) conjugated to a carrier protein. The Hib antigen may optionally be adsorbed onto aluminium phosphate or may be unadsorbed.

In an embodiment, the capsular polysaccharide of Haemophilus influenzae type b is conjugated to carrier molecule selected from tetanus toxoid, CRM 197 or diphtheria toxoid or outer membrane protein (OMP). Hib antigen of the present invention may be present in an amount of 5-25 mg, preferably 5 to 20 mg per dose conjugated to 10 to 25 mg of carrier protein.

IPV S 19 vims of the present invention may be produced by methods known in the art and may comprise a) revival of adherent cells into static containers; b) amplification of adherent cells in a stirred tank bioreactor containing microcarriers in a culture medium, c) detachment of cells inside the bioreactor and transferring the cell culture to another bioreactor, d) repeating the steps (b) and (c) at least twice, e) transferring the cells to production bioreactor having a volume of at least 100 liters, f) infecting the adherent cells in production bioreactor with S 19 polio virus, g) incubating the infected cell culture to propagate S19 virus for a period of at least 3 days, h) harvesting the S 19 polio vims, followed by inactivation and purification to produce inactivated polio vims in high yield.

The cell culture media used for amplification of adherent cells is a conventional culture media and is free of any protein of animal origin or even of any product of animal origin. The cell culture media may contain low-molecular- weight proteins at very low concentrations. Suitable media include VP SFM medium, Opti Pro™ semm-free (InVitrogen), Episerf (InVitrogen), Ex-Cell® MDCK (Sigma-Aldrich), Ex-Cell™ Vero (SAFC biosciences), MP- BHK® semm free (MP Biomedicals), SFC-10 BHK express semm-free (Promo cell), SFC-20 BHK express protein-free (Promo cell), HyQ PF Vero (Hyclone Ref. SH30352.02), Hyclone SFM4 Megavir, MDSS2 medium (Axcell biotechnology), Iscove's modified DMEM medium (Hyclone), Ham's nutritive media (Ham's F10, Ham's F12), Leibovitz L-15 medium (Hyclone), ProVero medium (Lonza) and Power MDCK medium (Lonza) that are free of any product of animal origin and which contain little or no proteins. A cell protection agent is usually added, when the protein content in the culture medium is low (e.g. < 15 mg/1 according to the Bradford method). The cell protection agent protects the cells against the shear forces that are exerted when the medium is subjected to stirring. Suitable cell protection agents that can be used in the process of the present invention may include polyvinyl alcohol polymers, polyethylene glycol, polyvinylpyrrolidone and poloxamers and the like.

The propagation of poliovirus is carried out in adherent Vero cell, PER.C6 cells, HEK293 cell or MRC-5 cells, preferably vero cells, which are obtained from WHO 10-87 cell bank. The Adherent cells are passaged in a culture medium, one or more times to amplify viral copy number, as well as to introduce vims into a living system for replication efficiency. In order to amplify cells and form industrial cell batches, a plurality of cell passages are carried out in separate bioreactors.

In one embodiment, the vaccine formulation of the present invention comprises inactivated rotavirus vaccine (IRV). The rotavirus strain 116E is generally used and it is a naturally occurring human-bovine reassortant. The methods of virus inactivation are known in the art and is preferably carried out by chemical or physical methods. Those methods may include heating at 50- 75 ^°C, preferably at 55-60 ^°C, for a period of 10-60 minutes, treatment with formaldehyde, glutaraldehyde and the like. The IRV component of the present invention may be present in an amount of 2.5-15 mg, preferably 5 to 10 mg per dose.

In an embodiment, the present invention provides a stable tetravalent vaccine composition, wherein each dose comprises:

a) Diphtheria toxoid (DT) present in the amount 5-35 LF,

b) Tetanus toxoid (TT) present in the amount of 2.5-30 LF,

c) acellular Bordetella pertussis antigen (aP) Pertussis toxoid (PT), FHA and pertactin (P69 or PRN) and each present in an amount of 3-30 mg, each or killed whole-cell pertussis in the amount of 5-50 IOU,

d) IPV S 19 Type 1 at a dose of 4 to 60 D-antigen units, S 19 Type 2 at a dose of 0.8 to 12 D- antigen units and S 19 Type 3 at a dose of 3.2 to 48 D-antigen units, and

e) one or more pharmaceutically acceptable carrier, preservatives and/or excipients.

In another embodiment, the present invention provides a stable pentavalent vaccine composition, wherein each dose comprises:

a) Diphtheria toxoid (DT) present in the amount 5-35 LF,

b) Tetanus toxoid (TT) present in the amount of 2.5-30 LF, c) acellular Bordetella pertussis antigen (aP) Pertussis toxoid (PT), FHA and pertactin (P69 or PRN) and each present in an amount of 3-30 mg, each or killed whole-cell pertussis in the amount of 5-50 IOU,

d) Hepatitis B surface antigen (HBS Ag) present in the amount 5-15 mg or Haemophilius influenza type b PRP-TT conjugate antigen (Hib-PRP-TT) is present in the amount 2- 15 mg/0.5 ml,

e) IPV S19 Type 1 at a dose of 4 to 60 D-antigen units, S19 Type 2 at a dose of 0.8 to 12 D-antigen units and S19 Type 3 at a dose of 3.2 to 48 D-antigen units, and

f) one or more pharmaceutically acceptable carrier, preservatives and/or excipients.

In yet another embodiment, the present invention provides a stable hexavalent vaccine composition, wherein each dose comprises:

a) Diphtheria toxoid (DT) present in the amount 5-35 LF,

b) Tetanus toxoid (TT) present in the amount of 2.5-30 LF,

d) Hepatitis B surface antigen (HBS Ag) present in the amount 5-15 mg,

e) Haemophilius influenza type b PRP-TT conjugate antigen (Hib-PRP-TT) is present in the amount 2-15 mg/0.5 ml,

f) IPV S19 Type 1 at a dose of 4 to 60 D-antigen units, S19 Type 2 at a dose of 0.8 to 12 D- antigen units and S19 Type 3 at a dose of 3.2 to 48 D-antigen units, and

g) one or more pharmaceutically acceptable carriers, preservatives and/or excipients.

In yet another embodiment, the present invention provides a stable multivalent vaccine composition, wherein each dose comprises:

a) Diphtheria toxoid (DT) present in the amount 15-30 LF,

b) Tetanus toxoid (TT) present in the amount of 5-15 LF,

c) acellular Bordetella pertussis antigen (aP) Pertussis toxoid (PT), FHA and pertactin (P69 or PRN) and each present in an amount of 3-30 mg, each or killed whole-cell pertussis in the amount of 10-30 IOU,

d) Hepatitis B surface antigen (HBS Ag) present in the amount 5-15 mg,

f) IPV S19 Type 1 at a dose of 4 to 60 D-antigen units, S19 Type 2 at a dose of 0.8 to 12 D- antigen units and S19 Type 3 at a dose of 3.2 to 48 D-antigen units, g) and optionally inactivated rotavirus (IRV) antigen present in the amount 2.5-15 mg, and h) one or more pharmaceutically acceptable carriers.

In yet another embodiment, the multivalent fully liquid vaccine composition further comprises Inactivated Japanese encephalitis (JE) virus. JE may be present in an amount of 5 to 20 mg, preferably 5 to 15 mg/ml.

In another embodiment, the single dose of the multivalent vaccine composition is 0.5 to 2 ml, preferably 0.5 to 1 ml.

In another preferred embodiment, the present invention provides a stable multivalent vaccine composition wherein each dose comprises:

a) Diphtheria toxoid (DT) present in the amount 15-30 LF,

b) Tetanus toxoid (TT) present in the amount of 5-15 LF,

c) acellular Bordetella pertussis antigen (aP) Pertussis toxoid (PT), FHA and pertactin (P69 or PRN) and each present in an amount of 3-30 mg, each or killed whole cell pertussis in the amount of 10-30 IOU,

d) Hepatitis B surface antigen (HBS Ag) present in the amount 5-15 mg,

e) Haemophilius influenza type b PRP-TT conjugate antigen (Hib-PRP-TT) is present in the amount 5-15 mg,

f) IPV S19 Type 1 at a dose of 40 D-antigen units, IPV S19 Type 2 at a dose of 8 D- antigen units and IPV S19 Type 3 at a dose of 32 D-antigen units,

g) optionally, inactivated rotavirus (IRV) antigen present in the amount 2.5-15 mg, and h) one or more pharmaceutically acceptable carriers.

In yet another preferred embodiment, the present invention provides a stable multivalent vaccine composition wherein each dose comprises:

a) Diphtheria toxoid (DT) present in the amount 15-30 LF,

b) Tetanus toxoid (TT) present in the amount of 5-15 LF,

d) Hepatitis B surface antigen (HBS Ag) present in the amount 5-15 mg,

f) IPV S19 Type 1 at a dose of 20 D-antigen units, IPV S19 Type 2 at a dose of 4 D- antigen units and IPV S19 Type 3 at a dose of 16 D-antigen units,

g) optionally, inactivated rotavirus (IRV) antigen present in the amount 2.5-15 mg, and h) one or more pharmaceutically acceptable carrier.

In yet another embodiment, the present invention provides a stable multivalent vaccine composition wherein each dose comprises:

a) Diphtheria toxoid (DT) present in the amount 15-30 LF,

b) Tetanus toxoid (TT) present in the amount of 5-15 LF,

d) Hepatitis B surface antigen (HBS Ag) present in the amount 5-15 mg,

f) IPV S19 Type 1 at a dose of 13.33 D-antigen units, IPV S19 Type 2 at a dose of 2.66 D-antigen units and IPV S19 Type 3 at a dose of 10.66 D-antigen units,

g) Inactivated rotavirus (IRV) antigen present in the amount 2.5-15 mg, and

h) one or more pharmaceutically acceptable carrier.

In another embodiment, the present invention provides a stable multivalent vaccine composition wherein each dose comprises:

a) Diphtheria toxoid (DT) present in the amount 15-30 LF,

b) Tetanus toxoid (TT) present in the amount of 5-15 LF,

c) acellular Bordetella pertussis antigen (aP) Pertussis toxoid (PT), FHA and pertactin (P69 or PRN) are present in the amount 3-30 mg, each or killed whole cell Pertussis in the amount of 10-30 IOU,

d) Hepatitis B surface antigen (HBS Ag) present in the amount 5-15 mg,

f) IPV S19 Type 1 at a dose of 4 D-antigen units, IPV S19 Type 2 at a dose of 0.8 D- antigen units and IPV S19 Type 3 at a dose of 3.2 D-antigen units,

g) Inactivated rotavirus (IRV) antigen present in the amount 2.5-15 mg, and

h) at least one or more pharmaceutically acceptable carriers.

In yet another embodiment, the pharmaceutically acceptable carrier includes adjuvants, excipients, preservatives, stabilizers, buffers, pH indicators, surfactants, solvents, diluents and the like. Suitable stabilizers include Ml 99 medium, amino acids like Alanine, Histidine, Proline, Glutamine, Glycine and the like. Suitable preservatives include 2-phenoxyethnaol, m- cresol, benzyl alcohol and benzoic acid and the like or a mixture thereof. The concentration of the preservative may range from of 0.001% to 2.0 % w/v (0.5 to lOmg). Suitable buffers include sodium phosphate, potassium phosphate, citrate buffer or histidine.

In yet another preferred embodiment, the present invention provides a stable multivalent vaccine formulation wherein each dose comprises:

a) Diphtheria toxoid (DT) present in the amount 15-30 LF,

b) Tetanus toxoid (TT) present in the amount of 5-15 LF,

d) Hepatitis B surface antigen (HBS Ag) present in the amount 5-15 mg,

f) IPV S 19 Type 1 at a dose of 4 to 60 D-antigen units, S 19 Type 2 at a dose of 0.8 to 12 D-antigen units and S 19 Type 3 at a dose of 3.2 to 48 D-antigen units,

g) optionally inactivated rotavirus (IRV) antigen present in the amount 2.5-15 mg, and h) 2-phenoxyethanol (2-PE) as preservative, wherein the concentration of 2-PE may range from 0.001% to 2.0 % w/v.

The present invention also relates to a process for the preparation of fully liquid hexavalent vaccine, comprising inactivated poliomyelitis virus S 19 strain, Diphtheria toxoid (DT), Tetanus toxoid (TT), killed whole-cell Bordetella pertussis, or two or more acellular Pertussis components (aP) (e.g. Pertussis toxoid (PT), filamentous haemagglutinin (FHA) and pertactin (PRN)); an antigen from Hepatitis B, a conjugate of the capsular polysaccharide of Haemophilus influenzae type B (Hib) and carrier protein;, which comprises the steps of :

(i) aseptically transferring HepB antigen, Diphtheria toxoid and Tetanus toxoid to a blending vessel containing aluminium phosphate,

(ii) the mixture of step (i) is stirred for 2 to 20 hrs,

(iii) wP or two or more acellular Pertussis components (aP) is transferred to adsorbed mixture of step (ii) to obtain DTHwP / DTHaP mixture, followed by adjusting the pH to 5.5 to 7.5,

(iv)S 19 the IPV bulk antigen is added to the mixture obtained in step (iii),

(v) optionally adjusting the pH and adding a preservative to the mixture obtained in step

(iv),

(vi)Hib conjugate is added to the above mixture and (vii) finally, the volume is adjusted with a saline solution to produce the hexavalent composition.

In yet another aspect, the pH of wP solution is adjusted 6 to 7 before adding to the DTHepB mixture.

In yet another aspect, the mixture containing DTwP-HepB-IPV is cooled to 0 to 10 °C before the addition of Hib conjugate.

In one embodiment, the S 19 IPV of the invention may be unadsorbed and manufactured using monovalent polio bulk antigens or trivalent poliovirus (TPV) bulk antigens containing S 19 Type 1, S 19 Type 2 and S 19 Type 3 strains.

In another embodiment, various antigens of the invention may be adsorbed separately or together as a mixture onto an aluminium salt such as aluminium hydroxide, aluminium phosphate and the like or mixture of both aluminium hydroxide and aluminium phosphate. The adsorbent may be prepared in situ or may be added during the manufacturing process.

In a preferred embodiment, the vaccine formulation of the present invention is free of thiomersal. The multivalent composition of the present invention is in fully liquid form or one or more components may present in lyophilized form. For example, the conjugate of Hib-PRP may be present in lyophilized form.

In yet another embodiment, the vaccine formulation of the present invention is in the form of kit comprising conjugate of Hib-PRP in lyophilized form in one vial and other vial containing Diphtheria toxoid (DT) Tetanus toxoid (TT), acellular Bordetella pertussis antigen (aP) or killed whole-cell Pertussis, Hepatitis B surface antigen (HBS Ag), IPV S 19 Type 1, S 19 Type 2 and S 19 Type 3 in liquid form.

In an embodiment, antigens in the vaccine composition of the present invention will be present in "immunologic ally effective amounts" i.e. the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention of disease. The dosage treatment may be a single dose schedule or a multiple-dose schedule (e.g. including booster doses).

The vaccine compositions of the present invention may be in the form of lyophilised powder or in aqueous form. The aqueous form may be in the form of solution or suspension. The vaccine composition may be filled in a vial, ampoule or they may be presented as pre filled syringes. The vaccine of the present invention may be a single dose or multi-dose vial. The single dose may preferably be a 0.5 ml vial or ampoule or Pre-filled syringe. In one embodiment, the vaccine of the present invention is administered to an adult, adolescent, toddler, infant or less than one-year-old human and may be administered by injection.

The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely for the purposes of illustration and are not intended to limit the scope of the invention. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.

Example 1: Fully liquid hexa valent formulation

The process steps involved in manufacturing the hexavalent formulation described above is given below:

i. HepB antigen, Diphtheria toxoid and Tetanus toxoid were added to a blending vessel containing aluminium phosphate gel and the mixture was stirred for 12-16 hrs, ii. the pH of wP was adjusted to 6.8-7.2 and added to the DTHepB mixture obtained in step (i), and the pH was adjusted to 6.2-6.5,

iii. to the above DTwP-Hep B mixture, S 19 IPV bulk antigen was added, followed by saline solution,

iv. a solution of 2-PE was added and the mixture was cooled at 2-8 °C,

v. to the mixture obtained in step (iv) HibTT was added,

vi. the volume was adjusted with normal saline solution, and vii. filled the solution in individual containers.

Example 2: Stability studies on formulation of Example 1

The stability and potency of the formulation prepared according to the Example 1 was studied under real-time and accelerated stability conditions.

The initial, 3 months real time (2-8°C) and the accelerated (25°C ± 2°C) stability study results are given in Table 1.

Rats were immunized with different dilutions of Hexavalent formulation and Reference Standard. The animals were bled on day 21. Sera was tested for IPV immunogenicity by Sera neutralization test and results are given in Table 2. The acceptance criteria for IPV in vivo potency is relative potency of test sample should not be significantly less than the reference vaccine.

Table 1: Results of stability studies performed on formulation of Example 1

Legend: NLT (Not less than); NMT (Not more than); PRP (polyribosyl ribitol phosphate); SHD (Single Human Dose); LCL (Lower Confidence Limit); UCL (Upper Confidence Limit); RP (Relative Potency)

The data shown above complies with the specification limits and indicates that the formulation is stable, and the antigens are potent.

Example 3: Fully liquid Pentavalent formulation

The process steps involved m manufacturing the Pentavalent formulation described above is given below :

iii. to the above DTwP-Hep B mixture, S19 IPV bulk antigen was added, followed by saline solution,

iv. a solution of 2-PE was added and the volume was adjusted with normal saline solution, stirred, and

v. filled in individual containers.

Example 4: Fully liquid tetra valent formulation

The process steps involved in manufacturing the Tetravalent formulation described above is given below :

i. Diphtheria toxoid and Tetanus toxoid were added to a blending vessel containing aluminium phosphate gel and the mixture was stirred for 12-16 hrs,

ii. the pH of wP was adjusted to 6.8-7.2 and added to the DT mixture obtained in step (i), and the pH was adjusted to 6.2-6.5,

iii. to the above DTwP mixture, S19 IPV bulk antigen was added, followed by saline solution,

v. filled in individual containers.

ADVANTAGES OF THE INVENTION

Multivalent combination vaccines provide protection against several diseases, simplify vaccine administration and allow for the administration of a number of vaccines without requiring additional health clinic visit and injections. Combination vaccines results in improved compliance in vaccination programs, fewer missed opportunities to vaccinate, improved record-keeping and tracking along with convenience and decreased costs for administration. The present invention provides for multivalent combination vaccines containing IPV antigens originated from S19 strain, which are equally or more immunogenic as compared to commercially available multivalent combination vaccine based on Salk and or Sabin strain. Global shortage in the supply of inactivated polio vaccine (IPV) has led to re- strategizing use of IPV in the form of fractional doses for vaccinating a larger population. The present invention provides means of recouping with this shortage as the containment requirements for manufacturing S19 IPV bulk antigen are less as compared to presently used Salk and Sabin bulk antigens. Consequently, IPV compositions of the present invention can be manufactured at low containment level independent of the herd immunization status of the country, the S19 strain is handled pre- and post-polio eradication, which would potentially lead to large scale production, economy of scale cost, and hence availability for immunization for all.

Claims

The claims:

1. A multivalent vaccine formulation comprising:

a. at least one inactivated poliovirus (IPV) S19 strain selected from a group comprising S19 Type 1, S19 Type 2 and S19 Type 3;

b. at least one antigen selected from a group comprising Diphtheria toxoid (DT), Tetanus toxoid (TT), whole-cell Bordetella pertussis (wP), acellular Pertussis (aP), Hepatitis B surface protein, Haemophilus influenzae Type b polysaccharide, Inactivated Japanese encephalitis (JE) virus and Inactivated Rotavirus 116E strain (IRV); and

c. at least one pharmaceutically acceptable carrier.

2. The vaccine formulation as claimed in claim 1, wherein IPV S19 Type-1 is a Mahoney strain or a Brunhilde strain.

3. The vaccine formulation as claimed in claim 1, wherein IPV S 19 Type-2 is a MEF-I strain.

4. The vaccine formulation as claimed in claim 1, wherein IPV S 19 Type-3 is a Saukett strain.

5. The vaccine formulation as claimed in claim 1, wherein acellular Pertussis (aP) antigens are selected from a group comprising Pertussis toxoid (PT), Filamentous Haemagglutinin (FHA), Pertactin (PRN), Fimbriae Type 2 and Fimbriae Type 3.

6. The vaccine formulation as claimed in claim 1, wherein Haemophilus influenzae Type b polysaccharide is conjugated to a carrier protein selected from a group comprising CRM197, Diphtheria toxoid (DT), Tetanus toxoid (TT) and outer membrane protein (OMP).

7. The vaccine formulation as claimed in claim 1, wherein the vaccine comprises inactivated poliovirus (IPV) S19 Type-1 at a dose of 4 to 60 D-antigen units.

8. The vaccine formulation as claimed in claim 1, wherein the vaccine comprises inactivated poliovirus (IPV) S19 Type-2 at a dose of 0.8 to 12 D-antigen units.

9. The vaccine formulation as claimed in claim 1, wherein the vaccine comprises inactivated poliovirus (IPV) S19 Type-3 at a dose of 3.2 to 48 D-antigen units.

10. The vaccine formulation as claimed in claim 1, wherein the vaccine comprises Tetanus toxoid (TT) at a dose of 2.5 to 30 Ff.

11. The vaccine formulation as claimed in claim 1, wherein the vaccine comprises Diptheria toxoid (DT) at a dose of 5 to 35 Ff.

12. The vaccine formulation as claimed in claim 1, wherein the vaccine comprises whole-cell Bordetella pertussis (wP) at a dose of 5 to 50 IOU.

13. The vaccine formulation as claimed in claim 1, wherein the vaccine comprises one or more acellular Pertussis (aP) antigens at a dose of 3 to 30 mg.

14. The vaccine formulation as claimed in claim 6, wherein the vaccine formulation comprises 5 to 25 mg Haemophilus influenzae Type b antigen conjugated to 10 to 25 mg carrier protein.

15. The vaccine formulation as claimed in claim 1, wherein Hepatitis B surface protein is present at a dose of 5 to 15 mg per dose.

16. The vaccine formulation as claimed in claim 1, wherein Inactivated Rotavirus 116E strain is present at a dose of 2.5 to 15 mg per dose.

17. The vaccine formulation as claimed in claim 1, wherein Inactivated Japanese encephalitis virus is present at a dose of 5 to 20 mg per dose.

18. The vaccine formulation as claimed in any one of claims 1 to 17, wherein one or more antigen is optionally adsorbed onto an aluminium salt selected from a group comprising aluminium hydroxide, aluminium phosphate and combinations thereof.

19. A hexavalent vaccine formulation comprising:

a. Inactivated Poliovirus S19 strain selected from S19 Type 1 Mahoney (MahPl) at a dose of 4 to 60 D-antigen units, S19 Type 2 MEF-1 (MEF2P1) at a dose of 0.8 to 12 D-antigen units and S 19 Type 3 Saukett (SktPl) at a dose of 3.2 to 48 D-antigen units;

b. Diptheria toxoid (DT) at a dose of 5 to 35 Lf;

c. Tetanus toxoid (TT) at a dose of 2.5 to 30 Lf;

d. Whole-cell Bordetella pertussis (wP) at a dose of 5 to 50 IOU;

e. Haemophilus influenzae Type b polysaccharide conjugated to tetanus toxoid at a dose of 2 to 15 mg per dose;

f. Hepatitis B surface antigen (HepB) at a dose of 5 to 15 mg;

g. an aluminum salt at a concentration of about 0.1 to 0.5 mg per dose;

h. 2-phenoxyethanol (2-PE) present at about 2 to 10 mg per dose; and

i. optionally, at least one pharmaceutically acceptable carrier.

20. A pentavalent vaccine formulation comprising:

b. Diptheria toxoid (DT) at a dose of 5 to 35 Lf;

c. Tetanus toxoid (TT) at a dose of 2.5 to 30 Lf; d. Whole-cell Bordetella pertussis (wP) at a dose of 5 to 50 IOU;

e. Hepatitis B surface antigen (HepB) at a dose of 5 to 15 mg;

f. an aluminum salt at a concentration of about 0.1 to 0.5 mg per dose;

g. 2-phenoxyethanol (2-PE) present at about 2 to 10 mg per dose; and

h. optionally, at least one pharmaceutically acceptable carrier.

21. A tetravalent vaccine formulation comprising:

b. Diptheria toxoid (DT) at a dose of 5 to 35 Lf;

c. Tetanus toxoid (TT) at a dose of 2.5 to 30 Lf;

d. Whole-cell Bordetella pertussis (wP) at a dose of 5 to 50 IOU;

e. an aluminum salt at a concentration of about 0.1 to 0.5 mg per dose;

f. 2-phenoxyethanol (2-PE) present at about 2 to 10 mg per dose; and

g. optionally, at least one pharmaceutically acceptable carrier.

22. The vaccine formulation as claimed in any one of claims 1 to 21, optionally comprising pharmaceutically acceptable adjuvants, excipients, preservatives, stabilizers, buffers, pH indicators, surfactants, solvents, residuals or diluents.

23. The vaccine formulation as claimed in any one of claims 1 to 22, wherein preservatives are selected from a group comprising 2-phenoxyethnaol, m-cresol, benzyl alcohol, benzoic acid and combinations thereof.

24. The vaccine formulation as claimed in any one of claims 1 to 23, wherein a single dose of the formulation is about 0.5 mL.