This is a National Phase Application filed under 35 U.S.C. 371 as a national stage of PCT/IB2017/055100, filed Aug. 24, 2017, and claims priority benefit from Indian Application No. 201621029037, filed Aug. 26, 2016, the content of each is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to stable combination vaccine(s) comprising a mixture of antigens for prevention and prophylaxis of infections caused by Rotavirus, poliomyelitis virus, Haemophilus influenzae, Corynebacterium diphtheriae, Clostridium tetani, Bordetella pertussis (whole cell) and Hepatitis B virus. The invention in particular relates to a stable multivalent combination vaccine comprising of significantly dose reduced Salk IPV or Sabin IPV (IPV) antigens and injectable heat inactivated Rotavirus antigen(s) obtained from Rotavirus (CDC-9) strains.

BACKGROUND OF THE INVENTION

The poliovirus invades the nervous system and can cause irreversible paralysis in a matter of hours. Three types of poliovirus exist globally i.e. Type 1, Type 2, & Type 3.

The prevalence of polio virus has largely been decreased by the use of Oral Polio Vaccine (OPV), based on live-attenuated Sabin polio strains. However, OPV has limitations for the post-eradication era. OPV contains circulating vaccine-derived polioviruses (cVDPVs) which are transmissible and can become neurovirulent (similar to wild polioviruses) resulting in vaccine associated paralytic poliomyelitis. Such strains can potentially re-seed the world with polioviruses and negate the eradication accomplishments. To prevent the emergence of circulating vaccine-derived polioviruses (cVDPVs), WHO's Strategic Advisory Group of Experts (SAGE) has recommended at least one dose of IPV along with Oral Polio Vaccine (OPV) in countries currently using OPV. (Ref: World Health Organization. Meeting of the Strategic Advisory Group of Experts on Immunization, November 2012—conclusions and recommendations. Wkly Epidemiol Rec 2013; 88:1-16; PMID:23311010).

Presently, IPV is made using wild polio strain i.e. Salk strain and the newer sabin strains which is made from the same attenuated Sabin strains as those used in the live attenuated OPV. The approved standard dose of polio vaccines delivered by intramuscular (IM) or deep subcutaneous (SC) injection contains D antigens as 40 Units of inactivated poliovirus type 1 (Mahoney), 8 units of inactivated poliovirus type 2 (MEF-I) and 32 units of inactivated poliovirus type 3 (Saukett) (e.g. Infanrix-IPV™). But IPV has high production costs as compared to OPV, mainly due to requirements for more virus per dose; additional downstream processing (i.e. concentration, purification and inactivation), and the related QC-testing; loss of antigen or poor recovery in downstream; and containment.

The production costs of IPV is currently estimated to be about 20-fold more expensive than OPV. The future global demand for IPV following eradication of polioviruses could increase from the current level of 80 million doses to 450 million doses per year. Therefore to reduce the final cost of IPV, efforts are undergoing to reduce the quantity of antigenic components i.e. to produce dose reduced IPV formulations.

Globally, Rotavirus is the leading cause of severe acute diarrhea and vaccine is found a promising solution to reduce the disease burden. It has been estimated that the number of rotavirus deaths in the world declined from 528 000 to 215 000 during thirteen years from 2000 to 2013, since rotavirus vaccine had been introduced in >60 countries . Out of that, an estimated 47 100 (22%) rotavirus deaths occurred in India in 2013. India, Nigeria, Pakistan, and Democratic Republic of Congo, these four countries accounted for approximately half (49%) of all estimated rotavirus deaths in 2013 .

In recent studies carried worldwide, it is observed that 4 serotypes of rotavirus i.e. G1, G2, G3 and G4 represents over 88% of the strains analyzed globally. The G1 serotype of rotavirus A is one of the most common forms of the virus which cause the disease worldwide, whereas serotype G9 viruses associated have been emerging since the late 1990s and now represent approximately 4% of global isolates. Vaccination against rotavirus-mediated disease is one strategy for addressing this significant health problem. The two currently licensed oral rotavirus vaccines, RotaTeq and Rotarix, which are very effective in reducing cases of severe diarrhea among children in developed and middle income countries, are much less efficacious (˜50%) in low income countries of Africa and Asia. [Refer Tate JE et al. “Sustained decline in rotavirus detections in the United States following the introduction of rotavirus vaccine in 2006. Pediatr Infect Dis J 2011; 30:S30-4; and Yen C et al. “Decline in rotavirus hospitalizations and health care visits for childhood diarrhea following rotavirus vaccination in El Salvador”. Pediatr Infect Dis J 2011; 30:S6-10]. In addition, the two current rotavirus vaccines have been associated with a risk of intussusception among vaccinated infants. [Refer Patel MM et al. “Intussusception risk and health benefits of rotavirus vaccination in Mexico and Brazil. N Engl J Med 2011; 364:2283-92 and Buttery JP et al. “PAEDS/APSU Study Group. Intussusception following rotavirus vaccine administration: post-marketing surveillance in the National Immunization Program in Australia”. Vaccine 2011; 29:3061-6].

One of the candidate Rotavirus vaccine of Bharat Biotech International is based on a 116E rotavirus strain, G9P[11] which is a naturally occurring reassortant containing one bovine rotavirus gene P[11] and ten human rotavirus genes. The protection offered by this vaccine during the first 2 years of life is against the array of circulating genotypes including G1P[8], G2P[4], G12P[6], G12P[8] and G9P[4].Thus it fails to provide cross protection for other strains like G3,G4,G5, G6,G8, G10, G11,G13 and G14. Also the efficacy of 116E based vaccine in the first 2 years of life has been modest (48 to 55%) as it is for other licensed vaccines. [Refer Nita Bhandari et al. “Efficacy of a monovalent human-bovine (116E) rotavirus vaccine in Indian children in the second year of life”; Vaccine. 2014 Aug 11;32 Suppl 1:A110-6]. Also, preclinical testing of several other intramuscularly administered IRV candidates that were based on G1, G3 or G6 have only shown partial protection.

Also inactivation of Rotavirus with β-propiolactone (BPL), an agent commonly used for the inactivation of many viruses, has been shown to cause severe damage to the integrity and biochemical composition of rotavirus particles. In addition, BPL-treated rotavirus showed reduced viral hemagglutinating activity and intramuscular injection with this material in mice evoked less neutralizing antibody than immunization with live virus. [Refer Offit PA et al “Noninfectious rotavirus (strain RRV) induces an immune response in mice which protects against rotavirus challenge”. J Clin Microbiol 1989; 27:885-8].

In many parts of the world, immunization with IPV has already replaced OPV so that in a few years, rotavirus vaccine will be the only vaccine administered orally adding to delivery costs. Several operational and logistic considerations thus favor an IRV for use in both developing and industrialized countries.

The appearance of dimers in D/T, seems to be a consequence of the detoxification process by formalin. The existence of dimers can affect the efficiency of the conjugation process—presumably through steric hindrance at the protein surface—leading to a loss of activity, and it is considered desirable that the level of monomers should not drop below 80%. Previously, it has been reported that T with at least 60% monomer can be obtained by using HIC followed by Ion exchange; T with 73% monomer content can be obtained by using only HIC Phenyl sepharose, T with 55% monomer content can be obtained by using only Ammonium sulphate. Therefore an alternative single step method is needed to obtain D/T having at least 80% monomer.

Combination vaccines comprises of two or more vaccines that could be given individually and put them into a single composition. Vaccinee gets the same protection as they do from individual vaccines given separately, but with fewer shots. A combination vaccine thus provides immunogenicity against large number of diseases and is always advantageous over the monovalent vaccines as the compliance is increased by reducing the number of separate vaccinations.

A heptavalent combination vaccine is being developed by Bharat Biotech International that consists of D, T, Acellular pertussis, Sabin IPV (type 1: 40 DU, type 2: 8 DU, type 3: 32DU), Single strain inactivated Rotavirus (G9 strain i.e. 116E strain), a conjugate Haemophilus influenza type b PRP conjugate to TT and a Recombinant Hepatitis B vaccine. As discussed earlier, such Heptavalent combination vaccine comprising of 116E based vaccine would fail to provide cross protection for other strains like G3, G4, G5, G6, G8, G10, G11, G13 and G14. Further the efficacy of said IRV component of combination vaccine in the first 2 years of life will be modest (48 to 55%) as it is for other licensed vaccines.

Another combination vaccine, Hexyon® (also called Hexacima® and Hexaxim®) from Sanofi Pasteur, contains aP. This vaccine is likely to be targeted for private markets in Europe and worldwide. Another hexavalent combination vaccine with aP antigen is currently in Phase III clinical studies, developed jointly by Merck and Sanofi Pasteur.

Currently, GSK's Infanrix Hexa® is the only globally marketed hexavalent pediatric combination vaccine containing IPV. This vaccine contains an acellular pertussis (aP) component and is presented in a syringe-plus-lyophilized vial format because of instability of the Hib component. The major barriers for use of Infanrix Hexa® (GSK), in a developing-country setting are the price of the vaccine product, the requirement for reconstitution of a lyophilized form, and concerns regarding effectiveness of an aP vaccine in the developing world. From a cost perspective, aP antigens have historically exceeded the cost of wP antigens by a factor of ten to more than 30 due to manufacturing differences and royalty costs. Thus, use of whole cell pertussis (wP) in multivalent combination vaccines intended for developing countries has become important both because of cost and emerging concerns about the long-term effectiveness of aP vaccines, especially in developing-country settings.

Several multivalent combination vaccines with wP and IPV are under development. However IPV antigens are not compatible with the common vaccine preservative thimerosal, a mercury-containing compound with antibacterial activity, which causes the polio capsid to lose its antigenicity. Thimerosal is used by many vaccine manufacturers in the inactivation of live B. pertussis organisms to make wP vaccine bulks, which is carried through into the final product, but also causes loss of antigenicity of IPV, and therefore IPV may need to be presented in a separate vial from thiomersal-containing wP to retain its potency over time.

GSK's whole-cell combination vaccine (DTwP-IPV-HBV//Hib) is reported to be in several early clinical trials. However the polio immunogenicity of this product was not as good as a comparator vaccine and it was found that the IPV dose would minimally need to be the same as the current standard IPV dose vaccine or may need to be increased.

Reduced-dose efficacious vaccine formulations which provide protection against infection using a lower dose of IPV antigen are desirable in situations where the supply of conventional vaccine is insufficient to meet global needs or where the cost of manufacture of the conventional vaccine prevents the vaccine being sold at a price which is affordable for developing countries. Also the exposure to lower dose of IPV; compared to the existing marketed formulations could be safer. A multivalent combination vaccine could simplify complex pediatric routine immunization schedules, improve compliance, and reduce delivery costs. However, IPV-containing multivalent vaccines have been a technical challenge for vaccine manufacturers since work began in the early 1990s to combine pediatric vaccines.

Instability of Hib antigen in the presence of aluminum adjuvant, more specifically aluminum hydroxide is a major technical issue. Thus the technical challenge that a manufacturer would need to resolve would be the avoidance of different aluminum adjuvants, if the vaccine bulks going into the combination already had been based on different aluminum chemistries. Thus the sequence of addition of antigen becomes an important factor, as mixing of incompatible antigens or adjuvants may lead to undesired physical appearances for the final product (such as extra precipitates and difficulty in re-suspension) further resulting in unacceptance of the final combination vaccine product [Refer Malecker et al 1996, “Factors affecting the ability of experimental vaccines to protect guinea pigs against lethal challenge with Diphtheria Toxin”; presented at WHO/IABS/NIBSC International meeting on the control and standardization of Acellular pertussis Vaccines, UK, Sep. 26-27 1996].

The currently known and available combination vaccines may not contain appropriate formulations of appropriate antigens in appropriate immunogenic forms for achieving desired levels of efficacy and immunogenicity in the susceptible human population, for a number of diseases in one shot. Most importantly, no multivalent combinations with dose reduced IPV (IPV), broadly cross-protective IRV and whole cell pertussis (wP) are commercially available. Given the above discussed scenario with respect to combination vaccines, there remains a distinct need for affordable all liquid multivalent combination vaccine(s) suitable for developing world providing equivalent or improved seroprotection for individual antigens devoid of antigenic interference.

SUMMARY OF INVENTION

The present invention relates to stable, immunogenic combination vaccine(s) comprising a mixture of antigens for prevention and prophylaxis of infections caused by Rotavirus, Poliomyelitis virus, Haemophilus influenzae, Corynebacterium diphtheriae, Clostridium tetani, Bordetella pertussis and Hepatitis B virus. The invention particularly provides a multivalent combination vaccine comprising of i) significantly dose reduced Salk IPV or Sabin IPV (IPV) antigens prepared by utilizing improved methods of formaldehyde inactivation and alum hydroxide adsorption resulting in maximum recovery of D-antigen and ii) Injectable heat inactivated Rotavirus antigen(s) obtained from Rotavirus (CDC-9) strains that provides a broad cross-protective immunity among human rotavirus strains, iii) Hib PRP-carrier protein conjugate having improved stability and immunogenicity wherein said Hib PRP-carrier protein conjugate is initially prepared by using novel conjugation process and subsequently blended at low temperature in presence of a stabilizer for minimizing free PRP release iv) whole cell pertussis antigen with improved immunogenicity and stability obtained by addition of whole cell pertussis antigen at a later stage in a blend thereby minimizing hydrolysis based degradation v) Homogenous fractions of Diphtheria toxoid and Tetanus toxoid obtained by removal of undesirable aggregates by using Gel Permeation chromatography. The process of making such stable and immunogenic vaccine compositions by i) individually adsorbing dose reduced IPV, IRV antigens on alum hydroxide and keeping other antigen(s) either unadsorbed or adsorbed on Alum Phosphate or Alum hydroxide , or a combination of both and ii) using a particular order of addition of antigens during blending is also disclosed. Further, present invention provides affordable combination vaccine(s) suitable for developing world providing equivalent or improved seroprotection for individual antigens devoid of antigenic interference.

