TITLE: Combination Rota-Polio vaccine formulations for oral administration

FIELD OF THE INVENTION

The invention relates to combination Rota-Polio vaccine formulations for oral administration. More particularly, the invention relates to stable combination Rota-Polio vaccine formulations for oral administration comprising human-bovine natural ressortants and naturally attenuated rotavirus strains and polio strains. The combination Rota-Polio vaccine formulations of the invention are stable for 12 months in both liquid and lyophilized form at -200 C storage temperature. The invention also relates to the method of preparation of such stable combination Rota-Polio vaccine formulations for oral administration.

BACKGROUND OF THE INVENTION

Vaccination is an important tool for handling healthcare programs both in developed and developing countries. The current global scenario calls for a more-efficacious, acceptable, cost-effective and reliable method of immunization for many fatal diseases. Today children receive vaccines against 11 diseases in the first 2 years of the life and may receive as many as 5 injections at a single office visit.

In 1980, infants only received diphtheria-tetanus-pertussis (DPT) vaccine and measles-mumps-rubella (MMR) vaccine by injection in the first two years of the life. Licensure of the Haemophilus influenzae type b (Hib) vaccines beginning 1988 added more injections to the schedule. In 1991, the advisory Committee on Immunization Practices (ACIP) recommended that all infants be immunized with 3 doses of hepatitis B virus vaccine (Hep B) by 18 months of age. The varicella vaccine was added to the immunization schedule in 1995 and a heptavalent pneumococcal conjugate vaccine for children younger than 5 years of age was added in 2000. Today children may receive 20 injections by the age of 2 years to complete their immunization series recommended by American Academy of Pediatrics (AAP), ACIP and the American Association of Family Physician (AAFP).

The availability of combined vaccines containing protective antigens against the majority of diseases for which universal immunization is recommended in infancy would simplify the implementation, increase the acceptance, reduce the global cost of immunization programs and improve disease control, while offering the possibility of disease elimination or even pathogen eradication. The desirability of combined vaccines is further enhanced, and made more urgent, because of the increasing number of diseases that can be prevented by vaccination. The complicated logistics of administering different vaccines that each requires several inoculations is a significant barrier to successful immunization of a population. Furthermore, interest in immunization is continuously gaining momentum since it is now generally recognized that vaccines are among the safest and most cost-effective medical interventions for infectious diseases that continue, in spite of the widespread use of efficacious antimicrobial drugs, to be an important cause of morbidity and mortality. This burden is likely to increase due to the development of antimicrobial resistance. Major research work is being done towards developing more polyvalent vaccines by adding antigens such as inactivated polio virus, conjugated Haemophilus influenzae type b polysaccharide and hepatitis B surface antigen to the diphtheria–tetanus–pertussis vaccine either in its ‘classical'' (whole-cell) or more purified (acellular) formulations.

However, development of combined vaccines involves more than the simple mixing of existing antigens. Possible incompatibilities or mutual interferences between the antigens themselves or between excipients, preservatives, adjuvants, residual contaminants, stabilizers and suspending fluids make it mandatory that each formulation be thoroughly tested for required immunogenicity, quality, stability, efficacy and safety.

Combination vaccines available for many years include diphtheria and tetanus toxoids and whole-cell pertussis vaccine (DTwP), measles-mumps-rubella vaccine (MMR), and trivalent inactivated polio vaccine (IPV). Combinations licensed in recent years in the United States include diphtheria and tetanus toxoids and acellular pertussis vaccine (DTaP), DTwP-Haemophilus influenzae type b (Hib) vaccine (DTwP-Hib), DTaP-Hib and Hib-hepatitis B (HepB) vaccine (Hib-HepB). In the future, combination vaccines might include increasing numbers of components in different arrays to protect against these and other diseases, including hepatitis A, Neisseria meningitidis, Streptococcus pneumoniae, and varicella.

Advantages of combining childhood vaccines include reducing the number of visits, injections and patient discomfort, increasing compliance, and optimizing prevention. As per the World Health Organization’s recommendations, routine infant immunization programs should also include vaccination against Haemophilus influenza type B (HIB) alongwith the combined diphtheria, tetanus, pertussis (DTP)-hepatitis B (HBV) vaccination. However, the effectiveness and safety of the combination vaccines needs to be carefully monitored to ensure their effectiveness and adoptability in various communities worldwide.

Among various routes of vaccine administration, oral vaccines are found to be a superior alternative of vaccination, more particularly for those infections which take oral-fecal route or propagate through gastro-intestinal track. The oral administration is not only preferred for needle phobic children, but needle administered vaccines are also associated with the problems of re-use, misuse and an occasional lack of proper sterilization of the syringe. Injectable vaccines also require the expertise of the person administering the vaccine, which sometimes may also be a severe limitation in mass immunization programs using injectable vaccines. Whereas, oral administration of vaccine requires little or no training at all, which not only helps in mass immunization but also reduces the significant cost of vaccination programs requiring trained professionals and syringes for vaccine administration Therefore, oral vaccines may provide an effective vaccination strategy, especially in developing countries.

Rotavirus infection is the major cause of diarrhea related deaths in infants and in young children. Every year Rotavirus gastroenteritis causes deaths of 300,000 to 600,000 infants and young children worldwide.

International health agencies have promoted the development of Rotavirus vaccines as the best method of prevention of infantile mortality associated with Rota viral gastroenteritis. In 1997 and again in 2000, the WHO recommended that all new Rotavirus vaccines should be tested in Asia and Africa and that this testing should be performed concurrently with the trials conducted in the US and Europe. By doing this safety and efficacy of vaccines might be demonstrated in poor developing countries early during development, thereby accelerating the availability of new vaccines to the children who are most in need of them. All Rotavirus vaccine strains reported till date are either natural, live human bovine naturally or artificially attenuated or genetically engineered human bovine strains with various combinations of VP4,VP7 and other genes of human, bovine Rotavirus strains. Rotavirus vaccines developed to date have been based on the Rotavirus strains that have been isolated from humans or animals and in vitro reassorted and adapted to cell culture, formulated for oral use. Both mono and multivalent animal based strains have demonstrated efficacy as candidate vaccines.

Poliomyelitis is an acute infection that involves the gastrointestinal tract and occasionally, the central nervous system. It is acquired by fecal-oral route. In the pre-vaccine era, infection with poliovirus was common, with epidemics occurring in the summer and fall in temperate areas. The incidence of poliomyelitis declined rapidly after the licensure of inactivated polio vaccine. Although a polio eradication program led to elimination of polio in the Western hemisphere, outbreaks of polio due to wild strains became prevalent. Clinical manifestations of polio infection range from asymptomatic to symptomatic including acute flaccid paralysis of a single limb to quadriplegia, respiratory failure and rarely death.

Polio is caused by three types of quite stable viruses namely Type-1, Type-2 and Type-3 belonging to the family enteroviruses. There are different polio vaccines available. These vaccines are trivalent containing a mixture of all the three types of poliovirus so as to confer immunity against all of them.

One type of polio vaccine is inactivated polio vaccine (IPV) and the other type is Live Attenuated vaccine based on Sabin strain. The OPV is commonly used because of the ease of availability of monovalent bulks, larger number of manufacturing facilities, ease of administration and most significantly the manufacture of OPV does not pose any risk of accidental release of virulent polio into the environment. Construction of vaccines against rhinoviruses and enteroviruses, particularly polio virus, was done by introduction of defined mutations into their genomes. These mutations attenuate the virulence of wild types and can further attenuate existing live attenuated vaccine strains thereby making them less likely to revert back to virulence.

The Oral Polio Vaccine (OPV) was developed in 1958 by Dr. Albert Sabin. Sabin attenuated the wild type poliovirus by passaging the virus in monkey kidney epithelial cells. The commonly used form of the oral polio vaccine is trivalent, which means that it contains live attenuated strains of the three serotypes of poliovirus. Trivalent OPV is characterized in vivo by efficient growth properties in the intestinal tract, unaltered immunogenic properties with respect to wild type progenitors, and attenuated neurovirulence after experimental intra-spinal injection into primates. This means that an individual immunized with trivalent OPV induces long-lasting (frequently life-long) protective immunity of the gastrointestinal tract to all known forms of poliovirus.

There are many biological and practical advantages to the Oral Polio Vaccine. Since attenuated vaccines are capable of transient growth, the OPV allows prolonged exposure of the immune system to the epitopes on the attenuated organisms, resulting in increased immunogenicity and memory-cell development. OPV also indirectly protects other susceptible individuals by secondary vaccination, which means that vaccinated individuals may spread the vaccine virus in the community and thereby inhibit the spread of the wild type virus if it occurs in the population. Significantly, OPV prevents the vaccinee from acting as a carrier of wild type poliovirus, and as stated, confers long-lasting immunity. The OPV is also easily administered by giving children a sugar cube or sugar liquid containing the vaccine, neither of which requires extensive medical training to be administered.

Various studies have been performed to ascertain the clinical interference between live polio vaccine and bovine rotavirus strains when given simultaneously.

In a clinical trial carried out in 3-month-old infants to assess whether concomitant oral administration of live polio and rotavirus RIT 4237 vaccines would reduce their immunogenicity as a result of mutual interference. One hundred and sixty breast-fed male and female infants were randomly allocated to four study groups to receive in a blind fashion the poliovirus vaccine, the RIT 4237 vaccine, a combination of both vaccines or a placebo preparation. Antibody titres were measured in pre- and post-vaccination serum samples by the ELISA test and the neutralizing antibody test (NT) for rotavirus and by the NT for polioviruses types 1 and 3. The percentage of subjects with immune responses to rotavirus in the placebo group was low, indicating the absence of wild rotavirus circulation in the population. Antibody responses against polio types 1 and 3 were found in about a quarter of the infants receiving a placebo because the study was performed during a polio vaccination campaign when vaccine viruses are known to circulate. The results showed that 73% of seroconversion was obtained when RIT 4237 was administered alone and that the responses to polioviruses types 1 and 3 were good. However, simultaneous administration of polio and RIT 4237 vaccines caused a significant reduction of the antibody response to rotavirus but not to polioviruses types 1 and 3. [1]

In another study a randomised, controlled trial of bovine rotavirus vaccine was undertaken in Gambian infants. Three doses were administered, from the age of ten weeks, concurrently with oral or killed polio vaccine. Pre-vaccination the rotavirus neutralizing antibody levels were high. 84/185 infants (45%) showed an increase in neutralizing antibody titre after receiving rotavirus vaccine, compared with 20/91 (22%) unvaccinated infants. Clinical rotavirus infection was detected in 24/78 (31%) children in the rotavirus/oral polio group, 34/83 (41%) children in the placebo/oral polio group, and 23/92 (25%) children in the rotavirus/killed polio group, giving an overall vaccine efficacy of 33% (95% CI 4-53%). RIT 4237 did not appear to reduce the severity of clinical infections. Most cases (92%) were caused by rotaviruses with short RNA electropherotypes. Serological responses to rotavirus vaccination appeared unaffected by the concurrent administration of oral polio vaccine. Lower types 1 and 3 polio antibody levels were found in children who received oral polio and rotavirus vaccines but the differences were not statistically significant. [2]

In another study done in Thailand, Rhesus-human reassortant tetravalent (RRV-TV) oral rotavirus vaccine was given at the same time as oral poliovirus vaccine (OPV) or inactivated parenteral poliovirus vaccine (IPV) to Thai infants at 2, 4 and 6 months of age. Sera for rotavirus antibody studies were taken prior to and one month after each vaccination. After the first dose of vaccine at 2 months of age, 37% of the infants receiving rotavirus vaccine with IPV but only 10% of those receiving it with OPV showed a seroconversion by rotavirus IgA ELISA antibody test (p<0.001). Likewise, neutralizing antibody seroconversion rates in initially seronegative subjects to rhesus rotavirus type 3 (RRV-3) after the first dose of RRV-TV vaccine were higher if the vaccine was given with IPV (74%) than if given with OPV (39%) (p=0.0069). After the second and third doses of vaccine, the rotavirus IgA ELISA and RRV-3-neutralizing antibody response rates were not different between groups. Development of neutralizing antibodies to human rotavirus serotypes 1, 2 and 4 in the first seven months of life in vaccinees receiving rotavirus vaccine with OPV tended to occur at a lower rate than in those receiving rotavirus vaccine with IPV but the antibody levels were not significantly different at 7 months of age. Poliovirus type 2 and type 3 antibody responses were not different in infants receiving the rotavirus vaccine with OPV as compared with infants receiving only OPV. The mean poliovirus type 1 antibody level was slightly but not significantly lower at 5 and 7 months of age in infants that received both rotavirus vaccine and OPV. [3] These results suggest that OPV is likely to interfere with the take of RRV-TV rotavirus vaccine but the interfering effect can largely be compensated for by giving multiple doses of RRV-TV vaccine or, possibly, by using a higher-titre rotavirus vaccine. Interference of RRV-TV vaccine with OPV may not pose a significant problem.

In another study, immunogenicity and reactogenicity of the oral rhesus rotavirus vaccine (RRV) were assessed among 72 infants (6 weeks old) in Lahore, Pakistan, from August to December 1985. Special emphasis was placed on the possible interaction or interference caused by giving RRV at the time infants received their first polio immunization. RRV was given to the infants at the same time as diphtheria-tetanus-pertussis (DTP), oral poliovirus vaccine (OPV), or inactivated poliovirus vaccine (IPV). The immune response to RRV was assessed by plaque-reduction neutralization 3 weeks after immunization and serum immunoglobulin (Ig) G and IgA antibody levels to poliovirus type 1 were tested by enzyme-linked immunosorbent assay (ELISA) after polio immunizations. Of the infants in the group given RRV with OPV, 50% had a two- to four-fold rise in neutralization titre against rotavirus, compared with 22% in the group given RRV with DTP and 20% in the group given RRV and IPV (P < 0•05). Interference by live oral polio vaccination in the response to RRV seems unlikely. No significant difference were observed in rates of seroconversion of IgG antibodies to poliovirus type 1 among infants aged 18 and 21 weeks who received RRV and OPV (81%), RRV with delayed OPV (67%), or RRV and IPV (59%). Administration of RRV was safe and was not associated with adverse reactions in the 6 weeks old infants. The low rate of seroconversion to rotavirus suggested that a more antigen-rich vaccine or multiple doses of the same vaccine could produce a better immune response. [4]
Successful co-administration of human rotavirus and oral poliovirus vaccines in Bangladeshi infants in a 2-dose schedule at 12 and 16 weeks of age. Co-administration of oral live-attenuated human rotavirus vaccine RIX4414 and oral polio vaccine (OPV) was assessed. Healthy infants were randomised to receive 2-doses of either: RIX4414 or placebo co-administered with OPV (12 and 16 weeks of age); or RIX4414 or placebo given 15 days after OPV. After vaccination, 56.5–66.7% of RIX4414 and 18.6% of placebo recipients had seroconverted for rotavirus IgA. No significant differences between RIX4414 groups with or without OPV co-administration were observed. No statistically significant differences were observed between groups for polio seroprotection rates. RIX4414 vaccine was immunogenic when co-administered with OPV and did not interfere with OPV seroprotection rates. [5]

Another study has been performed to determine whether an oral tetravalent rotavirus vaccine (RV-TV) can be safely co-administered with a combined diphtheria-tetanus-pertussis-Haemophilus influenzae type b vaccine (DTP/Hib) and oral poliovirus vaccine (OPV) to healthy infants without interfering with the immune responses to any of the component antigens. In the study, two hundred sixty-seven infants ages 2 to 3 months were randomly assigned in a double blind fashion to receive three doses of either placebo or RV-TV, each containing 4 × 105 plaque-forming units, concurrently with DTP/Hib and OPV at ~2, 4 and 6 months of age. Infants were followed for 5 days after each dose for the occurrence of adverse events and subsequently until 3 to 6 weeks after the third dose of RV-TV or placebo. Immune responses were assessed by measuring the post vaccination serum antibody titers to each component of DTP/Hib and OPV at 3 to 6 weeks after the third dose.

The results of the study showed that the percentage of infants who attained protective antibody titers and the distribution of antibody titers against diphtheria toxoid, tetanus toxoid and H. influenzae type b were not statistically different between RV-TV and placebo recipients. The distribution of antibody titers against different antigens of Bordetella pertussis (agglutinins, pertussis toxoid, filamentous hemagglutinin, fimbriae antigens and the 69-kDa outer membrane protein) was compared and no significant differences were found. The percentage of infants with detectable neutralizing antibodies against the three serotypes of poliovirus and the distribution of antibody titers was not statistically different between RV-TV and placebo recipients. There were no clinically meaningful differences in post vaccination reactions between RV-TV and placebo recipients.

The results of the study showed that three doses of RV-TV can be safely co-administered with three doses of DTP/Hib and OPV without diminishing an infant''s serum antibody responses to each component of these vaccines. Therefore RV-TV can be given at the standard childhood visits at 2, 4 and 6 months of age.

Further, administering an extra dose of live, attenuated virus vaccines to immunocompetent persons who already have vaccine-induced or natural immunity has not been demonstrated to increase the risk of adverse events. Examples of these include MMR, varicella, rotavirus, and oral polio vaccines.

All the studies in the prior art have shown that rotavirus vaccine can be safely co-administered with poliomyelitis vaccine without comprising the safety and efficacy of both the vaccines.

However, the recipient of the vaccines being infants and kids, co-administering the vaccines has less patient compliance, requires multiple clinic visits and chances of missed vaccination are very high. A combination vaccine comprising both rotavirus and polio vaccine would overcome this problem.

However, due to possible viral interference between live Rota and Polio viruses at the storage temperature and preserving culture / medium available in the prior art, it has not been possible till date to prepare a combination Rota-Polio oral vaccine either in liquid or lyophilized form.

OBJECT OF THE INVENTION

Primary object of the present invention is to provide combination Rota-Polio vaccine formulations for oral administration.

Another object of the invention is to provide combination Rota-Polio vaccine formulations for oral administration wherein such formulations are stable in both liquid as well as lyophilized form for atleast 12 months without reduction in immunogenicity of the vaccines.

Another object of the invention is to provide stable combination Rota-Polio vaccine formulations for oral administration wherein the rotavirus antigen is human bovine natural reassortant and naturally attenuated rotavirus strain selected from 116 E, (G9) [P11], I321(G10) [P11] and tetravalent UK Bovine Human rotaviruses.

Another object of the invention is provide stable combination Rota-Polio vaccine formulations wherein Polio vaccine comprises all three types i.e. Type-1, Type-2 and Type-3 poliomyelitis vaccines derived from Sabin.

Another object of the invention is to provide stable combination Rota-Polio vaccine formulations which are stable for atleast 12 months or more without any mutual interference between viral strains themselves and between the viral strains and pharmaceutically acceptable excipients.

A further object of the invention is to provide a method for preparation of such combination Rota-Polio vaccine formulations stable in both liquid as well as lyophilized form.

SUMMARY OF THE INVENTION

Novel combination Rota-Polio vaccine formulations for oral administration stable in liquid and lyophilized form for atleast 12 months at storage temperature of -200 C are disclosed. The combination Rota-Polio vaccine formulations comprise human-bovine natural ressortants and naturally attenuated rotavirus strains and polio strains. The rotavirus strains of the combination vaccine formulations are selected from rotavirus strains 116E (G9P11), I321(G10) [P11] and tetravalent UK Bovine Human rotaviruses. Poliomyelitis strains are live attenuated trivalent polio viruses comprising Type 1, Type 2 and Type 3 polio viruses. The combination vaccine formulations of the invention may be prepared and stored in liquid or lyophilized form.

The novel Rota-Polio combination vaccine formulations of the invention are stable for 12 months or more without any interference between the Rotavirus and Poliomyelitis strains and without any significant reduction in immunogenicity of the vaccines. The novel combination formulations of the invention remain stable with high titer of both rotavirus and poliomyelitis strains after 12 months when stored at temperature of -200 C.

A method of adaptation of human Rotaviruses that are human-bovine natural reassortants and naturally attenuated strains 116E(G9P11), I321(G10P11) and UK Bovine Human rotaviruses and other strains to Vero cells in order to get high titer is also disclosed. The invention also relates to the production of virus suspensions suitable for making stable combination Live Attenuated liquid and lyophilized formulations for oral administration in human beings.

The Rotavirus strain of 116E (G9)[P11], I321 (G10)[P11] and UK Bovine Human rotavirus confer substantial level of immunity in infants and young children. Also the ability of these strains to replicate in new born without causing disease in presence of higher titers of maternal antibody make them more promising live naturally attenuated monovalent Rotavirus vaccine candidate.

The present invention also relates to a cell substrate that can yield high titers of virus for vaccine production. The present invention provides a method for adaptation of Rotavirus to cell cultures. The method includes optimization of trypsin concentration for activation and in medium to get the virus harvests in 10 days useful to formulate live attenuated oral Rotavirus vaccine for human use.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the stability data of Rota -Polio combination vaccine at 250C for formulation 1 comprising rotavirus 116E equivalent to 105.5 FFU/0.5ml , polio type-1 equivalent to 106.7CCID50/0.5ml, polio type-2 equivalent to 105.7CCID50/0.5ml and polio type-3 equivalent to 106.5CCID50/0.5ml

Figure 2 shows the stability data of Rota-Polio combination vaccine at 2-80C for formulation 1 comprising rotavirus 116E equivalent to 105.5 FFU/0.5ml , polio type-1 equivalent to 106.7CCID50/0.5ml, polio type-2 equivalent to 105.7CCID50/0.5ml and polio type-3 equivalent to 106.5CCID50/0.5ml.

Figure 3 shows the stability data of Rota-Polio combination vaccine at -200C for formulation 1 comprising rotavirus 116E equivalent to 105.5 FFU/0.5ml, Polio Type-1 equivalent to 106.7CCID50/0.5ml, Polio Type-2 equivalent to 105.7CCID50/0.5ml and Polio Type-3 equivalent to 106.5CCID50/0.5ml.

Figure 4 shows the stability data of Rota-Polio combination vaccine at 250C for formulation 2 comprising rotavirus 116E equivalent to 105.5 FFU/0.5ml, Polio Type-1 equivalent to 106.1CCID50/0.5ml, Polio Type-2 equivalent to 105.0CCID50/0.5ml and Polio Type-3 equivalent to 105.9CCID50/0.5ml .

Figure 5 shows the stability data of Rota-Polio combination vaccine at 2-80C for formulation 2 comprising rotavirus 116E equivalent to 105.5 FFU/0.5ml, Polio Type-1 equivalent to 106.1CCID50/0.5ml, Polio Type-2 equivalent to 105.0CCID50/0.5ml and Polio Type-3 equivalent to 105.9CCID50/0.5ml.

Figure 6 shows the stability data of Rota-Polio combination vaccine at -200C for formulation 2 comprising rotavirus 116E equivalent to 105.5 FFU/0.5ml, Polio Type-1 equivalent to 106.1CCID50/0.5ml, Polio Type-2 equivalent to 105.0CCID50/0.5ml and Polio Type-3 equivalent to 105.9CCID50/0.5ml.

Figure 7 shows the stability data of Rota-Polio combination vaccine at -200C for formulation 3 comprising rotavirus I321 equivalent to 105.5 FFU/2.5ml, Polio Type-1 equivalent to 106.1CCID50/2.5ml, Polio Type-2 equivalent to 105.0CCID50/2.5ml and Polio Type-3 equivalent to 105.9CCID50/2.5ml.

Figure 8 shows the stability data of Rota-Polio combination vaccine at -200C for formulation 4 comprising rotavirus I321 equivalent to 105.5 FFU/0.5ml, Polio Type-1 equivalent to 106.1CCID50/0.5ml, Polio Type-2 equivalent to 105.0CCID50/0.5ml and Polio Type-3 equivalent to 105.9CCID50/0.5ml .

Figure 9 shows the stability data of Rota-Polio combination vaccine at -200C for formulation 5 comprising tetravalent UK Bovine Human rotavirus Serotype G 1, G 2, G 3 G 4 and Polio Type 1, Type 2 and Type 3.

DETAILED DESCRIPTION OF THE INVENTION

Detailed embodiments of the present invention are disclosed herein however, it is to be understood that the disclosed embodiments are merely exemplary of the invention, which can be embodied in various forms. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a basis for the claims and as a representative basis for teaching one skilled in the art to variously employ the present invention in virtually any appropriately detailed structure. Further, the terms and phrases used herein are not intended to be limiting but rather to provide an understandable description of the invention.

The present invention provides stable combination Rota – Polio vaccine formulations, wherein the combination vaccine formulations can be administered orally to infants and young children. Due to worldwide requirement and distribution of vaccines and diversified environmental conditions with varying temperatures at different places the novel combination Rota-Polio vaccine formulations of the invention were subjected to different storage temperatures for the stability test. The stability tests were performed at 250 C, 2-80 C and -200 C. Various stability tests established that the novel Rota-Polio combination formulations of the invention were stable at -200 C in both liquid as well as lyophilized form for atleast 12 months or more without any mutual interference between viral strains and without any interference between the viral strains and pharmaceutically acceptable excipients used as stabilizers in the formulations. In the stability test at temperature of -200 C, there was no significant drop in virus titers and therefore no reduction in immunogenicity of the individual virus strains after 12 months. The rotavirus strains in the novel combination vaccine formulations were selected from rotavirus 116E (G9)[P11], rotavirus I321(G10)[P11] and tetravalent UK Bovine Human rotaviruses. The poliomyelitis virus strains were live attenuated trivalent polio viruses comprising Type 1, Type 2 and Type 3 polio viruses.

Polio strains

The Polio vaccine in the combination vaccine of the present invention is derived from Sabin strain for effective immunization against poliomyelitis. The monovalent bulk suspensions are used for blending of trivalent oral polio vaccine by adding a stabilizer that confers stability to the preparation. In an essential embodiment of the invention, the polio viruses are stabilized to retain their shape, since the three dimensional structure of the viruses is highly important for their antigenicity.

Rotavirus 116E and I321

The human rotavirus strain 116E, a natural human-bovine reassortant, naturally attenuated rotavirus is characterized as a human G9 strain into which a single bovine VP4 gene, homologous to P[1l] gene segment is naturally introduced.

Rotavirus I321 G10[P11] is a bovine-human reassortant strain that was isolated from a large number of asymptomatic neonates in Bangalore, India in 1992-1993. Strain I321 was shown to possess 2 genes encoding the nonstructural proteins (NSPs) NSP1 and NSP3 of a human rotavirus and all other genes from a G10[P11] bovine rotavirus strain. The I321 VP7 aminoacid sequence was found to be 97.8% similar and 93.6% identical to the VP7 sequence of B223 , a serotype 10 bovine rotavirus. Serotype analysis using mAbs was specific for VP7 of different serotype 10 bovine rotavirus. Notable differences between the VP4 of I321 and B223 showed amino acid similarities and identities of 94.8% and 92.55 respectively. Notable difference between the VP4 of I321 and B223 include an additional arginine within the trypsin cleavage peptide of B223 that was not found in I321 and an additional cysteine at position 80 of B223 that is absent in I321. P type was found to represent a new and unique P serotype in humans.

Bharat Biotech International Ltd. (BBIL) obtained the human rotavirus strains, 116E and I321 from National Institute of Health (NIH) under the material transfer agreement with National Institute of Allergy and Infectious Diseases (NIAID), NIH, Bethesda, USA. The complete genomic sequence of rotavirus strains 116E and 1321 is reported. The original 116E (G9)[P11] and 1321 (G10)[1l] were adapted to grow in cell culture by passages in primary African green monkey kidney (AGMK) cells then in MA l04 cell substrate and later in Serially Passaged AGMK (SPAGMK). MA l04 and SPAGMK cell substrates are not approved by National Regulatory Authorities (NRA) for commercial vaccine production. Hence it is preferable to adapt 116E and I32 l and other rotavirus vaccine strains to approved, certified, licensed and fully characterized cell substrate like Vero cell substrate and/ or human diploid cells like MRC-5.

These two rotavirus vaccine strains individually have been prepared as pilot lots of monovalent oral rotavirus vaccine liquid formulations for clinical trials to be conducted in India.

UK Bovine Rotavirus

Scientists at the National Institute of Allergy and Infectious Diseases (NIAID) have made efforts on development of live attenuated Rotavirus vaccines capable of inducing immunity against each of the four epidemiologically important rotavirus serotypes. Reassortants, each containing the rotavirus serotype and the other ten genes from either a Rhesus or Bovine Rotavirus were produced and evaluated.

NIAID scientists have produced four human reassortant rotaviruse derived from the UK (Compton) Bovine Rotavirus strain which belongs to the same VP 7 serotype as the NCD V strain and the same VP4 serotype as the WC3 strain. These reassortants contain the VP7 gene from human serotypes 1, 2, 3 or 4 respectively and the remaining genes from the UK bovine strain.

The attenuated, quadrivalent human-bovine (UK) Rotavirus Reassortant Vaccine was evaluated in adults, 6 to 60 months old children and 1.5 to 5.9 month old infants.

HDBRV-1

The Bovine Rotavirus was isolated in Primary Calf Kidney cells from spontaneous diarrhea, a colostrum deprived calf in 1973. The Reassortant isolation, identification, amplification and plaque purifications were performed in primary or secondary AGMK cell cultures. Reassortants were recovered following mixed infection with the two parent viruses. Selection for the desired viral Reassortant was achieved by exposing progeny of the co-infected cultures to guinea pig hyper-immune antiserum prepared against the NCDV strain of bovine Rotavirus. This antiserum specifically neutralized the VP7 protein of the bovine rotavirus parent. The human rotavirus parent would not plaque under the conditions used. A plaque picked and amplified in AGMK cultures and fifth passage became the seed virus.

Genotype

This Reassortant contains the ninth gene from human rotavirus D strain encoding VP 7, a major neutralization protein. The remaining ten genes are derived from bovine rotavirus parent UK strain.

Serotype

This Reassortant has similar neutralization specificity to its human rotavirus parent serotype 1 ;a low level of cross-reactivity to antiserum directed at bovine rotavirus parent UK strain exists.

HDS1/BRV-1

The human Rotavirus DS-1 strain (serotype 2) was originally detected in the stool of a child hospitalized with diarrhea. The Reassortant isolation, amplification and plaque purifications were executed in the similar manner as above. The seed virus was designated HDS-1/BRV-1

Genotype

This Reassortant contains the eighth gene from human rotavirus DS-1 strain encoding VP 7, a major neutralization protein. The remaining ten genes are derived from bovine rotavirus parent UK strain.

Serotype

This Reassortant has similar neutralization specificity to its human rotavirus parent serotype 2; a low level of cross-reactivity to antiserum directed at bovine rotavirus parent UK strain exists.

HPBRV-2

The human virus was obtained from an infant hospitalized with diarrhea. The diarrheal stool was used as the source of the P strain serotype 3 virus. The Reassortant isolation, amplification and plaque purification were conducted in a similar manner as above.

Genotype

This Reassortant contains the eighth or ninth gene from human rotavirus P strain encoding VP 7, a major neutralization protein. The remaining ten genes are derived from bovine rotavirus parent UK strain.

Serotype

This Reassortant has similar neutralization specificity to its human rotavirus parent serotype 3; a low level of cross-reactivity to antiserum directed at bovine rotavirus parent UK strain exists.

ST3BRV-2

The human virus was obtained from the stool of a neonate with diarrhea. The virus was serially passaged 23 times in primary AGMK cell cultures before becoming the human parent virus. The Reassortant isolation, amplification and plaque purification were conducted in a similar manner as above.

Genotype

This Reassortant contains the ninth gene from human rotavirus ST3 strain encoding VP 7, a major neutralization protein. The remaining ten genes are derived from bovine rotavirus parent UK strain.

Serotype

This Reassortant has similar neutralization specificity to its human rotavirus parent serotype 4; a low level of cross-reactivity to antiserum directed at bovine rotavirus parent UK strain exists.

In the preferred embodiment of the invention, the attenuated poliomyelitis vaccines derived from Sabin for effective immunization against poliomyelitis were first stabilized by adding pharmaceutically acceptable stabilizers and the then stable formulations of Polio vaccines were prepared with different Rotavirus strains.

Examples

In order to establish the stability of the combination Rota-Polio vaccine formulations of the invention, five different oral formulations hereinafter named as Formulation-1, Formulation-2, Formulation-3, Formulation-4 and Formulation-5 with different combinations of the rotavirus strains and poliomyelitis strains were prepared. In every combination prepared and studied, one rotavirus was selected from rotavirus 116E, rotavirus I321 and UK Bovine rotaviruses (serotype-1, serotype-2, serotype-3 and serotype-4) in combination with the live attenuated poliomyelitis strains Type-1, Type-2 and Type-3. Different combination formulations were prepared with different titres of rotavirus and poliomyelitis strains and stability studies of all five formulations were carried out at 25oC, 2-80C and -200C for 12 months period.

The stability test results have shown that different combination formulations of the invention were not sufficiently stable for enough storage time at temperatures 25oC and 2-80C. Whereas, all combination Rota-Polio vaccine formulations were stable at -200 C for a period of 12 months or more without significant loss of titres. Therefore, in forthcoming examples, stability test results for the combination formulation comprising rotavirus strain 116E are explained in detail for the stability test results at temperatures 250C, 2-80 C and –200 C whereas, for other combination formulations comprising I321 and UK Bovine rotaviruses (Serotype 1, 2, 3 and 4), results are discussed only for the storage temperature of -200 C.

Stabilizers used

A combination of sugars, phosphates and glutamates were used for stabilizing the combination formulations. Divalent metal ions were also added for stability. Without being limited to the same, non-ionic surfactants can also be used in the present invention for stabilizing the formulations.

Sugars may be selected from sucrose, dextrose, trehalose, maltose, lactose, gentobiose, melibiose, cellobiose and turanose. In preferred embodiment of the invention, sucrose is used. Sugars can be used up to about 3.0M for preparation of the liquid combination vaccine of the invention. In the preferred embodiment of the invention, sucrose is used less than 3.0 M, preferably between 0.01 M to 2.8 M, even more preferably less than 2.5 M, most preferably between 0.01 M to 2.45 M.

Phosphates used in preparation of the formulations of the invention are up to about 3.0M. However, in one preferred embodiment of the invention, phosphates used were less than 1.0 M, preferably between 0.01 M to 0.9 M, more preferably less than 0.5 M, even more preferably between 0.01 to 0.4 M, most preferably between 0.01M to 0.3 M.

Sodium and potassium salts of the phosphates like sodium phosphates or potassium phosphates may be used in the invention whereas, such phosphates include but are not limited to monophosphates of sodium and potassium, polyphosphates and phosphorylated compounds. Addition of phosphates provides better stability to the formulations.

In the preferred embodiment of the invention, pH of the formulations was maintained between 5.0 to 8.0
Glutamates used in the invention are sodium or potassium salts of Glutamic acid. Glutamates may be used up to about 1.0M, preferably less than 1.0 M, more preferably between 0.01M to 0.9M, even more preferably less than 0.7M between 0.01M to 0.5M, most preferably between 0.01M to 0.45M.

Divalent metal ions are halides of divalent metal ions selected from MgCl2, CaCl2 and ZnCl2. However, in preferred embodiment of the invention, MgCl2 was used. Halides of divalent metal ions can be used to a concentration of about 3.0M, preferably less than 3.0M, more preferably between 0.001M to 2.0M or even less than 2.0 M, even more preferably between 0.001M to 1.5M, most preferably between 0.001M to 1.35M.

The non-ionic surfactants used in the invention are Polysorbates. Polysorbates may be selected from polysorbate-20, polysorbate-21, polysorbate-40, polysorbate-60, polysorbate-61, polysorbate-65, polysorbate-80, polysorbate-81, polysorbate-120, Triton X-100, NP 40 and Triton X 114.

In the preferred embodiment of the invention, polysorbate-20 and polysorbate-80 (tween20 and Tween80) are used in the concentration of about 0.001% to 0.6% w/v.

In another embodiment of the invention, polysorbate-21, polysorbate-40, polysorbate-60, polysorbate-61, polysorbate-65, polysorbate-81, polysorbate-120, Triton X-100, NP 40 and Triton X 114 may also be used in a concentration of about 0.01% to 0.3% w/v.

Different formulations of the combination Rota-Polio vaccine were prepared using vaccine titres as specified in the forthcoming examples. Sugars, Phosphates, Glutamates, Halides of divalent ions and non-ionic surfactants were used as stabilizers in the concentrations as specified above. Without being limited to, same stabilizers according to above specified concentrations may be used for each formulation. Other suitable pharmaceutically acceptable stabilizers may also be used in different embodiment of the invention.

Results obtained from the stability studies are explained hereinafter with reference to the drawings.

Example 1:

Formulation-1- Combination formulation of Rota-Polio vaccine with Rotavirus116E(G9)[P11] and Polio virus with following titres of Rotavirus and Poliomyelitis strains was prepared for 0.5ml oral dose. Wherein Polio vaccine is 0.7 log more when 0.5 ml is administered.

Table 1: Vaccine composition for Formulation -1

S.N. Virus strain Virus titre
1. Rotavirus116E (G9) [P11] bulk Equivalent to 105.5 FFU/0.5ml
2. Polio type 1 Equivalent to 106.7CCID50/0.5ml
3. Polio type 2 Equivalent to 105.7CCID50/0.5ml
4. Polio type 3 Equivalent to 106.5CCID50/0.5ml

The above combination was stabilized with SPG and QS with Magnesium chloride.
S= Sugar
P= Phosphate
G= Glutamate

Stability Test

Stability tests were performed for Formulation-1at 250C, 2-80C and -200C to establish the optimum temperature with maximum stability of the combination Rota-Polio oral vaccine formulation without any viral interference and drop in the titre.

Stability test at 250C

Stability test was done for Formulation 1 at 250C to find out the stability of the formulation-1. Test results showed that Rotavirus strain 116E (G9) [P11] was stable upto 4 weeks whereas, Polio strains (Type-1, Type-2 and Type-3) were stable only upto one week and there was remarkable drop in polio strains titres at the end of 4th week. The results were tabulated as given in following Table 1-A and also graphically represented in figure 1 of the accompanying drawings.

Figure 1 is the graphical representation showing titre drop for Rotavirus 116E(G9) [P11] and Polio Type 1, Type 2 and Type 3 at the end of 1 week, 2 weeks, 3 weeks and 4 weeks.

Table-1-A: Stability of Formulation-1 at 250C.

Strain 0day 1wk 2wk 3wk 4wk
Rota 116E(G9P11) 5.42 5.23 5.46 5.25 5.12
Polio Type 1 6.89 6.35 6.01 5.71 5.32
Polio Type2 5.75 5.51 5.23 4.89 4.45
Polio Type3 6.69 6.42 6.11 5.84 5.26

Figure 1 and Table 1-A show that at 250C Rotavirus 116E(G9) [P11] was stable even after end of 4 weeks but there was remarkable drop in stability of Polio strains Type 1, 2 and 3. Polio is stable only for 1week and dropped by 1.5 log loss in titer at the end of 4th week.

Stability test at 2-80C

Stability test was done for Formulation 1 at storage temperature of 2-80C to find out the stability of the formulation-1 at this temperature. Test results showed that Rotavirus strain 116E was stable upto 12 months at this temperature whereas, Polio strains were stable only upto 6 months and there was significant drop in the titres for all three strains of Polio vaccine at the end of 6 months. Following Table 1-B and figure 2 in the accompanying drawings show the stability test results for Formulation-1 at 2-80C.

Figure 2 is the graphical representation showing measured titres for Rotavirus 116E(G9) [P11] and Polio Type 1, Type 2 and Type 3 at the end of 1 month, 3 months, 6 months, 9 months and 12 months.
Table-1-B: Stability of Formulation-1 at 2-80C.

0day 1month 3months 6months 9months 12months
Rotavirus 116E(G9)[P11] 5.42 5.79 5.55 5.52 5.48 5.24
Polio Type1 6.89 6.65 6.52 6.32 5.26 4.54
Polio Type2 5.75 5.54 5.41 5.25 4.11 3.14
Polio Type3 6.69 6.54 6.34 6.02 5.37 4.83

Figure 2 and above Table 1-B show that at 2-80C Rotavirus 116E (G9) [P11] was stable upto the end of 12 months whereas there was significant drop in titres of Polio Type 1, 2 and 3 strains at the end of 6 months.

Stability test at -200C

Stability test was done for Formulation 1 at storage temperature of -200C to find out the stability of the combination vaccine at the given storage temperature. Test results showed that both Rotavirus 116E(G9) [P11] strain and Polio vaccine (all strains, Type-1, Type-2 and Type-3) were stable upto 12 months at this temperature. Following Table 1-C and accompanying figure 3 show the stability test results for Formulation-1 at -200C.

Figure 3 is the graphical representation showing measured titre for Rotavirus 116E(G9)[P11] and Polio Type 1, Type 2 and Type 3 at the end of 1 month, 3 months, 6 months, 9 months and 12 months.

Table-1-C: Stability of Formulation-1 at -200C.

0day 1month 3months 6months 9months 12months
Rota 116E(G9)[P11] 5.42 5.48 5.25 5.46 5.35 5.41
Type1 6.89 6.72 6.61 6.85 6.81 6.86
Type2 5.75 5.68 5.81 5.59 5.65 5.68
Type3 6.69 6.83 6.91 6.75 6.84 6.62

Conclusion of stability test for Formulation-1

Above Tables 1-A, 1-B, 1-C and Figures 1, 2 and 3 show that both the vaccines i.e. Rotavirus 116E(G9)[P11] and Polio vaccine (all strains including Type-1, type-2 and type-3) were stable for one year (12 months) at -200C. At -200C there was no significant drop in the titre of either Rotavirus strain or Polio strains. These results were sufficient to prove that at -200C the combination Rota-Polio oral vaccine formulation-1had no viral interference between the live naturally attenuated rotavirus strain and Polio virus strains present in the combination vaccine. The results also proved that there was no interference between the virus strains and the stabilizers The combination vaccine had the same efficacy and immunogenicity as they would produce when these vaccines i.e. Rotavirus vaccine and Polio vaccine are given separately or co-administered to the subject.

Example 2

In another example, combination vaccine Formulation-2 of Rota-Polio combination vaccine with Rotavirus 116E (G9)[P11] and Polio virus equivalent to following titre range of Rotavirus and Polio strains was prepared. Wherein Polio vaccine is equivalent to 0.1 mL titre when 0.5 ml is administered.

Table 2: Vaccine composition of Formulation 2

S.N. Virus strain Virus titre
1. Rotavirus116E(G9) [P11] bulk Equivalent to 105.5 FFU/0.5ml
2. Polio type 1 Equivalent to 106.1CCID50/0.5ml
3. Polio type 2 Equivalent to 105.0CCID50/0.5ml
4. Polio type 3 Equivalent to 105.9CCID50/0.5ml

The above combination was stabilized with SPG and QS with Magnesium chloride.
S= Sugar
P= Phosphate
G= Glutamate
Stability test of Formulation-2 at 250C

Stability test was done for Formulation 2 at 250C to analyze the stability of the Formulation 2 at 250 C. Test results showed that at this temperature, Rotavirus strain 116E (G9)[P11] was stable upto 4 weeks whereas, Polio strains were stable only upto one week and there was significant drop in the titre of Polio strains at the end of 4th week. Following Table 2-A and figure 4 in the accompanying drawings show the stability test results for Formulation-2 at 250C.

Figure 4 is the graphical representation showing titre drop for Rotavirus strain 116E (G9) [P11] and Poliomyelitis strains Type 1, Type 2 and Type 3 in Formulation 2 at the end of 1 week, 2 weeks, 3 weeks and 4 weeks at 250 C.

Table 2-A: Stability of Formulation-2 at 250C.

0day 1wk 2wk 3wk 4wk
Rotavirus 116E(G9P11) 5.49 5.42 5.31 5.45 5.25
Polio Type1 6.12 5.79 5.32 4.96 4.66
Polio Type2 5.25 4.82 4.58 4.31 3.85
Polio Type3 6.03 5.82 5.46 5.02 4.65

Table 2-A and Figure 4 show that at 250C Rotavirus 116E (G9) [P11] was stable even after end of 4 weeks but there was significant drop in stability of Polio strains Type 1, 2 and 3. Polio is stable only for 1week and dropped by 1.5 log in titer at the end of 4th week. The maximum titre loss was observed for Polio Type 2.

Stability test of Formulation-2 at 2-80C

Stability test was performed for Formulation 2 at storage temperature of 2-80C to find out the stability of the Formulation 2 at this temperature. Test results showed that Rotavirus strain 116E (G9)[P11] was stable upto 12 months at this temperature whereas, Polio vaccine was stable only upto 6 months and there was significant drop in the titre for all three strains of Polio vaccine at the end of 6 months. Following Table 2-B and figure 5 in the accompanying drawing shows the stability test results for Formulation-2 at 2-80C.

Figure 5 is the graphical representation showing measured titre for Rotavirus 116E(G9P11) and Polio Type 1, Type 2 and Type 3 at the end of 1 month, 3 months, 6 months, 9 months and 12 months.

Table 2-B: Stability of Formulation-2 at 2-80C.

0day 1month 3months 6months 9months 12months
Rotavirus 116E(G9)[P11] 5.49 5.58 5.42 5.53 4.72 4.02
Polio Type1 6.12 6.07 5.71 5.65 4.89 4.48
Polio Type2 5.25 5.17 4.92 4.75 4.18 3.69
Polio Type3 6.03 6.01 5.79 5.49 4.98 4.41

Table 2-B and Figure 5 show that at 2-80C Rotavirus 116E(G9)[P11) was sufficiently stable upto the end of 12 months but there was significant titre drop for all Polio strains including Polio Type 1, 2 and 3 at the end of 6 months.

Stability test of Formulation- 2 at -200C:

Stability test was done for Formulation 2 at storage temperature of -200C to find out the stability of the formulation at the given temperature. Test results showed that both Rotavirus 116E (G9) [P11] vaccine and Polio vaccine (all strains, Type-1, Type-2 and Type-3) were stable upto 12 months at this temperature. Following table 2-C and figure 6 in the accompanying drawings show the stability test results for Formulation-2 at -200C.

Figure 6 is the graphical representation showing measured titre for Rotavirus 116E(G9P11) and Polio Type 1, Type 2 and Type 3 at the end of 1 month, 3 months, 6 months, 9 months and 12 months.

Table 2-C: Stability of Formulation-2 at -200C.

0day 1month 3months 6months 9months 12months
Rotavirus 116E(G9P11) 5.49 5.51 5.31 5.53 5.61 5.49
Polio Type1 6.12 6.21 6.15 6.31 6.27 6.18
Polio Type2 5.25 5.35 5.28 5.15 5.32 5.24
Polio Type3 6.03 6.01 6.18 5.98 6.21 6.11

Conclusion of stability test for Formulation-2

Above Table 2-A, 2-B, 2-C and Figures 4, 5 and 6 show that both the vaccines i.e. Rotavirus 116E (G9) [P11]) and Polio vaccine (Type-1, type-2 and type-3) are stable for one year at -200C. At -200C there was no significant drop in the titre of either Rotavirus or Poliomyelitis. These results also proved that at -200C the combination Rota-Polio oral vaccine formulation had no viral interference between the live naturally attenuated rotavirus and polio virus strains present in the combination vaccine. The results also proved that there was no interference between the virus strains and the stabilizers. The combination vaccine has the same efficacy and immunogenicity as they would have produced if these vaccines were given separately or co-administered to the subject.

Example-3: Combination Rota-Polio formulation with Rotavirus strain I321

In another example, combination vaccine Formulation-3 of Rota-Polio combination vaccine with Rotavirus strain I321 and Polio virus strains Type-1, Type-2 and Type-3 equivalent to following titres was prepared

Table 3: Vaccine composition of Formulation-3

S.N. Virus strain Virus titre
1. Rotavirus I321 bulk Equivalent to 105.5 FFU/2.5ml
2. Polio type 1 Equivalent to 106.1CCID50/2.5ml
3. Polio type 2 Equivalent to 105.0CCID50/2.5ml
4. Polio type 3 Equivalent to 105.9CCID50/2.5ml
The above combination was stabilized with SPG and Qs to volume with MgCl2.
S= Sugar
P= Phosphate
G= Glutamate

Stability test of Formulation -3 at -200 C.

Stability test of Formulation 3 was performed at storage temperature of -200C to find out the stability of the formulation at the given temperature. Test results showed that both Rotavirus I321 and Polio vaccine (all strains, Type-1, Type-2 and Type-3) were stable upto 12 months at this temperature. Following table 3-A and figure 7 in the accompanying drawings show the stability test results for Formulation-3 at -200C.

Figure 7 is the graphical representation showing measured titre for Rotavirus I321 and Polio Type 1, Type 2 and Type 3 at the end of 1 month, 3 months, 6 months, 9 months and 12 months.

Table 3-A: Stability of Formulation-3 at -200C.

Virus strain 0Day 1 Month 3 Months 6 Momths 9 Months 12 Months
Rotavirus I321 5.65 5.61 5.49 5.53 5.39 5.59
Polio Type 1 6.22 6.18 6.28 6.11 5.98 6.15
Polio Type 2 5.21 5.05 5.11 5.23 5.18 5.12
Polio Type 3 5.88 5.95 5.95 5.91 6.01 5.99

Conclusion of stability test for Formulation-3

Tables 3-A and Figure 7 show that both the vaccines i.e. Rotavirus I321 and Polio vaccine (Type-1, type-2 and type-3) are stable for one year at -200C. At -200C there was no significant drop in the titre of either Rotavirus or Poliomyelitis. These results also proved that at -200C the combination Rota-Polio oral vaccine formulation had no viral interference between the live naturally attenuated rotavirus and polio virus strains present in the combination vaccine. The results also proved that there was no interference between the virus strains and the stabilizers.

The combination vaccine has the same efficacy and immunogenicity as they would have produced if these vaccines were given separately or co-administered to the subject.

Example-4

In another example, Formulation-4 of Rota-Polio combination vaccine with Rotavirus I321 and Polio virus (Type-1, 2 and 3) equivalent to following vaccine titres was prepared.

Table 4: Vaccine composition for Formulation-4

S.N. Virus strain Virus titre
1. Rotavirus I321 bulk Equivalent to 105.5 FFU/0.5ml
2. Polio type 1 Equivalent to 106.1CCID50/0.5ml
3. Polio type 2 Equivalent to 105.0CCID50/0.5ml
4. Polio type 3 Equivalent to 105.9CCID50/0.5ml

The above combination was stabilized with SPG and Qs to volume with MgCl2
S= Sugar
P= Phosphate
G= Glutamate

Stability test of Formulation-4 at -200 C.

Stability test of Formulation 4 was performed at storage temperature of -200C to find out the stability of the formulation at the given temperature. Test results showed that both Rotavirus I321 and Polio vaccine (all strains, Type-1, Type-2 and Type-3) were stable upto 12 months at this temperature. Following table 4-A and figure 8 in the accompanying drawings show the stability test results for Formulation-4 at -200C.

Figure 8 is the graphical representation showing measured titre for Rotavirus I321 and Polio Type 1, Type 2 and Type 3 at the end of 1 month, 3 months, 6 months, 9 months and 12 months.

Table 4-A: Stability of Formulation-4 at -200C.

Virus strain 0Day 1 Month 3 Month 6 Month 9 Month 12 Month
RotavirusI321 5.58 5.68 5.41 5.43 5.55 5.63
Polio Type 1 6.18 6.11 6.16 6.07 6.21 6.14
Polio Type 2 5.03 4.99 5.08 5.11 5.22 5.15
Polio Type 3 5.98 5.93 6.02 6.05 5.95 5.87

Conclusion of stability test for Formulation-4
Tables 4-A and Figure 8 show that both the vaccines i.e. Rotavirus I321 and Polio vaccine (Type-1, type-2 and type-3) are stable for one year at -200C without any significant drop in the titre of either Rotavirus or Poliomyelitis. These results also proved that at -200C the combination Rota-Polio oral vaccine formulation had no viral interference between the live naturally attenuated rotavirus and polio virus strains present in the combination vaccine. The results also proved that there was no interference between the virus strains and the stabilizers. The combination vaccine has the same efficacy and immunogenicity as they would have produced if these vaccines were given separately or co-administered.

Example-5: Combination Rota-Polio formulation with Tetravalent UK Bovine Rotavirus
In another example, Formulation-5 of Rota-Polio combination vaccine comprising tetravalent UK Bovine Reassortant with Serotypes 1, 2, 3, and 4 and the Polio virus strains Type-1, Type-2 and Type-3 equivalent to following titres was prepared

Table 5: Vaccine composition of Formulation-5

S.N. Virus strain Virus titre
1. Serotype G 1 Equivalent to 105.8 pfu/mL
2. Serotype G 2 Equivalent to 105.3 pfu/mL
3. Serotype G 3 Equivalent to 105.3 pfu/mL
4. Serotype G 4 Equivalent to 105.8 pfu/mL
5. Polio type 1 Equivalent to 106.1CCID50/mL
6. Polio type 2 Equivalent to 105.0 CCID50/mL
7. Polio type 3 Equivalent to 105.9 CCID50/mL
Above combination was stabilized with SPG and MgCl2. DMEM added to QS
S= Sugar
P= Phosphate
G= Glutamate

Stability test of Formulation-5 at -200 C.

Stability test of Formulation 5 was performed at storage temperature of -200C to find out the stability of the formulation at the given temperature. Test results showed that all serotypes of UK Bovine rotavirus and the Polio vaccine were stable upto 12 months at this temperature. Following table 5-A and figure 9 in the accompanying drawings show the stability test results for Formulation-5 at -200C.

Figure 9 is the graphical representation showing measured titre for tetravalent UK Bovine Rotavirus (Serotype 1, 2, 3 and 4) and Polio Type 1, Type 2 and Type 3 at the end of 1 month, 3 months, 6 months, 9 months and 12 months.

Table 5-A: Stability of Formulation-5 at -200C.

Strain 0 Day 1 Month 3 Months 6 Months 9 Months 12 Months
Serotype G1 5.83 5.79 5.81 5.82 5.76 5.78
Serotype G2 5.25 5.28 5.31 5.26 5.32 5.29
Serotype G3 5.28 5.34 5.27 5.35 5.31 5.36
Serotype G4 5.76 5.78 5.74 5.81 5.79 5.82
Polio Type 1 6.11 6.23 6.21 6.18 6.08 6.12
Polio Type 2 5.08 5.03 4.99 5.06 5.12 5.09
Polio Type 3 5.93 5.91 5.85 5.87 5.93 5.95

Conclusion of stability test for Formulation-5

Tables 5-A and Figure 9 show that all four serotypes of tetravalent UK Bovine rotavirus vaccine and the Polio vaccine (Type-1, type-2 and type-3) are stable for one year at -200C without any significant drop in the titre of either UK Bovine Rotavirus or Polio virus. These results also proved that at -200C the combination Rota-Polio oral vaccine formulation had no viral interference between the live naturally attenuated rotavirus and polio virus strains present in the combination vaccine. The combination vaccine has the same efficacy and immunogenicity as they would have produced if these vaccines were given separately or co-administered. In fact, at the end of 12 months, there was slight increase in the measured titre values for Rotavirus Serotype 2, 3, 4 and Polio type-1, 2 and 3. Therefore, the formulation of the invention was completely stable for upto 12 months without any negative interference between the rotavirus and polio virus when the formulation was kept at -200 C.

From the stability studies of Formulations 1, 2, 3, 4 and 5 at varied storage temperatures of 250 C, 2-80 C and -200 C, it has been observed that the combination Rota-Virus oral formulations of the invention are stable for atleast12 months or more at -200 C.

Further, in addition to stability and sustained immunogenicity, the combination oral vaccine formulations of the invention have better patient compliance, require less clinic visit and minimize the chances of missed vaccination.

It should be understood that the examples given in above tables are only for the understanding of a person skilled in the art and for illustration purposes, showing different embodiments of the invention only. The scope of the claims should not be limited to these formulations and examples, as several other combination vaccines may also be prepared in combination of the novel combination vaccine formulations of the invention without deviating from the spirit and scope of the invention.

References-

1. Combined vaccination with live oral polio vaccine and the bovine rotavirus RIT 4237 strain. Vodopija I et al, Vaccine 1986 Dec;4 (4):233-6.

2. TRIAL OF AN ATTENUATED BOVINE ROTAVIRUS VACCINE (RIT 4237) IN GAMBIAN INFANTS, P. Hanlon a, V. Marsh a, F. Shenton a, O. Jobe a, R. Hayes b, H.C. Whittle a, L. Hanlon a, P. Byass a, M. Hassan-King a, H. Sillah a, B.H. M''Boge c, B.M. Greenwood a, The Lancet, Volume 329, Issue 8546, Pages 1342 - 1345, 13 June 1987

3. Simultaneous administration of oral rhesus-human reassortant tetravalent (RRV-TV) rotavirus vaccine and oral poliovirus vaccine (OPV) in Thai infants. Migasena S, Simasathien S, Samakoses R, Pitisuttitham P, Sangaroon P, van Steenis G, Beuvery EC, Bugg H, Bishop R, Davidson BL, et al. Vaccine. 1995 Feb;13(2):168-74

4. Immunogenicity and reactogenicity of rhesus rotavirus vaccine given in combination with oral or inactivated poliovirus vaccines and diphtheria-tetanus-pertussis vaccine. F. Jalil et al, Trans R Soc Trop Med Hyg. 1991 Mar-Apr;85(2):292-6

5. Successful co-administration of a human rotavirus and oral poliovirus vaccines in Bangladeshi infants in a 2-dose schedule at12 and 16 weeks of age. Zaman K et al, Vaccine. 2009 Feb 25;27(9):1333-9. Epub 2009 Jan 20.

WE CLAIM

1. Novel combination Rota–Polio vaccine formulations for oral administration wherein the formulations are stable at -200 C for atleast 12 months or more in both liquid and lyophilized form.

2. Novel combination Rota-Polio vaccine formulations as claimed in claim 1, wherein said formulations comprise human-bovine natural ressortants and naturally attenuated Rotavirus strains, Sabin derived live attenuated Polio virus strains and stabilizers.

3. Novel combination Rota-Polio vaccine formulations as claimed in claim 2, wherein said Rotavirus strains are selected from Rotavirus 116E, Rotavirus I321 and tetravalent UK Bovine Human Reassortant Rotaviruses.

4. Novel combination Rota-Polio vaccine formulations as claimed in claim 2, wherein said Polio virus strains comprise Type-1, Type- 2 and Type-3 Poliomyelitis viruses.

5. Novel combination Rota-Polio vaccine formulations as claimed in claim 3, wherein said Rotavirus 116E titre is about 1x104 FFU/ml to 1x107 FFU/mL.

6. Novel combination Rota-Polio vaccine formulations as claimed in claim 3, wherein said Rotavirus I321 titre is 1x104 FFU/ml to 1x107 FFU/mL.

7. Novel combination Rota-Polio vaccine formulations as claimed in claim 3, wherein said tetravalent UK Bovine rotavirus titre is 1x104 FFU/ml to 1x107 FFU/mL.

8. Novel combination Rota-Polio vaccine formulations as claimed in claim 4, wherein said Poliomyelitis Type-1 titre is between 104 to 106.5 CCID50/mL.

9. Novel combination Rota-Polio vaccine formulations as claimed in claim 4, wherein said Poliomyelitis Type-2 titre is between 104.5to 106 CCID50/mL.

10. Novel combination Rota-Polio vaccine formulations as claimed in claim 4, wherein said Poliomyelitis Type-3 titre is between 105 to 106.3 CCID50/mL.

11. Novel combination Rota-Polio vaccine formulations as claimed in claim 2, wherein said stabilizers are selected from sugars, phosphates, glutamates, divalent metal ions and non-ionic surfactants.

12. Novel combination Rota-Polio vaccine formulations as claimed in claim 11, wherein said sugars are about 0.01M to 3.0M, phosphates are about 0.01M to 3.0M, glutamates are about 0.01M to 1.0M, divalent metal ions are about 0.001M to 3.0M and non-ionic surfactants are about 0.001% to 0.6%.

13. Novel combination Rota-Polio vaccine formulations as claimed in claim 12, wherein said sugars are selected from sucrose, dextrose, trehalose, maltose, lactose, gentobiose, melibiose, cellobiose and turanose.

14. Novel combination Rota-Polio vaccine formulations as claimed in claim 13, wherein said sugar is sucrose.

15. Novel combination Rota-Polio vaccine formulations as claimed in claim 14, wherein said sucrose is about 0.01M to about 2.45M.

16. Novel combination Rota-Polio vaccine formulations as claimed in claim 12, wherein said phosphates are selected from monophosphates, polyphosphates and phosphorylated compounds of sodium or potassium.

17. Novel combination Rota-Polio vaccine formulations as claimed in claim 16, wherein said phosphates are sodium or potassium compounds of monophosphates about 0.01M to 3.0M.

18. Novel combination Rota-Polio vaccine formulations as claimed in claim 16, wherein said phosphates are sodium or potassium compounds of polyphosphates and phosphorylated compounds about 0.01M to 0.3M.

19. Novel combination Rota-Polio vaccine formulations as claimed in claim 12, wherein said glutamates are sodium or potassium salts of glutamic acid.

20. Novel combination Rota-Polio vaccine formulations as claimed in claim 19, wherein said sodium or potassium salts of glutamic acid are about 0.01 M to about 0.45 M.

21. Novel combination Rota-Polio vaccine formulations as claimed in claim 12, wherein said divalent metal ions are halides of divalent metal ions selected from MgCl2, CaCl2 and ZnCl2.

22. Novel combination Rota-Polio vaccine formulations as claimed in claim 21, wherein said halides of divalent metal ions are about 0.001 M to 3.0 M.

23. Novel combination Rota-Polio vaccine formulations as claimed in claim 22, wherein said halide of divalent metal ion is MgCl2 about 0.001M to about 1.35M.

24. Novel combination Rota-Polio vaccine formulations as claimed in claim 12, wherein said non-ionic surfactants are polysorbates.

25. Novel combination Rota-Polio vaccine formulations as claimed in claim 24, wherein said polysorbates are selected from polysorbate -20, polysorbate-21, polysorbate-40, polysorbate-60, polysorbate-61, polysorbate-65, polysorbate-80, polysorbate-81, polysorbate-120, Triton X-100, NP 40 and Triton X 114.

26. Novel combination Rota-Polio vaccine formulations as claimed in claim 25, wherein said polysorbates are polysorbate -20 and polysorbate-80 about 0.001% to 6%, w/v.

27. Novel combination Rota-Polio vaccine formulations as claimed in claim 25, wherein said polysorbates are polysorbate-21, polysorbate-40, polysorbate-60, polysorbate-61, polysorbate-65, polysorbate-81, polysorbate-120, Triton X-100, NP 40 and Triton X 114 about 0.01% to 0.3% w/v.

28. Novel combination Rota-Polio vaccine formulations as claimed in claim 1, wherein pH of the formulations is about 5.0 to 8.0.

29. Novel combination Rota–Polio vaccine formulations for oral administration such as herein described in the description with reference to the accompanying examples, drawings, and tables.

Dated this 3rd day of June 2011.

AFZAL HASAN
Of Tempus Law Associates
AGENT FOR THE APPLICANT