Field of Invention

The invention relates to combined heptavalent vaccine with a single injection to provide better immunity for children. More particularly, the invention relates to development of combination heptavalent (IPV-IRV-Hib-HBsAg- DPT) vaccine for multiple immunogenicity in human beings, comprising Inactivated poliovirus (Sabin), Inactivated rotavirus, Haemophilus influenzae type b, Hepatitis B surface antigen, Diphtheria, Tetanus and Pertussis (wP)) vaccine.

Back ground of the invention

The current global scenario calls for a more-efficacious, acceptable, cost-effective and reliable method of immunization for many fatal diseases. Vaccination is an important tool for handling healthcare programs both in developed and developing countries. The number of recommended vaccination has increased significantly in recent years. Now-a-days, in some countries, at least 13 separate vaccinations are needed to vaccinate a child since birth to the age of 6 years. The availability of combination vaccines containing protective antigens against majority of diseases, for which universal immunization is recommended in infancy, helps in simplifying the implementation, increasing the acceptance, reducing the global cost of immunization programmes and improving disease control. Therefore, cost, convenience and compliance factors contribute to enhanced use of combination vaccines over monovalent vaccines. It can also increase the opportunities to vaccinate hand-to-reach patients or populations or frequent non-attendees (Rumke,1994).

The development of multivalent vaccine capable of protecting against diphtheria, tetanus, pertussis, poliomyelitis, hepatitis B and Haemophilus influenzae type b has been an evolutionary process which started in the 1950s when diphtheria and tetanus toxoids were first combined with inactivated whole cell pertussis(wP) vaccine (Mallet et al; 2004). More recently, to further facilitate paediatric vaccination programmes, combination vaccines have been introduced in which inactivated poliovirus (IPV) and/or Hib and Hepatitis antigen are added to the core DTwP or DTaP combination.

Although the eradication of poliomyelitis from the western world, and its global decline, is a result of successful use of oral polio vaccine (OPV) in most parts of the world (Plotkin,1995), the potential risk of vaccine -associated paralytic poliomyelitis(VAPP) with OPV, has led many counties to switch over to using IPV for routine immunization.

A detailed study on Immunogenicity and safety of a new liquid hexavalent combined vaccine compared with separate administration of reference licensed vaccines in infants was published by Mallet E, Fabre P, Pines E, et al. Pediatr Infect Dis J 2000;19:1119–27.

Some other prior art publications in the field are Plotkin SA. Inactivated poliomyelitis vaccine for the United States: a missed vaccination opportunity. Pediatr Infect Dis J 1995;14:835–9 and Rümke HC. Are different combined vaccines needed in different parts of Europe? A point of view from a Northern European country. Biologicals 1994; 22:425–7.

A combination vaccine which can provide immunogenicity against large number of diseases is always advantageous over the monovalent vaccines. We cannot reduce the number of immunizations required in infants and children to protect them from various fatal diseases but the compliance can be increased by reducing the number of separate vaccinations. A combination vaccine is advantageous over monovalent vaccine as it not only increases the compliance but it is cost effective and convenient also and also reduces the chances of missed vaccination. However, due to complications associated with the preparation of such combination vaccines like stability of such combination vaccines due to possible interaction between the antigens has always been a challenge before the scientist community.

Vaccines combining diphtheria-tetanus-pertussis antigens are available and widely used for over 60yrs. Expanded quadrivalent and pentavalent combination vaccines also have been developed that include Haemophilus influenzae type b vaccine. Pentavalent combinations that include DPT-Hib-HB are used in several developed and developing countries. Hexavalent vaccines (HPV included) have been used in several European countries. Such hexavalent vaccine available in the art can provide immunization only against six important childhood diseases with single injection.

However, in the prior art, it has not been possible to develop such a combination heptavalent vaccine which could be able to provide protection against poliovirus, rotavirus, Hib, Hepatitis B, Diptheria, Tetanus and Pertussis by single administration.

The present invention overcomes the limitations of the prior art by providing a liquid, combination heptavalent vaccine ( Inactivated poliovirus (Sabin)-Inactivated rotavirus- Haemophilus influenzae type b- Hepatitis B surface antigen- Diptheria-Tetanus-Pertussis(wP)) which permits vaccination against poliomyelitis, rota, Hib, hepatitis B, diphtheria, tetanus and pertussis by single injection.

Object of the invention

Primary object of the invention is to provide a combination heptavalent vaccine which induces immunogenicity in human beings against multiple diseases.

Another object of the invention is to provided a liquid, combination heptavalent vaccine which permits vaccination against poliomyelitis, rotavirus, Hib, hepatitis B, diphtheria, tetanus and pertussis by single injection.

Another object of the invention is to provide a liquid, combination heptavalent (IPV-IRV-Hib-HBsAg-DPT) vaccine comprising Inactivated poliovirus (Sabin), Inactivated rotavirus, Haemophilus influenzae type b, Hepatitis B surface antigen, Diphtheria, Tetanus and Pertussis (wP)) vaccine.

A further object of the invention is to provide a method for preparation of combination heptavalent (Inactivated poliovirus (Sabin)-Inactivated rotavirus-Haemophilus influenzae type b- Hepatitis B surface antigen- Diptheria-Tetanus-Pertussis(wP)) vaccine.

Summary of the invention

Present invention relates to a novel liquid, combination heptavalent (Inactivated poliovirus (Sabin)-Inactivated rotavirus-Haemophilus influenzae type b- Hepatitis B surface antigen- Diptheria-Tetanus-Pertussis(wP)) vaccine and a process for preparation of the same.

The combination heptavalent vaccine of the invention decreases the number of injections required for immunization, improves parental and child satisfaction, decreases pain, is more convenient, improves compliance with on-time immunization, improves record keeping and is more cost effective.

Heptavalent vaccine of the invention is a liquid, combination heptavalent vaccine comprising following antigens,
i) Three types of inactivated polio virus (type-1,2,3),
ii) Single strain inactivated rotavirus (116E), adsorbed
iii) A conjugate Haemophilus influenza type b
iv) Recombinant Hepatitis B vaccine, adsorbed
v) Diphtheria toxoid (DT), adsorbed
vi) Tetanus toxoid (TT), adsorbed
vii) Bordetella Pertussis, whole cell(wP)

General description of the invention

Present invention relates to a novel liquid, combination heptavalent (Inactivated poliovirus (Sabin)-Inactivated rotavirus-Haemophilus influenzae type b- Hepatitis B surface antigen- Diptheria-Tetanus-Pertussis(wP)) vaccine and a process for preparation of the same.

Heptavalent vaccine of the invention is a combination vaccine comprising following antigens,
viii) Three types of inactivated polio virus (type-1,2,3),
ix) Single strain inactivated rotavirus (116E), adsorbed
x) A conjugate Haemophilus influenza type b
xi) Recombinant Hepatitis B vaccine, adsorbed
xii) Diphtheria toxoid (DT), adsorbed
xiii) Tetanus toxoid (TT), adsorbed
xiv) Bordetella Pertussis, whole cell(wP)

The Heptavalent vaccine formulation of the invention is in liquid form.

The combination heptavalent vaccine of the invention decreases the number of injections required for immunization, improves parental and child satisfaction, decreases pain, is more convenient, improves compliance with on-time immunization, improves record keeping and is more cost effective.

Various antigens used in the heptavalent vaccine of the invention alongwith a brief description of method of their preparation would now be described in the following paragraphs alongwith their advantages and applications over the prior art.

i). Inactivated polio vaccine

The present oral live attenuated vaccine strains developed by sabin replicate in the human gut and give rise to viral strains of increased neurovirulence that on very rare occasions, approximately 1 case per 2.5 million doses, causes vaccine associated paralytic poliomyelitis.

In populations with low vaccine coverage and poor surveillance, vaccine-derived strains can silently circulate for long periods of time leading to poliomyelitis out breaks.

WHO views the exportation of large scale salk-IPV production technology, using wild type poliovirus for production into countries with limited resources and /or weak bio-safety regulatory environments where a significant number of population became fully susceptible to polio virus infection following the cessation of OPV use. Therefore, the inactivated polio vaccine IPV in the invention has been developed from live attenuated sabin strain.

Process of preparation of inactivated polio virus of the invention comprises following steps:

1.Chemical inactivation of live attenuated Sabin strain viral harvest type 1,2 and3.
2.Chemical inactivation was performed at 37degree Celsius for 12 days.
3.Suitable stabilizer was added during inactivation process to maintain the antigenicity and immunogenicity of D-antigen.
4.Concentration of viral harvest was performed by 100KDa cassettes followed by dialysis.
5.Purification of the concentrated harvest chromatography.
6.Antigen content is determined by ELISA using type specific polyclonal antibodies and type specific poly or monoclonal antibodies (MAbs) as detecting antibodies.

ii). Inactivated rotavirus vaccine

Rotavirus remains a prime killer of children due to diarrhea, dehydration and malnutrition in countries of Africa and Asian continent. Commercial vaccines have not been tested on target population in these poor countries. The existing live vaccine is not enough to induce immunity and not protecting the complete population. Some time it causes adverse events like intussusceptions also.

When mixed infections with more than one rotavirus strain occurs, the gene segments from the parental viruses may reassort independently due to genetic shift, producing reassortants of mixed parentage, a source of viral diversity.

The present invention provides a novel method for development of inactivated injectable rotavirus vaccine (IRV). The novel rotavirus vaccine developed by the novel method of the invention overcomes the post vaccination problems associated with the oral rotavirus vaccines known in the art.

The inactivated rota antigen is produced on the vero cell line using rota strain 116E. Production of the vaccine includes the following steps: Preparation of cell substrate, virus inoculation, virus harvest, virus purification, sterile filtration and inactivation. The virus was inactivated with different inactivating agents like heat, formaldehyde and betapropiolactone. Rota antigen was stored at deep freeze (-70 degree Celsius) with suitable stabilizers until formulation was carried out.

iii). Haemophilus influenza type b conjugate vaccine

Preparation of Haemophilus influenza type b conjugate vaccine involves the following steps:
1.Fermentation of haemophilus influenzae type b culture.
2.Extraction and purification of PRP.
3.Activation of PRP with cyanogen bromide and linked with adipic acid dihydrazide.
4.Coupling of purified tetanus toxoid.
5.Purification of conjugate by size exclusion chromatography.

iv). Hepatitis B surface antigen

The surface antigen of HBV is produced by culture of genetically-engineered yeast cells which carry the gene coding for major surface antigen of HBV. The HBS Ag is expressed in yeast cells and purified by Himax-technology.

v). Diphtheria toxoid:

Diphtheria toxoid is produced by purification of formaldehyde treated corynebacterium diphtheriae toxins.

vi). Tetanus toxoid:
Tetanus toxoid is produced by purification of formaldehyde treated purified clostridium tetani toxins.
vii). Pertussis antigen:

Bordetella pertussis culture is grown, concentrated, inactivated and used in the Heptavalent vaccine.

Table 1
S.No Antigenic components Vaccine formulation
A B C D
1
2
3
4a
4b
4c
5
6
7 Diphtheria toxoid (Lf/dose)
Tetanus toxoid (Lf/dose)
Pertussis-wP (OU/dose)
Inactivated polio virus type-1 (D-Ag/dose)
Inactivated polio virus type-2 (D-Ag/dose)
Inactivated polio virus type-3 (D-Ag/dose)
Inactivated rota virus protein (µg/dose)
Hepatitis B surface antigen (µg/dose)
Hib PRP-TT conjugate (µg/dose) 25
7.5
20
40
8
32
5
10
10 25
7.5
20
40
8
32
2
10
10 25
7.5
20
40
8
32
1.0
10
10 25
7.5
20
40
8
32
0.5
10
10

Table 1 shown above is a Vaccine formulation Design of heptavalent vaccine of the invention showing various antigen components in four different vaccine formulations A, B, C and D.

Dated this 3rd day of January 2011.

AFZAL HASAN
IN/PA-1328
Agent for the Applicant