GB1565657A - Combined vaccines against rubella and cutomegalic inclusion disease - Google Patents

Description

(54) COMBINED VACCINES AGAINST RUBELLA AND CYTOMEGALIC INCLUSION DISEASE (71) We, RECHERCHE ET INDUSTRIE THERAPEUTIQUES, RIT, of 13 rue du Tilleul, B-320 Genval, Belgium, a Belgian Body Corporate do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: This invention related to bivalent vaccines against rubella and cytomegalic inclusion disease (CMID).

Rubella virus is known to pass through the human placenta and to infect the foetus, and it can cause severe anomalies in new-born children.

Maternal circulatory antibodies against rubella virus can prevent this threat, and for many years different vaccines have been developed either as monovalent vaccines or as combined preparations with measles and/or mumps vaccines.

Examples of attenuated vaccines against rubella are vaccines containing the RA 27/3 strain, the HPV 77 DES strain, or the CENDEHILL strain.

However, rubella virus is not the only virus which is able to cause damage to the foetus.

Cytomegalovirus (CMV), which causes cyto megalic inclusion disease, is the most common cause of both mental retardation and neonatal microcephaly of viral origin, and it is estimated that among new-born children excreting this virus (i.e. from 1 to 4% of all new-born children), from 5 to 15% present character istics of mental retardation and that from 30 to 45% of nubile women do not have anti bodies against CMV.

This is the reason why, in several countries, investigators have been studying the innocuity and efficacy of vaccines against CMID.

Examples of virus strains used in these vaccines are the TOWNE 125 strain (Just. M., et al., Infection 3: 111-14, 1975) and a vaccinal strain derived from the Ad 169 strain (Elek S.D. et al.: The Lancet 1 (7845): 1-5, 1974). As both rubella virus vaccine and CMV vaccine have the same objective, i.e. protection of the foetus, and as inoculation with both vaccines is by the same route, it seems attractive to inoculate simultaneously both vaccines in the form of a combined vaccine and more particularly in the form of a live combined vaccine.

Furthermore, the use of combined vaccines requires less qualified medical staff and fewer injections than monovalent vaccines.

Interference between CMV and viruses such as myxoviruses, arboviruses, picornaviruses, herpes viruses and poxviruses has been demonstrated by in vitro experiments (Glasgow L.A., Infection and Immunity 9: 702-7, April 1974).

Similarly, rubella virus induces in vitro interference with heterologous viruses, e.g.

picornaviruses, myxoviruses and arboviruses (Parkman P.D., et al., J. Immunol 93 (4): 595-607, 1964).

In the study of combined vaccines, preliminary tests are thus needed to determine the possible interference factor in any combination of vaccines. We have found that in a combined live vaccine against rubella and CMID, simultaneous administration of both viruses does not affect the immunogenic response to each vaccine component.

According to the present invention there is provided a live combined vaccine against rubella and cytomegalic inclusion disease, in dosage unit form, comprising respective amount of vaccinal rubella virus and of vaccinal cytome galovirus corresponding at least to the immunogenic amounts of each virus. The attenuated viruses can be cultivated separately according to processes known to the art and mixed in adequate ratios, and the obtained bivalent vaccine is preferably freeze-dried after having been supplemented with a stabilizing composition as it is well known to the art. Examples of stabilizing compositions are those containing human serum albumin or polyvinylpyrrolidone.

As CMV is more labile in vitro than rubella virus, the operative conditions for the freezedrying of the bivalent vaccine are those required for the freezedrying of CMV.

As indicated above, the rubella virus strain, preferably in an amount of at least 103 TCIDso per dosage unit can be any vaccinal strain known to the art, e.g. the attenuated CENDHILL strain cultivated on primary rabbit kidney cell cultures, and the CMV strain, preferably present in an amount of at least 104 TCID50 , can be any vaccinal strain known to the art, e.g. the attenuated TOWNE 125 strain cultivated on human diploid cell line WI-38.

When bivalent vaccines according to the present invention are freeze-dried, they can be reconstituted by the addition of either water or any other diluent for the preparation of parenteral vaccines.

The so-obtained bivalent vaccines of the present invention can be administered intramuscularly or subcutaneously.

The following Examples illustrate the present invention.

EXAMPLE 1 To 375 ml. of the supernatant of a WI-38 cell culture containing about 1047TCIDso of CMV, TOWNE 125 strain, per millilitre is added the same volume of an aqueous solution containing sucrose (0.218 M), potassium monobasic phosphate (0.0038 M), sodium dibasic phosphate (0.0072 M), potassium glutamate (0.0049 M) and sodium ethylenediamine tetraacetate (EDTA) (0.2%) supplemented with human serum albumin (1%).

To 125 ml of the supernatant of a primary rabbit kidney cell culture containing about 103OTCID50 of rubella virus, CENDEHILL strain, per millilitre is added the same volume of an aqueous solution containing sucrose (200 gll.) and potassium glutamate (30 g/l.).

Both preparations are thoroughly mixed and the mixture is distributed into glass vials containing each one millilitre of combined vaccine in order to obtain dosage units administrable by intramuscular or subcutaneous routes.

Vaccination of 13 adults susceptible to both viruses demonstrated formation of antibodies against rubella virus, -demonstrated by hemagglutination inhibition test, and against CMVdetermined by immunofluorescense assay, from 6 to 8 weeks after vaccination.

EXAMPLE 2 To 270 ml. of the supernatant of a WI-38 cell culture containing about 104-8TCIDso of CMV, TOWNE 125 strain, per millilitre is added the same volume of an aqueous solution of sucrose (0.218 M), potassium monobasic phosphate (0.0038 M), sodium dibasic phosphate (0.0072 M), potassium glutamate (0.0049 M) and sodium EDTA (0 2to) supplemented with human serum albumin (loo) To 90 ml of the supernatant of a primary rabbit kidney cell culture containing about 104 3TCIDso per ml. of rubella virus, CENDEHILL strain, is added the same volume of an aqueous solution of sucrose (200 girl.) and potassium glutamate (30 girl.).

Both preparations are mixed and the mixture is distributed into glass vials, each containing one millilitre of combined vaccine which is then freeze-dried. The vials are then tightly stoppered.

The titres per dosage unit after freeze-drying of the bivalent vaccine are 104TCIDso and 1032TCIDso for CMV and the rubella virus respectively.

The vaccine can be administered by the intramuscular or subcutaneous route.

WHAT WE CLAIM IS: 1. A live combined vaccine against rubella and cytomegalic inclusion disease, in dosage unit form, comprising respective amounts of vaccinal rubella virus and vaccinal cytomegalicous corresponding at least to the immunogenic amounts of each virus considered separately.

2. A live combined vaccine against rubella and cytomegalic inclusion disease. in dosage unit form, comprising at least 103TCIDso of vaccinal rubella virus and at least 104TCIDso of vaccinal cytomegalovirus.

3. A live combined vaccine against rubella and cytomegalic inclusion disease according to claim 1 or claim 2, wherein the vaccine has been freeze-dried and it also includes a stabilizing composition for the viruses.

4. A live combined vaccine against rubella and cytomegalic inclusion disease according to any of claims 1 to 3, wherein the attenuated cytomegalovirus is the cytomegalovirus TOWNE 125 strain.

5. A live combined vaccine against rubella and cytomegalic inclusion disease, the vaccine being substantially as herein described in any of the Examples.

**WARNING** end of DESC field may overlap start of CLMS **.

Claims (5)

**WARNING** start of CLMS field may overlap end of DESC **. galovirus corresponding at least to the immunogenic amounts of each virus. The attenuated viruses can be cultivated separately according to processes known to the art and mixed in adequate ratios, and the obtained bivalent vaccine is preferably freeze-dried after having been supplemented with a stabilizing composition as it is well known to the art. Examples of stabilizing compositions are those containing human serum albumin or polyvinylpyrrolidone. As CMV is more labile in vitro than rubella virus, the operative conditions for the freezedrying of the bivalent vaccine are those required for the freezedrying of CMV. As indicated above, the rubella virus strain, preferably in an amount of at least 103 TCIDso per dosage unit can be any vaccinal strain known to the art, e.g. the attenuated CENDHILL strain cultivated on primary rabbit kidney cell cultures, and the CMV strain, preferably present in an amount of at least 104 TCID50 , can be any vaccinal strain known to the art, e.g. the attenuated TOWNE 125 strain cultivated on human diploid cell line WI-38. When bivalent vaccines according to the present invention are freeze-dried, they can be reconstituted by the addition of either water or any other diluent for the preparation of parenteral vaccines. The so-obtained bivalent vaccines of the present invention can be administered intramuscularly or subcutaneously. The following Examples illustrate the present invention. EXAMPLE 1 To 375 ml. of the supernatant of a WI-38 cell culture containing about 1047TCIDso of CMV, TOWNE 125 strain, per millilitre is added the same volume of an aqueous solution containing sucrose (0.218 M), potassium monobasic phosphate (0.0038 M), sodium dibasic phosphate (0.0072 M), potassium glutamate (0.0049 M) and sodium ethylenediamine tetraacetate (EDTA) (0.2%) supplemented with human serum albumin (1%). To 125 ml of the supernatant of a primary rabbit kidney cell culture containing about 103OTCID50 of rubella virus, CENDEHILL strain, per millilitre is added the same volume of an aqueous solution containing sucrose (200 gll.) and potassium glutamate (30 g/l.). Both preparations are thoroughly mixed and the mixture is distributed into glass vials containing each one millilitre of combined vaccine in order to obtain dosage units administrable by intramuscular or subcutaneous routes. Vaccination of 13 adults susceptible to both viruses demonstrated formation of antibodies against rubella virus, -demonstrated by hemagglutination inhibition test, and against CMVdetermined by immunofluorescense assay, from 6 to 8 weeks after vaccination. EXAMPLE 2 To 270 ml. of the supernatant of a WI-38 cell culture containing about 104-8TCIDso of CMV, TOWNE 125 strain, per millilitre is added the same volume of an aqueous solution of sucrose (0.218 M), potassium monobasic phosphate (0.0038 M), sodium dibasic phosphate (0.0072 M), potassium glutamate (0.0049 M) and sodium EDTA (0 2to) supplemented with human serum albumin (loo) To 90 ml of the supernatant of a primary rabbit kidney cell culture containing about 104 3TCIDso per ml. of rubella virus, CENDEHILL strain, is added the same volume of an aqueous solution of sucrose (200 girl.) and potassium glutamate (30 girl.). Both preparations are mixed and the mixture is distributed into glass vials, each containing one millilitre of combined vaccine which is then freeze-dried. The vials are then tightly stoppered. The titres per dosage unit after freeze-drying of the bivalent vaccine are 104TCIDso and 1032TCIDso for CMV and the rubella virus respectively. The vaccine can be administered by the intramuscular or subcutaneous route. WHAT WE CLAIM IS:

1. A live combined vaccine against rubella and cytomegalic inclusion disease, in dosage unit form, comprising respective amounts of vaccinal rubella virus and vaccinal cytomegalicous corresponding at least to the immunogenic amounts of each virus considered separately.

2. A live combined vaccine against rubella and cytomegalic inclusion disease. in dosage unit form, comprising at least 103TCIDso of vaccinal rubella virus and at least 104TCIDso of vaccinal cytomegalovirus.

3. A live combined vaccine against rubella and cytomegalic inclusion disease according to claim 1 or claim 2, wherein the vaccine has been freeze-dried and it also includes a stabilizing composition for the viruses.

4. A live combined vaccine against rubella and cytomegalic inclusion disease according to any of claims 1 to 3, wherein the attenuated cytomegalovirus is the cytomegalovirus TOWNE 125 strain.

5. A live combined vaccine against rubella and cytomegalic inclusion disease, the vaccine being substantially as herein described in any of the Examples.