Title:  Inactivated respiratory syncytial viral vaccines

United States Patent:  6,395,469

Assignee:  Aventis Pasteur Limited (Toronto, CA)

Filed:  June 7, 1995

An immunogenic composition capable of producing a respiratory syncytial (RS) virus specific immune response in a host immunized therewith comprises purified, inactivated RS virus which is substantially free from cellular and serum components and which is non-infectious, non-immunopotentiating, immunogenic and protective. The virus is grown on a vaccine quality cell line and harvested virus is purified under non-denaturing conditions to be substantially free from cellular and serum components. The purified RS virus is inactivated using .beta.-propiolactone, a non-ionic detergent, particularly n-octyl-.alpha.-D-glucopyranoside and n-octyl-.beta.D-glucopyranoside, or ascorbic acid. The immunogenic composition may be formulated as a vaccine for in vivo administration to a human host. The immunogenic composition also may be used in diagnostic applications.

SUMMARY OF THE INVENTION

The present invention provides a novel approach to the provision of such antigens and immunogens by inactivation of purified RS virus.

In one aspect of the present invention, there is provided a method of preparing an immunogenic composition capable of producing a respiratory syncytial (RS) virus specific immune response in a host immunized therewith, particularly a human host, which comprises a plurality of steps. The RS virus first is grown on an appropriate cell line and the virus harvested. The harvested virus is purified under non-denaturing conditions to produce a purified virus substantially free from cellular and serum components. The purified virus then is inactivated with an inactivating agent to provide a non-infectious, non-immunopotentiating and immunogenic RS virus. This RS virus then is formulated as an immunogenic composition.

The inactivating agent may be .beta.-propiolactone; a non-ionic detergent, including n-octyl-.alpha.-D-glucopyranoside and n-octyl-.beta.-D-glucopyranoside; or ascorbic acid.

The purifying step which is carried out on the harvested virus preferably may be effected by microfiltration to remove cell debris, tangential flow ultrafiltration to remove serum components, particularly employing an about 100 to about 300 kDa nominal molecular weight cut-off membrane, pelleting the ultrafiltered material by ultracentrifugation to further remove serum components and subjecting the pelleted material to sucrose density gradient centrifugation. Alternatively, the retentate from tangential flow ultrafiltration may be subjected to gel filtration followed by ion-exchange chromatography to further remove serum components.

This procedure provides a novel immunogenic composition capable of producing an RS virus specific immune response in a host immunized therewith which constitutes a further aspect of the present invention. Such immunogenic composition comprises purified, inactivated RS virus which is substantially free from cellular and serum components and which is non-infectious, non-immunopotentiating, immunogenic and protective, and a carrier therefor. The immunogenic composition may be formulated as a vaccine for in vivo administration to a human host for protecting the human from a disease induced by RS virus. The carrier for the immunogenic composition may comprise an adjuvant. The immunogenic composition may be formulated as a vaccine to be administered in an injectable form, intranasally or orally.

The present invention further provides a method of immunizing a host, particularly a human host, against disease caused by RS virus, which comprises administering to the host an effective amount of the immunogenic composition provided herein. The host immunized by such procedure may be selected from infants, young children, pregnant women, women of childbearing age, elderly individuals, immunocompromised individuals and other susceptible persons.

The inactivated RS virus provided herein also may be used as a diagnostic reagent for detecting infection by RS virus. Accordingly, the present invention further includes a method of determining the presence of antibodies specifically reactive with RS virus proteins in a sample, comprising the steps of:

(a) contacting the sample with the immunogenic composition of the invention to produce complexes comprising the non-infectious, non-immunogenic and immunogenic RS virus and any antibodies present in the sample specifically reactive therewith; and

(b) determining production of the complexes.

In addition, the present invention provides a method of determining the presence of RS virus proteins in a sample, comprising the steps of:

(a) immunizing a subject with the immunogenic composition of the invention to produce antibodies specific for RS virus proteins;

(b) contacting the sample with the antibodies to produce complexes comprising any RS virus proteins present in the sample and the RS virus protein-specific antibodies; and

(c) determining production of the complexes.

The present invention further provides a diagnostic kit for determining the presence of antibodies in a sample specifically reactive with RS virus proteins, comprising:

(a) the immunogenic composition of the invention;

(b) means for contacting the non-infectious, non-immunopotentiating and non-immunogenic RS virus with the sample to produce complexes comprising the non-infectious, non-immunopotentiating and immunogenic RS virus and any said antibodies present in the sample; and

(c) means for determining production of the complexes.

Having regard to the prior art difficulty with RS virus vaccine preparations, it is surprising that the procedures described herein provide immunogenic compositions which exhibits immunogenicity and protective ability while being non-infectious and non-immunopotentiating.

Claim 1 of 11 Claims

What we claim is:

1. A method of determining the presence of antibodies specifically reactive with respiratory syncytial virus (RSV) proteins in a sample, comprising the steps of:

(a) obtaining and preparing a sample suspected of containing RSV specific antibodies,

(b) obtaining and preparing a purified, inactivated RSV viral preparation by the steps of:

(i) crowing RSV virus on a cell line to produce a harvested virus,

(ii) harvesting the grown virus to produce a harvested virus,

(iii) purifying said harvested virus under non-denaturing conditions to produce a purified virus free from cellular and serum components, and

(iv) inactivating said purified virus with an inactivating agent,

(c) contacting the sample of step (a) with the purified, inactivated RSV viral preparation of step (b) as antigen under conditions which permit binding of antibody to antigen and the formation of an antigen antibody immune complex; and

(d) detecting said immune complex formation.

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