Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1995-06-07
2002-05-28
Scheiner, Laurie (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S007100, C422S067000, C424S211100
Reexamination Certificate
active
06395469
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to the field of immunology and, in particular, to inactivated respiratory syncytial (RS) virus vaccines.
BACKGROUND OF THE INVENTION
Human respiratory syncytial virus is the main cause of lower respiratory tract infections among infants and young children (refs. 1 to 3-a list of references appears at the end of the disclosure and each of the references in the list is incorporated herein reference thereto). Globally, 65 million infections occur every year resulting in 160,000 deaths (ref. 4). In the USA alone, 100,000 children may require hospitalization for pneumonia and bronchiolitis caused by RS virus in a single year (refs. 5, 6). Providing inpatient and ambulatory care for children with RS virus infections costs in excess of $340 million annually in the USA (ref. 7). Severe lower respiratory tract disease due to RS virus infection predominantly occurs in infants two to six months of age (ref. 8). Approximately 4,000 infants in the USA die each year from complications arising from severe respiratory tract disease caused by infection with RS virus and Parainfluenza type 3 virus (PIV-3). The World Health Organization (WHO) and the National Institute of Allergy and Infectious Disease (NIAID) vaccine advisory committees have ranked RS virus second only to HIV for vaccine development.
RS virus is a member of the Paramyxoviridae family of the pneumovirus genus (ref. 2). The two major protective antigens are the envelope fusion (F) and attachment (G) glycoproteins (ref. 9). The F protein is synthesized as a 68 kDa precursor molecule (F0) which is proteolytically cleaved into disulfide-linked F1 (48 kDa) and F2 (20 kDa) polypeptide fragments (ref. 10). The G protein (33 kDa) is heavily O-glycosylated giving rise to a glycoprotein of apparent molecular weight of 90 kDa (ref. 11). Two broad subtypes of RS virus have been defined: A and B (ref. 12) The major antigenic differences between these subtypes are found in the G glycoprotein (refs. 7, 13).
A safe and effective RS virus vaccine is not available and is urgently needed. Approaches to the development of RS virus vaccines have included inactivation of the virus with formaldehyde, isolation of cold-adapted and/or temperature-sensitive mutant viruses and isolation of the protective antigens of the virus. Clinical trial results have shown that both live attenuated and formalin-inactivated vaccines failed to adequately protect vaccinees against RS virus infection (refs. 14 to 16). Problems encountered with cold-adapted and/or temperature-sensitive RS virus mutants administered intranasally included clinical morbidity, genetic instability and overattenuation (refs. 17 to 19). A live RS virus vaccine administered subcutaneously also was not efficacious (ref. 20). Inactivated RS viral vaccines have typically been prepared using formaldehyde as the inactivating agent. Murphy et al. (ref. 21) has reported data on the immune response in infants and children immunized with formalin-inactivated RS virus. Infants (2 to 6 months of age) developed a high titre of antibodies to the F glycoprotein but had a poor response to the G protein. Older individuals (7 to 40 months of age) developed titres of F and G antibodies comparable to those in children who were infected with RS virus. However, both infants and children developed a lower level of neutralizing antibodies than did individuals of comparable age with natural RS virus infections. The unbalanced immune response, with high titres of antibodies to the main immunogenic RS virus proteins F (fusion) and G (attachment) proteins but a low neutralizing antibody titre, may be in part due to alterations of important epitopes in the F and G glycoproteins by the formalin treatment. Furthermore, some infants who received the formalin-inactivated RS virus vaccine developed a more serious lower respiratory tract disease following subsequent exposure to natural RS virus than did non-immunized individuals (refs. 15, 16). The formalin-inactivated RS virus vaccines, therefore, have been deemed unacceptable for human use.
Evidence of an aberrant immune response also was seen in cotton rats immunized with formalin-inactivated RS virus (ref. 22). Furthermore, evaluation of RS virus formalin-inactivated vaccine in cotton rats also showed that upon live virus challenge, immunized animals developed enhanced pulmonary histopathology (ref. 23).
The mechanism of disease potentiation caused by formalin-inactivated RS virus vaccine preparations remains to be defined but is a major obstacle in the development of an effective RS virus vaccine. The potentiation may be partly due to the action of formalin on the F and G glycoproteins. Additionally, a non-RS virus. specific mechanism of disease potentiation has been suggested, in which an immunological response to contaminating cellular or serum components present in the vaccine preparation could contribute, in part, to the exacerbated disease (ref. 24). Indeed, mice and cotton rats vaccinated with a lysate of HEp-2 cells and challenged with RS virus grown on HEp-2 cells developed a heightened pulmonary inflammatory response.
Furthermore, RS virus glycoproteins purified by immunoaffinity chromatography using elution at acid pH were immunogenic and protective but also induced immunopotentiation in cotton rats (refs. 22, 25).
There clearly remains a need for immunogenic preparations, including vaccines which are not only effective in conferring protection against disease caused by RS virus but also does not produce unwanted side-effects, such as immunopotentiation. There is also a need for antigens for diagnosing RSV infection and immunogens for the generation of antibodies (including monoclonal antibodies) that specifically recognize RSV proteins for use, for example, in diagnosis of disease caused by RS virus.
Art recognized approaches to the developments of RSV vaccines have been summarized in recent review articles (refs. 2, 31 to 35), none of which propose the development of an inactivated RSV vaccine.
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 &bgr;-propiolactone; a non-ionic detergent, including n-octyl-&agr;-D-glucopyranoside and n-octyl-&bgr;-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-infec
Ewasyshyn Mary Elizabeth
Klein Michel Henri
Sanhueza Sonia E.
Aventis Pasteur Limited
Parkin Jeffrey S.
Scheiner Laurie
Sim & McBurney
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