Merck + Co., Inc.

Other

William J McAleer

Edward H Wasmuth

William M Hurni

William J Miller

Henry Z Markus

Raymond E Spier

Maurice R Hilleman

E Dale Lehman

Roy A Machlowitz

Ted F Schaefer

Kenneth L Posch

Philip J Provost

Roy W Grabner

Edward M Scolnick

Clarence L Baugh

Paula A Giesa

Edward L Paul

Lynn T Callahan

Fred J Bailey

Robert Z Maigetter

D Eugene Wampler

Arthur Friedman

Abner J Schlabach

The invention comprises a lyophilized live herpes virus vaccine that comprises from about 0.5% to about 8% moisture.

Stable lyophilized live herpes virus vaccine

A quantitative assay for HAV comprises infecting at a temperature of from about 30.degree. to about 37.degree. C. a cell sheet with a dilution series of hepatitis A virus and then adding Newcastle disease virus (NDV) to the infected cell sheet and incubating the infected cell sheet in the presence of NDV at an elevated temperature for a time sufficient to permit a cytopathic effect (CPE) to become manifest, this time typically being at least about 4 days and preferably about 5 days. At an incubation temperature of from about 31.degree. to about 33.degree. C. the absence of CPE indicates the presence of HAV and the presence of CPE indicates the absence of HAV. At an incubation temperature of from about 34.degree. to about 36.degree. C. the absence of CPE indicates the absence of HAV and the presence of CPE indicates the presence of HAV.

Assay for hepatitis a virus

Hepatitus A virus is grown in Vero cells after passage in in vitro cell culture. Virus replication and continued passage in Vero cells requires extended incubation times, up to about four weeks, for early passages and incubation temperatures no higher than about 32.degree. C. Continued passage results in a significant decrease in incubation time and an increase in virus yield. Cultivation of hepatitus A virus in Vero cells meets the development requirements for an inactivated human vaccine.

Growth of hepatitus A virus in vero cells

Recombinant pre-S-HBsAg is purified by a rapid and efficient two step chromatographic process. Yeast cells expressing recobinant pre-S-HBsAg are disrupted, the cell contents are clarified and separated by polymerized human serum albumin affinity chromatography. The pre-S-HBsAg is further purified by hydrophobic interaction chromatography using butyl agarose. This process results in pre-S-HBsAg that is greater than 90% pure.

Purification of hepatitis pre-S antigens by polymerized serum albumin

The surface antigen protein of human Hepatitis B virus is synthesized in Saccharomyces cerevisiae as a 23,000-26,000 dalton polypeptide, essentially free of intermolecular disulfide bonds. This antigen is a poor immunogen in animals and man. No prior precedent or method exists for efficiently converting the non-disulfide bonded antigen to a fully intermolecular disulfide bonded particle. We describe the first example of such a conversion in vitro and show that the act of this conversion enhances the immunogenicity of the antigen about 10-fold. The in vitro conversion makes practical the production of hepatitis B surface antigen from microorganisms using recombinant DNA methods.

Immunogenic HbsAg derived from transformed yeast

HBcAb in a biological fluid is adsorbed on a surface which is then coated with BSA. The coated surface is then incubated first in the sample and then in the presence of radiolabelled HBcAb.

RIA assay for HBcAg

Hepatitis B surface antigen (HBsAg) is produced in vitro in high titer and purity from tissue cultures of cells that shed HBsAg. The cells are grown on hollow fiber capillary units having a molecular weight cut-off of about 10,000.

Methods of producing HBsAg

Hepatitis A virus is harvested from persistently infected cells, the cells are then trypsinized, replanted and a second harvest of hepatitis A virus is obtained. The cells can be trypsinized and replanted and harvested indefinitely.

Process for preparing hepatitis A virus

A diagnostic reagent for hepatitis A antibody is prepared by adhering hepatitis A antibody to a surface by non-specific adsorption followed by specific coupling of hepatitis A antigen to the antibody. This reagent is useful in an in vitro assay for hepatitis A antibody.

Hepatitis A antibody assay

An improved stabilized liquid live viral vaccine contains a live virus, partially hydrolyzed gelatin, a monosaccharide or disaccharide, a cell culture medium, L-glutamic acid, L-arginine and sufficient physiologically acceptable acidic buffer to maintain the pH at from about 6.0 to about 6.5.

Vaccine stabilizer containing L-glutamic acid and L-arginine

Semi-automated assays for viral infectivity and an assay for serum neutralizing antibody content are based on staining the cells and macroscopic reading of the cytopathic effect.

Macroscopic method for determining cytopathic effects in viral assay

A method for determining the end point in a viral assay comprising

Automated plate reader

Hepatitis A virus (HAV) is produced in high titer from tissue cultures grown on a hollow fiber capillary unit.

High titer production of hepatitis A virus

Hepatitis B surface antigen (HB.sub.s Ag) is produced in vitro in high titer and purity from tissue cultures of cells that shed HB.sub.s Ag by growing the cells with a first incubation at elevated temperature followed by a second incubation at a lower elevated temperature.

Growing community of inventors

Ambler, PA, United States of America

William J McAleer