GB1130715A - Improvements in or relating to vaccines based on mycoplasma pneumoniae - Google Patents

Abstract

1,130,715. Vaccines. MERCK & CO. Inc. 7 Feb., 1966 [12 Feb., 1965; 29 Dec., 1965], No. 5250/66. Heading A5B. [Also in Division C6] The specification relates to vaccines based on attenuated Mycoplasma pneumoniae, particularly to the Eaton strain of Mycoplasma pneumoniae referred to in the specification as Eaton pleuropneumoniae-like micro-organisms (Eaton PPLO). The Mycoplasma pneumoniae are cultivated on a medium of pH between 6.5 and 8.0 (preferably 7.2-7.4) comprising: (1) 0.1%-20% by weight of (i) egg allantoic fluid, (ii) a combination of albumin (human albumin, ovalbumin or lactalbumin hydrolysate), dextrose and arginine, or (iii) egg allantoic fluid in combination with albumin and/or dextrose and/or arginine; (2) a maximum of 0.025% by weight of yeast extract or a combination of cholesterol, lecithin and diphosphopyridine nucleotide; and (3) Hank's balanced salt solution. Sodium bicarbonate is employed for adjusting the pH of the medium. Vaccines are produced by inoculating viable Eaton PPLO into specified Medium I or Medium II (based on the above-defined compositions) as a 5-10% inoculum containing approximately 10<SP>5</SP>-10<SP>10</SP> organisms per millilitre, and incubating at about 37‹C in an aerobic atmosphere for a sufficient number of days, usually from 7 to 14, to raise the total population of the organisms in the growth medium to approximately 10<SP>6</SP>-10<SP>10</SP> per millilitre. The suspension containing the micro-organisms is then centrifuged at 20,000-50,000 r.p.m. and washed with buffered saline solution, and is then resuspended in phosphate buffered saline solution of pH 7.0-7.2, preferably to a 60-fold concentration, and inactivated. Inactivation methods mentioned are: (1) addition of formalin and incubation for 24 hours at 37‹C-the residual unbound formulin can be neutralized with sodium bisulphite and dialyzed' against saline solution for 48 hours, or it can be left untouched; (2) heating at 37‹C for 7-10 days; or (iii) addition of phenol. Residual proteins can be removed by centrifuging at 16,000 r.p.m. for 45-60 minutes. The vaccine suspensions are preserved by formalin solution and thimerosal. Quality control tests are performed with each vaccine formulation; the potency of the vaccine is determined by administration to animals and human beings and assaying their sera for antibody levels by complementfixation and serum neutralization techniques. There are described preparations of aqueous vaccine, of alum adsorbed vaccine and of an emulsified vegetable oil adjuvant vaccine in which the adjuvant is prepared by dispersing aluminium monostearate in a preformed mixture of peanut oil and mannide mono-oleate.