Influenza virus vaccines containing from 1 to 10 mg. of virus materials per cc. concentrated and purified from infectious allantoic fluids by means of one or two cycles of differential centrifugation and inactivated by different treatments have been prepared and subjected to laboratory tests. Suitable inactivation of the virus preparations with retention of full red cell agglutinating activity and immunizing potency in mice was achieved by treatment with minimal amounts of formaldehyde or ultraviolet light. Treatment with phenol or chloroform failed to cause adequate loss of virus activity. Excessive amounts of formaldehyde or of ultraviolet light were found to cause a loss in red cell agglutinating activity and in immunizing potency. Freezing resulted in the immediate loss of red cell agglutinating activity of the formalinized vaccine. Storage of the vaccines in the frozen state was accompanied by a gradual decrease in red cell agglutinating activity. Drying of the vaccines from the frozen state resulted in a loss of red cell agglutinating activity and, in the case of the formalinized vaccine, in a loss in immunizing potency. There appeared to be at least a rough correlation between red cell agglutinating activity and immunizing potency. The immunizing potency and red cell agglutinating activity of a purified formalinized vaccine containing 2 mg. of virus material per cc. were unchanged following 2 months' storage at 4° but were measurably decreased following storage for 2 months at 18 to 25° and at 37°. At equivalent dosages of virus material the immunizing potency of formalinized centrifugally purified virus, of formalinized virus purified by the red cell elution method, and of infectious allantoic fluid was not measurably different. The immunizing potency of a formalinized polyvalent vaccine containing centrifugally purified Lee, PR8, and Weiss influenza virus materials at concentrations of 5, 2.5, and 2.5 mg. per cc., respectively, was found to be essentially the same as that of a similar vaccine prepared commercially. In both cases the protection afforded against the Weiss strain appeared to be better than that against the Lee and PR8 strains. The commercially prepared vaccine is being subjected to clinical tests in man at dosage levels ranging from 0.01 mg. to 10 mg. The latter corresponds to a level approximately 100 times that of infectious allantoic fluid.
It was found that the bacterial contamination that frequently accompanies operation on a large scale can be controlled by the addition of one part per 10,000 of formalin plus one part per 100,000 of phenyl mercuric nitrate to the allantoic fluid immediately following harvesting, without affecting the quality of the vaccine. This procedure and the use of virus materials purified and concentrated by a single cycle of differential centrifugation by means of the Sharples centrifuge were found to be suitable for the production of influenza virus vaccines on a large scale. By means of this method influenza vaccines possessing 20 or more times the immunizing potency of infectious allantoic fluid and 10 or more times the immunizing potency of the usual commercial vaccine prepared by the red cell elution method can be manufactured rapidly on a very large scale with considerable ease and efficiency.