Alfred A. Tytell

Purification and Characterization of Chick Embryo Interferon

Summary Chick embryo interferon was purified from allantoic fluid of embryonated eggs infected with influenza A virus. Purification consisted essentially of acid precipitation to remove virus and extraneous protein, concentration and purification by precipitation with Zn++, column chromatography on CM-cellulose, and zone ionophoresis on pevikon. The interferon was purified 4500 times with respect to initial protein and one unit of interferon activity was 0.0042 μg protein in the chick embryo cell-EEE virus assay system used. Interferon was found to be a slightly basic protein of low molecular weight and free of nucleic acid as well as constituents giving absorption in the visible spectrum. A trace amount of carbohydrate was found. Detailed findings in the analyses are presented. The striking difference in properties of interferon from those described heretofore by other workers appears to lie in previous failure of purification. Both crude and purified chick interferon were active in suppressing Rous sarcoma development and NDV virus infection in susceptible chicks but were without therapeutic effect.

Purified and inactivated human hepatitis B vaccine: progress report.

Developments leading to the preparation and testing for safety and potency of a highly purified inactivated preparation of hepatitis B surface antigen are descirbed. Protective efficacy studies in chimps of a lot of the inactivated hepatitis B vaccine are currently and suitable for clinical trials in man.

Inducers of interferon and host resistance. VI. Antiviral efficacy of poly I:C in animal models.

Summary Studies were conducted in mice and chicks to measure the prophylactic and therapeutic efficacy of poly I:C in a variety of infections by both DNA and RNA viruses. Drug and virus were given by a variety of routes in a number of different regimens including variable drug and virus dosage. Marked prophylactic protection was obtained against PVM, Columbia SK, vaccinia, and parainfluenza 1 virus infections in mice. Poly I:C was therapeutic against PVM infection in mice when given as late as 3 days after infection and the duration of protective effect induced by poly I:C persisted for almost 7 days in the PVM system. Weak responses were noted against rabies, influenza B, and Rous sarcoma viruses, while the responses to influenza A, yellow fever, and Mareks agent were negative. The highly complex relationships between virus and drug administration will necessitate further study before optimal prophylactic and therapeutic regimens can be developed. The authors are indebted to Carolyn Saydah, Carol Bonoma, Kersti Young, Mary Ellen Davies, and John N. Armstrong, Jr. for technical assistance in these studies.

Serumless medium for cultivation of cells of normal and malignant origin.

Summary A serumless medium suitable for prolonged growth and repeated subculture of Walker carcinosarcoma 256, sarcoma 180, KB and human heart cells in vitro was described. In addition to amino acids, vitamins, minerals and glucose, it contained insulin, salmine sulfate, pyruvate, mucate, methyl oleate, folinic acid and lactalysate. The latter group of materials with exception of mucate was required for maximal growth rates, comparable to those on media containing dialyzed serum. Suspension cultures of Walker carcinosarcoma 256 were grown with continuous passage in the serumless medium.

Development and Chimpanzee Testing of a Vaccine against Human Hepatitis B

Summary Highly purified hepatitis B virus surface antigen (Australia antigen) purified by physical and chemical procedures from infected human plasma was used to prepare hepatitis B vaccine. The purified antigen was treated with formalin and the vaccine was tested exhaustively for safety by ordinary procedures and additionally in marmosets (for live hepatitis A virus) and in chimpanzees (for live hepatitis B virus). The vaccine was highly potent, inducing antibody in guinea pigs, grivet monkeys, and chimpanzees given three doses of vaccine containing up to 20 μg of hepatitis B antigen per dose. A protective efficacy trial was carried out in chimpanzees that were given three doses of vaccine subcutaneously and then challenged intravenously with 1000 chimpanzee infectious doses of human hepatitis B virus. All of five unvaccinated control animals developed hepatitis B virus an-tigenemia following challenge and all of six vaccinated animals were protected, including one animal that had failed to develop detectable antibody following vaccination.

Induction of interferon in human subjects by poly I:C.

Summary Poly I:C (rIn:rCn), a synthetic double-stranded polynucleotide previously demonstrated to be a potent inducer of interferon and host resistance to viral infection in cell culture and in animals, has been successfully used to induce interferon in human beings. Fourteen of 20 patients with advanced cancer developed interferon after a single intravenous administration of poly I:C. The interferon was identified by the usual criteria of pH stability, host species specificity, broad antiviral spectrum, inactivation by trypsin, and nonsedimentability under defined conditions. Several of the patients were capable of repeated induction of interferon by poly I:C at intervals of 3 to 7 days. Evidence of refractoriness to induction occurred only after repeated daily injections of poly I:C. The only consistent clinical manifestation of poly I:C administration was a febrile response. None of the patients tested developed demonstrable CF antibodies against either poly I:C or denatured DNA during the course of treatment.

The concentration and purification of poliomyelitis virus by the use of nucleic acid precipitation

Abstract The precipitation of poliovirus by nucleic acid at low pH represents a new, highly satisfactory method for the concentration of virus from large volumes of tissue culture filtrate. This method has been used as a first step in a procedure for the purification of poliovirus. Virtually pure preparations so obtained have permitted the redetermination of the extinction coefficient of poliovirus (E260190 = 81.6) and the determination of the specific refractive increment of the virus (Δn = 0.00174). Sufficient quantities of the virus became available through the use of this procedure for the determination of the elementary composition of the virus. Purified preparations of poliovirus obtained by the use of this method were inactivated with formaldehyde and were found to induce neutralizing antibodies in animals and man.

Hyperpotentiation by synthetic double-stranded RNA of antibody responses to influenza virus vaccine in adjuvant 65.

Summary Immunologic adjuvant 65 caused a strong enhancement of antibody response to influenza virus antigens in tests in animals. Complexed synthetic polynucleotides Poly I:C (rI:rC) and Poly A:U (rA:rU) produced comparatively weak potentiation of antibody response to these antigens and in certain instances, Poly A:U was inhibitory. Immunologic adjuvant 65 combined with the Poly I:C or Poly A:U acted synergistically to cause a hyperpotentiation of antibody response which greatly exceeded the additive effects of adjuvant and complexed polynucleotides tested singly. Such potentiation did not occur when the complexed polynucleotides were tested with alum adjuvant or when Poly I or Poly C was added alone. Poly I:C was generally more effective than Poly A:U. Maximal potentiation was demonstrated at 26 μg/ml of Poly I:C and there was no further enhancement when the concentration was raised to 260 μg/ml. Use of adjuvant 65 combined with complexed polynucleotides promises to contribute a new magnitude of immunizing capability to be obtained when used with influenza and other killed antigen preparations.

Inducers of Interferon and Host Resistance VII. Antiviral Efficacy of Double-Stranded RNA of Natural Origin

Summary Studies in cell cultures, rabbits, and mice demonstrated the prophylactic and therapeutic efficacy against viral infections and of interferon induction by a variety of double-stranded RNAs of natural origin including fungi, animal, plant, insect, and bacterial viruses. All were found active in microgram amounts. RNA and virus were given by a variety of routes in a number of different regimens including variable drug and virusdosage. The most extensive studies were conducted with double-stranded replicative form RNA from MU9 mutant of MS2 coliphage. Remarkably favorable effects were obtained against PVM, Columbia SK, vaccinia, and parainfluenza 1 virus infections. The polynucleotide was therapeutic in mice when given as late as 2 days following PVM virus, and prophylactic when given as long as 7 days before the virus infection. Preliminary test results showed activity of the DNA-RNA hybrid of the F1 DNA coliphage in inducing interferon and resistance to viral infection.

New Metabolizable Immunologic Adjuvant for Human Use. I. Development and Animal Immune Response.

Summary A new immunologic adjuvant, adjuvant 65, is described and the results of extensive tests of polyvalent influenza virus vaccine in the adjuvant are presented. The vaccine consists of a water-in-oil emulsion in which the adjuvant portion consists of peanut oil, mannide monooleate and aluminum monostearate. The adjuvant effect measured in terms of hemagglutination-inhibition antibody responses 28 days after vaccination in mice or guinea pigs is roughly equal to that obtained with Freunds incomplete mineral oil adjuvant. Adjuvant 65 influenza vaccine showed striking enhancement and long-term persistence of antibody in guinea pigs, rabbits and monkeys compared with the corresponding aqueous vaccines. The findings in histomorphologic-pathologic studies in animals and the clinical and serologic results obtained in large-scale studies in man are presented elsewhere. Adjuvant 65 shows promise of meeting the present urgent need for a safe and effective adjuvant for enhancing immunologic responses to viral and other vaccines and possibly also for treating allergic disease.