Nemes Mm

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.

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.

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.

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.

Characterization of Chick Embryo Interferon Induced by a DNA Virus

Summary Biochemical, biophysical and biological characterization was carried out previously for highly purified chick embryo interferon induced by an RNA virus, influenza A (RNA-interferon). The present report summarizes the findings in studies to characterize partially purified chick embryo interferon induced by a DNA virus, herpes simplex (DNA-interferon). DNA-interferon was shown to be a protein of low molecular weight (about 36,000) which is trypsin sensitive, relatively heat stable (70°C) and is stable over a wide range of pH (pH 1 to 11). The findings by others are reviewed and analyzed. Development of a new method for measuring isoelectric point based on pH elution profile from CM-sephadex established the isoelectric point for DNA- and RNA-in-terferon at pH near neutral. Both DNA-and RNA-interferon were active against vesicular stomatitis virus in the homologous chick cell culture system but showed no activity against this virus in mouse embryo, grivet kidney, bovine kidney or rabbit kidney cell cultures. The remarkable similarity of chick DNA-interferon to RNA-interferon, even though induced by basically different agents, is consistent with the concept that interferon is synthesized by resident host mechanisms rather than under the viral genetic code.

Relationship of Molecular Size of rIn:rCn (Poly I:C) to Induction of Interferon and Host Resistance

Summary Preparations of high molecular weight rIn:rCn were exposed to sonic radiation. A decrease in the average molecular weight of the polynucleotide complexes after treatment was accompanied by an exponential decrease in viscosity of the preparation, an increase in the sensitivity of the polynucleotide complex to degradation by pancreatic ribonuclease, a decrease in the thermal transition midpoint, and a decrease in the hyperchromicity observed during thermal transition. The lower molecular weight rIn:rCn complexes were tested for their capacity to induce interferon in rabbits and resistance against VSV infection in cell culture and PVM infection in mice. Sonic-treated preparations of rIn:rCn reduced to an average molecular weight of approximately 4.6 × 105 were markedly reduced in capacity to protect mice against PVM infection, compared with untreated rIn:rCn preparations containing complexes having an average molecular weight of approximately 7.8 × 106. The capacity of sonic-treated rIn:rCn to induce production of rabbit interferon and resistance to virus infection in cell culture was more resistant to molecular weight reduction. Significant decrease in activity was observed in rIn:rCn fractions with approximate molecular weights less than 1.2 × 105.

Influence of polyamines on induction of interferon and resistance to viruses by synthetic polynucleotides.

Summary Certain basic polyamines, such as neomycin, neamine, kanamycin, streptomycin, and spermine, added to poly I:C increased the T m. The degree of termal stabilization was influenced by the molecular ratio of polyamine to poly I:C and by the ionic strength. Complexing with certain of the polyamines markedly reduced the rate of degradation of poly I:C but not of poly C by pancreatic ribonuclease. Some of the polyamines potentiated induction of resistance by poly I:C of primary rabbit kidney cells but not RK-13 rabbit kidney line or primary grivet kidney cells to infection by vesicular stomatitis virus. Neomycin did not alter the activity of poly I:C in intact animals against pneumonia virus of mice or against parainfluenza 1 (Sendai) virus in mice and did not decrease the toxicity of the complexed polynucleotide for these animals. Added neomycin did not affect the induction by poly I:C of interferon in rabbits. Neomycin did not render the single-stranded RNAs, poly I and poly C, capable of inducing interferon in rabbits and poly C was not made resistant to RNase by neomycin. Neomycin suppressed rather than increased the uptake of poly I:C by primary rabbit kidney cells. The implications of the findings are discussed. The authors are indebted to J. N. Armstrong, Jr., C. Bonoma, M. Davies, C. Saydah, and K. Young for valuable technical assistance.

Multiple Molecular Species of Interferons of Mouse and of Rabbit Origin

Summary Interferons found present in mouse serum following intravenous injection of NDV virus were of 2 different kinds. Component A had a molecular weight of 38,000 and an isoelectric point of 7.7 while component B had a molecular weight of 23,500 and an isoelectric point of 7.4. Interferons induced in mouse spleen and L (MCN) cell cultures by the same virus had molecular weighty of 26,000 and 25,000, respectively, and an isoelectric point of 7.0. Rabbit spleen cell culture interferon had a molecular weight of 33,000 and an isoelectric point of 7.4. A second molecular moiety which appears in chick embryo interferon induced by WS influenza virus was found to have a molecular weight of approximately 50,000 in contrast to 38,000 for the more predominant component. The mouse serum interferons appeared less heat stable than chick interferon and component A more sensitive to high pH. The characteristics of these interferons were compared with the properties of the spectrum of interferons produced in various animal species by various microbial agents.

Influence of Size of Individual Homopolynucleotides on the Physical and Biological Properties of Complexed rIn:rCn (Poly I:C)

Summary Reduction in the size of rCn by sonic radiation caused a decrease in viscosity when complexed with rIn of high molecular weight but did not bring about a decrease in protective activity of the rIn:rCn against PVM or VSV viruses. Greater reduction in size of rCn by treatment with RNase resulted in progressive loss of capacity to complex with rIn and to protect against viral infections. The capacity of rIn:rCn to induce resistance to VSV virus in vitro and to PVM virus in vivo was more dependent upon maintaining a high molecular weight of rIn than of rCn. Activity was markedly diminished when the average molecular weight of rIn was below 1.9 × 105 and of rCn below 2.3 × 104.

EFFECTIVE TREATMENT OF EXPERIMENTAL HERPES SIMPLEX KERATITIS WITH NEW DERIVATIVE, 5-METHYLAMINO-2'-DEOXYURIDINE (MADU).

Summary A recently synthesized compound, 5-methylamino-2′-deoxyuridine (MADU) was shown to be highly active in suppressing infection with herpes simplex virus in vitro and to exert beneficial effect in therapeutic and prophylactic-therapeutic treatment of experimental keratitis in the rabbit eye caused by herpes simplex virus. The beneficial effect was equal to or only slightly less than that observed in comparative tests with 5-iodo-2′-deoxyuridine (IDU). The low toxicity of MADU for the rabbit eye, the high level of activity, the absence of halogen, and the apparent low level incorporation into the DNA of host cells justify experimental investigations in man in infections in which IDU would normally be employed. The practical and theoretical implications of use of MADU and IDU in the human being are discussed.