Field Ak

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.

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.

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.

The Effect of Altering the Size of Poly C on the Toxicity and Antigenicity of Poly I:C

Summary Appropriate reduction in size of poly C by degradation with ribonuclease prior to complexing with poly I effected marked reduction in toxicity for mice and antigenicity for rabbits of poly I:C without detectable reduction in in vivo or in vitro protection against viral infection. There was slight reduction in interferon-inducing capacity for rabbits. Such procedure adds another possible means for increasing the safety of poly I:C in eventual clinical application. The authors are indebted to Mary-Ellen Davies, Helen Perry, Carolyn Saydah and Carol Bonoma for excellent technical assistance.

Determination of Antibodies to Double-Stranded RNA by Enzyme-Linked Immunosorbent Assay (ELISA)

Abstract A sensitive enzyme-linked immunosorbent assay (ELISA) for antibodies to double-stranded RNA has been developed using polyinosinic acid . polycytidylic acid (I n C n ) as antigen. Bound antibody was detected using alkaline phosphatase-conjugated goat antiimmunoglobulin and ρ-nitrophenylphosphate as substrate. The assay, as demonstrated for rabbit, NZB/NZW mouse, and grivet monkey sera, was far more sensitive and reproducible than quantitative complement fixation. I n C n complexed with poly-L-lysine and adsorbed to polystyrene tubes resulted in optimal uptake of antibodies to double-stranded RNA. I n -C n without added poly-L-lysine or with other cationic substances adsorbed poorly, as measured by reduced antibody uptake. Specificity of the assay for antibodies to double-stranded RNA was demonstrated by reduction of antibody binding to antigen-coated tubes following adsorption of sera with double-stranded, but not single-stranded RNAs. Similar adsorption studies indicated that this method could be used also to assay for double-stranded RNA. Therefore, a simple ELISA can be used for quantitative determination of either antibodies to double-stranded RNA or double-stranded RNA antigen.

Antigenicity of double-stranded ribonucleic acids including poly I:C.

Summary Rabbits repeatedly injected intravenously with poly I:C developed antibodies reactive with poly I:C. However, oral or nasal administration of poly I:C to rabbits failed to stimulate detectable circulating antibodies. Intravenous injection of ICR mice, guinea pigs, and grivet monkeys also failed to stimulate a detectable circulating antibody. Combination of poly I:C with methylated bovine serum albumin enhanced the antigenicity of poly I:C in rabbits. Antibodies produced in response to poly I:C were primarily the IgM type, whereas those produced in response to poly I:C plus methylated bovine serum albumin were mainly the IgG type. Hyperimmunization with poly I:C resulted in shift to predominance of IgG antibodies. Rabbits with high antibody titers to poly I:C showed reduced serum interferon production in response to induction with poly I:C. However, blood leukocyte alterations and febrile responses to poly I:C in immunized and normal rabbits were essentially similar. Both the IgM and IgG antibodies to poly I:C were capable of neutralizing the capacity of poly I:C to induce interferon in unimmunized rabbits. Antisera reactive with poly I:C were similarly reactive with other double-stranded RNAs, were variably reactive with poly I, but were unreactive with other single-stranded RNAs or DNA. The authors are grateful to Dr. E. Piperno for advice concerning experiments on febrile responses. Skillful technical assistance was provided by W. P. M. Fisher, M. Johnston, M. E. Davies and H. Perry.

Demonstration of Double-Stranded Ribonucleic Acid in Concentrates of RNA Viruses

Summary Nucleic acid preparations from unpurified concentrates of Newcastle disease virus, Sindbis virus and Semliki Forest virus were found to contain up to 2% double-stranded RNA. Double-stranded RNA was identified by reaction with antisera specific for double-stranded RNA, capacity to protect primary rabbit kidney cells against virus infection, and relative ribonuclease resistance. The presence of such double-stranded RNA in virus preparations provides an explanation for how inactivated crude suspensions of single-stranded RNA virus virions may induce interferon in the absence of detectable viral replication. Serologically active double-stranded RNA was also detected in normal chick embryo cell nucleic acid extracts. This differed greatly from double-stranded viral RNA, however, since the serologic activity was more sensitive to destruction by ribonuclease than the viral dsRNA and it did not stimulate resistance to virus infection in primary rabbit kidney cells. The authors are indebted to W. P. M. Fisher, Marilyn Johnston, Mary-Ellen Davies, and Helen Perry for excellent technical assistance.