An integrated and comparative study of the antiviral effects and other biological properties of the polyinosinic-polycytidylic acid duplex and its mismatched analogues. III. Chronic effects and immunological features. (1976)

Extensive investigations were made of the chronic biological properties of the polyinosinic·polycytidylic acid (rI n ·rC n ) duplex, along with two duplexes having mismatched base pairs, rI n ·r(C 12 , U) n and rI n ·r(C 29 ,G) n , and a double-stranded RNA (BRL 5907) obtained from virus-like particles in Penicillium cultures. Earlier we had shown that mismatched polynucleotide duplexes are comparable in their antiviral properties to rI n ·rC n , but possess much less pronounced immediate secondary effects than the rI n ·rC n molecule. In this work the relative antiviral activities against encephalomyocarditis virus infection in mice of these four duplexes were compared and rI n ·r(C 29 ,G) n was found to be somewhat less active than the other double-stranded RNAs. In experiments on mice with a 7-day repeat dose, rI n ·rC n , and BRL 5907 caused a spectrum of toxic effects: a substantial loss of body weight, acute lymphopenia, and thrombocytopenia; on day 8, neutrophil leukocytosis, anemia, splenomegaly, and thymic atrophy were also noted. In contrast, rI n ·r(C 29 ,G) n was less toxic and did not promote thymic atrophy; rI n ·r(C 12 ,U) n effected even fewer secondary biological events since it did not promote anemia or a change in body weight, modify the neutrophil count, or induce the thymic atrophy, effects characteristic of rI n ·rC n and BRL 5907. All inducers, including the mismatched duplexes, induced some degree of thrombocytopenia, acute lymphopenia, and splenomegaly by day 8. Splenomegaly may be due to the mitogenic potential of double-stranded RNA on splenocytes, a reaction known not to be completely abrogated by purposeful mismatching of bases in RNA. Thus these studies established an order of capacity for triggering specific secondary toxic responses: BRL 5907 ≅ rI n ·rC n > rI n ·r(C 29 ,G) n [unknown] rI n ·r(C 12 ,U) n . Using radioactive rI n ·rC n as a probe, serum antibody was assayed in both mice and rabbits following repeated administration of the three synthetic double-stranded RNAs: rI n ·rC n; appeared the most immunogenic of the three. The order of immunogenic potential for mismatched duplexes, tested only by cross-reaction, was BRL 5907 > rI n ·rC n > rI n ·r(C 29 ,G) n [unknown] rI n ·r(C 12 ,U) n . Interestingly, antibody raised against the naturally occurring RNA could interact effectively with antibody raised specifically against rI n ·rC n , thereby supporting the notion that extensive cross-reactivity is shared by antibodies raised against the double-stranded RNAs with the various sequences. Companion studies in vitro , which evaluated the relative rates of polymer degradation with pancreatic RNase, confirmed that mismatching of the base pairs in the duplex RNA, as exemplified by rI n ·r(C 12 ,U) n and rI n ·r(C 29 ,G) n , would enhance the rate of polymer degradation. The natural double-stranded RNA (BRL 5907) was degraded much more slowly than the others. Thus this mechanism appears to be one of the factors in the differential effects observed in vivo . Together with our earlier work, the current results more firmly establish the importance of the kinetic term in modifying the therapeutic ratios of double-stranded RNAs in intact biological systems and illustrate how the spectrum of biological responses to double-stranded RNAs can be modulated by this strategy.