A soluble complex of poly I:C and poly-L-lysine (poly I:C/poly-L-lysine) has been prepared that induces high titers of circulating interferon in monkeys. By limiting the molar ratio of lysine to nucleotide to 0.5, a complex was formed that was soluble up to 2.0 mg poly I:C/ml of phosphate-buffered saline. Complexes of poly I:C with poly-L-lysine of various molecular weights, and in a constant ratio (0.5) of lysine to nucleotide, were evaluated for capacity to induce serum interferon in grivet monkeys. Substantial enhancement (10- to 100-fold) of the capacity of poly I:C to induce interferon in grivet monkeys was observed using poly I:C complexed with poly-L-lysines of molecular weight 10(4) daltons or greater. The poly I:C/poly-L-lysine as an effective inducer of interferon in grivet monkeys, rhesus monkeys, chimpanzees and marmosets. A high interferon titer (greater than 100 units/ml blood) was maintained in grivet monkeys by repeated daily administration of the complex. No long-term hyporesponsiveness was noted following repeat inductions over a period of months. The serum interferon produced in monkeys in response to poly I:C/poly-L-lysine resembled human leukocyte interferon in its biological characteristics.

A sensitive enzyme-linked immunosorbent assay (ELISA) for antibodies to double-stranded RNA has been developed using polyinosinic acid . polycytidylic acid (InCn) 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. In Cn complexed with poly-L-lysine and adsorbed to polystyrene tubes resulted in optimal uptake of antibodies to double-stranded RNA. In-Cn 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.

The etiology of immune polioencephalomyelitis (IPE) and the mechanisms of resistance to IPE induction were investigated in C58 mice. IPE was found to be induced by a lipid-solvent-sensitive, filterable replicating agent present in line Ib leukemic cell suspensions. IPE was serially transmitted in immunosuppressed mice with filtered extracts of spleens from diseased animals. The IPE-inducing activity of Ib cell extracts was abolished by chloroform or deoxycholate. Gel filtration of Ib cell extracts showed that the IPE agent has a molecular weight of at least 10(7). Electron microscopy of the active fractions from columns and of spinal cord extracts from mice with IPE revealed a virus-like particle, 40 nm in diameter, which is probably the IPE revealed a virus-like particle, 40 nm in diameter, which is probably the IPE agent. Administration of cyclophosphamide at various times after challenge increased the incidence of IPE in mice, suggesting that IPE is not autoimmune mediated. Immunosuppression resulted in maintenance of high levels of IPE agent in the central nervous system tissue, while immunization resulted in low levels. Moreover, immunized mice produced neutralizing antibodies. These data suggest that antibodies help restrict the amount of IPE agent in the nervous tissue, and that this restriction is required for resistance to IPE induction in C58 mice.

The relative contribution of thymus-derived lymphocytes (T-cells) and of macrophages to resistance to primary infection with herpes simplex virus type 1 (HSV-1) was studied in C58 mice. Resistance was dependent on macrophage competence, but was relatively independent of T-lymphocyte competence. Although aging mice became progressively more deficient in functional T-cells, as demonstrated by a decreasing resistance to transplanted line Ib leukemia and by declining responses to T-cell nitogens (concanavalin A and phytohemagglutinin), their resistance to HSV-1 increased with increasing age. Moreover, in mice that were made selectively deficient in T-cells by the combination of adult thymectomy and treatment with anti-thymocyte serum, resistance to HSV-1 did not correlate spleen and mesenteric lymph nodes. However, selective reduction of macrophages by intraperitoneal injection of silica resulted in enhanced susceptibility to HSV-1. Furthermore, in vitro suppression of HSV-1 plaque formation in mouse embryo fibroblast cells was obtained by cocultivation of infected fibroblast monolayers with peritoneal macrophages, but not with splenic lymphocytes, from adult mice. Macrophages from weanling mice failed to suppress the development of plaques, indicating that the increase in resistance to HSV-1 with age is a result of increased macrophage competence.

Thymopoietin, ubiquitin, and serum thymic factor, all of which induce T cell markers on lymphocytes, have been evaluated for their capacity to induce thymus-de-pendent activities in vivo. Multiple treatments over a period of weeks failed to restore either resistance to line lb leukemia or responses to T cell mitogens in T cell-deficient C58 mice. The findings suggest that these substances are ineffective in inducing thymus-dependent im-munocompetence that is meaningful in the intact animal.

A highly purified and inactivated vaccine was made of hepatitis B virus surface antigen. The vaccine was tested exhaustively for safety by ordinary procedures and additionally in chimpanzees and marmosets. It was highly potent and induced antibody in guinea pigs, grivet monkeys, and chimpanzees after three doses of vaccine were given subcutaneously. Chimpanzees given three doses of vaccine were protected against challenge with 1,000 chimpanzee-infectious doses of live human hepatitis B virus given intravenously in controlled studies. Tests of the vaccine for control of hepatitis B in man are to be carried out.

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 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 mug 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 antigenemia following challenge and all of six vaccinated animals were protected, including one animal that had failed to develop detectable antibody following vaccination.

The present report confirms the findings by Steinberg et al. (9) that repeated intraperitoneal injections of poly I:C (3 mug/g, three times per wk, 40-52 doses) enhanced the incidence and severity of glomerular lesions that occur spontaneously in NZG/NZW mice and also increased the development of circulating antibody against nucleic acids. This effect was minimal when only six intraperitoneal doses were given in 1 mug/g amount at weekly intervals. Intranasal administration of poly I:C (0.2 mug/g, three times per wk, 40 doses) or six doses of the drug (1 mug/g weekly) caused no apparent potentiation of glomerular response. ICR/Ha mice, which do not suffer from the spontaneously occurring disease, were uneffected by poly I:C treatment except for occasional development of antibody against poly I:C or DNA.

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.

Chemical vaccine containing di-N-acetylchitobiosyl poly(L-asparagine) prepared as described by Shier was assayed for its ability to prevent XS63 5 myeloma transplant tumors and 3-methylcholanthrene-induced tumors in BALB/c mice. The vaccine was ineffective.

SummaryPrimary rabbit kidney cells released measurable quantities of extracellular interferon within 2 hr after initiation of induction by poly I:C. This occurrence was accompanied by slow uptake of inducer in minute quantity. There was no preferential uptake of poly I:C by primary rabbit kidney cells compared to that of noninducing polynucleotides and nucleotides. Induced cells produced high concentrations of apparently unbound intracellular interferon which appeared to be the source of extracellular interferon. The dynamics of interferon production in primary rabbit kidney cell cultures induced by poly I:C involved two separate steps, viz., the synthesis of intracellular interferon and the release of this interferon into the extracellular medium.The authors are indebted to Mary-Ellen Davies and Kersti Young for valuable technical assistance.

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.

SummaryAppropriate 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.

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.