Double-stranded polynucleotides as interferon inducers.

A discussion of factors considered influential in making a polynucleotide an efficient inducer of interferon was presented. These factors were double-strandedness of the polynucleotides, the sugar moiety of the polynucleotides, thermal stability, resistance to enzymatic degradation, and molecular size of the polynucloetides. Recent developments concerning interferon induction during virus infection were also discussed.

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.

Safety assessment of poly I:C in NZB/NZW mice (38565).

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.

Resistance of C58 mice to primary systemic herpes simplex virus infection: macrophage dependence and T-cell independence.

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.

Viral etiology of age-dependent polioencephalomyelitis in C58 mice.

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.

Poly I:C/poly-L-lysine: potent inducer of interferons in primates.

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.

Vaccine against human hepatitis B.

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.

Development and chimpanzee testing of a vaccine against human hepatitis B.

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.

Replacement of animal serum proteins by human albumin for growth and interferon production by Namalva cells.

Growth and interferon production by human lymphoblastoid cells (Namalva) have been achieved by using medium with a complete substitution of human albumin for fetal bovine serum. Medium containing 0.3% human albumin supported exponential cell growth (minimum doubling time, 20 h) in Bilthoven 10-liter fermentors. Interferon induction with Sendai virus resulted in interferon yields of 0.5 x 10(4) to 1.0 x 10(4) interferon units per 10(6) Namalva cells per ml in RPMI 1640 medium containing 0.15 to 0.3% human albumin. Both cell growth and interferon production in medium containing human albumin were comparable to growth and interferon production in the same medium containing 10 and 5% fetal bovine serum, respectively.