Immunogenicity of liposomal malaria sporozoite antigen in monkeys: adjuvant effects of aluminium hydroxide and non-pyrogenic liposomal lipid A.

The immunogenicity of a recombinant protein (R32tet32) containing sequences from the tetrapeptide repeat region of the circumsporozoite protein of Plasmodium falciparum was enhanced by encapsulation in liposomes containing lipid A and adsorption of the liposomes with alum. The toxicities and efficacies of preparations containing different types and doses of lipid A were assessed by studying pyrogenicity in rabbits and adjuvanticity in monkeys. In each case liposomal lipid A was 25-fold to 200-fold less pyrogenic than free lipid A. Monophosphoryl lipid A, whether free or in liposomes, was the least pyrogenic of the three lipid A preparations tested. High antibody levels were obtained after immunization of rhesus monkeys with a formulation consisting of alum-adsorbed liposomes in which the liposomes contained R32tet32 and a strongly pyrogenic dose of native lipid A. Excellent antibody levels were also observed in monkeys immunized with a combination of R32tet32 encapsulated in alum-adsorbed liposomes containing non-pyrogenic doses of monophosphoryl lipid A and alum. The adjuvant effect was related to the dose of the lipid A in the liposomes, and the adjuvant effect was still strongly expressed despite suppression of the pyrogenic effect of lipid A. Antibody levels were considerably lower in monkeys immunized with liposomes lacking lipid A. It was concluded that a non-pyrogenic formulation of alum-adsorbed liposomes, in which the liposomes contained both lipid A and an encapsulated synthetic sporozoite antigen, shows considerable promise for inducing high titres of antibodies to sporozoites.

Prostaglandin and thromboxane in liposomes: suppression of the primary immune response to liposomal antigens.

Liposomes containing lipid A as adjuvant and also containing prostaglandin E2 or thromboxane B2 were examined for the ability to influence induction of humoral immunity against liposomal protein or lipid antigens in rabbits. The protein antigen consisted of cholera toxin that was bound to ganglioside GM1 on the surface of the liposomes. High titers of anti-cholera toxin antibodies were produced and IgM and IgG responses were detected. When the immunizing liposomes contained either prostaglandin E2 or thromboxane B2 as part of the lipid bilayer, the primary immune response, involving both IgM and IgG antibodies, was greatly reduced. The secondary immune response observed after a boosting immunization was not suppressed by liposomal eicosanoids. A similar inhibitory effect on the primary response was observed when liposomal lipid antigens were examined instead of cholera toxin. An inhibitory effect of liposomal prostaglandin E2 on the phagocytic uptake of opsonized liposomes by cultured macrophages was also observed, suggesting that liposomal eicosanoids can have direct local effects on macrophages that might influence the immune response to liposomal antigens.

Liposomes, lipid A, and aluminum hydroxide enhance the immune response to a synthetic malaria sporozoite antigen.

A liposome-encapsulated cloned protein (R32tet32) containing sequences from the tetrapeptide repeat region of the circumsporozoite protein of Plasmodium falciparum sporozoites was examined for immunogenicity with rabbits and monkeys. Effects of adjuvants were tested by encapsulation of the antigen in liposomes either lacking or containing lipid A and adsorption with aluminum hydroxide (ALUM). When rabbits were immunized with R32tet32 alone, a primary antibody response was not seen and a secondary response did not appear until 32 to 36 weeks after boosting. Immunization with ALUM-adsorbed R32tet32 resulted in a minimal primary antibody response. A moderate secondary antibody response was detected within 2 weeks after boosting, but antibody levels decreased to preimmunization levels 8 weeks after boosting. When R32tet32 was encapsulated in liposomes containing lipid A, strong primary and secondary antibody responses were observed. Strong primary and secondary responses also were obtained when R32tet32 was encapsulated in liposomes either containing or lacking lipid A and the liposomes were adsorbed with ALUM. The strongest antibody response was obtained by immunization with ALUM-adsorbed liposomes containing lipid A and R32tet32, suggesting that the adjuvant effects of liposomes, lipid A, and ALUM were additive or synergistic.

Liposome-encapsulated hemoglobin: oxygen binding properties and respiratory function.

Lipid bilayer membranes have been used to encapsulate hemoglobin and organic phosphates, resulting in a synthetic red cell with oxygen-binding properties very similar to red blood cells (RBCs). In addition to the oxygen-binding parameters, we report here an improvement in the lipid formulation that confers upon the liposome-encapsulated hemoglobin (LEH) a circulation retention half-time of 15 to 20 hours in mice. Cooperative oxygen binding of the encapsulated hemoglobin is retained and the oxygen-binding affinity can be maintained at or below the level of fresh whole blood for many weeks by co-encapsulation of an appropriate concentration of pyridoxal-5-phosphate. A comparison of the kinetics of oxygen exchange across the membranes of the synthetic red cells and RBCs indicates that the increased surface to volume ratio of the much smaller synthetic red cells, coupled with their somewhat lower concentration of hemoglobin, results in more rapid rates of oxygen uptake and release than are seen with RBCs. Efficacy studies have begun and we report here the successful transfusion of rats with LEH. After greater than 90% exchange, LEH transfused rats were able to maintain activity with normal respiratory and cardiac function parameters.

Long-acting intramuscular anesthetic regimen for swine.

A two-stage, long-acting, injectable anesthetic regimen which provided pain-free restraint for swine was developed using nine mixed-breed domestic pigs. Each animal was administered a first-stage drug combination consisting of meperidine hydrochloride and azaperone in the caudal thigh muscles, followed after 20 minutes by a second-stage group of injections consisting of ketamine hydrochloride combined with morphine sulfate in the same muscles of the opposite leg. A mean surgical anesthetic time of 60.6 +/- 18.6 minutes was achieved with this regime and it was found that total anesthetic time could be doubled by a second injection of the ketamine and morphine components alone. All animals had a rapid, uneventful recovery. This combination regime not only provided reliable, long-acting anesthesia but was administered easily, required no tracheal intubation and produced no significant changes in the animals' heart rate or body temperature during the anesthetic period.

An enlarged aggregate of apocrine glands on the chest of karyotype I owl monkeys.

A firm circumscribed umbilicated mass in the middle of the chest, overlying the cephalad portion of the sternum, was an incidental finding in the skin of three males and one female from a group of eight (four male and four female) aged karyotype I owl monkeys (Aotus trivirgatus) released to our laboratory from the breeding colony of the Walter Reed Army Institute of Research (WRAIR). Light microscopic examination of each mass revealed an enlarged aggregate of apocrine glands which was interpreted to be the result of hyperplasia. Some degree of enlargement of the aggregate of glands was observed on all adult male karyotype I owl monkeys recently examined at WRAIR.