Vaccination of experimental monkeys against Plasmodium falciparum: a possible safe adjuvant.

Owl monkeys (Aotus trivirgatus griseimembra) were effectively immunized against a human malaria parasite, Plasmodium falciparum. Two injections of antigen, primarily mature segmenters with fully developed merozoites, mixed with adjuvant (6-O-stearoyl-N-acetylmuramyl-L-alanyl-D-isoglutamine and liposomes) were administered intramuscularly at a 4-week interval. Approximately 2 weeks after the second vaccination, the monkeys were challenged with the homologous strain of P. falciparum. All immunized monkeys survived the challenge. The substitution of Freund's complete adjuvant is an encouraging step toward the development of an effective and safe vaccine for human malaria.

Partial protection of Plasmodium falciparum-vaccinated Aotus trivirgatus against a challenge of a heterologous strain.

Two Aotus trivirgatus griseimenbra monkeys which had been immunized with the merozoite-enriched FUP strain of Plasmodium falciparum were protected against a primary challenge with the homologous strain. The results described here show that these two monkeys were protected against a subsequent challenge with a heterologous strain (FVO) of P. falciparum. The unimmunized control monkey died of FVO infection by day 18.

Comparative studies on dihydrofolate reductases from Plasmodium falciparum and Aotus trivirgatus.

Dihydrofolate reductase (E.C. 1.5.1.3) from Plasmodium falciparum and from its host, the owl monkey (Aotus trivirgatus), were partially purified and characterized. The molecular weight of the parasite enzyme was estimated to be over 10 times as high as that of the host enzyme. The host enzyme had 2 pH optima whereas the parasite enzyme only one. The activity of the host enzyme was greatly stimulated by KCl and urea, while that of the parasite enzyme was inhibited at high concentrations of such chaotropic agents. Km of the parasite enzyme was significantly higher than that of the host enzyme. The parasite enzyme had much lower Ki for pyrimethamine than the host enzyme. Dihydrofolate reductases isolated from pyrimethamine-resistant and pyrimethamine sensitive strains of P. falciparum were found to be similar.

Plasmodium falciparum: comparative analysis of antigens from continuous in vitro cultured and in vivo derived malarial parasites.

A comparison of metabolically labeled proteins from continuous in vitro and in vivo derived Plasmodium falciparum revealed both similarities and differences. Metabolic labeling of synchronized cultures showed that the uptake of label increased as the parasites matured from the ring to the schizont stage in both cultures. Also, in both continuous in vitro and in vivo derived cultures, prominent high-molecular-weight proteins were synthesized during the late developmental stages. However, the continuous in vitro cultured parasites incorporated twice as much of the label at each stage as did the in vivo derived parasites. Immunoprecipitation with serum samples from vaccinated Aotus trivirgatus griseimembra monkeys revealed major differences involving protein antigens that migrated in the molecular weight regions of b (Mr = 152,000), c (Mr = 143,000), j (Mr = 82,700), and n (Mr = 57,400). These antigens were more readily detected in the continuous in vitro cultured schizonts than in the in vivo derived schizonts.

In vitro production and partial purification of Plasmodium falciparum antigen.

A simple technique for achieving high yields of Plasmodium falciparum parasites on a continuous basis is described. The technique is applicable in any laboratory. The culture apparatus is also simple and inexpensive and allows multiple cultures to be run simultaneously. A total of approximately 1-2 x 10(9) parasites can be harvested per culture flask per week requiring the use of only 40.0 ml of culture medium (RPMI 1640), 5.0 ml of human sera, and 2.0 ml of outdated human whole blood. P. falciparum parasites (segmenters containing individual merozoites) are cultured in vitro and concentrated 10-15 fold through the use of discontinuous bovine serum albumin gradient centrifugation.Commercial saponin is purified on a Sephadex G-25 column. The haemolytic effect of purified saponin related to human red blood cell concentration is studied. Preliminary observations on the action of some synthetic detergents and enzymes on human erythrocytes are also reported. Purified saponin is used to lyse red blood cells infected with in vitro cultured P. falciparum for the preparation of merozoite antigen. Further purification of parasite material is carried out by sucrose density gradient centrifugation.

Plasmodium falciparum: protein antigens identified by analysis of serum samples from vaccinated Aotus monkeys.

Serum samples from Aotus trivirgatus subsp. griseimembra monkeys obtained at different stages of a vaccination experiment were analyzed for total antibody titer to Plasmodium falciparum and were used for identifying protective antigens of the human malaria parasite. Total malarial antibody titers were higher in serum samples from protected monkeys (vaccinated with antigen in an adjuvant) than in those from unprotected monkeys (vaccinated with either antigen or adjuvant only). Parasite proteins were labeled with [3H]isoleucine, solubilized with nonionic detergent, and reacted with immune Aotus sera. Immunoprecipitates obtained were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Thirteen protein antigen bands in the molecular weight range 73,000 to 180,000 were resolved. Serum samples obtained from protected Aotus monkeys reacted more intensely with these proteins than samples from unprotected monkeys did. Evidence is presented that the protective antigen is not a single, normally nonimmunogenic, protein that is recognized only in protected monkeys. Rather, the present data indicate that a heightened immune response to multiple proteins correlated with in vivo protection to P. falciparum in Aotus monkeys. This finding may have a significant bearing on strategies for the development of a human P. falciparum vaccine.

Immunization of experimental monkeys against Plasmodium falciparum: use of synthetic adjuvants.

The replacement of Freund's adjuvant by a possible safe adjuvant for effective immunization of owl monkeys (Aotus trivirgatus griseimembra) against a human malaria parasite, Plasmodium falciparum, has been investigated. Experiments involved the use of two synthetic adjuvants: MDP (N-acetylmuramyl-L-alanyl-D-isoglutamine) and stearoyl-MDP (6-O-stearoyl-N-acetylmuramyl-L-alanyl-D-isoglutamine). In both cases, P. falciparum merozoites obtained through short-term in vitro cultivation were used as antigen. MPD was used as adjuvant in 5 owl monkeys; 2 control monkeys died and of the 3 experimental monkeys only 1 survived. In contrast, in another experiment where stearoyl-MDP was used as adjuvant, there was 100% protection of 4 immunized monkeys against a challenge with the homologous strain of P. falciparum. The results of the second experiment are encouraging for the development of an effective and safe vaccine for human malaria.

Induction of protective immunity to monoclonal-antibody-defined Plasmodium falciparum antigens requires strong adjuvant in Aotus monkeys.

Monoclonal antibodies to the major Plasmodium falciparum merozoite surface coat and rhoptry antigens were produced. A combination of the affinity-purified polypeptides with Freund complete adjuvant which was given three times completely protected an Aotus lemurinus azure (karotype VI) monkey against homologous challenge; however, immunization with the same polypeptides with a muramyl dipeptide derivative [MDP-Lys(L18)] did not protect a second Aotus monkey, even though comparable high antibody titers were induced.

Merozoite surface coat precursor protein completely protects Aotus monkeys against Plasmodium falciparum malaria.

Groups of Aotus (owl) monkeys were immunized with either the Plasmodium falciparum merozoite surface-coat precursor protein and its processing fragments or a complex of high molecular mass rhoptry proteins and challenged with a lethal infection of the homologous P. falciparum Uganda Palo Alto (FUP) strain. No patent parasitemia could be detected on thick blood films of monkeys immunized with the merozoite surface antigens; however, only one of three monkeys immunized with the rhoptry proteins was partially protected, while two required drug therapy. The experiment clearly demonstrates that the merozoite surface-coat precursor protein can completely protect Aotus monkeys against a lethal infection of the human malaria parasite.