Plasmodium falciparum: passive immunization of Aotus lemurinus griseimembra with immune serum.

Owl monkeys (Aotus lemurinus griseimembra) were immunized against Plasmodium falciparum by infection and drug cure. After challenge, 3 of 4 monkeys developed extended prepatent periods and low grade parasitemias followed by self cure. The fourth monkey did not develop a patent infection. Immune monkey serum passively transferred at the time of challenge conferred immunity to 20 naive monkeys. Immunity was characterized by extended prepatent periods in 19 monkeys, low levels of parasitemia (< or = 1%) followed by self cure in 12 animals, and lack of detectable infection in 3 recipient monkeys. Immune serum collected from monkeys undergoing repeated challenges afforded more protection than serum from singly infected monkeys. However, single doses of hyperimmune serum appeared to be as effective as multiple doses. Normal serum had no effect on the course of infection in 12 monkeys. These studies confirm that owl monkeys can be immunized by infection and cure and demonstrate that this immunity can, in large part, be transferred to nonimmune recipients with serum from immune donors.

Malaria vaccine research.

In our report "Activation of Raf as a result of recruitment to the plasma membrane" (3 June, p. 1463) (1), panels E and F of figure 1 on page 1464 were incorrect. The correct photographs appear below. In addition, the [See figure in the PDF file] second sentence of the legend to figure 1 should have read, "The Raf constructs were tagged at the COOH-terminus with a Glu-Glu epitope (MEYMPME) (24) for c-Raf, or at the NH(2)-terminus with both the Glu-Glu and the Myc (MEQKLISEEDL) (23) epitopes for RafCAAX"; the next-to-the-last sentence of the legend to figure 1 should have read, "The c-Raf constructs in (A through D) are Glu-Glu-tagged and were detected by using an anti Glu-Glu antibody, and the RafCAAX and Raf6QCAAX constructs used in E and F were detected by using the antibody to Raf COOH-terminal peptide"; and the third sentence of note 26 should have read, "After blocking with 5% milk in phosphate-buffered saline (M-PBS), cells were incubated with a mouse monoclonal antibody to Glu-Glu or a rabbit polyclonal antibody to a 20-amino acid COOH-terminal peptide of Raf-1 (Santa Cruz Biotechnology, Santa Cruz, California), washed, and incubated with donkey antibodies to mouse or rabbit IgG combined with Texas Red (Jackson) in M-PBS, washed, and mounted in FITC-Guard (Testog)."

The major merozoite surface protein as a malaria vaccine target.

Experts gathered for two days in the summer of 1992 at the National Institutes of Health and the Walter Reed Army Institute of Research to discuss the potential of a major merozoite surface protein (MSP-1) in malaria vaccine development. The participants came in an exemplary spirit of co-operation, sharing ideas and unpublished data toward the common goal of a malaria vaccine. Their conclusions are presented here by Carter Diggs, Ripley Bollou and Lou Miller.

Characterization of gp195 processed products purified from Plasmodium falciparum culture supernates.

Schizonts of the malaria parasite Plasmodium falciparum synthesize a 195 kDa surface glycoprotein (gp195) that is processed into several smaller products including one of 83 kDa, which, in the case of the Camp strain, is sequentially processed into 73 and 67 kDa products. gp195 and its processing intermediates larger than 83 kDa were not precipitated from culture supernates, but the 83 and 73 kDa products were precipitated by three monoclonal antibodies (McAbs). The 83 and 73 kDa products were affinity purified from culture supernates by adsorbing to McAb 7B2 coupled to Affigel 10 and eluting either with 0.2 N acetic acid, pH 2.8, or with 3 M potassium isothiocyanate (KSCN). The epitope recognized by McAb 7B2 was denatured by acid elution but could be regenerated by treating with 8 M urea followed by dialysis. The implications of renaturing antigens to regenerate epitopes should be considered in studies on the purification, function and immunogenicity of malaria antigens.

Plasmodium falciparum: immunogenicity of circumsporozoite protein constructs produced in Escherichia coli.

The immunogenicity of Plasmodium falciparum recombinant circumsporozoite protein constructs R16tet32, R32tet32, and R48tet32 in mice was examined by measuring antibody responses by enzyme linked immunosorbent assay, immunofluorescence, circumsporozoite precipitation, and inhibition of sporozoite invasion. All three constructs were found to be immunogenic when administered alone, but antibody responses were greater for the larger constructs, R32tet32 and R48tet32. Increased dose, boosting, and the use of adjuvants further augmented antibody responses. R32tet32 was found to be the most immunogenic of the three constructs, and high levels of protective antibodies were found to persist for at least 44 weeks when the construct was given with alum. Clinical trials with alum adjuvanted R32tet32 have now begun.

Monoclonal antibody characterization of the 195-kilodalton major surface glycoprotein of Plasmodium falciparum malaria schizonts and merozoites: identification of additional processed products and a serotype-restricted repetitive epitope.

The gp195 from Camp strain parasites was characterized with eight monoclonal antibodies (MAb) that recognize different epitopes on gp195 and three of its merozoite-associated processed products. Four MAb (3H7, 3B10, 7F1, and 4G12) reacted with different epitopes on the 45-kDa glycosylated product (gp45), shown by differences in their reactivities with soluble and immunoblotted gp45. One MAb (7H10) reacted with a conformational epitope probably formed as a result of the interaction of gp45 with a nonglycosylated 45-kDa product (p45). Three other MAb (3D3, 7B11, and 7B2) reacted with different epitopes on a nonglycosylated 83-kDa product (p83), shown by differences in their reactivities against various parasite isolates in immunofluorescent antibody assays. Immunoprecipitation of antigens that were pulse-labeled with [3H]isoleucine and chased with cold isoleucine showed that p45 and gp45 were processed products of gp195 and p83 was sequentially processed into smaller fragments of 73 and 67 kDa (p73 and p67). Immunoblots showed that the 7B11 and 7B2 epitopes were present on p83, p73, and p67, but that the 3D3 epitope was present only on p83 and p73. A two-site immunoassay showed the 3D3 epitope to be repetitive. The 3D3 and 7B11 epitopes were serotype restricted (present in seven and 24 of 33 isolates, respectively), but the other five epitopes were common to all isolates tested. The gp195 and its processed products have Mr that are consistent with the Mr of a number of antigens shown previously to be associated with the immune complexes that are formed when merozoites are agglutinated by antibodies contained in some growth inhibitory immune sera.

Plasmodium falciparum antigens synthesized by schizonts and stabilized at the merozoite surface by antibodies when schizonts mature in the presence of growth inhibitory immune serum.

Some immune sera that inhibit erythrocyte invasion by merozoites also agglutinate the merozoites as they emerge from rupturing schizonts. These immune clusters of merozoites (ICM) possess a surface coat that is cross-linked by antibody and is thicker than the surface coat associated with normal merozoites (NM) obtained from cultures containing preimmune serum. Analysis of metabolically labeled ICM and NM performed by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that washed ICM possessed immune complexes containing antigens representative of schizonts and merozoites. Characteristics of the immune complexes included: a) they were not soluble in pH 8 Triton X-100, b) they were soluble at an acid pH, and c) after pH neutralization they were precipitated by using staphylococcal protein A. Merozoite antigens having Mr of 83, 73, and 45 kDa were associated with immune complexes in ICM. The 83 and 73 kDa antigens were recovered in considerably larger quantities from ICM than from NM. Schizont antigens having Mr of 230, 173 (triplet), 152 (doublet), and 31 kDa were associated with immune complexes in ICM, and a 195 kDa antigen(s) from schizonts and merozoites was also present in the immune complexes. In addition, other antigens of Mr 113, 101, 65, and 51 kDa may have been immune complexed. These 15 antigens accounted for less than 30% of the schizont and merozoite antigens recognized by the immune serum. Immune complexes probably formed between antibodies and a) surface antigens of schizont-infected erythrocytes exposed to antibody before schizont rupture, b) surface antigens of merozoites and schizonts exposed during schizont rupture, and c) soluble antigens normally released during schizont rupture. The antibody components of the immune complexes may have prevented rapid degradation or shedding of some antigens from the merozoite surface. Allowing schizonts to rupture in the presence of inhibitory antibodies (to form ICM) is a useful approach to identifying exposed targets of protective immunity against malaria.

Decreased growth of Plasmodium falciparum in red cells containing haemoglobin E, a role for oxidative stress, and a sero-epidemiological correlation.

The in vitro growth of Plasmodium falciparum in red cells containing haemoglobin E (HbE) was studied at oxygen concentration of 5 to 20%, with and without antioxidants. Under all conditions, parasite growth decreased as the concentration of HbE increased as compared with growth in red cells containing only HbA. The decreases were proportionately greatest at the highest oxygen concentration. The antioxidant vitamin C partially reversed the decreases in growth observed in HbE-containing cells at 20% oxygen. South-east Asian refugees with HbAE or HbEE had high antimalarial IFA titres, indicative of exposure to malaria more frequently than did refugees with HbAA. The decreased growth of P. falciparum in HbE-containing red cells may reduce the severity of malaria infections, conferring a survival advantage and thus increasing the numbers of individuals with HbE in local areas of South-east Asia with high incidences of malaria.

Expression of Plasmodium falciparum circumsporozoite proteins in Escherichia coli for potential use in a human malaria vaccine.

The circumsporozoite (CS) protein of the human malaria parasite Plasmodium falciparum may be the most promising target for the development of a malaria vaccine. In this study, proteins composed of 16, 32, or 48 tandem copies of a tetrapeptide repeating sequence found in the CS protein were efficiently expressed in the bacterium Escherichia coli. When injected into mice, these recombinant products resulted in the production of high titers of antibodies that reacted with the authentic CS protein on live sporozoites and blocked sporozoite invasion of human hepatoma cells in vitro. These CS protein derivatives are therefore candidates for a human malaria vaccine.

Serotypes of Plasmodium falciparum defined by immune serum inhibition of in vitro growth.

In vitro growth inhibition assays were used to detect antigenic differences among geographically distinct strains of Plasmodium falciparum. Owl monkeys were immunized against the Camp and FCR-3/FMG strains of P. falciparum by infection, drug treatment, and rechallenge with homologous parasites. Camp-immune monkey serum was used to inhibit the in vitro growth of eight strains of P. falciparum. Inhibition was maximum for the homologous Camp strain (an average of 62% inhibition by 100 ml/litre Camp-immune serum). Four other strains were inhibited to a lesser degree, and three strains (FCR-3/FMG, FVO, and Smith) were not significantly inhibited by Camp-immune serum at concentrations as high as 400 ml/litre. FCR-3/FMG-immune serum at a concentration of 50 ml/litre caused significant inhibition of the FCR-3/FMG strain, but not the Camp strain. Thus Camp and FCR-3/FMG strains appear to bear distinct antigenic determinants recognized by the homologous, but not the heterologous, antiserum. Inhibition of in vitro growth by immune serum may be useful for serotyping P. falciparum and may have application in the selection of strains for inclusion in a malaria vaccine.

Parasitologic and immunologic studies of experimental Plasmodium falciparum infection in nonsplenectomized chimpanzees (Pan troglodytes).

Parasitologic, hematologic, and immunologic parameters were monitored in intact (nonsplenectomized), adult chimpanzees infected with a "chimp-adapted" strain of Plasmodium falciparum. Following primary and secondary injections of 10(9) P. falciparum-infected erythrocytes, each chimpanzee developed a low grade parasitemia (up to 1,000/mm3) and maintained the infection without evidence of eliminating the parasites. Hematologic and serum biochemical values, as well as the majority of immunologic parameters tested, remained unaltered in infected chimpanzees. However, 2 weeks after infection T cells from infected chimpanzees demonstrated an enhanced response in vitro to stimulation with the mitogen PHA, and monocyte phagocytic activity for antibody-coated erythrocytes (Fc-mediated phagocytosis) increased significantly. During malarial infection, apes developed a strong T cell proliferative response to P. falciparum antigens and monocytes showed enhanced phagocytic activity for P. falciparum-infected erythrocytes in the absence of immune serum. These results suggest that cellular immune mechanisms, especially macrophage activation, may help control, but not eliminate, P. falciparum malaria in chimpanzees.

Restriction endonuclease analysis of Leishmania kinetoplast DNA characterizes parasites responsible for visceral and cutaneous disease.

The kinetoplast DNA (kDNA) from promastigotes of Leishmania responsible for Old and New World cutaneous and visceral disease was characterized to determine if species and strains causing similar or different diseases could be identified. Restriction enzymes were used to digest kDNA into fragments that were separated into characteristic banding patterns after electrophoresis in agarose or linear gradient polyacrylamide gels. Hybridization was conducted with a 32P-kDNA probe and kDNA fragments transferred from agarose gels to nitrocellulose paper. Leishmania species causing cutaneous diseases in the New and Old Worlds all had different kDNA digest patterns. Visceralizing Leishmania from the New and Old Worlds also had different kDNA restriction fragment patterns although Leishmania donovani parasites with similar fragment patterns were isolated from several humans from central Kenya. Nucleotide sequences were shared among kDNA networks from L. donovani, Leishmania d. chagasi, Leishmania d. infantum, Leishmania tropica, and Leishmania major as determined by hybridization with a 32P-kDNA probe from L. donovani. However, no hybridization was detected between the L. donovani 32P-kDNA probe and kDNA from Leishmania aethiopica or Leishmania braziliensis panamensis. Leishmania characterization results for the same isolates from the published literature were compared and kinetoplast DNA analysis was found to be one of the most sensitive procedures for species and strain identification.

Trypanosoma rhodesiense: analysis of the genetic control of resistance among mice.

Inbred mouse strains differ in their resistance to infection with the human pathogen Trypanosoma rhodesiense. Of the strains tested, C57BL/6 (B6) mice were the most resistant, and BALB/c (C) mice were among the most susceptible. The genetic basis underlying the different susceptibility of these two strains was analyzed. (CXB6)F1 progeny of either sex were more resistant than the BALB/c parent. Also, the backcross of F1 mice to the susceptible male or female BALB/c parent resulted in 52.0% susceptible (i.e., death on or before day 24) progeny, as compared with only 0.64% susceptible F1 progeny. The data suggested that resistance was the dominant phenotype and that the resistant allele was carried by the B6 parent. The presence of another locus regulating resistance to death was suggested by the facts that only a small percentage of F2 mice were susceptible and that a number of F1 and F2 mice were more resistant than their B6 parent. The locus responsible for these phenomena was presumably hypostatic in nature and carried by BALB/c mice, and its effects were only evident in the presence of other resistance genes. In addition, the observation that many of the susceptible individuals among F2 and backcross mice were more resistant than the BALB/c mice suggested that other minor genes also modulated the response of mice to infection. A set of CXB recombinant inbred mice was tested as well, and the individual strains within this set could also be placed into four groups: susceptible, intermediate, resistant, or hyperresistant. These findings are compatible with the multigenic model suggested by the Mendelian analyses.

Localization of the major Plasmodium falciparum glycoprotein on the surface of mature intraerythrocytic trophozoites and schizonts.

The subcellular location of the major malarial glycoprotein in erythrocytes infected with schizonts of Plasmodium falciparum has been studied by two methods. In the first, glycoproteins were labelled with [3H]glucosamine or [3H]isoleucine during in vitro culture. Trypsin treatment of intact infected erythrocytes caused no major qualitative or quantitative changes in [3H]glucosamine labelled glycoproteins or [3H]isoleucine labelled proteins separated by sodium dodecylsulfate polyacrylamide gel electrophoresis. However, in the presence of Triton X-100 the labelled glycoproteins and proteins were completely cleaved by trypsin. In the second method, two monoclonal antibodies which specifically immunoprecipitate the major 195 kDa glycoprotein failed to react on indirect immunofluorescence with intact non-fixed schizont-infected erythrocytes, but reacted strongly with saponin released schizonts indicating specificity for the surface of mature intracellular parasites. Immunoelectronmicroscopy using ferritin-conjugated secondary antibody confirmed the location of the epitope(s) recognized by these monoclonals on the surface of intracellular parasites. Ferritin particles were not associated with knob-bearing erythrocyte membranes. The results indicate that only a small proportion or none of the 195 kDa glycoprotein is on the surface of the infected erythrocyte and that the largest proportion is expressed on the surface of mature intraerythrocytic parasites.

Plasmodium falciparum strain-specific human antibody inhibits merozoite invasion of erythrocytes.

The extent to which human antibodies involved in functional immunity react with antigenic determinants varying between different isolates or strains of the human malaria parasite Plasmodium falciparum will influence the design of vaccines against malaria. We identified nine immune sera from Cambodian refugees which blocked in vitro invasion of erythrocytes by merozoites of the Camp strain of P. falciparum and agglutinated Camp strain merozoites. However, none of these sera blocked invasion of erythrocytes by merozoites of the FCR-3 strain. We conclude that antibodies in these human sera recognized antigenic determinants present on the surface of viable merozoites of the Camp strain but not the FCR-3 strain. These parasite strains and in vitro assays can be used to analyze strain-specific functional immunity in humans.