Carter L. Diggs

Plasmodium falciparum: assessment of in vitro growth by [3H]hypoxanthine incorporation.

To evaluate rapidly Plasmodium falciparum growth in Vitro, [3H]hypoxanthine was added to parasite microcultures and radioisotope incorporation was measured. When culture parameters were carefully controlled, [3H]hypoxanthine incorporation was proportional to the number of parasitized erythrocytes present. Factors affecting [3H]hypoxanthine incorporation included initial parasitemia, duration of culture, duration of radioisotope pulse, parasite stage, concentration of uninfected erythrocytes, the use of serum or plasma to supplement growth, and the concentration of a variety of purines in the culture medium. The method described can be used to measure inhibition of P. falciparum growth by immune serum and has previously been used to study antimalarial drug activity in vitro.

The cellular and humoral immune response to Schistosoma mansoni infections in inbred rats. I. Mechanisms during initial exposure.

Fisher rats were infected with Schistosoma mansoni by skin exposure to penetrating cercariae. The subsequent development of immunity was ascertained through the rechallenge of animals with cercariae and the simultaneous quantitative assessment of the development of the worm burdens which resulted from each of the two infections. The basic nature of the immune response to rechallenge by S. mansoni was investigated through the adoptive transfer of cells or serum obtained from normal or preexposed animals. The results indicate that the rat develops a strong, immunologically mediated, augmented resistance to infection by S. mansoni which begins within 1 week of initial exposure. This response is reflected in both quantitative and qualitative changes in the development of the reinfecting population and has a general characteristic of an anamnestic or secondary response judged by a variety of immunologic criteria. The developing immunity apparently is both stimulated by, and directed exclusively against, the very early stages of infection. Although the mechanism of this immunity is not clear, it apparently involved the production of significant quantities of protective immunoglobulin.

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.

Serological cross reactivity between Plasmodium vivax and Plasmodium falciparum as determined by a modified fluorescent antibody test

Abstract The cross reactivity between P. vivax and P. falciparum was studied by an indirect fluorescent antibody (FA) technique in which homologous and heterologous antigen-antibody systems were used. Two modifications of the previously described test were employed: (1) the use of liquid nitrogen for the preservation of parasitized whole blood for use as the source of antigen, and (2) the use of Evans blue as a counterstain to decrease the intensity of nonspecific fluorescence. Six of 29 sera from natural P. vivax infections reactive with P. vivax antigen were also reactive with P. falciparum antigen. Eleven of 21 serologically positive sera from natural P. falciparum infections were reactive with P. vivax antigen, two of these with the P. vivax antigen only. Two groups of sera from human volunteers with either P. vivax or P. falciparum infections were titrated in parallel tests with homologous and heterologous antigen. In the first series the geometrical mean reciprocal titers (GMRTs) with P. falciparum, sera were 28.3 for the homologous antigen and 6.3 for the heterologous antigen. For the P. vivax sera the values were 17.2 and 9.3 for the homologous and heterologous cases, respectively. In the second series the values for the P. falciparum sera were 132 with the homologous antigen and 20.0 with the heterologous antigen; the P. vivax sera gave values of 30.0 and 11.9 with the homologous and heterologous antigens, respectively. A comparison of titers of blood collected on filter paper and matched serum samples from subjects with P. vivax or P. falciparum infections revealed GMRTs of 24.2 for the blood and 41.0 for the serum. Specificity tests of 246 sera from subjects presumptively free of malaria revealed 28 positive reactions. It is concluded that there is both a species-specific and a group-specific component in the reaction of these two organisms. The specificity of the reaction and the usefulness of the filter paper method of blood collection have been confirmed.

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.

Trypanosoma congolense: Natural and acquired resistance in the bovine

Abstract A total of 42 animals of various ages were infected with Trypanosoma congolense to investigate age resistance. Ten of eleven animals between 4 months and 1 year of age survived the infection without treatment. Two of eleven animals in the age range of 1 to 2 years also survived the infection whereas all 20 animals between 2 and 5 years of age died or needed treatment to survive. Young animals which needed no treatment to survive were refractive to challenge for at least 1 year after their last patent parasitemia. Older animals which required treatment to survive were also challenged at intervals after therapy. Three animals infected for 49 to 75 days before treatment were rechallenged 198 to 296 days later. Extensions in prepatent periods ranged from 5 to 13 days when compared to controls and the resulting infections were of a relapsing nature followed by self-cure. Effects of this disease on clinical parameters of previously infected animals were minimal. One animal infected for 196 days and rechallenged 501 days later had a prepatent period of 14 days as compared to 5 days for controls. This animal developed a brief relapsing infection followed by self-cure. Animals which were infected for periods of 41 to 77 days, received treatment, and were then rechallenged from 600 to 900 days later, showed some resistance to infection. Prepatent periods were extended from 1 to 3 days over those of control animals and although the resulting disease was severe, one of four animals self-cured without treatment. When animals which had self-cured primary challenges were rechallenged at periods up to 2 years later, they were completely refractory. When 12 animals which were presumed to be immune to syringe-passaged T. congolense were challenged by tsetse fly bite with the same strain of trypanosome, an appreciable immunity was evident. Five of twelve immune animals did not become patent while the other seven developed mild infections without severe clinical signs. All nine controls developed severe infections with eight requiring treatment to survive. When animals immune to the Trans-Mara I strain of T. congolense were challenged either by syringe or tsetse fly bite with a heterologous strain of T. congolense obtained from a different geographical area, no evidence of immunity was detected.

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.

Trypanosoma rhodesiense: variant specificity of immunity induced by irradiated parasites.

Abstract Rats immunized with irradiated Trypanosoma rhodesiense resisted infection with the homologous strain. When similarly immunized rats were challenged with parasites obtained from rhesus monkeys infected with the same strain, resistance depended on when parasites were obtained from the donor monkeys. Immunized rats challenged with trypanosomes obtained from a monkey during the first peak of parasitemia were solidly immune; immunized rats challenged with trypanosomes obtained from monkeys after their initial peak of parasitemia all succumbed to the challenging infection. These observations indicate that parasites of a variant antigenic specificity arose during the course of the monkey infections. Neutralization tests performed on the various isolates from rats and monkeys using antiserum obtained from immunized rats confirmed that the immunity produced by irradiated trypanosomes was variant specific.

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 berghei: Biological variation in immune serum-treated mice

Abstract Antiserum was obtained from mice which had been immunized with irradiated Plasmodium berghei parasitized erythrocytes and which survived subsequent challenge. This antiserum suppressed P. berghei infections in mice; parasitemia and mortality were delayed 7–8 days as compared to those of control animals. Parasites surviving in antiserum-treated animals were isolated by inoculation of blood into normal recipients. When antiserum was tested against this derived parasite population, there was no observable effect on parasitemia or mortality. The derived parasites also exhibited a decreased virulence for mice. This work confirms the previous observation that antiserum treatment can result in a biologically variant population of P. berghei .