K. N. Brown

Protective immunity to malaria: studies with cloned lines of Plasmodium chabaudi and P. berghei in CBA/Ca mice. I. The effectiveness and inter-and intra-species specificity of immunity induced by infection

Summary CBA/Ca mice were immunized by infection with cloned lines of Plasmodium berghei (isolates ANKA, KSP‐11). Plasmodium chabaudi chabaudi (AS, CB) or Plasmodium chabaudi adami (DS) and then challenged with either homologous or heterologous parasites. Protective responses were assessed in immune mice relative to the controls by their ability to (i) extend the time taken for the mean parasitaemia to reach a predetermined level (1% or 0·1%) (ii) reduce peak parasitaemia (iii) resolve the parasitaemia sooner and/or (iv) control or eliminate recrudescences. At both the inter‐ and intra‐species level, immunity appeared largely specific for the cloned line inducing it. At the interspecies level marginally effective cross‐immunity was sometimes evident, thus P. berghei KSP‐11 immune mice displayed some immunity against P. c. chabaudi AS, although immunity to this parasite was relatively ineffective against P. berghei ANKA or KSP‐11. Cross‐immunity was more apparent between the subspecies P. c. adami and P. c. chabaudi and between cloned lines of the latter parasite derived from the AS and CB isolates. These data reflect considerable inter‐ and intra‐species structural and immunogenic differences in certain antigens of parasitized erythrocytes and merozoites, which have been identified in a number of murine malarias and associated with protective immunity. Similar differences recently identified in the equivalent antigens of the human parasite P. falciparum may therefore have important implications for protective immunity in man.

Protective immunity to malaria: studies with cloned lines of rodent malaria in CBA/Ca mice. IV. The specificity of mechanisms resulting in crisis and resolution of the primary acute phase parasitaemia of. Plasmodium chabaudi chabaudi and P. yoelii yoelii

Summary Low numbers of parasites from cloned lines of the rodent malaria parasites, Plasmodium chabaudi chabaudi AS and P. yoelii yoelii A, injected into CBA/Ca mice produce acute but usually self–limiting infections. During crisis, i.e. 1–2 days after peak parasitaemia, ‘pre–immune’ mice experiencing such ‘background’ infections were reinfected intravenously with homologous parasites or parasites of heterologous strains or species. P. c. chabaudi AS pre–immune mice controlled an AS challenge with essentially the same kinetics as the background infection. Reinfection of AS pre–immune mice with the heterologous (CB and IP–PCI) P. c. chabaudi strains or P. chabaudi adami DS had little effect on the initial growth of these parasites, although eventually the parasitaemia was controlled. In contrast, a partial inhibitory effect on the growth of P. vinckei lentum DS was evident. Challenge with the non–lethal (A) or lethal (YM) variants of P. y. yoelii resulted in an increase in both the growth and virulence of these parasites. P. y. yoelii A pre–immune mice controlled a homologous challenge, but were less effective at controlling the YM variant. In addition, they were unable to clear rapidly a P. c. chabaudi AS or P. v. lentum DS challenge. Both the multiplication and virulence of P. berghei ANKA were enhanced. These findings demonstrate that resolution of the primary acute parasitaemia in P. c. chabaudi AS– and P. y. yoelii A–infected mice is predominantly mediated by species– and strain–specific mechanisms.

Protective immunity to malaria. Studies with cloned lines of Plasmodium chabaudi chabaudi and P. berghei in CBA/Ca mice. II. The effectiveness and inter- or intra-species specificity of the passive transfer of immunity with serum.

Summary Serum was obtained from CBA/Ca mice infected, reinfected or superinfected with parasites taken one or two syringe passages from cryopreserved reference stabilates derived from cloned lines of the AS or CB isolates of P.c. chabaudi. Serum was also collected from mice superinfected with parasites derived from a cloned line of P. berghei KSP‐11. When injected into normal syngeneic recipients subsequently challenged with homologous or heterologous parasites, these sera mediated some or all of the following modifications to the breakthrough parasitaemias which invariably occurred (i) an extension of the pre‐patent period (ii) an extension of the time taken for the parasitaemia to reach 2% (iii) a reduction of peak parasitaemia (iv) protraction of the initial peak of parasitaemia. These modifications were particularly evident with serum from superinfected mice and to a lesser extent with serum from animals reinfected once after recovery from a primary infection. Serum taken during the course of such a primary infection produced extended pre‐2% periods, other effects being only marginal. Serum mediated modifications produced by reinfection and superinfection serum appeared largely species‐specific with a limited degree of cross‐reactivity. Intraspecific specificity was also apparent with serum from P.c. chabaudi AS or CB reinfected or superinfected mice, although marginal cross‐immunity was again observed. When analysed by the fluorescent antibody technique on smears of methanol fixed parasitized erythrocytes, reinfection and superinfection sera were almost totally cross‐reactive both within and across species. Preliminary evidence that parasites breaking through the effects of these sera may constitute a phenotypic antigenic variant is presented and possible mechanisms for the parasitaemia modifying effects of the various sera discussed.

The binding of antibodies from Plasmodium berghei‐infected rats to isoantigenic and parasite‐specific antigenic sites on the surfaces of infected reticulocytes

Summary Ferritin‐labelling techniques at the ultrastructural level have shown that antiserum from August rats immune to P. berghei infection contains antibodies which bind to the surfaces of parasitized reticulocytes but not to uninfected cells. Two antibody specificities have been demonstrated by comparing antisera i absorbed with infected reticulocytes, ii absorbed with uninfected reticulocytes, and iii unabsorbed. Ferritin labelling was much increased with antiserum preabsorbed with uninfected reticulocytes, and also with heat‐inactivated serum, indicating a blocking effect on parasite‐specific antibody binding by cold‐reacting anti‐erythrocyte isoantibodies known to be present. Energy‐dependent aggregation, shedding and endocytosis of labelled material was observed at the surfaces of unfixed infected reticulocytes.

Protective immunity to malaria: studies with cloned lines of Plasmodium chabaudi chabaudi in CBA/Ca mice. III. Protective and suppressive responses induced by immunization with purified antigens

Summary The protective effect of affinity purified antigen has been investigated in an experimental model for malaria which shows a well marked recrudescence of parasitaemia, a feature of the disease in man. A monoclonal antibody (MoAb) recognizing an epitope common to two genetically distinct cloned lines of Plasmodium chabaudi (AS and CB). was used to purify a Mr250 000 polymorphic schizont antigen (PSA) from these parasites. The purified preparations were then examined for the presence of specific and cross‐reactive epitopes by immunoprecipilation with a panel of MoAb raised against P. chabaudi AS. When tested previously on smears of parasitized blood by immunofluorescence, or against lysates of parasitized erythrocytes by immunoprecipitation, most of these MoAb had been found to be AS specific. When either AS or CB affinity purified Mr 250 000 PSA was used as the target, these same MoAb immunoprecipitated both antigens, and in some cases, a number of associated polypeptides (AP) which co‐purify with the Mr 250 000 PSA. Subsequently, mice were immunized with either the purified AS or CB antigens in Freunds complete adjuvant (FCA). Pre‐challenge sera were compared by indirect immunofluorescence and immunoprecipitation. Sera from mice immunized with AS antigen reacted strongly with AS and cross‐reacted with CB parasite preparations. Pre‐challenge serum from CB antigen immunized mice reacted well with CB, but only faintly with AS preparations. In mice immunized with the AS antigen and then challenged with either AS or CB parasites, the initial parasitaemias were delayed in appearance and the height of the peak parasitaemia reduced, an effect which was most pronounced after challenge with homologous parasites. Only homologous challenge of the mice immunized with CB antigen produced statistically significant modification of the initial parasitaemia. In the immunized mice challenged with homologous parasites, the delayed appearance and slightly reduced peak of the primary parasitaemia was associated with delayed resolution of the patent parasitaemia and significant enhancement of the recrudescence.

Variabilityin parasite protein antigen structure and protecitive immunity to malaria

Summary Cloned lines of the rodent malaria parasite Plasmodium chabaudi (denoted AS and CB) have been used to investigate the strain specificity of immunity to malaria. One defined difference between these lines is their expression of serologically and structurally distinct forms of an M, 250Kd parasite-encoded antigen. This antigen is a member of a family of schizont/ merozoite-associated polypeptides which have been implicated in the induction of protective immunity to rodent, simian and human malaria parasites. CBA/Ca mice were immunized by either (a) purified P. chabaudi AS-250Kd antigen (b) chronic AS infection or (c) irradiated nonreplicating AS-parasitized erythrocytes. Post-immunization sera were examined by immunoprecipitation of 35 S-methionine-labelled parasites, and the mice challenged with either AS or CB parasites. On challenge, mice developed a parasitaemia, the level of which was determined in part by isolate specificity, but only mice in groups (b) and (c) later developed a response which transcended AS/CB differences. The implications of these findings for the nature of exposed parasite antigens and the induction of protective immunity to malaria is discussed.

The adoptive transfer of T-cell dependent immunity to Plasmodium chabaudi chabaudi in CBA/Ca mice is achieved only after superinfection of immune spleen cell donors.

Summary The transfer of spleen cells from CBA/Ca mice recovered from a P. c. chabaudi AS primary infection into irradiated syngeneic recipients conferred very poor protection. Neither elimination of Ly2 cells from immune spleen cells nor reinfection of the donors some days before transfer improved protection significantly. Significant protection was transferred with spleen cells from donors which had been infected 7 times prior to cell transfer. Transferred protection was reduced or eliminated by pretreatment of cells with anti‐Thy‐1 or anti‐L3T4 monoclonal antibodies but not with anti‐Ly2.

A single gene copy merozoite surface antigen and immune evasion

During the course of chronic malaria infection antigenic variants of a parasite antigen are expressed and exposed on the surface of infected erythrocyte membranes. There also exists a number of apparently invariant single gene copy blood‐stage antigens, exposed or non‐exposed, which have been shown to afford immunity under experimental conditions. To determine why the host, presented with invariant ‘protective’ antigens, is unable to control infections effectively, immunity to a representative single gene copy antigen, the merozoite surface protein 1 (MSP1) was investigated in Plasmodium chabaudi chabaudi AS, a murine model of chronic malaria. Immunization with monoclonal antibody affinity purified native MSP1 resulted in enhanced control of parasitaemia on challenge, irrespective of the parasite inoculum size; challenge with a single parasite, however, suggested that expansion of resistant parasite subpopulations was not occurring. Challenge of mice immunized with recombinant fusion proteins encoding N‐ or C‐terminal regions of the P.c. chabaudi AS MSP1 produced inconsistent effects, often parasitaemias were indistingishable from controls despite significant anti‐MSP1 antibody responses. The not unlikely contamination of MSP1 native preparations with erythrocyte (E) components was considered. Immunization with a mixture of the MSP1 C‐terminus recombinant polypeptide and a Triton X‐100 solubilized lysate of normal E resulted in enhanced control of parasitaemia, however, no effect was seen after administration of either component on its own. Co‐immunization of E with the N‐terminus polypeptide reversed the inhibition seen, on this occasion with this construct alone.