Antibody responses to surface antigens of Plasmodium falciparum gametocyte-infected erythrocytes and their relation to gametocytaemia.

An essential element for continuing transmission of Plasmodium falciparum is the availability of mature gametocytes in human peripheral circulation for uptake by mosquitoes. Natural immune responses to circulating gametocytes may play a role in reducing transmission from humans to mosquitoes. Here, antibody recognition of the surface of mature intra-erythrocytic gametocytes produced either by a laboratory-adapted parasite, 3D7, or by a recent clinical isolate of Kenyan origin (HL1204), was evaluated longitudinally in a cohort of Ghanaian school children by flow cytometry. This showed that a proportion of children exhibited antibody responses that recognized gametocyte surface antigens on one or both parasite lines. A subset of the children maintained detectable anti-gametocyte surface antigen (GSA) antibody levels during the 5 week study period. There was indicative evidence that children with anti-GSA antibodies present at enrolment were less likely to have patent gametocytaemia at subsequent visits (odds ratio = 0·29, 95% CI 0·06-1·05; P = 0·034). Our data support the existence of antigens on the surface of gametocyte-infected erythrocytes, but further studies are needed to confirm whether antibodies against them reduce gametocyte carriage. The identification of GSA would allow their evaluation as potential anti-gametocyte vaccine candidates and/or biomarkers for gametocyte carriage.

Inhibition of intraerythrocytic development of Plasmodium falciparum by proteinase inhibitors.

A group of inactivators of cysteinyl proteinases which function by covalent bond formation have been examined for their ability to inhibit the development of Plasmodium falciparum within red blood cells. The most effective of these caused inactivation of the parasite near 10(-8) M concentration. The range of inhibitory action varied with peptide structure in a manner characteristic of affinity labels for proteinases suggesting that the target of inhibition was an unidentified proteinase, probably of the cysteinyl type, but different from cathepsins B and L.

Sexual-stage-specific RNA expression of a new Plasmodium falciparum gene detected by in situ hybridisation.

A gene, Pf77, transcribed in the sexual stages of Plasmodium falciparum was isolated from a genomic expression library with a polyclonal antibody raised to gametocyte proteins. The entire coding region was obtained from a series of overlapping genomic and cDNA clones (from gametocyte RNA). A single open reading frame is present with no introns and no tandem repeat sequences. A Pf77 probe hybridised to a single transcript present in RNA prepared from purified gametocytes and could not be detected in RNA prepared from asexual blood stages. In situ hybridisation studies confirmed that the expression of Pf77 mRNA is sexual-stage-specific and in addition, showed that Pf77 mRNA is present only in female gametocytes during the vertebrate stages of the parasite's development.

Plasmodium falciparum sexual stage antigens: immunogenicity and cell-mediated responses.

Antibody and cell-mediated immune responses to the transmission-blocking target antigens of Plasmodium falciparum, Pfs 48/45, were determined in infected non-immune patients and in immune individuals from an endemic area. Characterization of the B cell epitopes with monoclonal antibodies showed that there were five regions identifiable but there could be interactions between them causing either competitive or enhancing effects. Sera from infected non-immune patients contained antibodies that would compete with one or more of the mAbs to the different epitopes. Immune responsiveness to purified Pfs 48/45 in P. falciparum-immune adults measured as lymphoproliferation, production of interferon-gamma, or as Pfs 48/45-specific antibody was very limited. This did not appear to be due to MHC class II restriction, to diversity in structure of the parasite antigens or to a failure of immunological memory. The antibody-response data were more consistent with down-regulation of immunity as a result of prolonged exposure to infection.

Primary structure and sexual stage-specific expression of a LAMMER protein kinase of Plasmodium falciparum.

We have isolated a LAMMER-like gene from Plasmodium falciparum by vectorette technique. The gene consists of 3316 bp encoding a protein 881 amino acids with a predicted molecular mass of approximately 106.7 kDa. The encoded protein, termed PfLAMMER, is composed of two distinct domains. The N-terminal domain is not related to any previously described protein kinases and has several interesting features including multiple consensus phosphorylation sites for a range of protein kinases, a number of RS/SR dipeptides, a large proportion of charged amino acids, two putative nuclear localisation signals and 14 copies of a tetramer DKYD repeats. The C-terminal domain is characteristic of a kinase in the LAMMER family with the highest homology to the Arabidopsis thaliana AFC3 kinase. Genomic restriction analysis showed that PfLAMMER is encoded by a single copy gene in the parasite genome. A single transcript of approximately 3800 nucleotides is expressed specifically in the sexual stage, indicating that PfLAMMER may be important in regulating the processes of sexual differentiation of the parasite.

Observations on early and late post-sporozoite tissue stages in primate malaria. I. Discovery of a new latent form of Plasmodium cynomolgi (the hypnozoite), and failure to detect hepatic forms within the first 24 hours after infection.

Previous work in this laboratory has demonstrated the ability of the immunofluorescence technique to detect pre-erythrocytic stages of the primate malaria parasite, Plasmodium cynomolgi bastianellii, in hepatic tissue obtained as early as 48 hours after sporozoite inoculation. In an attempt to visualize still earlier post-sporozoite stages, hepatic tissue obtained from a rhesus monkey infected with 12,000,000 sporozoites was examined at 2, 12, 24, and 48 hours after inoculation, employing antisera reactive with both invertebrate and vertebrate stages of the parasite. Tissue was also obtained at 7, 50, 102, and 105 days after sporozoite inoculation, and was examined for adequacy of the hepatic infection and for the presence of late exoerythrocytic schizonts. Although a new, previously unrecognized, uninucleate latent stage of 5 micrometer diameter (the "hypnozoite") was detected among large maturing schizonts in the 7-day and later biopsies, no intrahepatic parasites were found in tissue taken at 24 hours or earlier, despite the presence of up to 61 7-day schizonts and eight hypnozoites per 5 X 8 mm section. Pre-erythrocytic forms again were detected at 48 hours, although in far smaller numbers than expected on the basis of the density of parasites at 7 days after infection. The significance of these observations is discussed in the context of previous negative findings.

Conserved and variant epitopes of target antigens of transmission-blocking antibodies among isolates of Plasmodium falciparum from Malaysia.

Monoclonal antibodies (MAbs) directed against different epitope regions on three sexual stage-specific gamete surface proteins of Plasmodium falciparum, Pfs 25, Pfs 230, and Pfs 48/45, were used to study the genetic diversity of these epitopes among fresh isolates of P. falciparum from Malaysia, using immunofluorescence microscopy (IFA). Among 45 Malaysian isolates, one epitope of Pfs 25, designated region I, showed evidence of variable reactivity with MAbs among different isolates; the Pfs 25 epitope, region II, was universally recognized by MAbs in all isolates. Two apparently distinct epitope regions of Pfs 230 were defined by MAbs, one of which was universally recognized by MAbs among the 45 isolates; the other was conserved in all but three isolates. The epitope regions of gamete-surface protein Pfs 48/45, designated regions I, IIa, IIb, IIc, III, and IV, were examined for reactivity by IFA in 33 isolates. Epitope regions I, IIb, III, and IV were conserved in all isolates; regions IIa and IIc existed in variant forms.

Observations on early and late post-sporozoite tissue stages in primate malaria. II. The hypnozoite of Plasmodium cynomolgi bastianellii from 3 to 105 days after infection, and detection of 36- to 40-hour pre-erythrocytic forms.

Confirmation of the existence of a persistent, uninucleate, dormant pre-erythrocytic stage, the hypnozoite, of the relapsing simian malaria parasite, Plasmodium cynomolgi bastianellii, has been obtained by means of experiments involving the intravenous injection into susceptible monkeys of 48 to 85 x 10(6) sporozoites derived from mosquitoes of a different species and source than employed previously. The development of these hypnozoites was traced from 3 days until 105 days after sporozoite inoculation, employing a sensitive immunofluorescence technique followed by restaining with Giemsa. From an average mean diameter of 4 micrometers at 3 and 5 days, uninucleate hypnozoites grow to 5 micrometers at 7 days, then persist with little change until at least 105 days after infection. Strong evidence for the viability of these persistent forms was obtained by treatment of a host monkey with primaquine, which eliminated all trace of hypnozoites present 2 weeks before. Examination of hepatic tissue from a monkey injected with sporozoites 36 and 40 hours earlier revealed rare uninucleate pre-erythrocytic forms of 2.5-micrometers diameter. These early forms were present in hepatocytes in a density only approximately 1/30th of that expected on the basis of numbers of pre-erythrocytic stages found in the same animal's liver 7 days after infection. Nevertheless, subinoculation experiments appeared to rule out the circulation as a vehicle for dissemination of any putative early intermediate hepatotropic forms from another site.

Reduced efficacy of chemotherapy of Plasmodium chabaudi in T cell-deprived mice.

Immunologically intact and T cell-deprived CBA mice were infected with Plasmodium chabaudi and treated with chloroquine, pyrimethamine or quinine. Chloroquine and pyrimethamine rapidly reduced the levels of parasitaemia in both types of host, but whereas the normal mice remained free of blood parasites thereafter, the deprived mice suffered recrudescences of the infection. Quinine was therapeutically more effective in the intact mice than in the deprived mice early after initiation of treatment and, while the normal mice suffered a transient recrudescence after quinine, the deprived mice retained a high parasitaemia during and after treatment. The results indicate that the immune response may contribute to effective chemotherapy of malaria.

Increased severity of malaria infection in rats fed supplementary amino acids.

Infection with Plasmodium berghei malaria is severely inhibited in rats fed on a low protein diet. A range of amino acid supplements was added to a 4.2% casein diet to determine whether the relationship between level of infection and protein content could be attributed to the dietary amounts of the essential amino acids. Significant increases in levels of infection were achieved by supplementation with specific combinations of amino acids. Threonine was most effective in increasing the degree of parasitaemia but its effect was further enhanced when it was combined with dietary excess of certain other amino acids, notably valine, isoleucine and methionine.

Resistance to superinfection with Plasmodium berghei in rats fed a protein-free diet.

The development of resistance to reinfection with Plasmodium berghei was studied in rats in which the primary infection had been almost totally suppressed by feeding a protein-free diet (peak parasitaemia 0.5%; patent for only the first four days after inoculation) On Days 5, 9, 15, 23 and 28 after primary inoculation groups of animals were challenged with the same strain of parasite. At the same time the diet was changed to that of a 17% casein formula. The development of resistance as judged by the level of parasitaemia following challenge reached a significant level nine days after the primary inoculation and almost complete protection by Day 23 of the study. The protective activity was immunological since it could be transferred to other animals by a single intravenous injection of a suspension of spleen cells from infected donors. The study illustrates that infected animals experiencing severe protein malnutrition are still capable of mounting a substantial immune response to malaria.

Estimates of the infectious reservoir of Plasmodium falciparum malaria in The Gambia and in Tanzania.

Separate studies carried out in Farafenni, The Gambia and Ifakara, Tanzania in 1990-94 provided comparative data on population age structure, population gametocyte prevalences and gametocyte carrier infectivity. The percentage of the population estimated to be infective to mosquitoes was 5.5% and 3.8% in The Gambia and Tanzania, respectively. The age groups 1-4 years, 5-9 years, 10-19 years and 20 years or more comprised 17.5%, 21.7%, 22.2% and 37.9%, respectively, of the infectious population in The Gambia; the corresponding figures for Tanzania were 30.9%, 25.2%, 15.7% and 28.1%. These figures are in broad agreement with those from other published studies which estimated the infectious reservoir directly and suggest that adults contribute significantly to the infectious reservoir of malaria, particularly in areas of intense seasonal transmission. Control measures aimed at reduction of transmission may have only a limited effect in areas of moderate seasonal transmission if directed only at children.

Observations on early and late post-sporozoite tissue stages in primate malaria. III. Further attempts to find early forms and to correlate hypnozoites with growing exo-erythrocytic schizonts and parasitaemic relapses in Plasmodium cynomolgi bastianellii infections.

Rhesus monkeys were heavily infected with sporozoites of Plasmodium cynomolgi bastianellii in an attempt to demonstrate the site of invasion of sporozoites into tissue cells and their growth there. Further attempts were made to correlate the appearance and loss of hypnozoites with parasitaemic relapses. Hypnozoites were demonstrated and once again shown to decrease in numbers over 229 days during which time the infection showed parasitaemic relapses. Liver biopsies taken at two-day intervals for 12 days showed that hypnozoites decreased in numbers over-all and growing schizonts were demonstrated in the liver. At this time a parasite the size of a hypnozoite was seen with two nuclei and another was seen with an elongate, possibly dividing nucleus in one monkey. an attempt to find the location of the early intracellular exoerythrocytic forms in the liver at various times less than 40 hours after infection using smears and immunological staining with newly prepared anti-sera failed. Large numbers of sporozoites of P. knowlesi were also injected into a rhesus monkey the liver of which on the fifth day after infection showed no hypnozoites among 157 sections of growing schizonts and no parasites at all on the 42nd day after infection. In P. cynomolgi bastianellii infections parasites, mostly hypnozoites, were found in the liver up to 229 days after infection.

Malaria vaccines and the new malaria agenda.

The development of an effective malaria vaccine has taken many decades, but there is now a good chance that the first malaria vaccine will be licensed within the next few years. However, this vaccine (RTS,S) will not be fully effective, and more efficacious, second-generation vaccines will be needed. Good progress is being made in the development of potential vaccines directed at each of the three main stages of the parasite's life cycle, with a variety of different approaches, but many challenges remain, e.g. overcoming the problem of polymorphism in many key parasite antigens. It is likely vaccines that are effective enough to block transmission, and thus contribute to increasing drives towards malaria elimination, will need to contain antigens from different stages of the parasite's life cycle.