Antigenic variation of cloned Plasmodium fragile in its natural host Macaca sinica. Sequential appearance of successive variant antigenic types.

The course of infection of Plasmodium fragile in its natural host, the toque monkey Macaca sinica, consists of a primary peak of parasitemia followed by several distinct, successive peaks of lower parasitemia. In the S+ host, the late intraerythrocytic asexual developmental stages of P. fragile induce the expression of antigens on the surface of infected erythrocytes, which could be detected using the technique of surface immunofluorescence. Immunofluorescence using unfixed erythrocytes in suspension has shown that antigens are recognized by immune serum on the surface of the erythrocytes infected with more mature stages of the parasite. These antigens undergo variation, each successive peak of parasitemia being characterized by a different variant antigenic type (VAT). The appearance of the successive VATs occurs in a sequential manner, following the same order in different sets of animals. This constitutes the first example of a sequential expression of antigens in a malaria parasite; it indicates that, in P. fragile, antigenic variation is not the result of random mutations selected by antibody. Parasite-induced antigens on the surface of infected erythrocytes could not be detected in the S- host. However, when nonexpressing parasites from the S- host were transferred by blood passage into a naive S+ animal, they began to express antigens on the surface of infected erythrocytes within two erythrocytic cycles. We have demonstrated that the ability of S- parasites to switch to a particular VAT when passaged into a S+ animal changes during the course of an infection in the S- animal, indicating that, although surface antigens are not expressed, the processes leading to antigenic variation occurs even in the S- host. Antibodies directed against these surface antigens inhibit the growth of intra-erythrocytic parasites. The growth inhibition effects of antibodies are also variant specific, indicating that these variant surface antigens are functionally important for parasite survival.

Transient increase in circulating gamma/delta T cells during Plasmodium vivax malarial paroxysms.

The percentage of peripheral blood mononuclear cells (PBMC) bearing the CD3+ phenotype and the alpha/beta and gamma/delta T cell receptors (TCR) in PBMC were examined in Plasmodium vivax malaria patients and convalescents. The cells were labeled with monoclonal antibodies, stained with either fluorescence or phycoerythrin, and examined by ultraviolet (UV) microscopy. A highly significant increase in both the proportion and the absolute numbers of gamma/delta T cells (p < 0.005 and < 0.001, respectively, Student's t test) was observed in nonimmune P. vivax patients during clinical paroxysms compared to nonmalarial controls. These T cells, which normally constitute not more than 3-5% of PBMC, constituted < or = to 30% of PBMC during paroxysms in these nonimmune patients in whom the clinical symptoms were severe. A less significant increase of gamma/delta T cells were also observed in these nonimmune patients during infection, between paroxysms and during convalescence. In contrast, in an age-matched group of semi-immune patients resident in a malaria-endemic region of the country, in whom the clinical disease was comparatively mild, there was no increase in gamma/delta T cells either during infection, even during paroxysms, or convalescence. The severity of disease symptoms in patients as measured by a clinical score correlated positively with the proportion of gamma/delta T cells in peripheral blood (r = 0.53, p < 0.01), the most significant correlation being found between the prevalence and severity of gastrointestinal symptoms, nausea, anorexia, and vomiting, and the proportion of gamma/delta T cells (r = 0.49, p = 0.002). These findings suggest that gamma/delta T cells have a role to play in the pathogenesis of malaria, possibly in the general constitutional disturbances and particularly in gastrointestinal pathology in malaria.

Cytokines kill malaria parasites during infection crisis: extracellular complementary factors are essential.

Malaria infection crisis, at which the parasitemia drops precipitously and the parasite loses infectivity to the mosquito vector, occurs in many natural malaria systems, and has not been explained. We demonstrate that in a simian malaria parasite (Plasmodium cynomolgi in its natural host, the toque monkey), the loss of infectivity during crisis is due to the death of circulating intraerythrocytic gametocytes mediated by crisis serum. These parasite-killing effects in crisis serum are due to the presence in the serum of cytokines tumor necrosis factor and interferon gamma, which are produced by the host as a result of the malaria infection. The killing activity of each cytokine is absolutely dependent upon the presence of additional, as yet unidentified factor(s) in the crisis serum.

Transmission blocking immunity in Plasmodium vivax malaria: antibodies raised against a peptide block parasite development in the mosquito vector.

One approach towards the development of a vaccine against malaria is to immunize against the parasite sexual stages that mediate transmission of the parasite from man to mosquito. Antibodies against these stages, ingested with the blood meal, inhibit the parasite development in the mosquito vector, constituting "transmission blocking immunity." Most epitopes involved in transmission-blocking immunity depend on the tertiary conformational structure of surface antigens. However, one of the transmission-blocking monoclonal antibodies we have raised against Plasmodium vivax reacts with a linear epitope on both asexual stages and gametes. This monoclonal antibody (A12) is capable of totally blocking development of the parasite in the mosquito host when tested in membrane feeding assays with gametocytes from P. vivax-infected patients. Immune screening of a P. vivax lambda gt11 genomic expression library with A12 led to the isolation of a clone to which was mapped the six-amino acid epitope recognized by A12. Antisera raised in mice against a 12-mer synthetic peptide containing this epitope coupled to bovine serum albumin not only had high titers of antipeptide antibodies as measured by enzyme-linked immunosorbent assay, but in addition recognized the same 24- and 57-kD parasite components as A12 on Western blots and reacted with the parasite by immunofluorescence. When tested in membrane feeding assays, these antibodies have significant suppressive effects on parasite development in the mosquito.

Cloning and characterisation of a gene from Plasmodium vivax and P. knowlesi: homology with valine-tRNA synthetase.

We have previously described a lambdagt11 clone detected by immune screening with a monoclonal antibody (mAb) A12. This mAb is capable of completely blocking Plasmodium vivax transmission in the mosquito vector. An epitope recognised by A12 was mapped to six amino acids (aa) within the translated sequence of this clone. Here, we describe the complete sequence of the gene within which we mapped this epitope. Surprisingly, the translated sequence of the full-length open reading frame shows homology with that of valine-tRNA synthetases (Val-tRS) from other organisms. DNA cross-hybridisation with several of these species was observed by Southern blot. In addition, the corresponding gene has been obtained from the closely related simian malaria parasite, P. knowlesi. The two aa sequences show 66% identity and yet are very divergent from other Val-tRS sequences, apart from conserved blocks related to functional activity. Multiple sequence alignments reflect this dichotomy, as do predicted differences in antigenicity.

Plasmodium vivax merozoite surface protein 1 C-terminal recombinant proteins in baculovirus.

Recombinant proteins derived from the Plasmodium vivax merozoite surface protein 1 have been produced in the baculovirus expression system. These proteins correspond approximately to the Plasmodium vivax analogs of the 42-kDa or 19-kDa C-terminal processing products previously described for Plasmodium falciparum. Each was produced in two versions, either as a membrane-bound entity located on the cell surface and probably carrying a glycosylphosphatidylinositol addition, or as a secreted entity lacking a membrane anchor. Many native conformational epitopes appear to be accurately reproduced in these molecules. Both the 42-kDa and 19-kDa analogs can be N-glycosylated in the baculovirus system and the N-glycosylation appears to be necessary for efficient secretion of both the 42-kDa and 19-kDa recombinant proteins.

Anti-parasite effects of cytokines in malaria.

Cytokines induced during natural malaria infections, e.g., at crisis of a blood infection of Plasmodium cynomolgi, and during clinical paroxysms in human Plasmodium vivax infections, mediate killing of intra-erythrocytic blood stage malaria parasites. These cytokines, TNF and IFN-gamma, require additional, yet unidentified complementary factors that are present in "crisis" and "paroxysm" serum to kill intra-erythrocytic blood stage parasites. In contrast, cytokines, (mainly IFN-gamma) are able to effect killing of intra-hepatic stages of the parasite by themselves independent of serum complementary factors, suggesting that the mechanisms of killing may be different with respect to the two parasite stages. Cytokines also appear to be critical intermediates in mechanisms of clinical disease in malaria. Serum cytokine (TNF) levels and killing effects on blood stage malaria parasites were lower in patients who were exposed to endemic P. vivax malaria who had partial clinical immunity, than in non-immune patients. Evidence suggest that individuals acquire natural immunity to the disease by avoiding the induction of high levels of cytokines and complementary factors.

Human immune responses against sexual stages of malaria parasites: considerations for malaria vaccines.

Studies on the natural immune responses to the sexual stages of malaria parasites have been reviewed in the context of human malaria transmission-blocking vaccines. Antibodies against the sexual stages of the malaria parasite, gametocytes and gametes, are readily evoked by natural malaria infections. These antibodies that suppress infectivity at high concentrations can, at low concentrations, enhance the development of the parasite in the mosquito; however, because enhancing antibodies are prevalent during natural malaria infections, it is likely that a vaccine would rapidly boost these antibodies to blocking levels. The immunogenicity of sexual stage antigens appears to be constrained in the human host, probably due to T epitope polymorphism and MHC restriction in humans. These constraints apply mainly to those antigens that are sensitive targets of host immunity such as the gamete surface antigens and not to internal gamete antigens, indicating that antigenic polymorphism may have evolved in response to immune selection pressure. Evidence for immunosuppression of the host by exposure to endemic malaria is presented and its consequences on vaccine development are discussed.

Filarial infection of the breast.

The breast is a common site of filarial infection in females in Sri Lanka. We report our experience with 13 cases of filarial breast nodules, 12 containing adult worms and the other only microfilariae. In five of these cases the species was identified as Wuchereria bancrofti.

Enterobius vermicularis in ectopic sites.

We document six cases in which tissues were invaded by Enterobius vermicularis. These cases illustrate several mechanisms whereby the worms form granulomata in ectopic sites. In three cases, the worms passed through pre-existing breaches in the intestinal mucosa. In one case, a gravid worm migrated via the female genital tract to ther peritoneal cavity. In two other cases, male worms were found on the outer surface of the intestine, suggesting active penetration of the intestinal wall.

Diversity of Plasmodium vivax-induced antigens on the surface of infected human erythrocytes.

Antigens were demonstrated on the surface of Plasmodium vivax schizont-infected erythrocytes by an indirect immunofluorescence test using fresh unfixed infected erythrocytes from acute vivax malaria patients. Surface immunofluorescence was used to show that sera of P. vivax-infected individuals contain antibodies directed against these surface antigens. Thirteen different isolates were screened for reactivity of surface antigens with a panel of 8 heterologous human immune sera and an immune rabbit serum. Surface immunofluorescence was detected in several isolates with some but not all the human sera, and not all sera reacted with the "positive" isolates. These results indicate a high degree of polymorphism of the surface antigens of different P. vivax isolates. Sera from patients who had suffered multiple malaria attacks and the immune rabbit serum (which was raised by immunizing with 7 different isolates) recognized surface antigens on more isolates than sera from patients who had experienced only one attack of malaria, indicating that repeated exposure to the disease confers immunity against a spectrum of variants of a polymorphic malarial antigen(s) prevalent in nature.

Microfilarial granuloma of the breast in a patient with tropical pulmonary eosinophilia.

Microfilariae found in a breast nodule of a patient with tropical pulmonary eosinophilia were identified as Wuchereria bancrofti, confirming that the tropical pulmonary eosinophilia syndrome may be associated with infections caused by this species of filarial worm.

Infectious reservoir of Plasmodium vivax and Plasmodium falciparum malaria in an endemic region of Sri Lanka.

The infectious reservoir of Plasmodium vivax and P. falciparum in a malaria endemic region in Sri Lanka was defined in a population of 3,625 by directly feeding mosquitoes on a sample of infected individuals during a period of 17 months. The malaria case incidence in this population was concurrently monitored. P. vivax gametocyte densities were highest in the youngest age groups, and decreased steadily with increasing age. However, the infectivity per gametocyte appeared to be lower in the younger age groups than in the older ones. There was no significant correlation between the age of patients and their gametocyte densities for P. falciparum, to which this population was only recently exposed, nor was there a discernible trend in the infectivity per gametocyte in different age groups. The average infectivity of patients was lowest in the youngest (0-5 years) and the oldest (greater than 50) age groups. The contribution made by P. vivax patients in the different age groups to the reservoir of infection was estimated. Patients in the 6-25 year age groups made the largest contribution to the reservoir, followed by those in the 26-50 year age group. Patients in the youngest and the oldest age groups contributed least to the infectious reservoir. When population sizes in the different age groups were taken into consideration, the age groups between 6 and 50 years contributed almost equally to approximately 87% of the infectious reservoir. The reservoir of P. falciparum malaria was very small, being confined to 9% of the patients, and this appears to be a characteristic of epidemic malaria, as was the case with P. falciparum.

Demonstration of anti-disease immunity to Plasmodium vivax malaria in Sri Lanka using a quantitative method to assess clinical disease.

Clinical immunity to malaria was studied by quantifying the intensity of symptoms as well as by measurement of several hematologic indicators of pathology (the erythrocyte sedimentation rate [ESR], serum bilirubin, reticulocyte count, plasma tumor necrosis factor-alpha [TNF-alpha], and blood glucose levels) in 39 Plasmodium vivax malaria patients exposed to endemic malaria in southern Sri Lanka, and for comparison in 43 nonimmune patients who were residents of nonmalarious regions of the country. The intensity of 11 symptoms was scored numerically in all patients using a questionnaire. This clinical score was validated by introducing internal controls to the questionnaire, and by correlating it with the underlying pathology. Both the intensity of clinical disease as well as the degree of underlying pathology were found to be significantly lower in endemic area patients (mean clinical score = 8.8, median ESR = 8 mm) compared with the nonendemic area patients (mean clinical score = 19.0, median ESR 31.5 mm). Endemic area patients also had lower parasite densities (mean = 0.06%) than those from the nonendemic area (0.12%) (P < 0.05). However, at any parasite density, both clinical disease and pathology were significantly less in the endemic area patients (P < 0.001, for both clinical score and ESR), indicating that the clinical immunity seen in the endemic area patients was a true tolerance of parasites. Although plasma TNF-alpha levels were elevated in both groups of patients, they were significantly higher in the nonendemic area patients than in patients from the endemic area (P < 0.01). Furthermore, at comparable levels of plasma TNF-alpha, nonendemic area patients had both a higher intensity of clinical disease and an underlying pathology than those from the endemic area, suggesting that if TNF-alpha is indeed a mediator of clinical disease, the endemic area patients may be tolerant to its effects. Hypoglycemia was not observed in any of these P. vivax patients despite some with high levels of plasma TNF-alpha.

Rosetting: a new cytoadherence property of malaria-infected erythrocytes.

Plasmodium fragile infection of the toque monkey is a natural host-parasite association in which parasite sequestration occurs as during P. falciparum infection of humans. We have studied parasite sequestration of P. fragile and demonstrated the existence of a new property of cytoadherence of infected erythrocytes, "rosetting," which is defined as the agglutination of uninfected erythrocytes around parasitized erythrocytes. Rosetting in vitro and sequestration in vivo appear simultaneously as the parasite matures. The spleen plays a role in modulating cytoadherence; both sequestration and rosetting, which occur with cloned parasites from spleen-intact animals, are markedly reduced in splenectomized animals infected with parasites derived from the same clone. Sequestration and rosetting can be reversed by immune serum. Protease treatment of infected blood abolishes rosetting; however, if treatment is performed at an early stage of schizogony, rosetting reappears if parasites are allowed to further develop in the absence of protease. These results indicate that with P. fragile in its natural primate host, rosetting and sequestration are related to the presence on the infected erythrocyte surface of a parasite-derived antigenic component, the expression of which is modulated by the spleen.

Uninfected erythrocytes form "rosettes" around Plasmodium falciparum infected erythrocytes.

The human malaria parasite, P. falciparum, exhibits cytoadherence properties whereby infected erythrocytes containing mature parasite stages bind to endothelial cells both in vivo and in vitro. Another property of cytoadherence, "rosetting," or the binding of uninfected erythrocytes around an infected erythrocyte, has been demonstrated with a simian malaria parasite P. fragile which is sequestered in vivo in its natural host, Macaca sinica. In the present study we demonstrate that rosetting occurs in P. falciparum. Rosetting in P. falciparum is abolished by protease treatment and reappears on further parasite growth indicating that, as in P. fragile, it is mediated by parasite induced molecules which are protein in nature. P. vivax and P. cynomolgi, which are not sequestered in the host, did not exhibit rosetting. Rosetting thus appears to be a specific property of cytoadherence in malaria parasites.

Immunity to malarial antigens on the surface of Plasmodium falciparum-infected erythrocytes.

An indirect immunofluorescence test with fresh non-fixed infected blood as antigen was used to show that antibody in human sera from the Gambia recognized antigens on the surface of Plasmodium falciparum-infected human erythrocytes. Surface immunofluorescence was detected on 90% of erythrocytes infected with trophozoites and schizonts produced in continuous culture of isolates from the Gambia (FCR 3/K+), Brazil and Thailand. Fluorescence was equally strong with a Gambian parasite clone (FCR 3/K-) that lacked knobs, an ultrastructural modification of the erythrocyte membrane associated with parasite sequestration. Immunofluorescence could not be detected with an isolate from Uganda. The surface antigenicity of parasitized erythrocytes was eliminated by chymotrypsin and trypsin treatment. Fluorescence was specific for the surface of trophozoite- and schizont-infected cells on the condition that fresh erythrocytes were added to cultures every 4-5 days (subculture); if fresh erythrocytes were not added for over 2 weeks, a large percentage of non-infected erythrocytes also bound antibody. Normal erythrocytes incubated with media from these cultures also gave positive surface immunofluorescence. Thus, there are two types of antigenicity on erythrocytes: one expressed on infected erythrocytes and another passively absorbed from media to normal erythrocytes when parasites are not subcultured for long periods.

The impact of repeated malaria attacks on the school performance of children.

The impact of repeated malarial infections on the school performance of children was investigated in 571 school children 6-14 years of age in a malaria-endemic area in southern Sri Lanka where both Plasmodium falciparum and P. vivax infections are prevalent. Malaria infections confirmed by microscopy were monitored over a six-year period. School performance was assessed by two specially designed, school grade-specific, test papers for Sinhala language and mathematics. The scores for Sinhala language and mathematics for each school term test for the year 1997 were obtained. Malarial infections were a major predictor of children's performance in language and mathematics after controlling for parent's education, monthly family income, and house type. The education of the father predicted language scores but not mathematics scores. A child who experienced more than five attacks of malaria scored approximately 15% less than a child who experienced less than three attacks of malaria. The data suggest that repeated attacks of malaria have an adverse impact on the school performance of children.

Characteristics of malaria transmission in Kataragama, Sri Lanka: a focus for immuno-epidemiological studies.

Parasitological and entomological parameters of malaria transmission were monitored for 17 months in 3,625 residents in a Plasmodium vivax malaria endemic region in southern Sri Lanka; the study area consisted of 7 contiguous villages where routine national malaria control operations were being conducted. Malaria was monitored in every resident; fever patients were screened and 4 periodical mass blood surveys were conducted. An annual malaria incidence rate of 23.1% was reported during the period: 9.3% was due to P. vivax and 13.8% was due to P. falciparum; there had been a recent epidemic of the latter in this region, whereas the P. falciparum incidence rate in the previous 10 years had been negligible. There was a wide seasonal fluctuation in the malaria incidence, with the peak incidence closely following the monsoon rains. The prevalence of malaria due to both species detected at the 4 mass blood surveys ranged from 0.98% (at low transmission) to 2.35% (at peak transmission periods). Adults and children developed acute clinical manifestations of malaria. Entomological measurements confirmed a low degree of endemicity with estimated inoculation rates of 0.0029 and 0.0109 (infectious bites/man/night) for P. vivax and P. falciparum, respectively. Several anopheline species contributed to the transmission, and the overall man biting rates (MBR) showed a marked seasonal variation. Malaria at Kataragama, typical of endemic areas of Sri Lanka, thus presents characteristics of "unstable" transmission. Malaria was clustered in the population. There was a low clinical tolerance to P. falciparum malaria, to which most had only been at risk, compared to P. vivax, to which most had had a life-long exposure.