Sequential expression of antigens on sexual stages of Plasmodium falciparum accessible to transmission-blocking antibodies in the mosquito.

Plasmodium falciparum gametocytes contain specific antigens, some of which (Mr 230,000, 48,000, 45,000) are expressed on the surface of the newly emerged macrogamete. A different antigen (Mr 25,000) surrounds the surface of the ookinete and, although present to some extent in the developing gametocyte, is synthesized in high quantities by the macrogamete/zygote and expressed progressively on the transforming zygote surface. These antigens are targets of transmission blocking antibodies that are effective at two distinct points after gametogenesis: fertilization of the macrogamete and ookinete to oocyst development. The antigens involved in the fertilization blockade are the Mr 48 and 45 proteins, which are expressed on the macrogamete surface. The Mr 230 K coprecipitating protein probably plays no part in transmission block. mAb directed against the Mr 25 K ookinete surface protein blocked transmission without inhibiting ookinete formation, indicating that this protein has an important role in the transformation of ookinete into oocyst. A combination of mAb recognizing different epitopes on the same protein molecule acted synergistically in inhibiting oocyst formation. Using a mixture of two blocking mAb reacting against the Mr 48/45 and 25 K proteins, respectively, an additive blocking effect could be demonstrated.

Immunogold determination of Plasmodium falciparum circumsporozoite protein in Anopheles stephensi salivary gland cells.

The distribution of circumsporozoite (CS) proteins of Plasmodium falciparum sporozoites was observed during the passage of mature sporozoites in the hemocoel of Anopheles stephensi and during their entrance and sojourn in the salivary gland cells (SGC). The CS protein was visualized using a monoclonal antibody (3SP2) and immunogold labeling on ultrathin cryosections. In the hemocoel the sporozoites cease synthesizing CS protein, and some of it is shedded resulting in a patchy labeling pattern on the outer pellicular membrane. No internal labeling was observed. The sporozoites enter the SGC by puncturing the basal or lateral membrane. Inside the SGC, CS protein synthesis is turned on again; the Golgi system, nuclear envelope and all 3 pellicular membranes contain CS immunoreactivity. In the last phase of maturation, micronemes display abundant CS immunoreactivity. Rhoptries also show some immunogold labeling, but not as much as the micronemes.

Immunogold localization of circumsporozoite protein of the malaria parasite Plasmodium falciparum during sporogony in Anopheles stephensi midguts.

The occurrence of the circumsporozoite (CS) proteins of Plasmodium falciparum sporozoites was monitored during sporogonic development in Anopheles stephensi mosquitoes. Using a monoclonal anti-CS protein antibody (3Sp2) and immunogold labeling on ultrathin cryosections it was found that CS protein is synthesized in immature oocysts from day 6 onwards when there are not yet signs of sporozoite formation. The CS protein is rapidly incorporated in the oocyst plasmalemma, which subsequently invaginates into the parasite. In the oocyst only the external sporozoite membrane contains CS protein. The inner pellicle membranes, rhoptries and micronemes do not react with monoclonal antibody (MoAb) 3Sp2.

Malaria sporozoite penetration. A new approach by double staining.

To determine, whether a sporozoite is outside the hepatocyte membrane or internalized, a double staining test was carried out using, successively, antibody labeled with peroxidase and fluorescein. This test permits the quantification of sporozoite entry and outline sporozoite-hepatocyte interactions.

Identification of plasma membrane proteins involved in the hepatocyte invasion of Plasmodium falciparum sporozoites.

To determine whether surface proteins of hepatocytes might be involved in the sporozoite invasion, plasma membrane proteins were prepared from human livers with CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulphonate) and radiolabelled with 125I (Iodogen; 1,3,4,6-tetrachloro-3 alpha,6 alpha-diphenylglycoluril). The labelled proteins were incubated with Plasmodium falciparum sporozoites and cross-linked with DSP (dithio-bis-succinimidylpropionate). Radiolabelled proteins released by reduction after repeated washing of the sporozoite-complex were separated by SDS-PAGE and autoradiographed. Two human hepatocyte membrane proteins of 20 and 55 kDa were found to be involved in the initial binding of P. falciparum sporozoites. The electrophoretically purified 20- and 55-kDa proteins both inhibited the binding of the corresponding radiolabelled proteins to P. falciparum sporozoites and reduced the invasion of sporozoites in an in vitro assay. We propose that these 20-kDa and 55-kDa proteins represent putative human hepatocyte receptors for P. falciparum sporozoite invasion.

Phase I clinical trial of a recombinant malaria vaccine consisting of the circumsporozoite repeat region of Plasmodium falciparum coupled to hepatitis B surface antigen.

R16HBsAg is an experimental recombinant malaria vaccine consisting of 16 repeats of a four amino acid sequence (Asn-Ala-Asn-Pro or NANP) of the circumsporozoite (CS) protein of Plasmodium falciparum expressed as a fusion protein with the recombinant hepatitis B virus surface antigen (HBsAg) produced by yeast cells. Twenty male volunteers were experimentally vaccinated with the product, as well as with two doses of the commercial recombinant HBsAg vaccine Engerix B (Smith Kline Beecham Biologicals, Rixensart, Belgium) at intervals during a period of 18 months. No serious side effects were observed. Circulating antibodies to recombinant CS antigen (R32tet32) developed in all volunteers and persisted in most cases over ten months. Anti-HBs antibody production was poor initially, but a single dose of the commercial hepatitis B vaccine was sufficient to elevate these titers to high levels in all but two volunteers.

Detection of falciparum malarial forms in naturally infected anophelines in Cameroon using a fluorescent anti-25-kD monoclonal antibody.

Anopheles gambiae s.s. and An. funestus were sampled in houses located in a Plasmodium falciparum-holoendemic site in southern Cameroon. The midguts of female mosquitoes in half-gravid or gravid stages of blood digestion were incubated with a fluorescent monoclonal antibody directed against the P. falciparum zygote/ookinete surface protein Pfs25 and examined using a fluorescent light microscope. Malarial forms were detected in 11.6% of the half-gravid mosquitoes and in 0.0% of the gravid ones (P = 0.012). No difference in infections or the occurrence of malarial forms between An. gambiae and An. funestus was observed. Overall, 127 malarial forms were counted and distributed among round forms, retorts, and ookinetes in 77.2%, 9.5%, and 13.4%, respectively. Round forms include macrogametes, activating microgametocytes, and zygotes. The mean number of malarial forms per infected midgut was 2.16 and the maximum number observed was 13. In four anophelines, round forms, retorts, and ookinetes were simultaneously observed. Sporozoite rates were 5.7% for An. gambiae and 3.8% for An. funestus. In the human population, the gametocyte index for P. falciparum was 38% with a mean density of 1.11 gametocytes per microliter of blood. Differences concerning malarial forms in mosquito midguts were observed between houses (range percentage = 4.7--21.3%; mean range of forms per positive anopheline = 1.1--3.1). In each house, relationships existed between infected vectors and the gametocyte reservoir of their inhabitants. The role in transmission of people with very low gametocytemia, approximately one per microliter, as a reservoir of falciparum malaria in highly endemic areas, is emphasized.

Testing for anti-circumsporozoite and anti-blood-stage antibodies for epidemiologic assessment of Plasmodium falciparum infection in travelers.

The purpose of this investigation was to assess the role of serology for establishing incidences of Plasmodium falciparum malaria and of exposure to P. falciparum in epidemiologic studies of travelers using chemoprophylaxis. The design was a prospective cohort study involving 548 short-term Dutch travelers to areas endemic for P. falciparum malaria. Sera were collected before departure and, together with the medical history, 2-6 weeks after return. All sera were tested for anti-circumsporozoite (CS) antibodies by an R32tet32-ELISA; sera of subjects reporting febrile illness during travel or after return or with anti-CS responses were tested for anti-blood-stage antibodies by an indirect fluorescence antibody test (IFAT). Five subjects (0.9%) reported P. falciparum malaria confirmed by thick blood smear examination (documented cases) and six (1.0%) reported treatment for malaria without a documented diagnosis (presumptive cases). Conversions in the IFAT were detected in six subjects, including all five documented cases and one presumptive case. Anti-CS antibodies were detected in seven subjects (1.3%), including three documented cases and four of 442 subjects with no history of fever or malaria treatment (0.9%). Incidence rates per 1,000 person-months of travel (95% confidence interval) of infection with P. falciparum, whether or not suppressed by chemoprophylaxis, were 16.9 (8-31) for all destinations and 91.6 (33-200) for West Africa. In epidemiologic studies of P. falciparum malaria in travelers, testing for antibodies to blood stages can increase the sensitivity and specificity of case detection; testing for antibodies to sporozoites may be useful for the assessment of exposure to P. falciparum in travelers using chemoprophylaxis, but the sensitivity is limited.

Cellular response against exoerythrocytic forms of Plasmodium berghei in rats.

Rats were infected with Plasmodium berghei sporozoites, and 47, 51, and 57 hr later exoerythrocytic parasites were examined by electron microscopy. At 47 hr, approximately 30% of nearly mature exoerythrocytic parasites were degenerating and were surrounded by a cellular infiltrate of Kupffer cells, monocytes, monocyte-derived macrophages, and neutrophils. Neutrophils appeared to be actively ingesting electron-dense fuzzy parasite material which was normally present in the parasitophorous vacuole. By 51 hr other mononuclear cells penetrated with filopodia between the host hepatocyte and exoerythrocytic parasite, and directly into the exoerythrocytic parasite. Exoerythrocytic parasites that formed merozoites at 51 hr lacked any notable cellular infiltration.

Effect of chloroquine chemoprophylaxis during pregnancy on birth weight: results of a randomized trial.

To determine the effect of chloroquine chemoprophylaxis during pregnancy on birth weights, a randomized trial was carried out in 1987 and 1988 in Banfora, Burkina Faso (West Africa). Seven hundred forty-five randomly selected women treated with chloroquine sulfate were compared to with 719 controls who received no treatment. In spite of an unquestionable effect of chloroquine in preventing placental infection (4.1% infected placentas in the treated group versus 19.0% in the controls), the mean difference in birth weights between the two groups (6 g) was not significant. The difference in the proportion of low birth weight (LBW) newborn babies in two groups (16.3% versus 16.4%) was also not significant. However, there was a strong relationship between placental infection and birth weight (the mean birth weight difference between infected and uninfected placentas was 113 g, and the proportion of LBW babies was 26.0% in infected placentas versus 14.8% in uninfected placentas). The small difference in birth weights observed between the two groups may be due to the fact that the prevalence rate of placental infection is low and that prophylaxis is effective only on a portion of the subjects in the treated group. It may also indicate that malaria is only one of several risk factors responsible for LBW. The relatively small increase in birth weight, the expected poor acceptance of mass prophylaxis, and the spreading of chloroquine-resistant Plasmodium strains should be considered before extending malaria chemoprophylaxis to all pregnant women. It might be worth considering to limit prophylaxis to primigravidae.

Effect of the sickle cell trait status of gametocyte carriers of Plasmodium falciparum on infectivity to anophelines.

Insect-reared Anopheles gambiae were experimentally fed with the blood of naturally infected human volunteers carrying gametocytes of Plasmodium falciparum. Infection of at least one mosquito was successful in 86 experiments. For these gametocyte carriers, the hemoglobin types studied were AA (normal, n = 77), AS (heterozygous sickle cell, n = 8), and SS (homozygous sickle cell, n = 1). The mean of the percentages of infected mosquitoes by gametocyte carriers of AS hemoglobin was almost double that of carriers of AA: 30.4% versus 17.5%. The genetic protection in humans conferred by the beta(s) gene in its heterozygous form seems to be associated with an increasing effect on P. falciparum transmission from humans to mosquitoes. The epidemiologic and evolutionary aspects of this finding are discussed.

Inhibition of Plasmodium berghei liver schizont development and reduction of cytokine production capacity in rats by dietary fish oil supplementation.

Experimental primary infection with Plasmodium berghei in rats is known to be influenced by several cytokines. Dietary supplementation of n-3 fatty acids has been shown to influence cytokine production capacity and to protect mice from cerebral malaria. We investigated the effect of dietary fish oil (FO) supplementation on cytokine and nitric oxide production and liver schizont development in male brown Norway rats. Control groups were fed either a corn oil-supplemented diet (CO) or standard lab chow (LC). After six weeks on either diet, rats given supplementary FO had a significantly lower production of interleukin-1 (IL-1) and IL-6 after stimulation with lipopolysaccharide, and also had significantly lower numbers of liver schizonts compared with CO- or LC-fed animals. We conclude that in rats, an FO-supplemented diet reduces the production capacity of IL-1 and IL-6 and inhibits schizont development after intravenous inoculation of P. berghei sporozoites. Fish oil did not influence nitric oxide production by peritoneal macrophages.

Malaria transmission-blocking activity in experimental infections of Anopheles gambiae from naturally infected Plasmodium falciparum gametocyte carriers.

Experimental infections of anopheline mosquitoes were carried out with Plasmodium falciparum gametocytes from 65 naturally infected patients in Cameroon. A comparison was made between infections with blood containing autologous plasma and blood in which the plasma was replaced with plasma from a donor without previous malaria exposure. A lower infection rate was observed in 50 of 65 autologous plasma samples. Transmission was significantly blocked in 3 infections. This indicates that, in a population living in an area endemic for malaria, blood plasma factor(s) can reduce the transmission capacity of gametocyte carriers to mosquitoes.

Effect of gametocyte sex ratio on infectivity of Plasmodium falciparum to Anopheles gambiae.

Insectary-reared Anopheles gambiae were experimentally fed with the blood of 90 naturally infected human volunteers carrying gametocytes of Plasmodium falciparum. At least one mosquito was successfully infected in 74% of experiments. The probability that a gametocyte carrier was infective, the probability that a mosquito became infected, and the number of oocysts harboured were related to gametocyte density. The mean proportion of male gametocytes was 0.217 (i.e., 3.6 females for every male). Sex ratios differed significantly between gametocyte carriers. Variation in sex ratio was not related to the probability that a gametocyte carrier was infective. Among infective people whose sex ratio estimates were based on a reasonable number of gametocytes, sex ratio significantly predicted the proportion of infected mosquitoes and mean oocyst load, with infectivity rising as the proportion of the male gametocytes increased towards 50%. There was no indication that infectivity reached a peak at some intermediate sex ratio, as would be expected if random mating of gametes was the primary determinant of fertilization success. These results raise 2 interesting questions: why should higher sex ratios be more infective, and why is the observed population sex ratio lower than that which produces the greatest infectivity?

The production of mature gametocytes of Plasmodium falciparum in continuous cultures of different isolates infective to mosquitoes.

In vitro gametocytogenesis of Plasmodium falciparum was observed in all 22 isolates established in this laboratory. Gametocytes were produced in variable numbers--up to 3% of red cells--for a limited period of time after which this stage was seen only very sporadically. Complete maturation of microgametocytes in vitro was obtained in all 14 of the isolates that were tested for exflagellation. Up to 88.2% of membrane-fed Anopheles stephensi were infected from material produced in culture. It was also possible to infect A. gambiae and A. freeborni. Addition of fresh red cells and serum to culture material promoted infectivity of gametocytes. Gametocyte infectivity declined rapidly with time in the membrane feeders held at 38 degrees C.

Study of the distribution of circumsporozoite antigen in Anopheles gambiae infected with Plasmodium falciparum, using the enzyme-linked immunosorbent assay.

Anopheles gambiae, experimentally infected with Plasmodium falciparum, were dissected 14 days later for microscopical detection of sporozoites and oocysts. The head, salivary glands, thorax, midgut, legs, ovaries, Malpighian tubules, the remainder of the abdominal tissues and the dissection fluid of each mosquito were examined by a two-site enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of circumsporozoite antigen (CS ag). 19 mosquitoes had CS ag in at least one of the specimens examined. Very large individual variability was observed in the presence and/or quantity of CS ag in the various parts. 7 mosquitoes were ELISA-positive in all 9 specimens; the salivary glands and thorax contained most CS ag, whereas the Malpighian tubules and ovaries contained the least; all the thoraces contained CS ag, even that of one mosquito of which the salivary glands lacked both sporozoites and CS ag; of 17 ELISA-positive salivary glands, 15 were found to contain sporozoites. The existence of free antigen associated with sporozoites, and the limitations of the ELISA technique in demonstrating the infectivity of a malaria vector, are discussed.

The disappearance of Dutch malaria and the Rockefeller Foundation.

Sixty years ago Professor Nico Swellengrebel wrote his famous book 'Malaria in the Netherlands' (Swellengrebel and de Buck, 1938). At that time tertian malaria was still endemic, with its epidemic ups and downs. Malaria disappeared as recently as 1960 and the Rockefeller Foundation (RF) contributed substantially to this effect. The Rockefeller Archives proved a valuable source of anecdotal information, which puts the scientific publications of the Dutch malariologists in a more vivid perspective. Following the course of history, first the already existing links with the RF are explained along with some peculiarities of tertian malaria in the Dutch temperate climate. The emergence of a new epidemic during the war years and the implication of new tools and principles for control as advocated by the RF are described. The subsequent shriveling of the vector population and the disappearance of malaria are presented, along with some details about the reluctance of WHO to declare the Netherlands malaria-free. Finally, recent unrest about possible return of malaria is put into perspective.

The Dutch school of malaria research.

An epidemic of tertian malaria in some coastal areas of The Netherlands resulted in the setting up of official measures in 1920. A scientific and a propaganda commission were charged with control. Efforts were made to reduce mosquito populations by adult and larval spraying. After the discovery that infected mosquitoes were to be found only inside houses, control operations were focussed against adult mosquitoes. Some later discoveries resulted in a more effective control. a) Spraying ditches with Paris green did not prevent adult mosquitoes from entering the control area. b) Anopheles maculipennis turned out to be a complex of species, with A. atroparvus as the vector. The latter preferred brackish water and did not go into full hibernation. The closing of the Zuyder Sea and the expected desalinization gave hope for less suitable conditions for the vector. c) Plasmodium vivax normally had an incubation period of 8 months. d) Pyrethrum was an effective but short-lasting insecticide. e) Healthy parasite carriers could infect mosquitoes. This knowledge was applied through an extensive system of investigation, including spleen examination of schoolchildren. Suspected houses were sprayed bimonthly from August to November, during which period infected mosquitoes were likely to be present. This system worked extremely well, and during the next epidemic from 1943 to 1947 the thus treated towns remained virtually free of malaria! DDT became available and was either sprayed in suspected houses as before, or through wide-spread coverage of all houses. The epidemic subsided whatever method employed and not only due to the use of DDT. The number of cases even went down to the point of no return and the last case of Dutch malaria was recorded in 1959. The wealth of experience on house-spray control, parasite and mosquito biology and experimental malaria of the Dutch malariologists has had its impact on the international bodies engaged in the battle against malaria.

Malaria transmission-blocking activity in the plasma of Plasmodium falciparum gametocyte carriers in Cameroon.

Experimental infections of Anopheles gambiae were carried out with Plasmodium falciparum gametocytes from 65 naturally infected patients in Cameroon. A comparison was made between infections with blood containing autologous plasma and blood in which the plasma was replaced by plasma from donors without previous malaria exposure. A lower mosquito-infection rate was observed in 50 out of 65 autologous plasma samples. The transmission was completely blocked in 8 infections, whilst belonging exposures to heterologous plasma led to infected mosquitoes. Evidence is shown that blood plasma factors of gametocyte carriers from a population living in a malaria-endemic area are able to reduce transmission capacity.

Comparison of serological tests and bio-assay for malaria transmission blocking capacity in field sera.

Competition ELISAs have been developed for natural transmission blocking antibodies. Approximately 50% of the sera blocking in the conventional mosquito feeding experiments, gave positive results in these competition ELISAs. Attempts to adapt competition ELISAs to a field application have been partly successful.